M. Gooz - Chronic Kidney Disease-Intech (2012)
M. Gooz - Chronic Kidney Disease-Intech (2012)
M. Gooz - Chronic Kidney Disease-Intech (2012)
Edited by Monika Gz
Chronic Kidney Disease
Edited by Monika Gz
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Chronic Kidney Disease, Edited by Monika Gz
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Contents
Preface IX
Chapter 1 ADAM Proteases as Novel Therapeutic Targets
in Chronic Kidney Disease 3
Monika Gz
Chapter 2 Severity and Stages of Chronic Kidney Disease 13
Syed Ahmed and Gerard Lowder
Chapter 3 The New Kidney and Bone Disease: Chronic Kidney Disease
Mineral and Bone Disorder (CKDMBD) 25
Igor G. Nikolov, Ognen Ivanovski and Nobuhiko Joki
Chapter 4 The Prevalence of Renal Osteodystrophy in Chronic
Renal Failure Patients in Urban Niger Delta of Nigeria 47
U. R. Onyemekeihia, C. O. Esume,
E. Unuigbe, E. Oviasu, L. Ojogwu
Chapter 5 Relationships Among Renal Function,
Bone Turnover and Periodontal Disease 73
Akihiro Yoshihara and Lisdrianto Hanindriyo
Chapter 6 Sarcoidosis and Kidney Disease 87
Tulsi Mehta, Anirban Ganguli and Mehrnaz Haji-Momenian
Chapter 7 Origins of Cardiorenal Syndrome
and the Cardiorenal Connection 107
L. G. Bongartz, M. J. Cramer and J. A. Joles
Chapter 8 Sub-Types and Therapeutic
Management of the Cardiorenal Syndrome 123
Margot Davis and Sean A. Virani
Chapter 9 Atherosclerotic Renovascular Disease 149
Gen-Min Lin, Chih-Lu Han, Chung-Chi Yang
and Cheng-Chung Cheng
VI Contents
Chapter 10 Pharmacologic Adjuvants to Reduce Erythropoietin
Therapy Dose in Anemia of Chronic Kidney Disease
and End Stage Renal Disease 161
Adeel Siddiqui, Aqeel Siddiqui and Robert Benz
Chapter 11 Molecular Mechanisms of
Nephro-Protective Action of HE-86 Liquid
Extract in Experimental Chronic Renal Failure 175
Li-qun He, Dong Feixia, Qiang Fu and Jun Li
Chapter 12 The Effects of Asymmetric Dimethylarginine (ADMA),
Nitric Oxide (NO) and Homocysteine (Hcy) on Progression
of Mild Chronic Kidney Disease (CKD): Relationship
Between Clinical and Biochemical Parameters 197
A. Atamer, S. Alisir Ecder, Y. Atamer,
Y. Kocyigit, N. Bozkurt Yigit and T. Ecder
Chapter 13 Neutrophil Activation and Erythrocyte
Membrane Protein Composition in Stage 5
Chronic Kidney Disease Patients 209
Elsio Costa, Lus Belo and Alice Santos-Silva
Chapter 14 Assessing Iron Status in CKD Patients:
New Laboratory Parameters 225
Elosa Urrechaga, Lus Borque and Jess F. Escanero
Chapter 15 Exogenous Fluorescent Agents for
the Determination of Glomerular Filtration Rate 251
Raghavan Rajagopalan and Richard B. Dorshow
Chapter 16 Modern Surgical Treatments of
Urinary Tract Obstruction 261
Bannakij Lojanapiwat
Chapter 17 Extra-Anatomic Urinary Drainage
for Urinary Obstruction 281
Michael Kimuli, John Sciberras and Stuart Lloyd
Chapter 18 Percutaneous Nephrostomy 297
Rameysh D. Mahmood, Lee Yizhi and Mark Tan M.L.
Chapter 19 Unusual Vascular Access for
Hemodialysis Therapies 315
Cesar A. Restrepo V
Chapter 20 The Role of Nephron-Sparing Surgery
(NSS) for Renal Tumours >4 cm 329
Amlie Parisel, Frederic Baekelandt,
Hein Van Poppel and Steven Joniau
Contents VII
Chapter 21 Benign Prostate Hyperplasia
and Chronic Kidney Disease 347
Ricardo Leo, Bruno Jorge Pereira and Hugo Coelho
Chapter 22 Asymptomatic Bacteriuria (ASB),
Renal Function and Hypertension 377
Suzanne Geerlings
Chapter 23 Sleep Disorders Associated with
Chronic Kidney Disease 385
Robert L. Benz, Mark R. Pressman and Iqbal Masood
Chapter 24 The Allo-Immunological Injury
in Chronic Allograft Nephropathy 401
I. Enver Khan, Rubin Zhang,
Eric E. Simon and L. Lee Hamm
Chapter 25 Prevention and Regression of Chronic
Kidney Disease and Hypertension 415
Hiroyuki Sasamura
Chapter 26 Health-Related Quality of Life in Chronic Renal
Predialysis Patients Exposed to a Prevention
Program Medelln, 2007-2008 431
Carlos E. Yepes Delgado, Yanett M. Montoya Jaramillo,
Beatriz E. Orrego Orozco and Daniel C. Aguirre Acevedo
Preface
Chronic kidney disease is an increasing health and economical problem in our world.
Obesity and diabetes mellitus, the two most common cause of CKD, are becoming
epidemic in our societies. Education on healthy lifestyle and diet is becoming more
and more important for reducing the number of type 2 diabetics and patients with
hypertension. Education of our patients is also crucial for successful maintenance
therapy. There are, however, certain other factors leading to CKD, for instance the
genetic predisposition in the case of polycystic kidney disease or type 1 diabetes,
where education alone is not enough.
When the first angiotensin converting enzyme inhibitor, Captopril, was developed in
1975 it changed not only the treatment of hypertension, but of diabetic nephropathy
and other chronic kidney diseases. In the past forty years we did not have such a
breakthrough in the treatment of CKD. However, several valuable discoveries were
made which greatly enhanced our understanding of the role of nitric oxide and
mechanisms responsible for anemia and CKD-related bone diseases. Most certainly,
dialysis techniques have developed greatly over the past seventy years and have
become available for a wide range of people. Furthermore, advanced surgical
procedures and tools were developed in the past years to resolve ureteral obstructions
originating from stones or prostate hypertrophy. These modern techniques are
discussed in our book along with currently accepted procedures for kidney cancers.
How can we further improve the treatment of CKD patients? Besides prevention, the
most important aim would be to constantly look for, and try to understand the
mechanistic details of disease development and progression. Perhaps no other disease
is as complex and complicated as CKD since the symptoms result from the constant
interaction of multiple organ systems as is the case with cardiorenal syndrome, CKD-
related anemia, and bone diseases. Because of the interdisciplinary nature of the
disease, we need continuous communication between nephrologists, surgeons, and
basic scientists, since only our joint approach can lay down the foundation of the next
(bio)medical breakthrough. The chapters of our book introduce readers to this
enthusiastic approach.
I would like to thank all of our contributors for their valuable time and expertise and
for the high quality chapters which provide a greatly enjoyable reading experience. I
X Preface
also would like to thank my family and friends who supported me during the editing
process: my Mom and Dad, Pal and Adam, and Carol of course. I dedicate this book to
you.
Monika Gz, MD PhD
Medical University of South Carolina
Charleston, SC,
USA
1
ADAM Proteases as Novel Therapeutic Targets
in Chronic Kidney Disease
Monika Gz
Medical University of South Carolina, Charleston, SC
USA
1. Introduction
More than 20 million Americans suffer, and ultimately die, from chronic kidney disease
(CKD). Based on data from the National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK), the yearly cost of dialysis treatment of patients with end stage renal
disease (ESRD) is currently $35 billion [1], and this number is predicted to rise as the US
population ages and more people develop obesity, metabolic syndrome, and diabetes. CKD
is associated with progressive renal fibrosis and inflammation, and currently there is no cure
for the disease.
The most common primary illnesses which result in end stage renal disease (ESRD) are
diabetes (~37%), hypertension (~24%), glomerulonephritis (~15%), cystic kidney diseases
(~4.7%) and urologic diseases (2.5%) [1]. There were 111,000 new ESRD patients diagnosed
in 2007 and out of a total of ~500,000 ESRD patients 368,500 people received dialysis
treatment in the same year. Dialysis patients have poor quality of life due to high
hospitalization rate (458/1000 patients in 2008), high morbidity and mortality (~20%) [1].
Presently, kidney transplant is the only option for these patients to have a close to normal
life. According to the US Renal Data System 2010 [1] however, out of the ~85,000 patients
awaiting transplant about 18,000 will receive kidney since the amount of available organs
did not increase significantly above this number for several years.
Angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers
(ARBs) are widely used to attenuate the development of cardiovascular diseases and
support renal function in CKD patients. However, novel therapeutic targets are desperately
needed to effectively treat CKD and slow down disease progression.
Currently, there are about 2,000 clinical trials worldwide addressing some aspects and/or
co-morbidities of CKD [2]. These include treatment of anemia, hypertension, secondary
hyperparathyroidism, depression and inflammation among others. So far increasing
frequency and quality of dialysis did not show advantages in survival rate [2]. Similarly,
treatments targeting hypercholesterolemia [3] and hyperhomocysteinemia [4] or the usage
of statins [5] failed to increase significantly the survival of ESRD patients.
In recent years, we and others obtained exciting new data on the pathophysiological role of
the disintegrin and metalloenzyme ADAMs in renal fibrosis and CKD. This chapter is
dedicated to summerize these discoveries and discuss their significance and potential role in
the future treatment of patients with renal diseases.
Chronic Kidney Disease
2
2. Physiology of ADAMs and ADAMTS
ADAMs (a disintegrin and metalloenzymes) and ADAMTS (ADAMs with thrombospondin-
1-like domains) are membrane-bound multidomain proteins similar to snake venom
metalloenzymes and disintegrins. Both groups have pro-, metalloenzyme-like, disintegrin-
like and cysteine-rich domains, but compared to ADAMs ADAMTS do not possess
cytoplasmic or transmembrane regions. Catalytically active ADAMs are Zn
2+
-dependent
endopeptidases and are best known for their sheddase activity. They cleave epidermal
growth factor ligands, cytokines and their receptors, adhesion molecules and the infamous
amyloid precursor protein among others [6]. ADAMs participate in interreceptor crosstalk
between G protein coupled receptors (like angiotensin receptors [7], bradykinin receptors [8]
and serotonin receptors [9]) and members of the tyrosine kinase receptors (epidermal
growth factors receptor, tumor necrosis factor receptor) by shedding membrane-bound pro-
forms of tyrosine kinase ligands (Figure 1). ADAMs are indispensable for normal
development, cell proliferation and growth however, at the same time, they can drive
pathological cell division and inflammation and have major role in the development of
several proliferative and inflammatory diseases [8]. Some of the ADAMs have mutation in
their so-called hemopexin-domain (HEXXHXXGXXH) which is responsible for the Zn
2+
-
binding of the protein. These ADAMs are catalytically inactive and may have a role in cell-
matrix and cell-cell interactions rather than in proteolytic processes [11].
Fig. 1. ADAMs participate in inter-receptor crosstalk: triple membrane spanning signalling.
AII: angiotensin-II, BK: bradykinin; GPCR: G protein-coupled receptor; mGF: membrane-
bound growth factor, sGF: soluble growth factor; EGFR: epidermal growth factor receptor.
ADAM Proteases as Novel Therapeutic Targets in Chronic Kidney Disease
3
ADAMTSs are secreted proteins which anchor to extracellular matrix molecules through
their thrombospondin-1 domain [12] and are involved in proteolytic cleavage of
proteoglycans [13], and of the von Willebrand factor [14]. Both protein families can have
significant contribution to CKD progression.
2.1 Expression of ADAM enzymes in the normal kidney
There are several ADAM and ADAMTS proteins which expression was shown in the human
or murine kidney by various techniques. Histochemical analysis showed that ADAM9 was
expressed in the nephron: both in the glomerulus and in tubular epithelial cells [15].
Expression of a short form of the enzyme lacking the cytoplasmic region was also reported
in the kidney [16]. ADAM10 expression was first shown in chick kidney [17], in mouse
kidney of mesenchymal origin [18] and later in humans in the distal tubule, in the
connecting tubule, in the principal cells of the collecting duct and in the thick ascending
limb of Henle [19]. ADAM11, which is known as a disintegrin metalloenzyme primarily
expressed in the central and peripheral nervous system, was also expressed in the epithelial
cells of the collecting duct at a low level [20]. Since ADAM11 is differentially expressed
during development, it may have an important role in normal kidney morphogenesis. There
is also data on the expression of ADAM13 mRNA in the developing mouse kidney [21].
ADAM17 is a disintegrin metalloenzyme which is ubiquitously expressed in almost all
mammalian cells. It is present in the kidney [22] and its expression is upregulated in various
renal diseases in humans [23]. The mRNA of ADAM19 was present in developing human
kidney, and in the endothelial cells and in cell of the distal tubules of the adult kidney [23].
Expression of ADAM31, another proteolytically active disintegrin metalloenzyme was also
identified in the epithelium of the convoluted tubuli [24]. High mRNA level of mouse
ADAM33 was also shown in the kidney [25]. Since this protein is catalytically inactive, it
may have a role in cell-cell interaction and communication.
Of the ADAMTS proteins ADAMTS-1 is expressed at high levels in the adult mice kidney
[26], and in situ hybridization showed high level of ADAMTS-1 in the epithelia of the
developing kidney [27]. In the rat higher level of ADAMTS-1 was observed in the adult
animals compared to newborns, and expression pattern of the protease was restricted to the
renal medulla and the principal cells of the collecting ducts in the kidney [28]. ADAMTS-5
was observed in glomerular mesangial cells [29]. ADAMTS-9 [30] and ADAMTS-10 [18] are
highly expressed in the developing and adult kidney, respectively, similarly to human
ADAMTS-14, -15, -16 [31] with no known function at the present. ADAMTS-13 was shown
in healthy human kidney samples and in kidneys of patients with thrombotic
thrombocytopenic purpura by real-time PCR and immunohistochemistry. ADAMTS-13 was
present in the glomeruli as well as in the tubuli [32]. Also, various transcripts of ADAM16
were shown in the developing human and rat kidneys [33, 34].
2.1.1 ADAM and ADAMTS in kidney development - what we learned from knockout
studies
There is very few data available on the role of ADAMs and ADAMTS enzymes in kidney
development. There is evidence that expression pattern of ADAMTS-1 [27] and ADAM10
[35] and ADAM13 [21] changes in the kidney during development and that ADAMTS-9 is
Chronic Kidney Disease
4
highly expressed in the mesenchyme of the developing kidney [30]. However, as of present,
there is no detail about how knocking down ADAMs influence kidney development.
Targeted knockout of Adamts-1 in mice showed that the enzyme has an important role in
kidney development. Deletion of exon 2 (encoding part of the metalloenzyme domain)
resulted in lack of ADAMTS-1 protein in mice and high perinatal lethality of the animals
due to kidney malfunction [36]. In these animals both the cortical and medullary areas were
reduced with concomitant increase in the caliceal space. Another group found that lack of
the whole metalloenzyme domain (deletion of exon 2-4) rendered ADAMTS-1 catalytically
inactive which resulted in enlarged renal calices and fibrosis of the uteropelvic junction [37].
These animals also developed bilateral hydronephrosis and papillary atrophy shortly after
birth [38]. Since normally there is a high level of ADAMTS-1 expressed in the epithelium of
the collecting ducts and of the uteropelvic junction, and because the phenotype greatly
resembles to symptoms of the human uteropelvic obstruction, these animals can be good
models for this genetic disease.
These data also show that targeting strategies can greatly influence the evolving
phenotypes.
3. ADAMs and ADAMTSs in chronic kidney diseases
3.1 ADAMs in diabetic nephropathy
There is increasing evidence on the pathophysiological role of ADAM17 (TACE), ADAM19,
ADAMTS-13 in CKD.
ADAM17 is a most well-studied sheddase enzyme. It was originally identified as the tumor
necrosis factor (TNF)- converting (or activating) enzyme [22] or TACE. It cleaves cell
surface molecules, most importantly cytokines and growth factors [39]. By activating EGFR
ligands and TNF- ADAM17 has a central role in inflammatory and proliferative processes
both of which have crucial role in the development of CKD (Figure 2).
Fig. 2. Role of ADAM17 in CKD.
ADAM Proteases as Novel Therapeutic Targets in Chronic Kidney Disease
5
Besides initiating inflammation, TNF has important pathophysiological role in insulin
resistance (reviewed in [40]). After activation by ADAM17, the soluble homotrimer of TNF
activates the TNF receptor and downstream signaling molecules. Activation of the MAP
kinase pathway initiates serine phosphorylation of the insulin receptor substrate (IRS)
intracellularly. Being phosphorylated on serine inhibits tyrosine phosphorylation of the IRS
which results in insensitivity of the insulin receptor to extracellular insulin and contributes
the development of diabetes (Figure 3).
Fig. 3. Mechanism of TNF-induced insulin resistance
Chronic Kidney Disease
6
High glucose was also shown to promote heparin-binding growth factor (HB-EGF)
shedding through ADAM17 activation, however the exact mechanism is unknown [41].
Since ADAM17 activates secretion of TNF, pharmacological inhibitors of the enzyme were
tested on blood glucose regulation in animal model of non-obesity-related insulin resistance
(fructose-fed rats). ADAM17 inhibitor restored the animals insulin resistance [42]. In
another study, animals heterozygous for ADAM17 (+/-) proved to be relatively protected
from high-fat diet-induced obesity and diabetes [43].
A close structural relative of ADAM17, ADAM10 is involved in shedding of RAGE: receptor
for advanced glycation end products [44]. Since soluble RAGE can block pathophysiological
processes initiated by RAGE, ADAM10 activation may slow down development of diabetes.
As of today, we do not have data on the pathophysiological role of ADAMTS enzymes in
diabetes mellitus.
3.2 ADAMs in renal transplant dysfunction and ischemia reperfusion injury
In vitro studies modelling mechanisms of transplant rejection showed that the mRNA
expression of ADAM17 was upregulated in the kidney and that the protein expression of
the enzyme was localized next to TNF receptor II. This suggested that ADAM17 may
antagonize the effect of TNF by shedding of its receptor during transplant rejection and
therefore higher ADAM17 activity might be beneficial [45]. On the other hand, ADAM17
also co-localized with HB-EGF in experimental ischemia-reperfusion injury which suggested
that increased shedding of the growth factor may have contributed to the observed fibrotic
injury [46]. Pharmacological inhibitors targeting ADAM17 activity reduced renal tissue
injury associated with reperfusion. This confirmed that the increased enzyme activity was a
cause rather than the consequence of the tissue injury [47].
Another ADAM enzyme, ADAM19 was also implicated in allograft nephropathy however,
we do not know any mechanistic details of its actions [48].
3.3 ADAMs in renal fibrosis
Renal fibrosis is a manifestation of several pathological processes. Glomerular fibrosis can
be induced by over-activation of the renin-angiotensin system, and the developing fibrosis
and inflammation can be successfully attenuated by ADAM17 inhibitors in animal models
of the injury [7]. We showed previously that serotonin-induced mesangial cell proliferation,
which is an important component of glomerular fibrosis, can be inhibited by knocking down
ADAM17 expression and inhibiting the enzyme activity [9]. On the other hand, we also
found that ADAM17 can protect glomerular function by decreasing podocyte permeability
through inducing re-arrangement of the zonula occludens protein ZO-1 [8]. These data
suggest that depending on the cellular context the enzyme can have different effect on the
renal function. Nonetheless, inhibitors of ADAM17 decreased infiltration of macrophages
both in the glomeruli and in the interstitium in models of kidney fibrosis [7, 46] proving that
targeting ADAM17 can be beneficial for preserving renal function.
There is very few data available on ADAMTS enzymes and renal fibrosis. Unilateral ureteral
obstruction in rat induced upregulation of ADAMTS-1 in the tubular epithelial cells. Further,
ADAM Proteases as Novel Therapeutic Targets in Chronic Kidney Disease
7
secreted ADAMTS-1 of cultured epithelial cells decreased proliferation of a tubular fibroblast
cell line which suggested that ADAMTS-1 may have anti-fibrotic effect [49].
3.4 ADAMs in polycystic kidney disease (PKD)
Autosomal-recessive polycystic kidney disease (AR-PKD) is one of the most common genetic
disorders of the kidney results in end-stage renal disease. This disease leads to rapid
enlargement of the kidney through massive cysts formation. The main pathogenic process in
cyst development is the overactivation of the mislocalized EGFR in the cystic apical epithelia
(for review see [50]). Excessive shedding of the pro-proliferative growth factor, transforming
growth factor (TGF) was also observed. Since secretion of TGFis regulated by ADAM17,
therapeutic potential of ADAM17 inhibitors were explored and established in the bpk murine
model of AR-PKD [51]. In a later study, the role of TGF was not confirmed even if ADAM17
inhibitors were beneficial for attenuating cyst development in AR-PKD [52].
3.5 Thrombotic thrombocytopenic purpura (TTP)/ haemolytic-uremic syndrome (HUS)
Thrombotic thrombocytopenic purpura/haemolytic uremic syndrome are often considered
variants of a disease characterized by microangiopathic haemolytic anaemia [53]. Platelets
are consumed by spontaneously developing microscopic thrombosis. ADAMTS-13, the
enzyme which normally processes the very large von Willebrand factor (vWF) is missing
[54] or disabled [55, 56] in this disease. Therefore, the very large vWF capture circulating
platelets and initiates microthrombi formation. The red blood cells passing through the
damaged arteries experience excessive shear stress which leads to haemodialysis. Besides
purpura and anaemia there are often fever and neurologic symptoms present and the
disease can lead to both acute kidney failure and CKD [57, 58]. Interestingly, a recent study
which investigated plasma level of vWF in patients with chronic kidney disease of different
origin found decreased level of vWF-cleaving protease [59]. Level of vWF was higher in
stage IV patients compared to stages II and III, but whether the increased vWF contributed
to the worsening of CKD is currently not known.
4. ADAMs in kidney cancer
Several ADAM enzymes were upregulated at the message level in human renal cell
carcinomas. Compared to normal tissue mRNA levels of ADAM8, -17, -19, -28 as well as
ADAMTS-2 were upregulated. Interestingly, mRNA level of ADAMTS-1 did not change
[60]. In other studies, ADAM10 [61] and ADAM9 expression was increased in renal cancer
cells and associated with tumor progression [62] suggesting that expression of these enzyme
may be used as tumor markers. ADAM15 and -17 contributed to the migratory potential of
kidney cancer cells through activation of the EGFR [63] and ADAM17 silencing disabled the
capability of renal carcinoma cells to form in vivo tumors [64]. Therefore these enzymes
seem to have direct role in renal cancer pathophysiology.
5. Conclusion
ADAM and ADAMTS families include growing number of metalloenzymes which have
important role in kidney development and are indispensable to normal kidney function.
Chronic Kidney Disease
8
Lack or overactivation of certain ADAM enzymes (especially ADAM17 and ADAMTS-13)
can have major pathophysiological role in development of various type of CKD. Therefore,
targeting these enzymes can be an exciting novel therapeutic approach in the future and a
new hope for CKD patients.
6. Acknowledgment
This work was partly supported by the Paul Teschan Research Fund of the Dialysis Clinic
Incorporated.
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Chronic Kidney Disease
12
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2
Severity and Stages of Chronic Kidney Disease
Syed Ahmed and Gerard Lowder
Internal Medicine, Harbor Hospital, Baltimore,
USA
1. Introduction
Nearly ten years ago Nephrologists began using asystem of classification for chronic kidney
disease (CKD). This was established in 2002 by the Kidney Disease Outcome Quality
Initiative (KDOQI) to estimate kidney function in a given patient regardless of the etiology
of the primary insult to the kidneys. Physicians were able place their patients in stages from
mild disease to end stage renal disease (ESRD).CKD is defined as glomerular filtration rate
(GFR) below 60 ml/min per 1.73 m
2
for 3 months or more.
Each stage served as a mile marker on lifes road for the patient with CKD. The natural
history of CKD usually is a steady decline in kidney function, as found in the relationship
between the reciprocal of serum creatinine values and time. A percentage of patients do not
follow this linear pattern, suggesting either worsening or improvement in their kidney
function. Factors which may cause worsening of CKD in such individuals are often
infections, dehydration, poor control of systemic blood pressure and exposure to
nephrotoxins, in particular nonsteroidal anti-inflamatorydrugs and radiocontrast agents.
Other individuals who do not follow the steady decline may actually show improvement in
their GFR. The potential to improve the natural history of CKD is through tight blood
pressure control and inhibition of rennin-angiotensin-aldosterone system.
2. Stages of chronic kidney disease
The early stages of kidney dysfunction are often clinically silent, especially when the
condition is only slowly progressive and symptoms are nonspecific. Stages 1 & 2 show
decreased kidney function without signs or symptoms of disease although the estimated
GFR is less than 120 ml/min per 1.73 m
2
but greater than 60 ml/min per 1.73 m
2
. The rate of
progression is influenced by a wide range of factors which may or may not have the
potential of modification and varies among different individuals and with the underlying
cause of nephropathy.When the patient enters Stage 3 he or she has lost approximately half
their kidney function. It is less likely for the kidney disease to progress unless more than
50% of the nephron function is lost. For example, individuals with a solitary kidney after
unilateral nephrectomy for living kidney donation usually do not progress to
CKD.Increased risk of natural progression with less than 50% of nephron loss can occur in
persons of African ancestry with hypertensive nephrosclerosis. In 2008, the U.K National
Institute of Health and Clinical Excellence (NICE) sub divided the stage 3 into 3A and 3B
with estimated GFRs of 45 to 59 ml/min per 1.73 m
2
and 44 to 30 ml/min per 1.73 m
2
Chronic Kidney Disease 14
respectively. The NICE CKD guideline also suggested adding the suffix p to the stages in
proteinuric patients.It has generally been assumed that the majority of patients with CKD
stages 3B to 5 eventually progress to ESRD. A Canadian study showed the natural history of
CKD stages 3 and 4 to be variable and reflecting the patients risk factor profile.Stage 4 may
present with hyperkalemia or problems with salt and water retention. The kidneys are no
longer able to adjust to abrupt changes in sodium, potassium and fluid intake (or loss). Prior
to initiation of renal replacement therapy, the patients appetite may decrease, accompanied
by weight loss and a decrease in the serum albumin. In CKD clinics, with patients seen at
frequent intervals, the goal is to initiate dialysis before the patient becomes malnourished.
Stage Description GFR (ml/min/1.73m
2
)
1 Kidney damage with normal or GFR 90
2 Kidney damage with mild GFR 60-89
3 Moderate GFR 3A 45 59
3B 30 - 44
4 Severe GFR 15-29
5 Kidney Failure < 15 (or dialysis)
The suffix p to be added to the stage in patients with proteinuria > 0.5 g/24h
Table 1. Stages of CKD.
Two commonly used formulas to calculate creatinine clearance are the Cockcroft-Gault
formula and MDRD formula.
Cockcroft-Gault formula:
140 0.85
72
Age Mass Kgs if female
GFR
SerumCr
Modification of diet in renal disease (MDRD) formula:
1.154 0.203
186 . 1.212 0.742 GFR SCr Age if black if female
3. Risk factors
It is estimated that by 2030,more than 2,000,000 Americans will need dialysis or
transplantation. Who are these patients? What risk factors do they have?
Low birth weight individuals with a decreased number of nephrons, the elderly
population losing 1 ml/min/year after the age of 30 and Americans of African descent
with hypertension, are several groups of individuals at risk.About one half of patients
starting dialysis in America have diabetes mellitus, with hypertension the second largest
group. Autoimmune disorders, infections, kidney stones, cystic kidneys and
toxins/medications round out the list. Microalbuminuria may indicate systemic
endothelial dysfunction and may be associated with a prothrombotic state. Insulin
resistance is mediated in part by aldosterone; blocking the receptor attenuates
cardiovascular and renal injury.
The risk factors can be classified as those that increase the risk of development of kidney
disease and those that increase the risk of adverse outcomes associated with CKD. The
Severity and Stages of Chronic Kidney Disease 15
factors which increase the risk for CKD are further classified into susceptibility and
initiation factors; whereas factors which effect adverse outcomes are classified as
progression factors and end stage factors. The association between variables and disease
may be due to chance, a non-causal relation or may signify a true risk factor.
3.1 Risk factors for development of CKD
1. Susceptibility Factors
A susceptibility factor is one that increases susceptibility to kidney damage following
exposure to an initiation factor. An ideal study design to study these factors would be to
identify a population of individuals who are free of kidney disease and are exposed to an
initiation factor and follow them for a period of time.
2. Initiation Factor
An initiation factor is one that directly initiates kidney damage in an individual who is
susceptible to kidney damage. An ideal study design for identification of initiation factors is
a prospective cohort study. This would involve identification and follow up of a group of
individuals free of kidney disease at baseline, with known susceptibility factors and with or
without exposure to initiation factors, for the development of kidney disease.
3.2 Risk factors effecting adverse outcome of CKD
1. Progression Factors
Progression factors worsen the kidney damage caused by initiation factors and lead to
further decline in kidney function. Indicators of progression may include progression of
microalbuminuria to overt proteinuria or reduced GFR, rate of decrease of GFR, or
development of kidney failure necessitating dialysis or transplantation.
2. End-Stage Factors
End stage factors are those that exacerbate the morbidity and mortality associated with
kidney failure. Examples of indicators of mobidity include hospitalizations, poor quality of
life measures, and cardiovascular disease complications.
3.3 Risk factors for progression of chronic kidney disease
1. Proteinuria
Proteinuria is associated with faster rates of CKD progression. It contributes to nephron
loss; filtered proteins are reabsorbed by the proximal tubular cells. Tubular cell contents
may leak into the interstitium. This can cause macrophage infiltration and
inflammatory mediators produced by them. The MDRD study showed proteinuria to be
the strongest predictor of kidney disease progression in non diabetic patients. The REIN
study done in non diabetic patients with proteinuria, showed the protein excretion rate
to be the best single predictor of GFR decline to ESRD. This finding was independent of
the initial insult.
The US Collaborative Study in type 1 diabetic patients with >500mg proteinuria/day
and serum creatinine values of 2.5mg% or less showed a 50% reduction in the risk of
combined endpoints (death, dialysis, transplantation) in patients treated with an ACE
inhibitor.
Chronic Kidney Disease 16
Risk Factor Definition Examples
Susceptibility
factors
Increase susceptibility to
kidney damage
Older age, family history of chronic kidney
disease, reduction in kidney mass, low
birthweight, U.S. racial or ethnic minority
status, low income or education
Initiation
factors
Directly initiate kidney
damage
Diabetes, high blood pressure,
autoimmune diseases, systemic infections,
urinary tract infections, urinary stones,
lower urinary tract obstruction, drug
toxicity
Progression
factors
Cause worsening kidney
damage and faster decline in
kidney function after
initiation of kidney damage
Higher level of proteinuria, higher blood
pressure, poor glycemic control in
diabetes, smoking
End-stage
factors
Increase morbidity and
mortality in kidney failure
Lower dialysis dose (Kt/V), temporary
vascular access, anemia, low serum
albumin level, late referral
Table 2. Risk Factors for Chronic Kidney Disease and its Outcomes.
The IDNT Study looked at type 2 diabetic patients treated with placebo, ibesartan or
amlodipine. The ARB outperformed the placebo group and calcium channel patients in
reaching doubling of the serum creatinine, ESRD, death by 20% and 23% respectively.
2. Hypertension
Blood pressure should be lowered to <120/80.
Patients with blood pressure 120-129/80-84 have a 1.6 fold greater risk of developing
ESRD and those with pressure >210/120 have a 4.2 fold risk of ESRD.
The MRFIT study showed that hypertension was an independent risk factor for the
development of ESRD.
3. Smoking cessation- smoking is a risk factor in the progession to kidney failure
Hallan, S & Orth, S. KI 2011.157
4. Glycemic control
Blood pressure control is more important with progression of CKD in the diabetic
patient, whereas hyperglycemia is important with the initiation of diabetic
nephropathy.
5. Management of dyslipidemia
LDL stimulates mesangial cell proliferation and the synthesis of proinflammatory
molecules.
No large study is available to show that control of lipids is effective in slowing the
progression of CKD. The SHARP study showed that CKD patients receiving
simvastatin and ezetimibe had approximately 15% fewer strokes and MIs.
4. Mechanism of progression
The characteristic structural change in CKD is scarring associated with glomerulosclerosis,
tubulointerstitial fibrosis, and vascular sclerosis. After this initial insult the kidney goes
down on one of the two paths, healing and functional recovery or scarring with loss of
Severity and Stages of Chronic Kidney Disease 17
kidney function progressing to CKD. It is less known what leads the kidney to which
pathway.
Healing primarily occurs in Acute Kidney Injury (AKI) and acute interstitial nephritis, when
treatment is instituted early in its course. Healing is also a hallmark of acute post infectious
glomerulonephritis. Renal function typically recovers within few weeks of acute nephritic
process.Chronic kidney damage on the other hand is usually induced by diabetes,
hypertension, chronic glomerulonephritits, or chronic exposure to infections or
nephrotoxins, progress to scarring with loss of function and CKD. (Fig. 1)
Fig. 1. Progression of initial kidney injury.
Renal cell injury results in loss of glomerular capillaries and cellular elements are replaced
by extracellular matrix and fibrous tissue. Acute severe glomerulonephritis damages the
capillaries and endothelium whereas sub-acute and chronic glomerulonephritis affect the
mesangium or the podocytes. Progressive renal scarring is associated with progressive
tubular cell loss and atrophy.
4.1 Role of intrinsic renal cells in kidney damage
Endothelium: Damage to the protective anticoagulant and anti-inflamatory endothelial
capillary lining in acute glomerulonephritis, transforms it into a pro-inflammatory surface
leading to accumulation of inflammatory cells and platelets within golmerular capillaries as
well as the stimulation of mesangial proliferation. Glomerular endothelial damage can also
be due to a metabolic insult as in diabetes or a physical hemodynamic stress as in
hypertension.
Mesangium: Mesangial cells respond to injury either with death, transformation,
proliferation and migration,or synthesis and deposition of extracellular matrix (ECM).
Scarring is usually characterized by uncontrolled mesangial proliferation and excessive
deposition of mesangial matrix. This process is driven by a number of growth factors like
transforming growth factor 1 (TGF1), platelet derived growth factor (PDGF), and
fibroblast growth factor (FGF).
Chronic Kidney Disease 18
Podocytes: After an injury to the podocytes, the glomerular basement membrane is exposed
to the parietal epithelial cells leading to the formation of capsular adhesions and segmental
glomerulosclerosis. This may lead to misdirected filtration with accumulation of amorphous
material in the glomerular space. Misdirected filtration causes disruption of the glomerular-
tubular junction resulting in atubularglomeruli. It may also contribute to tubular atrophy
and interstitial fibrosis. Thus podocytes help in conserving the structural integrity of the
glomerulus by forming a protective membrane over the basement membrane.
Tubular cells: As mentioned earlier, after the initial insult the tubular cells may undergo
healing and recover renal function, but repeated insults stimulate epithelial mesenchymal
transformation of tubular cells to myofibroblastic phenotype with excessive deposition of
ECM. Thus tubular injury can lead to renal fibrogenesis.
Vascular cells: Vascular sclerosis is an intergral feature of renal scarring and is associated
with progressive kidney failure in glomerulonephritis. Hyalinosis of afferent arterioles, in
diabetes, and damage to the post-glomerular arteriole and peritubular capillaries cause
interstitial ischemia and fibrosis.
Fig. 2. Role of Intrinsic Cells in Kidney Damage.
4.2 Role of extrinsic cells in kidney damage
Infiltration of inflammatory cells into the glomeruli and the renal interstitium is the
hallmark of glomerulosclerosis and tubuloiterstitial fibrosis.
Severity and Stages of Chronic Kidney Disease 19
Platelets and coagulation: Platelets and their release products within the damaged glomeruli
stimulate a coagulation cascade which activate the mesangial cells to induce sclerosis.
Thrombin stimulates glomerular TGF-1 leading to production of mesangial ECM and
inhibition of metalloproteinases.
Lymphocytes, Monocytes-Macrophages, Dendritic cells play important role in the formation
of glomerulosclerosis by causing inflammation.
Fig. 3. Deposition on of ECM within and around the glomerulus.
Fig. 4. Glomerular hypercellularity due to proliferation of intrinsic glomerular cells and
intracapillary leukocytes.
Chronic Kidney Disease 20
Fig. 5. Capillary tufts almost replaced by the fibous tissue forming glomerular scarring.
Fig. 6. Immunofluorescent stain shows deposition of coarsely granular deposits of
complement C3.
4.3 Role of angiotensin II, hypertension and hyperfiltration
With progression of kidney disease the afferent arteriole tone decreases to a much larger
extent than the efferent tone. As a result intra-glomerular pressure rises leading to
hyperfiltration. Angiotensin II aides in hyperfiltration through its vasoconstrictor effect
predominantly on the efferent arteriole. Apart from its hemodynamic effects, Angiotensin II
acts directly on the glomerular membrane. It acts on the angiotensin II receptors on the
surface of the podocytes, altering their permselective property, by contracting the foot
processes. This allows proteins to escape in the urinary space.
Angiotensin II also induces proliferation ofglomerular cells and fibroblasts. It acts on AT1
receptors on tubular cells causing hypertrophy, which results in increased synthesis of
collagen type IV. It increases macrophage activation and phagocytosis responsible for the
inflammatory component associated with CKD.
Severity and Stages of Chronic Kidney Disease 21
4.4 Role of proteinuria
Proteinuria is not only a marker of kidney damage, but also contributes to nephron damage.
Filtered proteins are reabsorbed from the proximal tubule. Damaged tubular basement
membrane causes leakage of tubular content into the interstitium, thereby causing
macrophage infiltration. Macrophages produce inflammatory mediators thus mounting an
immense inflammatory reaction inside the renal interstitium.
Fig. 7. Focal segmental and global Glomerulosclerosis and nephron loss is a vicious circle
ultimately leading to proteinuria.
5. Pathology of CKD
Fibrosis in the kidneys initiated by a variety of insults may not be a uniform process.
Progressive disease in diabetic patients may be related to endothelial nitric oxide deficiency
with resultant endothelial dysfunction.The eventual pathology of the above mentioned
series of events lead to two major histologic characteristic of CKD, focal segmental
glomerulosclerosis and tubulointerstitial fibrosis. An initial insult to the kidneys will cause
nephron loss.The remaining nephrons work harder to compensate for the lost nephrons
Chronic Kidney Disease 22
(compensatory hypertrophy). This leads to hemodynamic changes including glomerular
hypertension and hyperfiltration. There is reduced afferent arteriolar resistance and
intraglomerular pressure rises with increased filtration by the remaining nephrons. The
intrinsic and extrinsic cells contribute to sclerosis as mentioned above contributing to the
focal and segmental glomerulosclerosis.
Tubulointerstitial injury results from ischemia of tubule segments downstream from
sclerotic glomeruli. Acute and chronic inflammation in the adjacent interstitium, and
damage of pericapillary blood supply also contribute to tubular injury. The above events
along with proteinuria eventually lead to tubulointerstitial fibrosis.
Angiotensin II increases vascular tone (predominantly post-glomerular) and affects
intraglomerular pressure. The increased pressure alters the structure of the pores in the
glomerular basement membrane (GBM) and increases proteinuria.
5.1 Clinical manifestation and management
What is the best way to manage these individuals? In the outpatient setting, achecklist for
each patient ensures that each individuals needs are met. A list of ten commandments for
the CKD patient is:
1. Estimate the GFR and stage the patients CKD.
2. Round up the usual suspects. Diabetes and hypertension account for almost of the
patient population. Urinalysis, serologies, sonography and biopsy (if necessary) to
make the diagnosis.
3. Fix what you can. Discontinue NSAIDs, correct volume depletion and treat BPH (men)
and bladder dysfunction (women).
4. Treat hypertension. Goal of therapy is <130/80.
Use ACE, ARB, both, renin blockers, calcium channel blockers, aldosterone antagonists,
loop diuretics as needed.
5. Measure (spot urine protein /creatinine) and treat proteinuria. The goal is<300mg/day.
Maximize the dose of an ACE inhibitor, then add an ARB at full dose and increase to
reach goal. Loop diuretics are essential to manage edema fluid and offset the
development of hyperkalemia. Renin blockers and aldosterone antagonists are added
with monitoring of the patients potassium and creatinine. If the potassium rises to
greater than 5.5 meq/l or if the serum creatinine increases more than 30% above
baseline, dosages will need to be decreased.
6. Treat anemia of CKD with an ESA if there is no blood loss and iron stores are adequate.
Check thyroid function, B-12, folic acid levels. The target Hgb is >10g/dl. Parenteral
iron may be needed to keep the TSAT > 25%.
7. Give base supplements to correct metabolic acidosis. Untreated acidosis causes
osteopenia and muscle catabolism, along with the release of calcium and phosphorous
from bone. Sodium bicarbonate is replaced at 0.5-1.0 meq/kg/day.
Treat hyperurricemia with allopurinol if the eGFR is >30 ml/min.
8. Phosphate binders, precursor vitamin D and active D (when necessary). We are using
both calcium and non-calcium containing binders in our clinic. We try to keep serum
calcium levels less than or equal to 9.5 mg%. Vitamin D2 and 3 are used in patients with
25(OH)D levels less than 30 ng/ml. Active vitamin D is used to control elevated iPTH
levels and the effects of secondary HPT.
Severity and Stages of Chronic Kidney Disease 23
9. Have a nutritionist help patients maintain caloric intake. Protein restriction is difficult
and may lead to malnutrition in patients with already poor appetites. We encourage
protein supplementation in our CKD patients. The phosphorus level will increase,
however, we try to maintain the patients albumin predialysis or pretransplantation.
Patients are started on a 2 gram potassium diet and educated about avoidance of foods
high in potassium. Loop diuretics + base supplements aid in the management of
hyperkalemia. Resin exchange binders are reserved for values greater than 6 as they
cause diarrhea, bicarbonate loss and may worsen acidemia and further increase the
serum potassium value.
10. Education and preparation for hemodialysis or peritoneal dialysis. See if acandidate is
available for transplantation. We encourage patients to have a fistula constructed after
they have attended the education class and decide to do in center or home
hemodialysis. These are patients generally in late stage 3 CKD.
Diabetic patients should maintain euglycemia, insulin requirements may decrease as CKD
progresses. Metformin should be avoided and glipizide isthe preferred oral agentbecause it
is not downgraded to a metabolite excreted by the kidneys.
6. Summary
CKD will remain a health concern into the future. CKD clinics managing patients in a
coordinated fashion with nutritionists and surgeons will improve lives. Better blood
pressure control with diminution of proteinuria will slow the progress of established
disease. Attention to acidemia and hyperruricemia will also be beneficial. New insights into
the pathogenesis and treatment of diabetes may help manage the number one cause of
kidney failure in America.
7. References
[1] Primer on Kidney Disease, 5
th
Edition, Greenberg et al. editors, Saunders (2009).
[2] Comprehensive Clinical Nephrology, 4
th
edition, Jurgen Floege; Richard J. Johnson, John
Feehally
[3] Brenner and Rectors The Kidney, 8
th
Edition.
[4] Pathologic Basis of Diseases, Eighth Edition. Robins and Cotran
[5] Tuttle, K. Relationship between cardiovascular disease and albuminuria in hypertension.
The Heart Institute of Spokane, Spokane, Waashington.
[6] Sowers J, Whaley-Connell A, Epstein M. The Emerging Clinical Implications of the Role
of Aldosterone in the Metabolic Syndrome and Resistant Hypertension. Annals of
Internal Medicine 150,776-783(2009).
[7] Rennke, Helmut.Glomerular Adaptations to Renal Injury: The Role of Capillary
Hypertension in the Pathogenesis of Focal and Segmental Glomerulosclerosis.
Advances in Nephrology 15,15-26(1988).
[8] Boor, P, Ostendorf, T andFroeje, J. Renal Fibrosis: Novel Insights into Mechanisms and
Therapeutic Targets. Nature Reviews in Nephrology 6,643-656 (2011).
[9] Carrero, Juan Jesus and Stenvinkel, Peter. Novel Targets for Slowing CKD Progression.
Nature Reviews in Nephrology 7,65-66(2011).
Chronic Kidney Disease 24
[10] Nakagama T, Tanabe K, Grant MB, Kosugi T, Croker B, Johnson R and Li Qiuhong.
Endothelial Dysfunction as a Potential Contributor in Diabetic Nephropathy.
Nature Reviews in Nephrology 7,36-44(2011).
[11] Baines R and Brunskill NJ.Tubular Toxicity of Proteinuria.Nature Reviews in
Nephrology 7,177-180(2011).
[12] Peralta C.Detection of Chronic Kidney Disease with creatinine,cystatin c and urine
albumin-to-creatinine ratio and association with progression to ESRD and
mortality. JAMA 305,1545-1552 (2011).
[13] Tonnelli M. Using proteinuria and estimated GFR to classify risk in patients with CKD:
a cohort study. Annals of Internal Medicine 154,12-21(2011).
[14] Levin A, Djurdjev O, Beaulieu M, Er L. Longitudinal follow-up and outcomes among a
population with chronic kidney disease in a large managed care organization.Arch
Intern Med. 2004 Mar 22;164(6):659-63.
3
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral
and Bone Disorder (CKDMBD)
Igor G. Nikolov
1
, Ognen Ivanovski
2
and Nobuhiko Joki
3
1
University Clinic of Nephrology, Medical Faculty - Skopje,
2
University Clinic of Urology, Medical Faculty - Skopje,
3
Division of Nephrology, Toho University Ohashi Medical Center, Tokyo,
1,2
Republic of Macedonia
3
Japan
1. Introduction
Kidney is one of the most important organs in the regulation of mineral metabolism
(Fukagawa et al., 2006). Chronic kidney disease (CKD) is a worldwide public health
problem that affects 5% to 10% of the world population, with increasing prevalence and
adverse outcomes, including progressive loss of kidney function, cardiovascular disease,
and premature death (Eknoyan et al., 2004). Calcium and phosphorus are fundamentally
important in a wide array of biological functions. Abnormalities in calcium, phosphorus,
parathyroid hormone (PTH), and vitamin D metabolism (usually referred to as disordered
mineral metabolism) are common in patients with (CKD) (Block et al., 1998). Cardiovascular
disease is the leading cause of death in patients with CKD (London et al., 2003). It has been
shown that in individuals with kidney failure on maintenance dialysis who are younger
than 65 years, cardiovascular mortality is 10 to 500 times higher than in the general
population, even after adjustment for sex, race, and presence of diabetes (Foley RN et al.,
1998). Disturbances in mineral metabolism are common complications of CKD and an
important cause of morbidity and decreased quality of life. Importantly, increasing evidence
suggests that these disturbances are associated with changes in arterial compliance,
cardiovascular calcification, bone disorders and all-cause and cardiovascular mortality
(Palmer SC et al., 2005, Drueke et al., 2010). Traditionally, when defining bone diseases in
CKD patients, this group of disorders has been usually termed renal osteodystrophy.
However, beside strictly defined, the term renal osteodystrophy means only bone
abnormalities. Recently, the KDIGO (Kidney Disease: Improving Global Outcomes)
conference group agreed that the definition of renal osteodystrophy should be only specific
to bone pathology found in patients with CKD (Moe S. et al., 2006). It has been concluded
that renal osteodystrophy is one component of the mineral and bone disorders that occur as
a complication of CKD. It has been proposed that the evaluation and definitive diagnosis of
renal osteodystrophy requires performing a bone biopsy. Histomorphometry is not essential
for clinical diagnosis, but should be performed in research studies. There was an agreement
that histomorphometric results are to be reported by use of the standard nomenclature
Chronic Kidney Disease
26
recommended by the American Society for Bone and Mineral Research (Parfitt et al., 1987),
and investigators would supply primary measurements used to report any derived
parameters. Based on all of this a new term has been proposed and coined Chronic kidney
disease mineral and bone disorder (CKD-MBD) willing to describe the systemic
consequences of mineral metabolism disturbances in CKD patients which can no longer be
considered restricted only to bone disease. CKD-MBD defines a triad of interrelated
abnormalities of serum biochemistry, bone and the vasculature associated with CKD. The
adverse effects of high serum phosphorus and an increase of serum calcium due to calcium
overload which are present late in CKD are important component of CKD-MBD as well as
vascular changes. Furthermore, to clarify the interpretation of bone biopsy results in the
evaluation of CKD-MBD, it has been proposed to use three key histologic descriptorsbone
turnover, bone mineralization, and bone volume (so called TMV system) with any
combination of each of the descriptors possible in a given specimen. The TMV classification
scheme provides a clinically relevant description of the underlying bone pathology, as
assessed by histomorphometry, which, in turn, helps to define the pathophysiology, and,
thereby, probably to guide the therapy (Moe S. et al., 2006).
2. CKD MBD and biochemical abnormalities
The initial evaluation of CKD-MBD should include laboratory for calcium (it has been
proposed either ionized or total corrected for albumin), phosphorus, PTH, alkaline
phosphatases (total or bone specific), bicarbonate, as well as imaging for soft-tissue
calcification. Epidemiologic studies from the early 1990s have demonstrated that an increase
in serum phosphorus and in calcium x phosphorus product are associated with poor
outcomes in CKD patients. The association of elevated serum phosphorus and calcium and
increased mortality in these patients has been confirmed in several recent studies. If
inconsistencies exist in the biochemical markers (eg, high PTH but low alkaline
phosphatases), unexplained bone pain, or unexplained fractures are present, a bone biopsy
would be strongly indicated (London and Drueke, 1997; London et al., 2003; Neves et al.,
2007; Bucay et al., 1998).
2.1 Calcium
Serum calcium is tightly controlled in healthy individuals, within a narrow range, usually
2.22.6 mmol/l, with a minimal, diurnal variation. In patients with CKD, serum calcium
levels fluctuate more, because of altered homeostasis and concomitant therapies. Serum
calcium levels are routinely measured in clinical laboratories using colorimetric methods in
automated machines. In patients with CKD stage 5D, there are additional fluctuations in
association with dialysis-induced changes, hemoconcentration, and subsequent
hemodilution. Moreover, predialysis samples collected from dialysis patients after the
longer interdialytic interval during the weekend, as compared with predialysis samples
drawn after the shorter interdialytic intervals during the week, often contain higher serum
calcium levels (Tentori et al., 2008). It has been shown that the serum calcium level is a poor
reflection of overall total body calcium. Only 1% of total body calcium is measurable in the
extracellular compartment while the most important part of calcium is stored in the bones.
Serum ionized calcium, generally 4050% of total serum calcium, is physiologically active,
while non-ionized calcium is bound to albumin or anions such as citrate, bicarbonate, and
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral and Bone Disorder (CKDMBD)
27
phosphate, and is therefore not physiologically active. In the presence of hypoalbuminemia,
there is an increase in ionized calcium relative to total calcium; thus, total serum calcium
may underestimate the physiologically active (ionized) serum calcium. The most commonly
used formula for estimating ionized calcium from total calcium is the addition of 0.2 mmol/l
for every 1 g decrease in serum albumin below 40 g/l. Unfortunately, recent data have
shown that it offers no superiority over total calcium alone and is less specific than ionized
calcium measurements. In addition, the assay used for albumin may affect the corrected
calcium measurement.
2.2 Phosphorus
It has been shown that inorganic phosphorus is critical for numerous normal physiological
functions, including skeletal development, mineral metabolism, cell-membrane
phospholipid content and function, cell signaling, platelet aggregation, and energy transfer
through mitochondrial metabolism. Owing to its importance, normal homeostasis maintains
serum concentrations between 0.811.45 mmol/l. The terms, phosphorus and phosphate, are
often used interchangeably, but strictly speaking, the term phosphate means the sum of the
two physiologically occurring inorganic ions in the serum, and in other body fluids,
hydrogenphosphate (HPO4
2
) and dihydrogenphosphate (H2PO
4
). However, most
laboratories report this measurable, inorganic component as phosphorus. Unlike calcium, a
major component of phosphorus is intracellular, and factors such as pH and glucose can
cause shifts of phosphate ions into or out of cells, thereby altering the serum concentration
without changing the total body phosphorus. Phosphorus is routinely measured in clinical
laboratories with colorimetric methods in automated machines. Serum phosphorus levels
reach the lowest level in the early hours of the morning, increasing to a plateau at the
afternoon, and further increasing to a peak late in the evening (Portale et al., 1987).
Hyperphosphatemia occurs as a consequence of diminished phosphorus filtration and
excretion with the progression of CKD. Decreased phosphorus excretion can initially be
overcome by increased secretion of parathyroid hormone (PTH), which decreases proximal
phosphate reabsorption (Slatopolsky and Delmez, 1994). Hence, phosphorus levels are
usually within normal range until the GFR falls below approximately 30 ml/min, or stage
IV. CKD according to the National Kidney Foundation Kidney Disease Outcomes Quality
Initiative (NKFK/DOQI) classification (National Kidney Foundation: K/DOQI). In more
advanced stages of CKD, the blunted urinary excretion of phosphorus can no longer keep
pace with the obligatory intestinal phosphate absorption, resulting in hyperphosphatemia.
Therefore, it is not surprising that the majority of patients with CKD stage 4 and stage 5
have a significant hyperphosphatemia (Block et al., 1998). It has been shown that in patients
with advanced CKD high serum calcium, phosphate, and calcium-phosphate product levels
are associated with unaccountably high rates of cardiovascular disease (Ganesh et al., 2001;
Stevens et al., 2004; Slinin et al., 2005). Moreover, it has been shown also that these
derangements in mineral metabolism could occur as well during the early stages of CKD
(Slatopolsky and Delmez, 1994).
2.3 Parathyroid hormone
The parathyroid gland plays an important role in the regulation of mineral homeostasis by
effects trough other organs such as the kidney and bone. Fluctuation in extracellular calcium
Chronic Kidney Disease
28
ion levels is sensed by the parathyroid calcium-sensing receptors (CaSRs) and subsequently
regulates the synthesis and secretion of parathyroid hormone (PTH) (Felsenfeld et al., 2007).
PTH acts on the bone to increase the efflux of calcium and phosphate, and acts on the kidney
to reduce urinary calcium excretion, inhibit phosphate reabsorption, and stimulate the
production of 1,25-dihydroxyvitamin D (1,25(OH)2D). PTH is cleaved to an 84-amino-acid
protein in the parathyroid gland, where it is stored with fragments in secretory granules for
release. When it is released, the circulating 184-amino-acid protein has a half-life of 24 min.
The hormone is cleaved both within the parathyroid gland and after secretion into the N-
terminal, C-terminal, and middle region fragments of PTH, which are metabolized in the liver
and in the kidneys. Enhanced PTH synthesis/secretion occurs in response to hypocalcemia,
hyperphosphatemia, and/or a decrease in serum 1,25-dihydroxyvitamin D (1,25(OH)2D),
whereas high serum levels of calcium or calcitrioland, as recently shown, of Fibroblast
growth factor 23 (FGF-23)suppress PTH synthesis/secretion. The extracellular concentration
of ionized calcium is the most important determinant of the minute-to-minute secretion of
PTH, which is normally oscillatory.
In patients with CKD, this normal oscillation is somehow altered. Over the past few decades
there has been a progress in development of sensitive assays in order to measure PTH.
Initial measurements of PTH using C-terminal assays were inaccurate in patients with CKD
because of the impaired renal excretion of C-terminal fragments (and thus retention) and the
measurement of these probably inactive fragments. The development of the N-terminal
assay was initially thought to be more accurate but it also detected inactive metabolites. The
development of a second generation of PTH assays, the two-site immunoradiometric
assaycommonly called an intact PTH assayimproved the detection of full-length
(active) PTH molecules. In this assay, a captured antibody binds within the amino terminus
and a second antibody binds within the carboxy terminus. Unfortunately, recent data
indicate that this intact PTH assay also detects accumulated large C-terminal fragments,
commonly referred to as 784 fragments; these are a mixture of four PTH fragments that
include, and are similar in size to, 784 PTH (Gao and D'Amour 2005). In
parathyroidectomized rats, the injection of a truly whole 1- to 84-amino-acid PTH was able
to induce bone resorption, whereas the 7- to 84-amino-acid fragment was antagonistic,
explaining why patients with CKD may have high levels of intact PTH but relative
hypoparathyroidism at the bone-tissue level (Slatopolsky et al., 2000; Malluche et al., 2003;
Huan et al., 2006). Thus, the major difficulty in accurately measuring PTH with this assay is
the presence of circulating fragments, particularly in the presence of CKD. Unfortunately,
the different assays measure different types and amounts of these circulating fragments,
leading to inconsistent results. More recently, a third generation of assays has become
available that truly detect only the 1- to 84-amino-acid, full-length molecule: whole or
bioactive PTH assays. There are differences in PTH results when samples are measured in
plasma, serum, or citrate, and depending on whether the samples are on ice, or are allowed
to sit at room temperature.
PTH and vitamin D have been shown to influence cardiac and vascular growth and function
experimentally in human subjects with normal renal function. Because of increased
prevalence of hyperparathyroidism and altered vitamin D status in CKD, these alterations
have been considered to contribute to the increased prevalence of cardiovascular disease
and hypertension seen in this patient population (Slinin Y et al., 2005).
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral and Bone Disorder (CKDMBD)
29
2.4 Vitamin D (25(OH)D)
The parent compounds of vitamin DD
3
(cholecalciferol) or D
2
(ergocalciferol)are highly
lipophilic. They are difficult to quantify in the serum or plasma. They also have a short half-
life in circulation of about 24 h. These parent compounds are metabolized in the liver to
25(OH)D
3
(calcidiol) or 25(OH)D
2
(ercalcidiol). Collectively, they are called 25(OH)D or 25-
hydroxyvitamin D. The measurement of serum 25(OH)D is regarded as the best measure of
vitamin D status, because of its long half-life of approximately 3 weeks. In addition, it is an
assessment of the multiple sources of vitamin D, including both nutritional intake and skin
synthesis of vitamin D. There is a seasonal variation in calcidiol levels because of an increased
production of cholecalciferol by the action of sunlight on skin during summer months. The
gold standard of calcidiol measurement is high performance liquid chromatography (HPLC),
but this is not widely available clinically. This is because HPLC is time consuming, requires
expertise and special instrumentation, and is expensive. In early 1985, Hollis and Napoli
developed the first radioimmunoassay (RIA) for total 25(OH)D, which was co-specific for
25(OH)D
2
and 25(OH)D
3
. The values correlated with those obtained from HPLC analysis, and
DiaSorin RIA became the first test to be approved by the Food and Drug Administration for
use in clinical settings (Hollis and Napoli, 1985). Another method now carried out is liquid
chromatography- tandem mass spectrometry (LC-MS/MS). Similar to HPLC, the LC-MS/MS
method also has the ability to quantify 25(OH)D
2
and 25(OH)D
3
separately, which
distinguishes it from RIA and enzyme-linked immunosorbent assay technologies. This
method is very accurate and has been shown to correlate well with DiaSorin RIA (Saenger et
al. 2006; Tsugawa et al., 2005). It has been suggested recently that the assays for 25(OH)D are
not well standardized, and the definition of deficiency is not yet well validated. At best,
clinicians should ensure that patients use the same laboratory for measurements of these
levels, if carried out. The most appropriate vitamin D assays presently available seem to be
those that measure both 25(OH)D
2
and 25(OH)D
3
. Presently, approximately 2050% of the
general population has low vitamin D levels, irrespective of CKD status. However, the
benefits from replacing vitamin D have not been documented in patients with CKD,
particularly if they are taking calcitriol or a vitamin D analog.
2.5 Vitamin D (1,25(OH)
2
D)
1,25(OH)2D is used to describe both hydroxylated D2 (ercalcitriol) and D3 (calcitriol)
compounds, both of which have a short half-life of 46 h. Furthermore, in patients with
earlier stages of CKD and in the general population, mild-to-moderate vitamin D deficiency,
or partly treated vitamin D deficiency, is frequently associated with increased levels of
1,25(OH)2D. Thus, even accurate levels can be misleading. The serum levels of 1,25(OH)2D
are uniformly low in late stages of CKDMBD, at least in patients not treated with vitamin D
derivatives (Andress et al., 2006). It has not been recommend a routine measurement of
1,25(OH)2D levels, as the assays are not well standardized, the half-life is short, and there
are no data indicating that the measurement is helpful in guiding therapy or predicting
outcomes (KDIGO).
2.6 Alkaline phosphatases
Alkaline phosphatases (ALP) are enzymes that remove phosphate from proteins and
nucleotides, functioning optimally at alkaline pH. Measurement of the level of total ALP (t-
Chronic Kidney Disease
30
ALP) is a colorimetric assay that is routinely used in clinical laboratories in automated
machines. The enzyme is found throughout the body in the form of isoenzymes that are
unique to the tissue of origin. Highest concentrations are found in the liver and bone, but
the enzyme is also present in the intestines, placenta, kidneys, and leukocytes (Iba K et al.
2004). Specific ALP isoenzymes to identify the tissue source can be determined after
fractionation and heat inactivation, but these procedures are not widely available in clinical
laboratories. Bone-specific ALP (b-ALP) is measured with an immunoradiometric assay.
Elevated levels of t-ALP are generally due to an abnormal liver function, an increased bone
activity, or bone metastases. Levels are normally higher in children with growing bones
than in adults, and often are increased after fracture. In addition, t-ALP and b-ALP can be
elevated in both primary and secondary HPT, osteomalacia, and in the presence of bone
metastasis and Pagets disease. In patients with CKDMBD alkaline phosphatise may be
used as an adjunct test, but if values are high, then liver function tests should be checked. t-
ALP could reasonably be used as a routine test to follow response to therapy. The more
expensive testing for b-ALP can be used when the clinical situation is more ambiguous.
Testing for t-ALP is inexpensive and therefore may be helpful for following patients
response to therapy or determining bone turnover status when the interpretation of PTH is
unclear. The use of b-ALP, an indicator of bone source, may provide additional and more
specific information, although it is not readily available (Iba K et al. 2004).
3. CKD MBD and bone abnormalities
Disorders of mineral metabolism are also associated with abnormal bone structure. It has
been shown that the gold standard test for bone quality is its ability to resist fracture under
strain. In animal models, this resistance can be directly tested with three-point bending
mechanical tests. Bone quality is impaired in CKD, as the prevalence of hip fracture is
increased in dialysis patients compared with the general population in all age groups.
Dialysis patients in their forties have a relative risk of hip fracture that is 80-fold higher than
that of age-matched and sex-matched control subjects. Furthermore, hip fracture in dialysis
patients is associated with a doubling of the mortality observed in hip fractures in
nondialysis patients (Coco M and Rush H., 2000; Alem et al., 2000). It has been shown that
risk factors for hip fracture in CKD patients include age, gender, duration of dialysis, and
presence of peripheral vascular disease. There are also analyses that found race, gender,
duration of dialysis, and low or very high PTH levels as risk factors for hip fracture. It has
been reported that both hip and lumbar-spine fractures occur independent of gender and
race in CKD patients. Other risk factors for abnormal bone identified in studies from the
general population are also common in CKD, including smoking, sedentary lifestyle, and
hypogonadism (Alem et al., 2000). These factors are likely to increase the risk of bone
fragility and fractures in CKD but have not been well evaluated. Extremes of bone turnover
found in patients with CKD have significant impact on fragility and are likely additive to
bone abnormalities commonly found in the aging and sedentary general population
(Vassalotti et al., 2008; Melamed et al., 2008).
3.1 Classification of renal osteodystrophy by bone biopsy
Bone biopsy is performed to understand the pathophysiology and course of bone disease, to
relate histological findings to clinical symptoms of pain and fracture, and to determine
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral and Bone Disorder (CKDMBD)
31
whether treatments are effective. The traditional types of renal osteodystrophy have been
defined on the basis of turnover and mineralization as follows: mild, slight increase in
turnover and normal mineralization; osteitis fibrosa, increased turnover and normal
mineralization; osteomalacia, decreased turnover and abnormal mineralization; adynamic,
decreased turnover and acellularity; mixed, increased turnover with abnormal
mineralization. It has been suggested recently that by performing bone biopsies in patients
with CKD the most important parameters which should be determined are bone turnover,
bone mineralization, and bone volume (TMV) (Moe et al., 2009).
3.1.1 Bone turnover
In CKD patients a spectrum of bone formation rates varies from abnormally low to very
high. Other measurements that help to define a low or high turnover (such as eroded
surfaces, number of osteoclasts, fibrosis, or woven bone) tend to be associated with the
bone-formation rate as measured by tetracycline labeling. This is the most definite dynamic
measurement, hence it was chosen to represent bone turnover. It should be noted that an
improvement of a bone biopsy cannot be determined on the basis of a simple change in the
bone-formation rate, because the restoration of normal bone may require either an increase
or a decrease in bone turnover, depending on the starting point (Melsen and Moselkilde,
1978).
3.1.2 Bone mineralization
It is a parameter which reflects the amount of unmineralized osteoid. Mineralization is
measured by the osteoid maturation time or by mineralization lag time, both of which
depend heavily on the osteoid width as well as on the distance between tetracycline labels.
The classic disease with an abnormality of mineralization is osteomalacia, in which the
bone-formation rate is low and the osteoid volume is high. Some patients have a modest
increase in osteoid, which is a result of high bone formation rates. They do not have
osteomalacia because the mineralization lag time remains normal. The overall
mineralization, however, is not normal because unmineralized osteoid is increased.
3.1.3 Bone volume
Bone volume contributes to bone fragility and is separate from the other parameters. The
bone volume is the end result of changes in bone-formation and resorption rates: if the
overall bone formation rate is higher than the overall bone resorption rate, the bone is in
positive balance and the bone volume will increase. If mineralization remains constant, an
increase in bone volume would also result in an increase in BMD and should be detectable
by dual-energy X-ray absorptiometry (DXA). Although both cortical and cancellous bone
volumes decrease in typical idiopathic osteoporosis, these compartments are frequently
different in patients with CKD. In dialysis patients with high PTH levels, the cortical bone
volume is decreased but the cancellous volume is increased. (Lindergard et al., 1985).
3.2 Bone markers
Generally, two different types of bone markers are used to determine the bone
patophysiology:
Chronic Kidney Disease
32
3.2.1 Collagen based bone markers
Active osteoblasts secrete pro-collagen type I, and the pro-peptides at both C-terminal and
N-terminal ends are immediately cleaved and can be measured in the circulation. The
collagen molecules are then covalently bonded through pyridinoline cross-linking. The
fragments containing these pyridinoline links (at both the C-terminal and N-terminal ends
of the peptides) are released during bone resorption: carboxyterminal (CTX) and
aminoterminal (NTX) cross-linking telopeptide of bone collagen, respectively. These
collagen-based markers have been studied in normal populations, where there are
significant but moderate correlations with bone-formation/resorption rates. These markers
are usually increased after bone fracture (Urea and De Vernejoul, 1999; Ivaska et al., 2007).
3.2.2 Non collagen type of bone markers
Osteoblasts secrete other proteins that have been used to assess their function, including b-
ALP, osteocalcin, osteoprotegerin, and receptor activator for nuclear factor kB ligand.
Osteoclasts secrete tartrate-resistant acid phosphatase. Osteocytes secrete FGF-23 in
response to phosphate and calcitriol. High levels of FGF-23 are seen in patients with CKD,
but this is a new measurement, and clinical significance remains to be determined. Some of
these markers are excreted by the kidneys, so in CKD, the serum concentrations may merely
represent accumulation instead of bone turnover (Rogers and Eastell, 2005).
Renal phosphate excretion is physiologically regulated mainly by proximal tubular cells,
which express Na/Pi Type II cotransporters at their apical membrane that control
phosphate reclamation. Renal phosphate reabsorption is mediated primarily through the
Na/Pi IIa co-transporter, whereas approximately one-third of phosphate ions are
reabsorbed through the Na/Pi IIc cotransporter. FGF-23 mediates its phosphaturic effect by
reducing the abundance of the Na/Pi IIa cotransporter in proximal tubular cells (Baum et al.,
2005). In animal studies, transgenic mice over-expressing human or mouse FGF-23 have
severe renal phosphate wasting because of suppression of renal Na/Pi cotransporter activity,
whereas FGF-23 inactivation leads to hyperphosphatemia (Liu et al., 2006). In addition, FGF-
23 may inhibit gastrointestinal phosphate absorption by reducing intestinal Na/Pi IIb
cotransporter activity in a vitamin D dependent manner (Liu et al., 2006). In CKD patients,
circulating FGF-23 levels gradually increase with renal function declining. Although the
increase in FGF-23 is most pronounced in patients with advanced CKD, it may begin at a
very early stage. Apparently, FGF-23 and PTH stimulate phosphaturia in a similar manner
by reducing phosphate reclamation through Na/Pi IIa cotransporters. Nonetheless, PTH is
not indispensable for FGF-23 activity, as the phosphaturic effects of FGF-23 are maintained
in animals after parathyroidectomy (Liu et al., 2006). In CKD patients, the increase in FGF-23
starts with modestly impaired estimated glomerular filtration rate, when serum phosphate
levels are still within the normal range CKD (KDOQI stages 23), whereas FGF-23 levels
increase by more than 100-fold in advanced CKD (KDOQI stage 5) compared with healthy
controls (Imanishi et al., 2004). However, this is inconsistent with the observation that there
is no increase in the accumulation of degraded FGF-23 in advanced CKD. These data instead
favor a mechanism involving increased FGF-23 secretion as the cause of elevated FGF-23
levels. Instead of decreased renal clearance, an end organ resistance to the phosphaturic
stimulus of FGF-23 may exist because of a deficiency of the necessary Klotho cofactor
(Kurosu et al., 2006). Moreover, higher FGF-23 levels in CKD may reflect a physiological
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral and Bone Disorder (CKDMBD)
33
compensation to stabilize serum phosphate levels as the number of intact nephrons declines.
As a result, FGF-23 increases urinary phosphate excretion and decreases gastrointestinal
phosphate absorption directly and through inhibition of 1a-hydroxylase and reduction of
circulating calcitriol levels indirectly. Oversecretion of FGF-23 allows the body to maintain
phosphate levels within a physiological range until very advanced CKD stages (Miyamoto
K et al. 2004).
4. CKD MBD and vascular calcification
Tissue calcification is a complex and highly regulated process in bone and teeth, and also at
extraosseous sites. The most threatening localization of unwanted calcification is at vascular
sites, where it may manifest as both medial and intimal calcification of arteries. Studies in
the general population have identified calcification in most of atherosclerotic plaques.
Calcification seems to be a part of the natural development of atherosclerotic plaques, with
extensive calcification associated with late-stage atherosclerosis. In the general population,
atherosclerotic plaque calcification is associated with cardiovascular events such as
myocardial infarction, symptomatic angina pectoris, and stroke. Medial calcification causes
arterial stiffness, resulting in an elevated pulse pressure and increased pulse wave velocity,
thereby contributing to left ventricular hypertrophy, dysfunction, and heart failure.
Furthermore, an advanced calcification of the heart valves may lead to dysfunction
contributing to heart failure and a risk of endocarditis development (Vliegenthart et al. 2002;
Vliegenthart et al. 2002).
4.1 Different types of vascular calcification
It is generally well recognized that the prevalence of calcification increases with progressively
decreasing kidney function and is greater than that in the general population. Cardiovascular
calcification is associated with increased frequency of major cardiovascular diseases, and could
be of predictive importance for adverse clinical outcomes, including cardiovascular events and
death (Foley RN et al., 1998). There is an increased prevalence of cardiovascular calcification in
patients even at early stages of CKD. Thus, an important percentage of CKD patients are at
high risk of cardiovascular events from vascular calcification. Two patterns of vascular
calcification have been described: namely intimal and medial calcification. In the general
population, an elevated coronary artery calcium (CAC) score almost exclusively reflects the
atherosclerotic disease burden. In two small autopsy studies, it became apparent that, in
dialysis patients, CAC is also predominantly localized in the coronary intima, whereas the
medial calcifications observed in a minority of such patients seemed to be adjacent to plaque
areas just beneath the internal elastic lamina. Although the coronary vascular bed may differ
considerably from other arteries with regard to the calcification process and its manifestations,
the same group observed a pure medial calcification in the coronary arteries during the early
stages of CKD (Schwarz et al., 2000). A pure medial calcification, in the absence of intimal
disease, was also observed in epigastric arteries obtained from dialysis patients at the time of
renal transplantation (Amann K., 2008;).
4.2 Promoters and inhibitors of calcification
Vascular calcification is the result of passive and active processes, as is bone mineralization.
It has been shown that that normal extracellular phosphate concentration is required for
Chronic Kidney Disease
34
bone mineralization, while lowering this concentration prevents mineralization of any
extracellular matrix. However, simply raising extracellular phosphate concentration is not
sufficient to induce pathological mineralization, because of the presence in all extracellular
matrices of pyrophosphate, an inhibitor of mineralization (Riser et al., 2011). They further
showed that extracellular matrix mineralization normally occurs only in bone because of the
exclusive coexpression in osteoblasts of Type I collagen and of tissue non-specific alkaline
phosphatase (Tnap), an enzyme that cleaves pyrophosphate. Pyrophosphate probably is the
most important non-protein inhibitor of vascular calcification. Its extracellular concentration
is strictly regulated by several enzymes. It is generated by PC-1 nucleotide triphosphate
pyrophosphohydrolase and metabolized to inorganic phosphate by nucleotide
pyrophosphatase/phosphodiesterase (NPP1), in addition to Tnap. Its hydrolysis to
inorganic phosphate actually transforms it from a calcification inhibitor to a promoter. In
addition to pyrophosphate other inhibitors are also present locally in VSMCs, including
matrix-gla protein (MGP) and Smad6 proteine (Lomashvili et al., 2008; Rutsch et al., 2001;
Johnson et al., 2005).
4.3 Contribution of experimental models in vascular calcification
Arterial calcification assessed by all the available imaging studies cannot accurately
differentiate calcification that is localized to the intima from calcification in the media
adjacent to the internal elastic lamina, or in the medial layer (Figure 1 and 2). Thus, there is
neither definitive evidence to suggest that isolated medial calcification is distinct from the
calcification that occurs in the natural history of atherosclerosis nor is there definite proof
against it. Experimental and ex vivo studies suggest that the vascular smooth muscle cell
may be critical in the development of calcification by transforming into an osteoblast-like
phenotype (Giachelli CM, 2004). Elevated phosphorus, elevated calcium, oxidized low-
density lipoprotein cholesterol, cytokines, and elevated glucose, among others, stimulate
this transformation of vascular smooth muscle cells into osteoblast-like cells in vitro using
cell-culture techniques. These factors likely interact at the patient level to increase and/or
accelerate calcification in CKD. Given the potential complexity of the pathogenesis and the
inability of radiological techniques to differentiate the location of calcification, the approach
to all patients with calcification should be to minimize atherosclerotic risk factors and
control biochemical parameters of CKDMBD. In addition, the pericyte in the media and
adventitia may have a role in the secretion of vascular calcification-inducing factors
(Giachelli et al., 2004). The stimulus for such a transformation may depend on the location of
calcification within the artery wall (Figure 2A and 2B). For example, in intimal lesions,
atherosclerosis may be the most important stimulus. However, in patients with CKD and
medial calcification, there may be additional, or additive, factors potentially explaining why
medial calcification of the peripheral arteries can be seen without intimal changes and is
more common in CKD than in the non-CKD population (Moe et al., 2003).
Over the past decade, several animal studies have provided evidence for an accelerated
progression of atherosclerosis in association with the uremic state. We and others have used
the apolipoprotein e knockout (Apoe/) mouse with superimposed CKD and observed that
in this experimental model of severe hypercholesterolemia the development of
atheromatous lesions was greatly enhanced compared with the rate of lesion development
in nonuremic Apoe/ mice (Massy et al., 2005; Ivanovski et al., 2005). Additionally, in our
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral and Bone Disorder (CKDMBD)
35
Fig. 1A. Intima and media calcification by radiography. a) Femoral artery intimal
calcification; b) Femoral artery medial calcification; c) Pelvic artery medial calcification; d)
Iliac arteries mixed calcification. (London et al. 2003).
Fig. 1B. Coronary artery calcification by Electron beam computed tomography (EBCT), (scan
courtesy of Pr P. Raggi).
a)
c)
b)
d)
Chronic Kidney Disease
36
A
B
Fig. 2. Localization of different types of vascular calcification in humans. A) Intimal; B)
Medial; (London et al. 2003).
The New Kidney and Bone Disease:
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37
model, accelerated calcification of the aortic wall both in the intima and the media, (Figure
3A and 3B, respectively) occurred in the absence of hypertension, and fetuin a deficiency
greatly enhanced intimal calcification. Similar observations have been made using another
hyper cholesterolemic animal model of severe atherosclerosis with superimposed CKD,
namely the LDL receptor knockout mouse model (Mathew et al. 2007; Davies et al. 2005). Of
note, the first cardiovascular changes observed in early stages of CKD in Apoe/ mice as
well as in wild type mice were left ventricular hypertrophy, altered left ventricular
relaxation and increased aortic stiffness in the absence of identifiable morphological changes
of the vessel wall. The observed cardiac and aortic abnormalities were not associated with
the degree of aortic calcification or the level of serum total cholesterol, but with the extent of
subendothelial dysfunction and the severity of CKD. Our findings have revealed that the
cardio vascular lesions observed in early stage of acute kidney injury are likely functional.
Although the above experimental findings need to be confirmed by additional studies in the
clinical setting, they open up the possibility of attenuation of atherosclerosis and even
reversal by adequate therapeutic strategies. Findings from experimental observations favor
the existence of two different types of vascular disease linked to CKD, namely early
arteriosclerosis, in the absence of atherosclerosis, and the acceleration of already existing or
subsequently developing atherosclerosis by the uremic state (Drueke and Massy, 2010).
Finally, a rare but very severe form of medial calcification of small (cutaneous) arteries is
calciphylaxis, also called calcific uremic arteriolopathy. This complication is strongly
associated with CKD-related disturbances of mineral metabolism, including secondary HPT,
in approximately one-third of cases. It is characterized by ischemic, painful skin ulcerations
followed by superinfections, and is associated with high mortality. Relationships with
dysregulated calcification inhibitors (fetuin-A and matrix Gla protein) have been implicated
in the pathogenesis of calciphylaxis (Schoppet et al., 2008; Suliman et al., 2008), but because
of the relatively low incidence of the disease, no conclusive data are available to firmly
comment on the nature of the disease process or to allow generalizable treatment options to
be recommended.
4.4 Management of patients with vascular/val vular calcification
Recently it has been confirmed that cardiovascular calcification development and
progression can be influenced by treatment. Given that vascular calcification is associated
with increased cardiovascular risk, and that the pathogenesis seems to be related to CKD
MBD abnormalities and atherosclerosis, it is appropriate to evaluate and modify both. CKD
MBD longitudinal studies have also shown that the progression of vascular calcification to
be modifiable by the choice of phosphate binders. Aluminum-containing phosphate binders
have been widely abandoned because of serious adverse effects including adynamic bone
disease, microcytic anemia, dementia, and death (Alfrey et al., 1976). They were initially
replaced by calcium-containing, aluminum-free phosphate binders. Subsequently, several
studies showed that the high amounts of calcium ingested with these binders were
associated with vascular calcification whose progression could be slowed by the calcium-
free, aluminum-free binder sevelamer (Block et al., 2005; Chertow et al., 2002; London et al.,
2008). The Treat-to-Goal study compared sevelamer-HCl to calcium-containing phosphate
binders, analyzing the progression of coronary artery and aortic calcification (by EBCT) in
prevalent HD patients over 1 year. Although calcification scores progressed with calcium-
Chronic Kidney Disease
38
(a)
(b)
Fig. 3. Extent and localization of different types of atherosclerotic lesion calcification in
apoE/ mice with CRF. von Kossa staining. a) solid type of plaque calcification,
magnification 25; b) non-plaque calcification, magnification25. (Phan et al. 2008).
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral and Bone Disorder (CKDMBD)
39
containing phosphate binders, treatment with sevelamer-HCl was associated with a lack of
calcification progression (Chertow et al., 2002). A similar design was used, and the results
showed more calcification progression in patients treated with calcium based binders
compared with sevelamer-HCl in the Renagel in New Dialysis Patients study, which studied
incident HD patients who were randomized within 90 days after starting dialysis treatment
(Block et al., 2005). The Calcium Acetate Renagel Evaluation-2 study showed that the use of
sevelamer-HCl and calcium acetate was associated with equal progression of CAC when
statins were used to achieve a similar control of the serum low-density lipoprotein
cholesterol in the two study arms (Qunibi et al., 2008). Interestingly, in Calcium Acetate
Renagel Evaluation-2, the combination of sevelamer- HCl and atorvastatin was actually
associated with a higher progression rate of CAC than that in Treat-to-Goal, instead of
showing an amelioration of CAC progression with the combination of calcium acetate and
statin. It is difficult to reconcile these differences, although one potential explanation is that
the Calcium Acetate Renagel Evaluation- 2 study patient population had a higher number of
cardiovascular risk factors than did that of the Treat-to-Goal study (Floege J., 2008).
Although abnormalities of calcium phosphate homeostasis have long been linked with
dysfunction of large arteries in these patients, more recent studies have suggested a role in
the pathogenesis of atherosclerosis in smaller, critical arteries, most notably the coronary
arteries (London et al., 2003). Coronary artery calcification (CAC) is a strong predictor of
atherosclerotic disease in the general population. It has been recognized that most
population studies measuring CAC did not necessarily exclude individuals on the basis of
kidney function and thus include variable numbers of CKD patients. In general, this
literature evaluating the general population supports the view that CAC is part of the
development of atherosclerosis and occurs almost exclusively together with atherosclerosis
in human arteries. It seems that calcification occurs early in the atherosclerotic process;
however, the amount of calcification per lesion has a variable relationship with the
associated severity of luminal stenosis. The relationship between the degree of calcification
in an individual lesion and the probability of plaque rupture is unknown. In the general
population, the overall coronary calcium score can be considered as a measure of the overall
burden of coronary atherosclerosis. The American College of Cardiology/American Heart
Association document indicates that the relationship between CAC and cardiovascular
events in the CKD population is less clear than that in the non-CKD population because of a
relative lack of informative studies and the possibility that medial calcification may not be
indicative of atherosclerotic disease severity. The almost exclusive relationship between
magnitude of calcification and atherosclerosis burden is controversial in CKD patients
(Amann, 2008), in contrast to the situation in the general population. Antiatherosclerotic
strategies using statin treatment have been shown to have a beneficial impact on the
atherogenic profile, atheroma progression, and cardiovascular events in patients with no
known CKD (Nissen et al. 2004). In our experimental model, we have shown that statins had
a beneficial effect on uremia enhanced vascular calcification in apoE knock out mice with
chronic kidney disease. This effect was observed despite the absence of changes in uremia
accelerated atherosclerosis progression, serum total cholesterol levels or osteopontin and
alkaline phosphatase expression. This observation opened the possibility of a cholesterol
independent action of statins on vascular calcification via a decrease in oxidative stress
(Ivanovski et al., 2008). In CKD patients, there are no data on the effects of statins on arterial
Chronic Kidney Disease
40
calcification, as compared with those of placebo. Even worse, the 4D study failed to show a
benefit of atorvastatin treatment on the outcome of diabetic dialysis patients. Studies in
progress like SHARP (Study of Heart and Renal Protection) and AURORA (A Study to
Evaluate the Use of Rosuvastatin in Subjects on Regular Hemodialysis: An Assessment of
Survival and Cardiovascular Events) failed to show a better understanding of the benefits of
correcting atherosclerotic risk factors on cardiovascular events and mortality in patients
with CKD stages 35 and 5D (Baigent et al., 2003).
An association of vascular calcification with high phosphate intake has so far not been
directly demonstrated in uremic patients, probably owing to the fact that it is difficult, if not
impossible, to assess phosphate (protein) intake in a quantitative manner over prolonged
time periods. Indirect evidence for a role of oral phosphate, however, has recently been
provided by Russo et al (Russo et al., 2007). They showed that in patients with CKD stage 3-
5, coronary artery calcification score progressed significantly over a time period of 2 years,
in association with a significant increase in phosphaturia. Many pharmaco-epidemiologic
studies have shown a survival benefit in CKD patients receiving active vitamin D
derivatives, as compared to those who did not receive such treatments. Finally, let us not
forget that association does not imply causation. We clearly need randomized prospective
trials showing that active reduction of serum phosphorus, PTH, or alkaline phosphatases
and normalization of serum calcium leads to an improvement in patient outcomes, and that
specific treatments given to the patients improve outcome, as compared to either placebo or
other treatments (Drueke and McCarron, 2003).
To date, there are no published prospective studies in humans that have evaluated the
impact of calcimimetics or calcitriol and vitamin D analogs on arterial calcification.
However, a recent observational study showed a U-curve type of relationship between
serum 1,25(OH)2D3 and arterial calcification in children and adolescents with CKD stage
5D. No such association existed between serum 25(OH)D and arterial calcification. In one
study in adult patients with CKD stage 5, no independent association of serum 25(OH)D or
1,25(OH)2D3 levels with arterial calcification was observed, (London et al., 2007). Although
the authors of another report identified an association between 25(OH)D deficiency and the
magnitude of vascular calcification (Matias et al., 2009). The experimental data supporting
less toxicity of vitamin D analogs compared with calcitriol are not completely consistent
across studies, but, in general, support the claim that there is reduced calcification with
equivalent PTH lowering with different vitamin D analogs (Lopez et al., 2008). Experimental
studies showed differential effects of calcimimetics and calcitriol on extraosseous
calcification, the former being neutral or protective, the latter being a dose-dependent risk
factor for calcification. In our studies, we have analysed the role of chronic renal failure
(CRF) on the arterial wall changes including atherosclerosis and vascular calcifications in
CRF apoE-/- mice experimental model (Massy, Ivanovski et al. 2005). Furthermore, we have
studied the effect of different non-calcium (Phan et al., 2005) and calcium phosphate binders
(Phan et al., 2008) and role of control of phosphatemia on vascular calcification and
atherosclerosis (Ivanovski et al. 2009). We have also showed for the first time that the
phosphate binder La carbonate is capable of preventing both uremia-enhanced vascular
calcification and atherosclerosis in experimental model of CKD (Nikolov et al., 2011). These
effects were comparable to those of sevelamer on vascular calcification and atherosclerosis,
as previously reported by us for sevelamer-HCl in this model (Phan et al., 2008).
The New Kidney and Bone Disease:
Chronic Kidney Disease Mineral and Bone Disorder (CKDMBD)
41
5. CKD MBD summary
Mineral and bone disorders are complex abnormalities that cause morbidity and decreased
quality of life in patients with CKD. To enhance communication and facilitate research, a
new term has been established, CKDMineral and Bone Disorder (CKD-MBD), to describe
the syndrome of biochemical, bone, and extraskeletal calcification abnormalities that occur
in patients with CKD. Also, it has been recommended that the term renal osteodystrophy be
used exclusively to define alterations in bone morphology associated with CKD. The latter
can be further assessed by histomorphometry, with results reported on the basis of a
classification system that includes parameters of turnover, mineralization, and volume. The
international adoption of the proposed uniform terminology, definition, and classification to
describe these two disorders caused by CKD enhanced communication, facilitated clinical
decision making, and can promote the evolution of evidence based clinical-practice
guidelines worldwide. This issue of Advances in CKD further describes the clinical
manifestations and pathophysiology of CKD-MBD. The optimal management of CKD-MBD
(Chronic Kidney Disease Mineral and Bone Disorder) should be achieved without
increasing the risk of metastatic calcification, including that of blood vessels.
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Calcification Study. Eur Heart J.; 23(20): 1596-1603.
4
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in
Urban Niger Delta of Nigeria
U. R. Onyemekeihia
1
, C. O. Esume
2
, E. Unuigbe
3
, E. Oviasu
3
, L. Ojogwu
3
1
Renal Unit, Department of Medicine, Central Hospital Warri, Delta State
2
Department of Pharmacology and Therapeutics, Delta State University, Abraka
3
Department of Medicine, University of Benin Teaching Hospital, Benin City
Nigeria
1. Introduction
Chronic renal failure (CRF) is defined as a progressive and persistent deterioration in renal
function with serum creatinine consistently greater than 175mol (Adelakun and Akinsola,
1988). It occurs as the termination of many chronic renal diseases, and is an important cause
of morbidity and mortality in Africa (Kadiri, 2001). End stage renal failure may be defined
as creatinine clearance of less than 10mls/minute or sustained plasma creatinine
concentration above 500pmol/l (Ojogwu, 2001).
In United Kingdom the prevalence of chronic renal failure is approximately 600 individuals
per million population per year (0.06%). The incidence of end-stage renal failure is of the
order of 200 per million population per year (0.02%) (Baker, 1999).
In Nigeria, although accurate figures are not available, the size of the problem has been
estimated using hospital admission records (Kadiri, 2001). Hospital admission rates of CRF
in South West Nigeria were reported as between 6.7%-8%
(Akinsola et al, 1989; Kadiri and
Arije, 1999). However, much earlier reports by Adetuyibi et al showed that CRF accounted
for 11.4% of deaths on the medical wards of a major teaching hospital in the region
(Adetuyibi et al, 1976).
Chronic glomerulonephritis and hypertension account for majority of CRF cases in Nigeria,
with diabetes mellitus, obstructive uropathy and autosomal dominant polycystic kidney
disease accounting for smaller proportions
(Akinsola et al, 1989). Chronic interstitial
nephritis is thought not to be a common cause of CRF in Nigeria and other parts of Africa
(Gold et al 1990, Ojogwu 1990, Mate-Kole et al 1990). Once established, chronic renal
impairment tends to progress inexorably to end-stage renal failure, but the rate of
progression depends on the underlying aetiology: for example chronic glomerulonephritis
leads to a more rapid deterioration compared with chronic tubulointerstitial nephropathies
(Baker, 1999). Chronic renal failure is associated with widespread complications, and renal
osteodystrophy (ROD) is one of such complications
(Hartmut and Marie-Claude 1990). ROD
develops in the early stages of loss of the excretory functions of the kidney, and can begin
many years before its symptoms and radiological changes appear in adults (Hartmut and
Chronic Kidney Disease
48
Marie-Claude 1990). Symptoms of ROD are seen only in about 10% of pre-dialysis patients,
but when they have been on dialysis for several years, 90% of them will have symptoms
(Sanchez 2001). When glomerular filtration rate (GFR) falls to 50% of normal, more than 50%
of patients exhibit abnormal bone histology. As much as 90% of patients with end-stage
renal failure on maintenance haemodialysis have abnormal bone history.
The bone disorders associated with chronic renal failure are; Osteitis Fibrosa cystica due to
secondary hyperparathyroidism, osteomalacia, osteoporosis. Adynamic osteopathy, skeletal
microglobulin amyloid deposit, aluminum related low turnover bone disease and mixed
forms of ROD. Osteitis Fibrosa is the commonest form of ROD (Hartmut and Marie-Claude
1990). All these increase the morbidity and mortality in patients with CRF. The prevalence
of the different types of ROD may vary depending on aluminum exposure, treatment with
Vitamin D metabolites, dietary intake, and whether or not is undergoing dialysis (Hartmut
and Marie-Claude 1990).
The diagnosis of ROD can either be by invasive or non invasive methods. The invasive
methods include: bone biopsy after double tetracycline labeling, scintigraphical scan studies,
computed tomography and bone densitometry (Sanchez 2001). A definitive diagnosis of
ROD can only be made with bone biopsy. The non invasive methods employ the use of
serum markers of bone metabolism, including bone-specific alkaline phosphatase (bap), pre
collagen type 1 carboxy1- terminal extension peptide (PICP), osteocalcin, pyridinoline
(PYD), tartrate resistance acid phospatase (TRAPE) and intact parathyroid hormone (IPTH),
and skeletal x-ray (Sanchez 2001). Indeed, detection of biochemical makers such as serum
bap can predict the presence of ROD. Serum bap is a specific and sensitive marker that is
used to evaluate the degree of bone remodeling in uraemic patients (Sanchez 2001, Urena et
al, 1991). Also intact PTH and several relatively new bone markers such as PYD and PICP
are of immense value in the non-invasive diagnosis of ROD. In patients that do not have
liver disease, parathyroid hormone and alkaline phosphatase are less expensive and non-
invasive alternatives for evaluation of ROD (Urena et al, 1991). These biochemical markers
have the added advantage of allowing for repeated measurements, and therefore make
possible the study of short term changes in bone turn over and the effect of treatment (Coen
et al 1998). They may be used to predict the risk of fracture (independently of bone loss),
Rate of bone loss and also the response to therapy (Coen et al 1998).
In developing countries like Nigeria, these non-invasive and relatively less expensive
methods for evaluating bone changes in CRF patients will be a very useful alternative to the
invasive and relatively more expensive method used in developed counties.
Because of paucity of data, the prevalence of ROD in Nigeria is not known, however the
prevalence of ROD in University of Nigeria Teaching Hospital Enugu using skeletal x-ray
was reported to be 3.35% (Odenigbo, 2003). With the increase in the number of patients with
CRF requiring or undergoing dialysis across Nigeria, it has becomes necessary to study the
extent of ROD in CRF patient with or without dialysis.
2 Methodology
2.1 Place of study
The study was done at the University of Benin Teaching Hospital (UBTH)) Benin City,
which is a 420-bedded tertiary hospital with Renal Unit that offers dialysis. Majority of
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
49
patients come from Edo State (where the hospital is situated), Delta, Anambra, Ondo, and
Oyo States.
2.2 Type of study
The study was prospective, descriptive, Clinico-pathological and hospital based.
2.3 Subjects
The study group was made up of consecutive chronic renal failure patients attending the
University of Benin Teaching Hospital (UBTH).
2.4 Inclusion criteria
1. Ultrasonographic findings of bilaterally shrunken kidneys of less than 9cm bipolar
diameter.
2. Persistently elevated serum creatinine concentration above 175 mol/l.
3. Patients aged between 18years and 65years.
The subjects were recruited after obtaining informed consent from them (and /or relations
when necessary). Also ethical approval was sought and obtained from the Ethical
Committee of the UBTH.
2.5 Exclusion criteria
Exclusion from the study included:
1. Patients aged below 18years
2. Those who have had or are on vitamin D therapy
3. Those with chronic liver disease
4. Those who indulge in excessive alcohol intake
5. Post menopausal women.
6. Bed ridden patients.
7. Patient with metastatic bone disease.
2.6 Sample size
Fishers formula for determining sample size was used. This is:
Z2Pq
N
d2
n= number of sample size
p= prevalence of the problem =0.06%
q= 1-P
z= 95% confidence interval =1.96
d= level of precision =0.05
n= 86.7=87
However since this was a pilot study, sample size of approximately 50% of above (that is 50)
was used.
Chronic Kidney Disease
50
A total of 115 patients were screened by:
Taking of a detailed history and physical examination at the initial contact with the patients
with view to determining whether the patients had features suggestive of CRF and also
whether patients met the inclusion criteria. Those with obvious exclusion criteria were
dropped from the study at this level. This resulted in the exclusion of 55 subjects.
Twenty-four hours urine was collected and used for estimation of creatinine clearance and
24hours urinary calcium. On the morning of the test (8.00am), patients emptied their
bladder and discarded the urine. Subsequently, urine passed was put into a clean container
until 8.00am the next day. At the end of the urine collection, 5mls of blood was collected
from the patients for estimation of serum creatinine. The creatinine clearance was calculated
by using the formula: UV/P, where V is the urine flow rate (mls/min). Normal values of
creatinine clearance was taken as 105-150mls/min while 24 hours urinary calcium was taken
as 100-300 mg/dl. Where it was necessary radiograph of the chest and lumbo-sacral spines
were carried out to rule out metastatic bone lesion. An abdominal ultrasonographic scan
was carried out. After these evaluations a total of 52 subjects were studied having met the
inclusion criteria.
2.7 Study design
The eventual 52 subjects who satisfied the inclusion criteria were required to complete a
researcher administered questionnaire which includes age, sex, occupation, dietary habits,
clinical symptoms of ROD, frequency and regularity of dialysis. Physical examination was
performed by the investigator.
Ten milliters (10ml) of venous blood was drawn from the remaining 60 patients from a
suitable vein with loose fitting tourniquet. The blood sample was used to assay for serum
alkaline phosphatase levels, bone specific alkaline phosphatase levels, serum albumin levels,
calcium and phosphate levels.
Osteocalcin, hydroxyproline, parathyroid hormone and calcitriol assays were not carried out
because of lack of necessary facilities. However, a surrogate for parathyroid hormone was
taken as total serum alkaline phosphatase.
Glomerular filtration rate was determined by 24hour urinary creatinine clearance. Also
24hour urine calcium was determined from the 24 hr urine. Plain x-rays of the wrist/
phalanges of both hands and / or lumbo-sacral spine to include the pelvis was carried out
looking out for features of osteodystrophy. Bone histology, though more sensitive in the
diagnosis of ROD than radiographic evidence could not be done in live subjects because
consent for the procedure was refused. 20 of the CRF patients died during the study.
However, out of these, only 14 died in the hospital and the corpse deposited in the
mortuary. Post mortem bone biopsies were done on 10 of the 14 bodies whose relations gave
consent for the post mortem after obtaining consent from relations.
40 patients without the exclusion criteria and who did not have the have renal failure but
attending out -patient clinic of UBTH, whose ages ranged from 18-65years were used as
controls.
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
51
MATERIAL AND METHODS
2.8 Apparatus
2.8.1 X-ray machine Watson RO1
This is a standard machine with good resolution quality.
The procedure was carried out by an experienced radiographer using the posterior- anterior
positions.
The film was read and reported by an experienced consultant Radiologist.
2.8.2 Ultrasound machine
Sonoace 1500 (Medison) 3.5 MHz sector probe was used. This is a standard machine with
good resolution.
Renal ultrasound scan was carried out by an experience Sonographer
2.9 Serum phosphate estimation
This was done using Fiske-Subbarow method. This is a colorimetric method using Fiske-
Subbarow reagent.
The composition of the reagent is a follows:
Ammonium molybdate -7nM
Sulphuric acid 1.7N
Iron (11) Sulphate -8Mm
2.9.1 Principle
The phosphate ion reacts with Molybdate to produce Phosphomolybdate, which is finally
reduced to a molybdenum blue, which is photo metrically measured.
2.9.2 Technique
1. Three test tubes were labeled as: Blank BL
Standard ST
Sample -SA
2. 0.1ml of patients blood sample was added to SA. 0.1ml of standard was added to ST.
To all 3 test tubes were added 3.0ml of the reagent.
3. The content of each of the test tubes were mixed properly and allowed to stand for 10
minutes at room temperature (20-25
0
C).
4. Reading was done using a spectrophotometer set at 650nM wavelength.
5. The concentration of inorganic phosphate in patient sample was calculated using the
formula.
Inorganic phosphate (mg/dl) =
SA O.D 4
ST O.D
O.D = Optic Density
Normal value of serum phosphate was taken as 2.4- 4.5 mg/dl.
Chronic Kidney Disease
52
2.10 Measurement of total serum calcium
The Cresolphtalein- Complexone method (CPC) was used for the determination of total
serum calcium. This is a standard Colorimetric method. This like most calcium assays
measures the total serum calcium although it is only the free calcium which constitutes 50-
65% of total calcium that is biologically active.
2.10.1 Principle
CPC forms a violet colored complex with calcium. The absorbance of the colour produced
was measured in a colorimeter using a Spectrophotometer at 575nM wavelength measured
in a colorimeter using a spectrophotometer at 575nM wave length. Interference from
magnesium was reduced by including 8-hydroxquinoline in the working CPC reagent. The
Ethanediol in the reagent suppresses the ionization of the 0- Cresolphtalein and helps to
give a clear solution. A correction was made when the patients serum albumin level was
below 40g/l using this formula:
Corrected serum albumin =
2
albumin g /l Ca
40
40
2.10.2 Method
1. Two sets of four tubes were labeled as follows:
B-reagent blank, standard (2mmol/l)
C - Control; p - patient.
2. 5mls of CPC reagent was added into each tube using a pipette
3. The following were added into each set of tubes using a pipette as follows:
B. - 0.05mls distilled water
S - 0.05mls standard (2mmol/1)
C- 0.05mls Control serum
p - 0.05mls patient serum
The contents of each tube were mixed for several seconds.
4. Using clean Cuvettes, the absorbance of the solution were read in a colorimeter using a
spectrophotometer set at 575nM. The instrument was zero with a blank solution in tube
B.
5. The concentration of Ca
2+
in the patients sample was calculated with the following
formula:
Ca
2+
(mmo1/L) =
AT
2
AS
AT= Absorbance of test or control
AS= Absorbance of standard
Serum calcium values of 8.5 10.5mg/dl was taken as normal.
2.11 Measurement of serum alkaline phosphatase
This was done using the kind and king method (1954). This is a standard colorimetric
method.
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
53
2.11.1 Princile
4- amino antipyrine gives a red purple colour with compounds containing a phenolic group
in the presence of alkaline oxidizing agents.
2.11.2 Technique
2mls of buffer substrate was measured into each of the 2 test tubes and placed in water bath
at 37
0
C for a few minutes. To one of the test tube (patients test tube) was added 0.lml of
serum and the tubes were incubated for exactly 15 minutes. They were removed from the
bath and 0.8ml of 0.5M sodium hydroxide and 1.2ml of 0.5M (the blank) was added to both
tubes, 1ml of amino- antipyrine reagent and 1ml of potassium ferricyanide were added. For
the standard, 1ml of buffer and 1ml of phenol standard containing 0.01mg of phenol was
taken. For the standard blank, 1.1ml of buffer and 1ml of water was taken to both tubes, and
then sodium hydroxide, bicarbonate amino-antipyrine and ferricyanide was added as
above. It was read with a spectrophotometer set at 520 millimicrons. Serum alkaline
phosphatase (King- Amstrong unit per 100mls) was derived from the formula:
Serum alkaline phosphatase = Reading of unknown - Reading of blank x 10
Reading of standard - Reading of standard blank
Total serum alkaline phosphatase values of 25-95 IU/L was taken as normal
2.12 Determination of bone specific alkaline phospatse
This was done by curve-fitting of inhibition kinetic as popularized by (Statland et al, 1972).
2.12.1 Principle
After the determination of the total serum alkaline phosphatase as described above, the
serum is heated for 13 minutes at 56
0
c to inactivate the bone- type isoenzyme. The sera are
now read as in the determination of the total serum alkaline phosphatase using a
spectrophotometer set at 520 millimicron wavelength.
The concentration of serum alkaline phosphatase excluding the bone isoenzyme was
determined as was done for total alkaline phosphatase using a spectrophotometer set at
520millimicron wavelength. The bone isoenzyme was determined by subtracting this value
from the total alkaline phosphatase. Values of bone specific alkaline phosphatase level
greater than 50% of total serum alkaline phosphatase shows significant contribution from
bone iso-enzyme.
2.13 Bone histology (post mortem)
2.13.1 Procedure
1. The autopsy specimen biopsy was taken from the pelvic bone.
2. Decalcification: The piece of bone biopsied was decalcified by emersion in 10% nitric
acid for 2 days.
3. Dehydration: the decalcified bone was dehydrated by passing the bone through
ascending grades of alcohol. (70%, 90%, 100%) respectively.
Chronic Kidney Disease
54
4. Clearing of excess alcohol: to rinse off excess alcohol that could be in the tissue, it was
rinsed with xylene or toluene.
5. Impregnation with paraffin wax: the tissue was then impregnated with paraffin wax,
and subsequently embedded into paraffin block.
6. Cutting into sections: the tissue embedded into paraffin block was cut into section of
about 5 micron thick.
7. The cut section was then placed on a slide and allowed to dry for a minimum of 30 min.
8. Staining process: heamatin and eosin stains were used.
9. Reading of slide: the slide was read and reported by an experienced histopathologist.
2.14 Statistical analysis
Data analysis was done using SPSS package, and the storage was in Microsoft excel. Data
are expressed in tabular, bar chart and prose forms. Mean standard deviation and
percentages of all data were derived. Odd ratio was used to measure strength of association
between ROD and their relative risk.
The t-test and chi-squared test were used to determine the differences in means of the CRF
group and control.
P value of less than 0.05 was regarded as significant.
3. Results
3.1 Characteristics of subjects studied
A total of 115 patients were screened for the study. 52 of them were studied, having met the
inclusion criteria. This was made up of 30 (58%) males and 22 (42%) females. 40 age and sex
matched controls, made up of 22 (55%) males and 18 (45%) females were also studied. The
age range of the study population was 18 65 years. 3 (5%) of the CRF patients were in the
age range less than 30 years, 16 (30%) were in the 31 40 years age range, 13 (25%) and 14
(26%) were in the age range 41 50 years and 51 60 years respectively, while 6 (11%) were
in the age range greater than 60 years. The peak incidence of CRF was in the 31 40 years
age range. The mean age of the CRF patients and controls were 42.5 + 11.6 years and 40.4 +
11.3 years respectively (refer to table 1).
AGE (YEARS) CRF GROUP (n = 52) CONTROL GROUP (n = 40)
Frequency (%) Frequency (%)
<30
31 40
41 50
51 60
>60
3 (5%)
16 (30%)
13 (25%)
14 (26%)
6 (11%)
7 (17%)
12 (30%)
10 (25%)
7 (17%)
4 (10%)
Table 1. Age and sex distribution of both CRF and control groups.
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
55
Characteristics CRF Group (n=52)
Mean + SD
Control Group
(n=40) Mean +SD
P
Value
Sex
Age (Yrs)
Renal sizes (cm)
Right
Left
Creatinine clearance (mls/min)
Total alkaline phosphatase (iu/l)
BAP (iu/l)
Serum calcium (mg/dl)
Serum phosphate (mg/dl)
24 hours urine calcium (mg/dl)
Serum creatinine (mg/dl)
Blood urea (mg/dl)
Serum protein (gm/dl)
M (30), F (22)
42.5 + 11.6
8.33 + 0.5
8.16 + 0.5
9.8 + 6.7
129.4 + 21.6
83.12 + 21.6
6.9 + 2.3
6.1 + 1.9
1.7 + 2.37
7.16 + 2.4
123.8 + 39.8
3.2 + 0.60
M (22), F (18)
40.38 + 11.3
11.99 + 0.32
11.92 + 0.33
126 + 7.3
43.73 + 8.31
21.8 + 4.11
9.12 + 0.5
3.20 + 0.6
3.8 + 1.76
0.8 + 0.15
24.4 + 5.6
4.46 + 0.52
>0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
>0.05
<0.05
<0.05
>0.05
Table 2. Characteristics of CRF and control group.
In the age ranges <30 years and 31 40 years, the mean creatinine clearance were 7 + 1.83
mls/min and 12.6 + 2.16 mls/min respectively. In the age ranges 41 50 years and 51 60
years, the mean creatinine clearances were 8.6 + 3.2mls/min and 8.2 + 2.6mls/min
respectively. In the age range >60 years, the mean creatinine clearance was 11.8 +
5.2mls/min. Creatinine clearance was lowest in the <30 years age range. This is represented
in figure1.
3.2 Symptoms of renal osteodydtrophy (ROD)
The symptoms suggestive of ROD in the study population include bone pain and pruritus.
7(14%) of the CRF group had symptoms. This was made up of 5(71%) that had bone pain
and 2(29%) that had pruritus. 5(12%) of the control group had bone pain. None had
pruritus. The symptom of bone pain was commoner in males compared to females. Of those
that had bone pain, 4 (80%) were males while 1 (20%) was a females. Pruritus was equally as
common in both sexes (1 male and 1 female).
The entire patients that had pruritus in the CRF group had elevated serum alkaline
phosphatase, hyperphosphataemia and elevated calcium-phosphate product. There was a
statistically significant correlation between bone pain and creatinine clearance (r =-0.3), such
that bone pain occurred more commonly in patients with end stage renal disease (ESRD).
Chronic Kidney Disease
56
Only 1(1%) patient in the CRF group had radiological evidence of Rugger Jersey Spine.
Radiological evidence of osteoarthritis was found in 3(60%) and 2(40%) of the patients that
had bone pain in the CRF and control group respectively (see Table 3).
Patients Characteristics
Sex Age (Yrs) Serum Ca
(mg/dl)
Serum P04
(mg/dl)
Serum
Alk
Phosp
(iu/l)
Ca x P04
mg
2
/dl
2
X-ray
Findings
Crcl
(mls/min)
Symptoms
1.
2.
3.
4.
5.
6.
7.
M
M
M
F
F
M
M
40
46
52
48
56
62
64
9.8
6.4
4
10.2
4.5
5.6
7.7
14
9.9
5.4
9
13
9.9
6.5
142
156
128
106
136
99
127
137.2
64.4
21.6
19.8
58.5
64.4
48.1
-
RJS
OA
OA
OA
OA
*
12
10.6
12.6
9.6
7.2
8.5
16
Pruritus
Pain
Pain
Pruritus
Pain
Pain
Pain
RJS = Rugger Jersey spine, PO4 = Phosphate, Ca = Calcium, Alk Phosp = Alkaline phosphate, Crcl =
Creatinine clearance, OA = Osteoarthritis.
Table 3. Characteristics of CRF patients symptomatic of ROD.
3.3 Creatinine clearance of subjects
The mean creatinine clearance in the CRF and control groups was 9.8 + 6.7mls/min and
126.2 + 7.4 mls/min respectively. There was a statistically significant difference between
both means (p < 0.05).
Table 4 shows the distribution of subjects according to creatinine clearance. In the CRF
group, 47 (90%) had ESRD, with creatinine clearance < 15mls/min, 4 (8%) had creatinine
clearance of 15 29mls/min, and 1 (2%) had creatinine clearance 30 59mls/min. All
controls had creatinine clearance > 95mls/min (see Table 4).
Creatinine clearance (mls/min) Frequency
CRF group
n = 52
Control group
n = 40
Frequency % Frequency %
> 95
30 59
15 29
<15
-
1
4
47
-
2
8
90
40
-
-
-
100%
-
-
-
Table 4. Distribution of subjects according to creatinine clearance.
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
57
3.4 Renal ultrasonographic scan of subjects
The mean kidney sizes in the CRF group was 8.2 + 0.5cm and 8.3 + 0.5cm for the right and
left kidneys respectively, while that of the control was 12.10 + 0.33cm and 12.02 + 0.33cm for
the right and left kidneys respectively. 52 (100%) patients in the CRF group had shrunken
kidneys (<11cm).
There was a weak positive correlation between renal sizes and creatinine clearance, with r =
0.12 and r = 0.17 for the right and left kidneys respectively (see Table 5).
Renal sizes (cm) Frequency
CRF GROUP
(n = 52)
CONTROL GROUP
(n = 40)
Right Left Right Left
Small size (<9cm)
9 10.9cm
Normal size
(11 12cm)
Large size >12cm
51
1
-
-
51
1
-
-
-
-
40
-
-
-
40
-
Table 5. Renal sizes of subjects on abdominal ultrasonographic scan.
Normal kidney size: 11 12cm (Brenner and Rector. The kidney vol.1, 6
th
edition 2000).
3.5 Serum calcium of subjects
The mean serum calcium in the CRF and control groups was 6.9 + 2.3mg/dl and 9.12 +
0.53mg/dl respectively. This difference between the means was statistically significant
(p<0.05).
Table 6 shows the pattern of serum calcium concentration in the study population. In the
CRF group, 37 (71%) patients had hypocalcaemia (<8.5mg/dl), 3 (6%) had hypercalcaemia
(>10.5mg/dl, while 12 (23%) had normal calcium levels (8.5 10.5mg/dl). In the control
group, 39 (97%) patients had normal calcium levels, while only 1 (3%) had hypocalcaemia.
None in the control group had hypercalcaemia. There was a weak positive correlation
between serum calcium and total alkaline phosphate in the CRF group (r = 0.04), such that
as the serum alkaline phosphate was increasing, the serum calcium was decreasing.
Amongst the CRF patients with total serum alkaline phosphate of <25iu/l, the mean serum
calcium was 6.4 + 1.6mg/dl, while in the group with total serum alkaline phosphate of 25
95iu/l, the mean serum calcium was 7.9 + 3.0mg/dl, and in the group with total serum
alkaline phosphate of >95iu/l, the mean serum calcium was 6.8 + 1.8mg/dl. There was an
insignificant correlation between serum calcium and creatinine clearance (r = 0.007).
Amongst the CRF patients with creatinine clearance of <15mls/min, the mean serum
calcium was 5.2 + 2.4mg/dl, while in the group that had creatinine clearance of 15
29mls/min, the mean serum calcium was 6.3 + 2.1mg/dl, and in the group with creatinine
clearance of >30mls/min, the mean serum calcium was 8.4 + 1.6mg/dl. Mean serum calcium
tends to be lower in ESRD patients (see table 6).
Chronic Kidney Disease
58
Serum Calcium (mg/dl) CRF group
n = 52
Control group
n = 40
Hypocalcaemia
(<8.5mg/dl)
Normal levels
(8.5 10.5mg/dl)
Hypercalcaemia
(>10.5mg/dl)
37 (71%)
12 (23%)
3 (6%)
1 (3%)
39 (97%)
-
Table 6. The distribution of subjects according to serum calcium.
3.6 Serum phosphate of subjects
The mean serum phosphate in the CRF group was 6.1 + 2.0mg/dl, and this was significantly
higher than the mean serum phosphate of 3.2 + 0.6mg/dl in the control group (p<0.05).
Table 7 shows the pattern of serum phosphate in both the CRF and control groups. In the
CRF group, 41 (79%) patients had hyperphosphataemia, while 11 (21%) had normal serum
phosphate levels. No patient had hypophosphataemia. In the control group, all the 40
(100%) patients had normal serum phosphate levels. There was insignificant but positive
correlation between serum phosphate and creatinine clearance (r = 0.1) and bone pain (r =
0.4). (See Table 7)
Serum phosphate
(mg/dl)
CRF group
n = 52
Control group
n = 40
Hypophosphataemia
(<2.4mg/dl)
Normal phosphate
Level (2.4 - 4.5mg/dl)
Hyperphosphataemia
(>4.5mg/dl)
-
11 (21%)
41 (79%)
-
40 (100%)
-
Table 7. Pattern of serum phosphate in subjects.
Amongst the CRF patients with serum alkaline phosphatase of <25iu/l, the mean serum
phosphate was 5.1 + 0.9mg/dl, while in the group with serum alkaline phosphatase of 25
95iu/l, serum phosphate was 6.5 + 1.2mg/dl, and in the group with serum alkaline
phosphatase of >95iu/l the mean serum phosphate was 6.2 + 1.4mg/dl. Thus, there was a
weak but negative correlation between serum phosphate and total serum alkaline
phosphatase (r 0.15), such that when total serum alkaline phosphatase was increasing, the
serum phosphate also increased.
Amongst the CRF patients with creatinine clearance of >30mls/min, the mean serum
phosphate was 4.4 + 1.2mg/dl, while in the groups with creatinine clearance of 15
29mls/min and <15mls/min (ESRD), the mean serum phosphate was 6.6 + 1.1mg/dl and
6.2 + 1.3mg/dl respectively. As the creatinine clearance tended towards ESRD, the serum
phosphate rises. There was a positive correlation between serum phosphate and creatinine
clearance (r = 0.10).
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
59
3.7 Serum alkaline phosphatase of subjects
The mean total serum alkaline phosphatase in the CRF group was 129.4 + 21.6iu/l, while
that of the control was 43.73 + 8.3iu/l. There was a statistically significant difference
between both means (p<0.05). 41 (79%) of the CRF group had elevated total serum alkaline
phosphatase levels, 8 (15%) had normal levels, 3 (6%) had low levels, while all controls had
normal levels. Of the 41 CRF patients that had elevated total serum alkaline phosphatase
levels, all (100%) had >50% of their alkaline phosphatase levels, from bone isoenzyme (bone
specific alkaline phosphatase). Only 1 (2%) CRF patient had radiological evidence of ROD
(Rugger Jersey Spine). Total serum alkaline phosphatase correlated positively with
creatinine clearance (r = 0.06) and bone pain (r = 0.4).
Amongst the CRF group with creatinine clearance of >30mls/min the mean total alkaline
phosphatase was 105 + 6.1iu/l, while in the groups with creatinine clearance of 15
29mls/min and <15mls/min (ESRD), the mean alkaline phosphatase were 124 + 4.6iu/l and
143 + 5.6iu/l respectively (see Table 8).
Total serum alkaline
phosphatase (iu/l)
CRF group, n = 52
Frequency (%)
Control group, n = 40
Frequency (%)
Increased levels (>95iu/l)
Normal levels (25 95iu/l
Decreased levels (<25iu/l)
41 (79%)
8 (15%)
3 (6%)
-
40 (100%)
-
Table 8. Distribution of CRF and control groups according to serum alkaline phosphatase.
3.8 Calcium and phosphate products of CRF subjects
The mean calcium x phosphate products in the CRF group was 42.8 + 21.6mg
2
/dl
2
while
that of the control was 28.21 + 2.4mg
2
/dl
2
. There was a statistically significant difference in
both means (p<0.05). Table 9 shows distribution of calcium x phosphate product amongst
the CRF patients. 3 (5%) of the CRF patients had calcium x phosphate product >70mg
2
/dl
2
.
This was made up of 2 (66%) males and 1 (34%) female. 6 (11%) patients had their calcium x
phosphate product between 52 70mg
2
/dl
2
. This was made up of 2 (33%) males and 4 (67%)
females. 43 (82%) patients had their calcium x phosphate products <52mg
2
/dl
2
. This was
made up of 26 (60%) males and 17 (40%) females. All 3 patients who had calcium x
phosphate product >70mg
2
/dl
2
died during the period of study and showed evidence of
ROD on postmortem bone biopsy.
Normal Ca x P04 product = <70mg
2
/dl
2
.
3.9 Urinary calcium excretion in study group
The mean 24 hours urinary calcium in the CRF group was 68.4+ 12.8mg/dl, and 162 +
40.4mg/dl in the control group. This difference in means is statistically significant (p<0.05).
There was an insignificant correlation between 24hours urinary calcium and creatinine
clearance, and serum calcium (r =-0.16) and (r =0.02) respectively. There was a statistically
significant correlation between 24hours urinary calcium and total serum alkaline phosphatase
(r= 0.38) but no significant correlation with sex (r =0.79), age (r=0.46) or bone paint (r =0.23).
Chronic Kidney Disease
60
MALES FEMALES
Serial
number
Serum
P04
(mg/dl)
Serum
Ca
(mg/dl)
Ca x P04
product
(mg
2
/dl
2
)
Serial
number
Serum
Ca
(mg/dl)
Serum
P04
(mg/dl)
Ca x P04
product
(mg
2
/dl
2
)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
6
5
4.2
6
8.9
8.5
3
6.2
9.7
6.8
5
5.4
9.9
6.5
5.4
7.3
8.6
8.6
4.9
5.4
5.4
14
7
13
9
6.5
8.3
3
5.6
5.4
5.1
4.8
5.8
7.3
4.9
5.3
4.1
5.8
4.8
3.2
2.7
8.7
6.5
7
4
4.3
6
3
7.3
7.1
3.2
9.8
6.9
4.5
12.5
5.9
60.1
6.1
5.8
5.9
30.6
24.0
24.4
43.8
43.6
45.1
12.3
36.0
46.6
21.8
13.5
47.0
64.4
45.5
21.6
31.4
51.6
25.8
48.1
34.8
17.3
17.3
137.2
48.3
58.5
112.5
38.4
50.6
17.4
37.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
6
8.7
5
9.8
10.6
9.2
4
5.6
3.7
6
6.2
6.5
5.7
6.8
7.6
7.8
4.8
5.8
10.2
4.6
7.1
4.9
5.2
3.8
6.5
5.8
6.4
7.5
11.1
4.9
4.4
6.4
7.4
8.6
7.8
7
4.8
4.2
6.4
6.5
5.8
10.2
4.6
7.1
31.2
33.1
32.5
56.8
67.8
69.0
44.4
27.4
16.3
38.4
45.9
55.9
44.5
47.6
36.5
32.8
30.7
37.7
53.4
91.8
35.0
34.8
Mean 6.99 5.83 42.02 6.84 6.47 43.79
Std Dev. 2.5 2.1 26.4 1.9 1.9 16.8
Table 9. Serum Calcium, Serum phosphate and calcium x phosphate product of CRF patients.
3.10 Radiological evidence of ROD
Of the patient that had symptoms of ROD, 5 (71%) had bone pain while 2 (29%) had
pruritus. Only 1 (20%) of the 5 patients that had bone pain showed radiological evidence of
Rugger-Jersey spine (see appendix 1), constituting 9%of the CRF group. Table 10 shows the
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
61
characteristics of the only patient with radiological evidence of Rugger Jersey spine. In the
control group, 5 (21%) had bone pain. Of these, 2 (40%) constituting 5% of the control group,
had radiological evidence of osteoarthritis while Radiological evidence of osteoarthritis was
found in 3(60%) patients that had bone pain in the CRF group.
APPENDIX 1: X-RAY OF THE SPINE SHOWING RUGER-JERSEY APPEARANCE.
NOTE THE ALTERNATING BANDS OF HYPO-DENSITY AND HYPERDENSITY
INDICATING OSTEOPOROSIS AND OSTEOSCLEROSIS RESPECTIVELY.
Chronic Kidney Disease
62
Age
(Years)
Sex RS
(cm)
Left Right
Serum Ca
(mg/dl)
Alk
Phosp
(iu/l)
Serum
P04
(mg/dl)
Ca x P04
(mg
2
/dl
2
)
Crcl
(mls/min)
54 M 7.8 7.6 6.5 156 9.9 64.4 6.0
(Rugger Jersey Spine).
Crcl = Creatinine clearance, Alk phosp = Alkline phosphatase,
P04 = Phosphate, Ca = Calcium, RS = Renal Size.
Table 10. Characteristics of the only patient with radiological evidence of ROD.
3.11 Histological evidence of rod on postmortem bone biopsy
10 Postmortem bone biopsies were carried out. This was made up of 7 (70%) males and 3
(30%) females. 9 (90%) had histological evidence of ROD, while 1 had normal bone
histology. Of the 9 that had histological evidence, 6 (66%) were males while 3 (34%) were
females. 6 (66%) had Osteitis Fibrosa. This was made up of 4 (50%) males and 2 (34%)
females. 2 (22%) had Osteomalacia, 1 each (50%) of male and female, while 1 (12%) male had
evidence of mixed type ROD (see Appendices 2-4). All the patients who had histological
evidence of ROD had their creatinine clearance < 15mls/min (ESRD). 8 (88%) of the patient
with histological evidence of ROD did not have any radiological evidence of ROD.
All the patients that had bone histological evidence of ROD had elevated total serum
alkaline phosphatase and serum phosphatase. There is a positive correlation between
histological evidence of ROD and total serum alkaline phosphatase and serum phosphate, (r
= 0.04) and (r = 0.036) respectively. There was no correlation between histological evidence
of ROD and symptoms of ROD (r = 0.48), see table 11.
Patients Gender Age
(Year)
Histological
type
Calcium
(mg/dl)
Phosp
(mg/dl)
Ca x P04
(mg
2
/dl
2
)
Total Alk.
Phosp
(iu/L)
1
2
3
4
5
6
*7
8
9
M
M
M
F
M
M
F
M
F
46
48
52
46
39
36
48
40
54
OF
OM
OF
OM
Mixed type
OF
OF
OF
OF
6.8
12.5
5.3
7.5
8.7
9.8
10.2
6.1
6.4
9.9
9.0
8.5
9.2
5.4
14.0
9.0
8.3
10.6
64.4
112.5
45.1
69.0
47.0
137.5
91.8
50.6
67.8
156
140
122
134
138
152
98
106
146
* Patient 7 had normal histology
OF = Osteitis Fibrosa, OM = Osteomalacia
Table 11. Characteristics of patients with bone histological evidence of ROD.
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
63
APPENDIX 2: PHOTOMICROGRAMS OF BONE HISTOLOGY SHOWING OSTEITIS
FIBROSA, OSTEOMALACIA AND OSTEITIS FIBROSA IN PATIENTS 1, 2 AND 3
RESPECTIVELY.
Chronic Kidney Disease
64
APPENDIX 3: PHOTOMICROGRAMS OF BONE HISTOLOGY SHOWING
OSTEOMALACIA, MIXED TYPE ROD AND OSTEITIS FIBROSA IN PATIENT4, 5 AND 6
RESPECTIVELY.
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
65
APPENDIX 4: PHOTOMICROGRAMS OF BONE HISTOLOGY SHOWING NORMAL
HISTOLOGY, OSTEITIS FIBROSA, OSTEITIS FIBROSA AND OSTEITIS FIBROSA IN
PATIENT 7, 8, 9 AND 10 RESPECTIVELY.
Chronic Kidney Disease
66
Patients Gender Age
(Year)
Histological
type
Calcium
(mg/dl)
Phosp
(mg/dl)
Ca x P04
(mg
2
/dl
2
)
Total Alk.
Phosp
(iu/L)
1
2
3
4
5
6
*7
8
9
M
M
M
F
M
M
F
M
F
46
48
52
46
39
36
48
40
54
OF
OM
OF
OM
Mixed type
OF
OF
OF
OF
6.8
12.5
5.3
7.5
8.7
9.8
10.2
6.1
6.4
9.9
9.0
8.5
9.2
5.4
14.0
9.0
8.3
10.6
64.4
112.5
45.1
69.0
47.0
137.5
91.8
50.6
67.8
156
140
122
134
138
152
98
106
146
* Patient 7 had normal histology
OF = Osteitis Fibrosa, OM = Osteomalacia
Table 11. Characteristics of patients with bone histological evidence of ROD.
4. Dicussion
4.1 Findings of the study
This study was carried out to determine the prevalence of ROD in chronic renal failure in
Benin City.
The main findings of the study suggest that:
1. Osteitis Fibrosa is the commonest type of ROD.
2. There is a correlation between histological evidence of ROD and biochemical maker
(Alkaline Phosphatase).
3. The yield of ROD using radiological examination is low in our chronic renal patients.
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
67
4. Radiological and biochemical evidence of ROD seems to be more prevalent in severe
chronic renal failure (ESRD).
5. ROD may be more prevalent in males.
6. There is no correlation between symptoms of ROD and biochemical or radiological
evidence of ROD. This suggests that many patients may have ROD with no symptoms.
7. Hypocalcaemia and hyperphosphataemia is prevalent in our CRF patients.
4.2 Osteitis fibrosa is the commonest type of rod
Osteitis Fibrosa is a form of high turnover bone disease as a result of hyperparathyroidism.
PTH assay was not done because of lack of facility in our center. However serum alkaline
phosphatase was used as a surrogate. 78% of the CRF patients had raised levels of total
serum alkaline phosphatase which correlate well with PTH levels and histological features
of secondary hyperparathyroidism. This is in agreement with the work done by Duursma et
al, (1975); Ritz et al (1974) and Hruska et al (1978). 66% of patients had Osteitis Fibrosa on
histology. This finding agrees with the work of Jarava et al (1996) who found bone
histological evidence of Osteitis Fibrosa cystica in 17 (85%) out of 20 haemodialysis patients
in England. Our findings also agrees with that of shin et al (1999) who found Osteitis Fibrosa
as the commonest type of ROD in predialysis patients in Canada (44%). This finding
contradicts that if Coen et al (1996) who found mixed type ROD as the commonest type in
predialysis CRF patient in England.
4.3 There is correlation between histological evidence of rod and serum alkaline
phoshatase
In this study, it was found that 90% of patient had histological evidence of ROD on
postmortem bone biopsy. This agrees with the finding Sanchez (2001), who found that 90%
of patients with ESRD on maintenance dialysis have abnormal bone histology. Majority of
patients are either predialysis or those who were not dialyzing adequately. It is known that
once a patient start on maintenance, the prevalence of ROD increases. One of the
contributing factors being aluminum deposition (from dialysate fluid), it means that the
prevalence may even be higher if our patients are dialyzed adequately. In our study we
found that all the patients that had histological evidence of ROD had elevated serum
alkaline phosphatase levels. This finding may possibly be pointing to the fact that serum
alkaline phosphatase can be used as a surrogate of parathyroid hormone as a predictor of
ROD in our patients. This agrees with the finding of Urena et al, (1991) that in the absence of
liver disease, serum alkaline phosphatase can be used to predict the presence of ROD. The
finding is also in agreement with that of Duursma et al and Ritz et al who found that plasma
alkaline phosphatase levels correlates with histological features of secondary
hyperparathyroidism (HPTH).
4.4 The yield of rod using radiological examination is low in our chronic renal failure
patients
In our study, we found only 2% of ROD using radiological examination. This agrees with
Odenigbo (2003) who found 3.35% of ROD in Enugu using radiological examination. In this
study, radiological evidence of ROD was not found in all 9(100%) patients who had
histological evidence of ROD on postmortem bone biopsy. This agrees with the finding of
Chronic Kidney Disease
68
Hodsons et al, (1981) that there is a disparity between the radiological and histological
evidence of ROD. In a study in Germany, Hodsons et al found only 7(41%) patients with
radiological evidence of ROD out of 17 with histological evidence of ROD. Micheal et al
(1998) found radiological features of ROD in 35% of CRF patients in ESRD.
There are some reasons for the low prevalence of ROD using x-rays. Firstly, the
conventional techniques for x-ray contribute. Meama et al
(1972) noted the phalanges to be
normal in 67% if uremic patients using conventional techniques for X-ray films, and only 8%
showed subperiosteal erosion. With the introduction of better films and the use of
magnification techniques, only 26% appeared normal while 29% for exhibited subperiosteal
erosion. There is no facility for magnification technique in the center where the study was
done. Secondly, it has been reported that more than 50% of bone can be lost without any
evidence in a radiograph, because only the cortical bone is clearly noted, and an important
loss of cancellous bone should occur before radiological feature of ROD can be appreciated
(Poznanki, 1993). Perhaps the fact that CRF patients in our environment have infrequent
haemodialysis and do not live long enough for these changes to be detected on x-ray studies
may be contributory to the low yield of ROD using radiological examination.
4.5 Radiological and biochemical evidence of rod is more prevalent in esrd patients
In our study, the only patient who had radiological evidence of ROD had a creatinine
clearance of 6mls/min. 90% of the CRF patients had creatinine clearance <15mls/min (ESRD).
The entire patients who had creatinine clearance <15mls/min had elevated serum alkaline
phosphatase levels. Theses finding agree with the findings of Coen et al (1996) that adynamic
bone disease is commoner n early stages of renal failure, while Osteomalacia and Osteitis
Fibrosa cystica tend to occur as resistance to PTH develops, a situation which occurs in ESRD.
4.6 ROD may be more prevalent in males
In this study, the one patient who had radiological evidence of ROD was a male. Also, of the
9 patients that had histological evidence of ROD, 6(66%) were males, while 3(34%) were
females, with a male- female ratio of 2:1, this finding is in contrast to the finding of
Odenigbo et al (2003) in a study carried out at Enugu where ROD was found to be more
prevalent in females. The finding also contradicts that of Couttenye et al (1997) who showed
that women seem to develop hyperparathyroidism whereas men seems to more frequently
develop aplastic bone disease. The reason why men in this study showed evidence of ROD
more than women may be due to the fact that there were more men in this study,
particularly in the group of 10 patients that had postmortem biopsy. However, the number
of patients studied was small for a general statement to be made on gender difference.
4.7 There is no correlation between symptoms of rod and biochemical or radiological
evidence
In the study, 7 (14%) of the CRF subjects had symptoms suggestive of ROD. Of these, 5(71%)
had bone pain while 2 (29%) had radiological evidence of ROD (Rugger Jersey spine),
while 3 (80%) had radiological features of osteoarthritis. This agrees with the finding of
Odenigbo, who reported that out of the 11 patients who had bone pain, none had
radiological evidence of ROD, but all patients who had radiological evidence of
The Prevalence of Renal Osteodystrophy in
Chronic Renal Failure Patients in Urban Niger Delta of Nigeria
69
Osteoarthritis (Odenigbo 2003). This study also agrees with the work of Harowin et al (1987)
who found a high incidence of joint symptoms and radiological abnormalities in his group
of Canadian patient, not necessarily due osteodystrophy.
4.8 Hypocalcaemia and hyperphosphataemia is prevalent in our crf patients
In the study the mean serum calcium of CRF subject was 5.83 2.1mg/dl. 37(71%) of CRF
subjects had hypocalcaemia (<8.5mg/dl). This finding agrees with that of Slatoposky et al
(1986). Calcium supplementation is a known modality for the treatment of hypocalcaemia.
The mean serum phosphate of CRF patients in this was 6.12.0mg/dl. 41 (79%) had
hyperphosphataemia (>4.5mg/dl). This agrees with finding of Slotoposky et al (1986) who
demonstrated hyperphosphataemia even in moderate CRF. Dietary phosphate restriction
and phosphate binding are effective methods of control of hyperphosphataemia.
4.9 Conclusion and recommendations
4.9.1 Conclusion
The findings of this study suggest that ROD which is a complication of chronic renal failure
does exist in our environment. The study has also shown that Osteitis Fibrosa is the
commonest type of ROD, and that ROD may be commoner in males. The study showed that
in majority of patients with ESRD there is biochemical evidence. This finding may possibly
be pointing to the fact that clinical features are a poor guide to the presence of ROD. Before
now, it was thought that ROD hardly existed in our chronic renal failure patients, because
they did not live long enough to manifest it. Though the findings of this study they agree
with that, going by the low incidence of ROD using clinical symptoms and radiological
methods, it is possible that in the nearest future, ROD may become more prevalent in on
society. This is because there is now an increase in the availability of dialysis in many
centers across the Nation, with possibility that many CRF patients may live long enough to
develop ROD. The findings of this study suggest that serum alkaline phosphatase assay, a
surrogate of parathyroid hormone, may be a good guide to the presence of ROD in our CRF
patients. Majority of patients had hypocalcaemia and hyperphosphataemia.
4.9.2 Recommendations
It is hereby recommended that:
1. In all chronic renal failure patients, ROD should be anticipated. Serum calcium,
phosphate, alkaline phosphatase should be done routinely.
2. Dietary restrictions of phosphate should be enforced in our chronic renal failure patient
as well as the use of phosphate binders,
3. Calcium supplementation should be routinely part of the management of our chronic
renal failure patients.
4. Control of hyperparathyroidism in our chronic renal failure patient will be an integral
part of management of CRF patients.
4.9.3 Limitation of the study
This study was faced with some limitations. It was not possible to carry out bone biopsies
for live patients because of lack of consent from the patients. However, postmortem bone
Chronic Kidney Disease
70
biopsy was carried out instead; it was also not possible to assay parathyroid hormone
because of lack of facility for its assay. In its place, serum alkaline phosphatase was used as a
surrogate. There is no doubt that alkaline phosphatase is influenced by several factors and
so is non-specific. It was also not possible to measure serum and tissue aluminum in the
study.
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Chronic Kidney Disease
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50.
5
Relationships Among Renal Function,
Bone Turnover and Periodontal Disease
Akihiro Yoshihara and Lisdrianto Hanindriyo
Division of Preventive Dentistry,
Department of Oral Health Science,
Graduate School of Medical and Dental Sciences,
Niigata University,
Japan
1. Introduction
Chronic renal failure (CRF) is defined as a progressive decline in renal function associated
with a reduced glomerular filtration rate. The most common causes are diabetes mellitus,
glomerulonephritis and chronic hypertension (Proctor et al., 2004).
The clinical signs and symptoms of renal failure are collectively termed uremia. CRF
affects most body systems, and the clinical features are dependent upon the stage of renal
failure and the systems involved.
Oral manifestations of CRF and related therapies:
a. Gingival enlargement
Gingival enlargement secondary to drug therapy is the most commonly reported oral
manifestation of renal disease. It can be induced by cyclosporine and/or calcium
channel blockers (Somacarrera et al., 1994; Kennedy and Linden 2000).
b. Oral hygiene and periodontal disease
The oral hygiene of individuals receiving hemodialysis can be poor. Deposits of
calculus may be increased (Epstein et al., 1980; Gavalda et al., 1999). There is no good
evidence of an increased risk of periodontitis (Brown et al., 1989; Thorstensson et al.,
1996; Naugle et al., 1998), although premature bone loss has been reported (Locsey et
al., 1986). Localized suppurative osteomyelitis, secondary to periodontitis, was
observed in individuals receiving hemodialysis (Tomaselli et al., 1993).
c. Xerostomia
Symptoms of xerostomia can arise in many individuals receiving hemodialysis (Kho et
al., 1999; Klassen and Krasko, 2002). Possible causes include restricted fluid intake, side-
effects of drug therapy and/or mouth breathing (Porter et al., 2004).
d. Oral malodor/bad taste/halitosis
Uremic patients may have an ammonia-like oral odor (Kho et al., 1999), which also
occurs in about one third of individuals receiving hemodialysis. CRF can give rise to
altered taste sensation, and some patients complain of an unpleasant and/or metallic
taste or a sensation of an enlarged tongue (Kho et al., 1999).
Chronic Kidney Disease
74
e. Mucosal lesions
A wide range of oral mucosal lesions, particularly white patches and/or ulceration,
have been described in individuals receiving dialysis and allografts (Proctor et al.,
2004).
f. Oral malignancy
Kaposis sarcoma (KS) can occur in the mouths of immunosuppresed renal transplant
recipients (Farge, 1993). Any increased risk of oral malignancy in CRF probably reflects
the effects of iatrogenic immunosupression, which increases the risk of virally-
associated tumors, such as KS or non-Hodgkins lymphoma (Proctor et al., 2004).
g. Oral infections
Candidosis, angular cheilitis has been described in up to 4% of hemodialysis and renal
allograft recipients (King et al., 1994; Klassen and Krasko, 2002). Other oral candidal
lesionssuch as pseudomembranous (1.9%), erythematous (3.8%), and chronic atrophic
candidosis (3.8%)have been reported in allograft recipients (King et al., 1994).
Viral infection, prior to the availability of appropriate anti-viral drugs (e.g., acyclovir,
gancyclovir, and valacyclovir), about 50% of renal allograft recipients, who were
seropositive for herpes simplex, experienced recurrent, severe, and prolonged HSV
infections (Armstrong et al., 1976). However, in recent years, the use of effective anti-
herpetic regimes has significantly reduced the frequency of such infection (Kletzmayr et
al., 2000; Squifflet and Legendre 2002).
h. Dental anomalies
Delayed eruption of permanent teeth has been reported in children with CRF (Wolff et
al., 1985; Jaffe et al., 1990). Enamel hypoplasia of the primary and permanent teeth (Kho
et al., 1999; Koch et al., 1999; Al Nowaiser et al., 2003) with or without brown
discoloration can also occur (Wolff et al., 1985).
i. Bone lesions
A wide range of bone anomalies can arise in CRF. These reflect a variety of defects of
calcium metabolism including, loss of hydroxylation of 1-hydroxycholecalciferol to
active vitamin D (1,25-dihydroxycholecalciferol), decreased hydrogen ion excretion
(and resultant acidosis); hyperphosphatemia, hypocalcemia and resultant secondary
hyperparathyroidism and interference with phosphate metabolism by dialysis (Nadimi
et al., 1993).
Orofacial features of renal osteodystrophy due to hyperparathyroidism include bone
demineralization, decreased trabeculation, decreased thickness of cortical bone,
ground-glass appearance of bone, metastatic soft-tissue calcifications, radiolucent
fibrocystic lesions, radiolucent giant cell lesions, lytic areas of bone, jaw fracture (due to
trauma or during surgery) and abnormal bone healing after extraction. Orofacial
features of renal osteodystrophy related to tooth and periodontium include delayed
eruption, enamel hypoplasia, loss of the lamina dura, widening of the periodontal
ligament, severe periodontal destruction, tooth mobility, drifting, pulp calcification and
pulp narrowing (Damm et al., 1997; Okada et al., 2000; Klassen and Krasko, 2002).
2. The relationships among osteoporosis, renal function and periodontal
disease
Osteoporosis is the most common metabolic bone disease among the elderly, and the
incidence of osteoporotic fractures obviously increases with age (Honig, 2010). In addition,
Relationships Among Renal Function, Bone Turnover and Periodontal Disease
75
elderly people often experience periodontal destruction. Because bone loss is a common
feature of periodontitis and osteoporosis, both diseases may share some common etiologic
factors (Offenbacher, 1996). The final expression of periodontitis is governed by complex
interactions among host, microbial and environmental factors occurring within an intricate
cellular mosaic (Offenbacher, 1996).
In addition, CRF is associated with marked disturbances of bone structure and metabolism,
and there is a slowly progressive loss of renal function over months or years (Ruggeneti,
1998). A significant decrease in bone mineral density after transplantation is a serious
finding (Huang & Sprague, 2009). It is well known that impaired renal function increases
osteoclast activity leading to bone turnover, and this may influence bone metabolic
parameters (Couttenye et al., 1999; Cirillo et al., 1998). There is a growing body of evidence
indicating that impaired renal function is associated with disrupted regulation of vitamin D
(Rix et al., 1999; Hamdy et al., 1995). Whereas some systemic factors that contribute to loss of
bone mass and periodontal progression have been identified, we hypothesized that renal
function is associated with bone metabolism, and thus is also associated with periodontal
disease. To test this hypothesis, it is essential to evaluate the relationships among bone
turnover, renal function and periodontal disease.
We initiated a longitudinal interdisciplinary study on aging (the Niigata Study) in 1998 to
examine the many links between oral health and general health and well being. In the
present report, we reviewed the relationship between bone metabolism and periodontal
disease, taking renal function into consideration, in elderly Japanese subjects from the
Niigata Study.
3. Principal findings from the Niigata Study
3.1 Outline of the Niigata Study
According to a registry of residents, questionnaires were sent to all 70-year-olds among the
4,542 inhabitants of Niigata City in Japan. Participants were informed of the purpose of the
survey, and the overall response rate was 81.4%. After dividing the residents into groups of
males and females, 600 individuals (the screened population) were randomly selected in
order to have approximately the same number of male and female participants in the study.
Follow-up surveys were carried out every year in June from1998 to 2008 (11 times in 10
years), using the same methods that were used at baseline. All subjects were Japanese and
did not require special care for their daily activities. Since age influences bone metabolism,
renal function and periodontal disease, subjects were restricted to 70 years old at baseline
(Ando et al., 2000).
3.2 Osteoporosis and periodontal disease
In addition to a strict age requirement, other study inclusion criteria included the following:
blood sugar < 140 mg/dL with no history of diabetes, more than 20 teeth remaining, non-
smokers, and no history of medication use for osteoporosis. There were 184 subjects among
the screened population that met all the inclusion criteria.
We utilized data on bone mineral density (BMD) of the heel, which we measured using an
ultrasound bone densitometer (Fig. 1, Achilles Bone Densitometer
TM
, Luner Corporation,
Chronic Kidney Disease
76
Fig. 1. Outline of the analysis between Osteoporosis and periodontal progression.
AAL: Additional attachment loss.
USA) (Lunar Corporation, 1991). Ultrasound densitometry enables the measurement of the
physical properties of bone, specifically BMD. The ultrasound measurement contains two
criteria, the velocity (speed of sound, SOS) and frequency attenuation (broadband
ultrasound attenuation, BUA) of a sound wave as it travels through a bone.
Stiffness is a
clinical index combining SOS and BUA, and is calculated by the following formula: (BUA
50) 0.67 + (SOS 1380) 0.28.
Stiffness is indicated in the bone densitometer monitoring device as the percentage of the
value for a normal younger population. Osteopenia was defined as a stiffness that was
85% for males and 69% for females. Follow-up clinical surveys were done by measuring
the clinical attachment level after 3 years. Clinical attachment level is the amount of space
between attached periodontal tissues and a fixed point, usually the cementoenamel junction.
A measurement used to assess the stability of attachment as part of a periodontal
maintenance program (Fig. 2). There were 179 subjects included in the final analysis, and all
of these subjects participated in both the baseline and the follow-up examinations.
We measured the number of progressive sites that had 3 mm of additional attachment loss
over 3 years (Fig. 2). After dividing the subjects into an osteopenia group (OG) and a no-
osteopenia group (NOG), we evaluated the number of progressive sites that had 3 mm of
additional attachment loss over 3 years by two-way analysis of variance (ANOVA).
The respective mean number of progressive sites for the OG and NOG were 4.75.5 and
3.33.0 in females, and 6.99.4 and 3.42.8 in males. The difference in the mean number of
progressive sites between the OG and NOG was statistically significant by ANOVA after
controlling for gender (Fig. 3, p = 0.043) (Yoshihara et al., 2004).
Relationships Among Renal Function, Bone Turnover and Periodontal Disease
77
Fig. 2. Clinical attachment level and periodontal disease progression.
A, B = Clinical attachment level
B-A = Additional attachment loss
Fig. 3. Relationship between number of progressive sites with 3mm additional attachment
loss and stiffness by gender.
The number of subjects: stiffness 69 (n=74) and >69 (n=19) for female, 85 (n=65) and >85
(n=22) for male.
OG: Osteopenia group, NOG: No-osteopenia group.
Chronic Kidney Disease
78
3.3 Bone metabolism and periodontal disease
A total of 398 subjects who turned 70 in 1998 had annual dental examinations. We selected
148 of these 398 subjects (79 males and 69 females) for participation in the study because
they had one or more teeth, were not taking any medicine or supplements for bone
disorders (tamoxifen, anabolic steroids, bisphosphonate, or estrogen), and did not have a
diagnosis of fracture based on an X-ray assessment by a physician. The subjects blood was
taken in the morning of the dental examination. Urine was collected over 24 hours (07:00 to
07:00 AM the day after the dental examination). During the day that urine was collected,
usual food and fluid intake were ingested. Biochemical parameters of bone turnover were
measured, including urinary deoxypyridinoline (U-DPD) (nM/nM*Cr) as a bone resorption
marker, and serum osteocalcine (S-OC) (ng/mL) and serum bone alkaline phosphatase (S-
BAP) (U/L) as bone formation markers. U-DPD data were corrected by the urinary
creatinine concentration measured by a standard colorimetric method.
We categorized subjects by tertiles according to the percentage of sites with 6 mm clinical
attachment level (6+ mm CAL). S-OC, S-BAP, and U-DPD were evaluated by analysis of
covariance (ANCOVA) adjusted for smoking habit (0: none, 1: past or current). Differences
in the distribution of bone turnover markers according to the percentage of sites with 6+
mm CAL per person are shown in Table 1. S-OC was significantly lower in the third tertile
than in the first and second tertiles after adjusting for smoking habit (males: p = 0.007,
females: p = 0.042, ANCOVA) (Yoshihara et al., 2009).
% of sites with 6mm attachment level
Males Females
1st 2nd 3rd p value* 1st 2nd 3rd p value*
Serum osteocalcin
(ng/ml)
8.5 4.5 6.8 2.7 5.7 1.8 0.007 9.9 2.8 9.3 2.4 9.1 3.5 0.042
Serum bone alkaline
phosphatase (U/L)
22.2 5.9 23.3 7.4 21.1 6.2 0.212 29.3 10.8 28.9 8.1 27.4 11.2 0.752
Urinary
deoxypyridinoline
(nM/nM*Cr)
4.8 1.0 4.4 1.2 4.0 1.0 0.055 6.6 1.4 6.8 1.4 6.3 1.7 0.664
* ANOCOVA adjusted for smoking habits.
Table 1. Relationship between % of sites with 6mm attachment level and bone metabolism
markers controlling for confounding factors by multiple regression analysis.
3.4 Renal function and periodontal disease
We randomly selected 145 subjects among 398 healthy elderly subjects. All subjects were
aged 77 years at the time of the renal function study in 2005. We evaluated the relationship
between bone turnover markers and periodontal disease, taking renal function into
consideration. Correlations among renal function and bone metabolism markers for
periodontal disease, including the number of remaining teeth and smoking habit, were
evaluated using multiple regression analysis.
Relationships Among Renal Function, Bone Turnover and Periodontal Disease
79
To evaluate the relationship between periodontal disease and renal function markers
(volume of urine per 24 hours [mL/day], creatinine clearance per 24 hours [L/day]) or bone
metabolism markers (U-DPD [nM/nM*Cr] and S-OC [ng/mL]), multiple linear regression
analysis was performed. For the final model, the confounding independent variables that
had p-values less than 0.05 according to the statistical association with the percentage of
sites with 6+ mm CAL by Pearson correlation coefficients, ANOVA, or chi-square test, were
selected. Results of multiple linear regression analysis between the percentage of sites with
6+ mm CAL and renal function markers after controlling for confounding factors are shown
in Table 2. Creatinine clearance for 24 hours was positively associated with the percentage of
sites with 6+mm CAL (sta. coef. = 0.26, p = 0.015). Furthermore, S-OC showed a negatively
independent association with the percentage of sites with 6+vmm CAL after adjustment for
the confounding factors (sta. coef. = -0.27, p = 0.006, Table 3) (Yoshihara et al, 2007).
Dependent variable
% of sites with 6mm attachment level
Independent variables Sta. Coef ().* p value
Number of remaining teeth -0.46 <0.001
Creatinine clearance for 24 h (L/day) 0.26 0.015
Volume of urine for 24 h (ml/day) 0.01 0.956
Smoking habit 0.08 0.500
Gender -0.17 0.121
Use of interdental brushes or dental floss -0.01 0.893
Constant 0.074
Creatinine (g/day) in urine per 24h/creatinine (g/L) in serum.
* Standardized coefficient.
Table 2. Relationship between % of sites with 6mm attachment level and renal function
markers controlling for confounding factors by multiple regression analysis.
Dependent variable
% of sites with 6mm attachment level
Independent variables Sta. Coef ().* p value
Number of remaining teeth -0.47 <0.001
Serum osteocalcin (ng/ml) -0.27 0.006
Urinary deoxypyridinoline (nM/nM*Cr) -0.04 0.688
Smoking habit -0.10 0.406
Gender 0.10 0.481
Use of interdental brushes or dental floss -0.01 0.861
Constant <0.001
* Standardized coefficient.
Table 3. Relationship between % of sites with 6mm attachment level and bone metabolism
markers controlling for confounding factors by multiple regression analysis.
Chronic Kidney Disease
80
The results showed that the subjects in the OG had a higher number of progressive sites for
additional attachment loss than the subjects in the NOG. This three-year longitudinal study
clearly demonstrated that BMD is a risk factor for periodontal disease progression in an
elderly population. In addition, according to our findings on linkage with BMD, there are
some systemic factors that contribute to both loss of bone mass and periodontal disease
progression (Kshirsagar, 2005). Systemic factors of bone remodeling may also modify the
local tissue response to periodontal disease. The BMD of the mandible is affected by the
mineral status of the skeleton and also by diseases that cause generalized bone loss
(Davidovich, 2005). The mouth and face are highly accessible parts of the body, and reflect
changes that occur internally. For the clinician, the mouth and face provide physical signs
and symptoms of local and generalized disease. During routine oral examinations,
periodontal disease including maxillary/mandibular general bone loss may be diagnostic of
early osteoporotic changes in the skeleton. Some systemic factors of bone remodeling also
modify the local tissue response to periodontal disease.
Osteoporosis and low renal function contribute to loss of bone mass. We were able to
identify a weak but clear relationship between CAL and S-OC. There was a significant
association between CAL and 24-hour creatinine clearance, which is a renal function marker.
These findings suggest that S-OC is a valid marker of bone turnover when evaluating
periodontal disease. It has been assumed that S-OC is associated with not only bone
turnover but also low renal function. Periodontal conditions, including bone metabolism,
may be affected by low renal function. The systemic bone metabolism, which might be
affected by low renal function, is associated with periodontal disease.
4. Association between chronic renal failure and periodontal disease
Based on several studies, CRF and periodontal disease can have reciprocal effects (Fig. 3).
CRF and renal therapy can greatly influence the dental management of renal patient.
Moreover, chronic adult periodontitis can contribute to the overall systematic inflammatory
burden and may therefore influence the management of the end-stage renal disease (ESRD)
patient on hemodialysis maintenance therapy (Craig, 2008).
4.1 Possible association of chronic renal failure with periodontal disease
CRF can cause several changes that influence oral conditions such as decreased salivary
flow rate, increase salivary urea level and calculus accumulation (Torres et al., 2010). CRF
may have an effect on the periodontal status of an individual through several possible
mechanisms.
a. A major clinical consequence of CRF is uremic syndrome (uremia). This condition
leads to an immune dysfunction possibly caused by defects in lymphocyte and
monocyte function, which in turn may increase the rate of gingival inflammation
(Craig, 2008).
b. Several studies have found an increasing level of plaque formation, calculus, gingival
inflammation and also decreasing saliva excretion, which can be considered together as
reduced oral hygiene (Yoshihara et al., 2007). The intense psychological and time
demands that are associated with hemodialysis in patients with ESRD may account for
reduced oral hygiene (Craig, 2008).
Relationships Among Renal Function, Bone Turnover and Periodontal Disease
81
Fig. 4. The mechanism between low renal function and periodontal disease.
c. CRF has an important effect on vitamin D metabolism (Yoshihara et al., 2007). Since
vitamin D is metabolized in the liver and kidney, the presence of CRF will
automatically disturb vitamin D metabolism. Vitamin D is metabolized by kidney to
its active metabolite, 1,25-dihydroxyvitamin D3. This substance subsequently
interacts with vitamin D nuclear receptor in the intestine, bone and kidney. The
functions of this substance are to regulate bone metabolism, immune response and
also cell proliferation and differentiation. Regarding bone metabolism, vitamin D
controls the availability of calcium phosphate by regulating the excretions of
hormones such as the parathyroid hormone (PTH) (Souza et al., 2007). CRF may
disrupt the regulation of PTH which may leads to hyperparathyroidism condition
and increased rate of bone disease (Yoshihara et al., 2007). Vitamin D also contributes
in the synthesis of bone matrix proteins such as type-I collagen, alkaline phosphatase,
osteocalcin and osteopontin (Souza et al., 2007). Osteocalcin may exist in the
circulating blood and undergo local accumulation in some parts of the body.
Osteocalcin has been postulated to have a role in both bone resorption and
mineralization and is currently considered the most specific marker of osteoblast
function. The serum level of this protein is considered to be a marker of bone
formation. Serum osteocalcin is presently considered a valid marker of bone turnover
Chronic Kidney Disease
82
when resorption and formation are coupled and a specific marker of bone formation
when formation and resorption are uncoupled (Bullon et al., 2005). Osteocalcin has
also been found in the gingival crevicular fluid (GCF). Several studies found an
increased level of serum osteocalcin in subjects with CRF. Moreover, the level of GCF
osteocalcin was found to be significantly associated with periodontal disease, since
there was an association with pocket depth, clinical attachment level and bleeding on
probing (Bullon et al., 2005). Therefore, it might be reasonable to explain an effect of
CRF on periodontal disease by its effect on bone metabolism (especially alveolar
bone) which is specifically marked by the level of serum osteocalcin and/or GCF
osteocalcin.
4.2 Possible association of periodontal disease with chronic renal failure
Periodontal disease may have an effect on CRF and also the treatment of CRF. Periodontal
disease may have an effect on CRF through several possible mechanisms.
a. Moderate to severe periodontal disease may increase the serum level of C-reactive
protein (CRP). CRP is an acute phase protein and systemic marker of inflammation
which is also a major risk predictor for cardiac disorder and all other mortality cause of
CRF persons (Craig, 2008). Several studies have reported periodontal disease to be
associated with elevated CRP as well as other serum components of the acute phase
response including decreased high density lipoprotein cholesterol, low density
lipoprotein cholesterol, blood glucose and decreased peripheral blood neutrophil
function and count (Muntner et al., 2000). Since all these factors are also risk factors for
CRF, it might be justifiable to assume that periodontal disease may be considered as a
predisposing factor and/or marker of CRF. Moreover, periodontal disease in a person
with CRF has a strong tendency to increase the possibility of the complication of
coronary atherosclerosis.
b. Several reports found that periodontitis may also contribute to the systemic
inflammatory burden in ESRD. The level of IgG antibody particularly to
Porphyromonas gingivalis correlated with elevated serum CRP (Muntner et al., 2000).
Therefore, it is also important to consider an effective periodontal therapy in order to
reduce the level of serum CRP which might eventually decrease the inflammatory
burden of ESRD or CRF.
On the other hand, there are also some studies which failed to find the type of correlations
mentioned above (Kitsou et al., 2000; Marakoglu et al., 2003; Duran et al., 2004; Bots et al.,
2006). It is acknowledged that differences in research design, measurement methods
instruments used, and other factors may have resulted in different findings. Therefore, it
is still relevant and reasonable to execute further research using a more sophisticated
and well-designed method to elucidate the relationship between CRF and periodontal
disease.
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6
Sarcoidosis and Kidney Disease
Tulsi Mehta, Anirban Ganguli and Mehrnaz Haji-Momenian
Department of Medicine, Washington Hospital Center,
Washington DC,
USA
1. Introduction
Sarcoidosis is an illness of granulomatous inflammation with multi-organ association. While
most individuals exhibit pulmonary pathology, renal involvement is not without prevalence
or significance. This chapter will review the current epidemiology of the disease and explore
the two major pathways in the pathogenesis of renal sarcoidosis, mainly granulomatous
deposition and deranged calcium management. With these concepts addressed, further
inquiries into intrinsic renal disease will be provided along with explanations of
renovascular complications, obstructive nephropathy, and transplant pathology. Each
ailment will be accompanied by common presentation, more detailed pathophysiology,
appropriate diagnostics, and current treatment recommendations. This chapter will seek to
purvey a comprehensive but concise exploration of renal sarcoidosis.
2. Epidemiology & susceptibility
Sarcoidosis can affect a wide range of racial and ethnic groups but it has high prevalence in
northern European countries, Japan, and the United States
1
. Certain countries have skewed
incidences, for example: black Americans are three times more likely than white Americans
to develop the disease (Iannuzzi et al. 2007). However, across the racial and ethnic groups,
females are more prone to the illness than males (Iannuzzi et al. 2007). The disease manifests
itself typically in patients less than 50 years of age and mainly in the third of fourth decade
of life (Iannuzzi et al. 2011). A patient with a first degree relative with the disease has a five-
fold increase of developing sarcoidosis. Nevertheless, this risk still does not exceed 1%
(Iannuzzi et al. 2011). Patient susceptibility also increases with certain associations of
genetics and environmental factors. Discoveries into HLA gene products and the
butyrophilin-like2 (BTNL2) gene are the latest areas of genetic interests (Iannuzzi et al.
2007). A variety of environmental triggers including wood-burning stoves, tree pollen,
inorganic particles, insecticides, and mold have also been scrutinized in addition to
mycobacteria and propionibacteria antigens (Iannuzzi et al. 2007, 2011)
.
In fact, combinations
of genetic and environmental activators have also been examined, for example: HLA-DQB1
and water damage or high humanity in the workplace (Iannuzzi et al. 2007). However, it
seems that a ubiquitous number of agents may initiate a similar immunologic pathway that
is pathognomonic for sarcoidosis.
Chronic Kidney Disease
88
3. Manifestations & pathogenesis
Sarcoidosis mainly affects the pulmonary system, with an over 90% occurrence rate in the
afflicted, presenting as mostly hilar lymphadenopathy but also including pulmonary
hyperternsion and obstructive and restrictive airway disease
(Iannuzzi et al. 2011). Other
major organ systems disturbed include the skin, the eye, the heart, and the nervous system
with approximately 25 to 30% involvement
(Iannuzzi et al 2011). Renal sarcoidosis is in fact
rare with exact number relating prevalence difficult to come by. Unfortunately, the etiology
for nephron-related disease is quite vast and it has been hard to delineate pure renal
manifestations from simple metabolic disturbances
(Berliner et al 2006). In order to
understand the extent and pathogenesis of renal involvement, two central pathways for
nephron insult has been validated including granulomatous deposition and deranged
calcium management. While these pathways are by no means the only two routes of renal
involvement, they are the most significant and the overriding themes for renal insult.
3.1 Granuloma formation
Many aspects of this process still require elucidation yet strong evidence reveals that
granuloma formation centers on T cells reacting with unclear triggers and certain gene
products to illicit cascades that either lead to complete resolution of inflammation or to
irreversible fibrosis (Iannuzzi et al. 2007). Specifically, antigen presenting cells including
macrophages with susceptible HLA or BTNL2 gene products present triggers including
organic, inorganic, and infectious agents to the CD4 T cell. Once initiated, numerous
peripheral cytokines, interleukins, and immune modulators steer T cells into a T Helper 1 or
T Helper 2 response; where with the former, resolution of inflammation is more probable
but with the later, fibrosis and irreversible damage is more probable
(Iannuzzi et al. 2007,
2011). This deposition of macrophages, giant cells, and T helper cells form the
pathognomonic, non-caseating granulomas that defines sarcoidosis
(Casella and Allon 1983)
See Figure 1. In renal disease, these granulomas are primarily in the cortex but may also be
found in the medulla or capsule
(Casella and Allon 1983). This process is the basis for
granulomatous interstitial nephritis, which will be further discussed subsequently.
3.2 Deranged calcium management
Despite the granulomatous inflammation that marks sarcoidosis, deranged calcium
homeostasis has a greater effect on the kidneys than the invasive granulomas themselves.
Activated pulmonary macrophages express 1- hydroxylase, which has important
implications in maintaining appropriate levels of calcium in the body. In normal physiology,
calcium balance is attained through the intricate interactions of parathyroid hormone (PTH),
calcium, phosphorus, and Vitamin D. PTH upregulates renal 1- hydroxylase, a cytochrome
P450 enzyme located in the proximal tubule, to metabolize 25-hydroxy vitamin D to 1, 25-
dihydroxy vitamin D, the bioactive form of Vitamin D, also known as calcitriol. Calcitriol, in
turn, promotes calcium absorption in the intestines, kidneys, and bones. When calcium
levels are adequate, normal physiological negative feedback mechanisms halt the PTH and
calcitriol cycle. However, in sarcoidosis, extra-renal production of 1- hydroxylase
inappropriately increases calcitriol levels thereby increasing serum calcium and decreasing
PTH. Unlike its renal equivalent, the granulomatous 1- hydroxylase is immuned from the
normal negative feedback mechanisms of hypercalcemia and is therefore unregulated,
Sarcoidosis and Kidney Disease
89
(Iannuzzi MC et al. N Engl J Med 2007;357:2153-2165.)
Fig. 1. Hypothesized Immunopathogenesis of Granuloma Formation.
causing disturbed calcium homeostasis. This not only causes hypercalcemia, hypercalciuria
and possibly subsequent nephrolithiasis and nephrocalcinosis, which itself is the most
common cause of progressive renal failure. The clinical consequences of each imbalance
range from trivial presentation to overt pathology including dehydration, renal colic, and
end-stage renal disease. Diagnosis may be established by laboratory findings,
ultrasonography, and computed tomography. General treatments incorporate adequate oral
hydration, minimization of dietary calcium and vitamin D, avoidance of UV light exposure,
and possibly corticosteroid therapy
(Sharma 1996).
Chronic Kidney Disease
90
Hypercalcemia may cause decrease glomerular filtration rate by vasoconstricting the afferent
arterioles and thereby decreasing renal blood flow
(Berliner et al 2006). Additionally, it may
cause tubular necrosis, tubulointerstitial non-granulomatous inflammation with calcium
deposits ultimately causing nephrocalcinosis and chronic kidney disease (Berliner et al 2006).
Hypercalciuria, which is three times as more common as hypercalcemia, predisposes patients
to calcium oxalate nephrolithiasis, which may ultimately lead to obstruction or chronic
pyelonephritis (Berliner et al 2006 and Sharma 1996). Renovascular complications as well as
obstructive nephropathy will also be further discussed subsequently.
4. Obstructive nephropathy
Abnormal calcium metabolism is a well known feature of sarcoidosis. Hypercalcemia and
hypercalcuria is related to endogenous vitamin D. It is suggested that excess vitamin D may
result in increased intestinal calcium absorption and consequent hypercalcemia,
hypercalcuria and renal calculi. Hypercalcuria is defined as using excretion of 300 mg/day
in men or 250 mg/day in women, about 2-5% healthy adults exhibit hypercalcuria.
Hypercalcuria is the most common renal manifestation. It is caused by glomerular filtration
of excess blood calcium and suppression by high calcitriol levels on PTH activity. It affects
50% of patients with sarcoidosis, often with an insidious onset because most patients remain
normocalcemic. Sharma suggests that 10% of patients with sarcoidosis are diagnosed with
hypercalcemia whereas 30% of patients with sarcoidosis show an increase in serum calcium.
(Sharma, 1996)
In 1988, Foster described eight patients where he described extra uveitis may be the
presenting sign of sarcoidosis. It was the first study that suggested that there may be
unexpected presenting signs of sarcoidosis. (Foster, 1988) One of these symptoms may be
nephrolithiasis. In a study from Italy, the charts 618 patients with histologically proven
sarcoidosis was reviewed in 1978-92 in order to identify nephrolithiasis as a presenting
feature of sarcoidosis. (Rizzato et al 1995) The authors concluded that calculi were the
presenting feature of sarcoidosis in 6 out of 618 patients (1%) and was the first manifestation
of disease in 14 (2. 2%) of the patients. In another 9 patients who presented with pulmonary
involvement, persistent hematuria or pyuria led to discovery of stones via ultrasound or
intravenous pyelography. Given that this is an uncommon disease, there is a very small
chance that a physician seeing a patient for the first time with a new kidney stone will later
prove to be is sarcoidosis. In the literature, the overall prevalence of nephrolithiasis is 10% in
patients with sarcoidosis. (Muther et al 1981 and Rizzato 1995) The incidence of 2.2%
exceeds more than 20 times the expected yearly rate of renal calculi in the general
population (36 per 100, 000 in women and 123 in men in Rochester (Johnson et al 1979), 122
in California
(Hiatt et al 1982) and 68 in Kyoto Osaka. (Yoshida and Okada, 1990) In course
of chronic sarcoidosis, approximately 10-13.8% of patients have at least 1 asymptomatic
stone. (Lebacq, 1970)
Treatment of hypercalcuria involves minimization of dietary calcium and Vitamin D,
avoidance of UV exposure, and dietary oxalate restriction. This is because an increase in
intestinal calcium absorption caused by excess in 1, 25 dihydroxyvitamin D may result in an
increase in urinary oxalate excretion especially if diet is low in calcium. Overabsorption of
calcium leaves less of this divalent cation to complex with oxalate in the proximal intestine
so more oxalate is delivered to the colon in which anion is hyperabsorbed. Corticosteriods
Sarcoidosis and Kidney Disease
91
are usually necessary to normalize these parameters as they can decrease inflammatory
activity and reduce calcitriol syntheses.
Retroperitoneal lymph nodes can enlarge sufficiently to cause urethral obstruction.
(Frailly et al 1990). Sarcoidosis has also been shown to be responsible for bilateral
hydronephrosis on the basis of retroperitoneal lymph node enlargement, with resolution
after corticosteroid treatment. (Miyazaki 1995).
5. Glomerular diseases associated with sarcoidosis
Glomerular involvement in sarcoidosis is not very common. The spectrum of commonly
reported glomerular diseases include focal segmental sclerosis, membranous
glomerulonephritis (GN), mesangioproliferative glomerulonephritis, mesangiocapillary
glomerulonephritis, IgA nephropathy and crescentric glomerulonephritis. (Sheffield 1997)
The exact mechanisms of glomerular disease in sarcoidosis are not known. Due to the
absence of a consistent glomerular pathology and a well described etiological pathway,
most cases are believed to be coincidental associations. Broadly speaking, abnormalities in
both the humoral and cellular immune system in sarcoidosis contribute to the development
of immune complex type glomerulonephritis which also explains why immunoglobulin
and complement deposition are commonly observed in renal biopsies in sarcoidosis. (Gobel
et al 2001).
5.1 Membranous glomerulonephritis
Overall, membranous glomerulonephritis (MGN) is the most commonly reported
glomerular pathology. Amongst 39 cases of glomerular diseases reported in sarcoidosis,
Vanhille et al found that 13 were MGN, largely occurring late in the course of overt disease.
(Vanhille et al 1986) Khan et al. described a 56-yr-old woman with pulmonary sarcoidosis
who developed heavy proteinuria. A renal biopsy revealed both interstitial granulomas and
membranous glomerulonephritis. (Khan et al 1999) Rarely patients may be diagnosed to
have sarcoidosis during the work up for secondary causes of nephrotic syndrome.
Dimitriades et al. described a 13-yr-old girl who presented with the nephrotic syndrome and
renal biopsy showed membranous nephropathy. (Dimitriades 1999) Typical subepithelial
deposits were found with electron microscopy. Bilateral hilar adenopathy was present,
which suggested sarcoidosis. The diagnosis was confirmed by a bone marrow biopsy, which
disclosed noncaseating granulomas. The patient was treated with corticosteroids and
cyclophosphamide, and her condition stabilized. In an experimental study, Maruyama et al,
induced subepithelial deposits in pigs injected with heterologous antibodies to angiotensin
converting enzyme (ACE). Confocal microscopy showed co localization of the granular
deposits of ACE and anti ACE goat IgG on the outer aspect of glomerular basement. The
authors conjectured that a similar autoimmune process may cause membranous GN in
sarcoidosis. While traditionally idiopathic MGN is steroid resistant, most cases of MGN
associated with sarcoidosis seem to respond to high dose steroid therapy especially if there
is coexistent granulomatous interstitial nephritis (GIN) (Khan et al 1999). Others used pulse
methylprednisolone plus oral cyclophosphamide to show remission of the nephrotic state.
(Dimitriades et al 1999) See Figure 2. for histology of membranous nephropathy in
sarcoidosis.
Chronic Kidney Disease
92
Fig. 2. (A) Immunofluorescence shows granular IgG deposits along the glomerular basement
membrane consistent with membranous glomerulonephritis. (B) Left forearm biopsy with
epithelioid granulomas. A star-shaped asteroid body is visible within a giant cell.
Magnifications: x800 in A (IgG); x500 in B (hematoxylin and eosin). Gobel U et al. JASN
2001;12:616-623
5.2 Minimal change disease
Nephrotic syndrome due to minimal change disease (MCD) also has been described in
patients with sarcoidosis. Mundlein et al, described a patient with Graves disease with
steroid dependent MCD who achieved complete remission with cyclophosphamide.
(Mundlein et al 1996) Patient was subsequently diagnosed to have typical chest findings of
pulmonary sarcoidosis. In contrast, Parry and Falk described a case of longstanding
pulmonary sarcoidosis that later went on to develop steroid resistant MCD not responding
Sarcoidosis and Kidney Disease
93
to high dose steroids or cyclophosphamide. (Parry et al 1997) The patient had to be started
on cyclosporine which was given for a year and a sustained remission was attained.
Spontaneous occurrence and remission of heavy proteinuria coinciding with the relapse of
the disease is also well described. (Mery 2005) The authors postulated that there is a
functional and transient increase of glomerular permeability to proteins secondary to release
of vascular permeability factor like lymphokines by activated T cells.
5.3 Crescentic glomerulonephritis
Crescent Glomerulonephrits (GN) has also been frequently reported in patients with
sarcoidosis and co-existing ANCA associated vasculitis. ANCA are autoantibodies found in
some autoimmune diseases, recognized by their reactivity with cytoplasmic antigens in
neutrophils; two groups are recognized: c-ANCA, reacting with proteinase 3, is found in
polyangiitis and Churg-Strauss syndrome; p-ANCA, reacting with myeloperoxidase is
found in Wegener granulomatosis. Auinger et al described a patient with rapidly
progressive glomerulonephritis and hepatosplenomegaly with no prior diagnosis of
sarcoidosis whose renal biopsy showed crescentic GN. (Auinger et al 1997) Diagnosis of
sarcoidosis was made with raised angiotensin converting enzyme (ACE) levels and both
liver and kidney biopsies showing interstitial noncaseating granulomas. Patient was started
on high dose steroids with which renal function improved. Subsequently, the patient
developed anti- myeloperoxidase (MPO) antibodies. In contrast, Ahuja et al reported a
patient with crescentic GN in the setting of Wegener's granulomatosis (WG). (Ahuja et al
1996). Patient responded well to long term oral cyclophosphamide treatment. Subsequently,
the patient developed biopsy-confirmed pulmonary sarcoidosis months later. Given such
close associations, it is believed that these sarcoidosis and granulomatous vasculitis like WG
may have some common mechanisms. See Figure 3.
5.4 Other glomerular diseases
Rare associations of sarcoidosis with post-infectious GN have also been noted. Michaels et
al. described two patients with sarcoidosis : one with recent history of pneumonia and
other with elevated antistreptolysin O titres who developed acute renal failure with active
urinary sediments and nephrotic range proteinuria (Michaels et al 2000). Biopsies
disclosed diffuse endocapillary proliferative GN with hump-like epithelial deposits. Both
patients responded well to corticosteroids with resolution of proteinuria and azotemia.
Similarly IgA nephropathy (IgAN), coexisting with sarcoidosis is not unusual given the
wide prevalence of IgAN. Taylor and Nishiki described a case of IgAN in sarcoidosis
typically presenting as nephritic syndrome that responded well to steroids. (Taylor at el
1996 and Nishiki et al 2010) Renal amyloidosis (AA type) has also noted in patients with
long standing sarcoidosis with the classical presentation of steroid resistant nephrotic
syndrome with slow progression to end stage renal disease. (Tchenio et al. 1996 and
Rainfray et al 1988).
6. Tubulointerstitial diseases
After excluding abnormalities affecting calcium homeostasis, tubulointerstitial diseases
are the most commonly encountered renal abnormalities in sarcoidosis. They are
Chronic Kidney Disease
94
Fig. 3. (A) Roentgenogram showing bilateral hilar adenopathy in a patient with sarcoidosis.
(B) Extracapillary glomerulonephritis with crescent formation. Magnification, x500 (periodic
acid-Schiff). Gobel U et al. JASN 2001; 12:616-623
histopathologically described as granulomatous interstitial nephritis (GIN). Approximately
20% of patients with sarcoidosis show granulomatous inflammation in the kidney (Sheffield
1997) although values range from 15 to 40% (Mery 2005) reflecting differences in the
indication for renal biopsies. In many instances, patients may be clinically silent and GIN
may present with concomitant findings with well known clinicopathological syndromes.
The variability in incidence of GIN also reflects sampling error in detecting scarce
granulomas especially in inadequate biopsy specimens.
Overall GIN is a rare histologic diagnosis seen in 0. 5 and 0. 9% of native renal biopsies and
0. 6% of renal transplant biopsies (Joss et al 2007). Possible etiologies include medications,
infections, sarcoidosis, Sjogrens syndrome, crystal deposits, paraproteinemia, Wegeners
granulomatosis and idiopathic causes. Drugs implicated include anticonvulsants,
antibiotics, nonsteroidal anti-inflammatory drugs, allopurinol, and diuretics. Mycobacteria
and fungi are the main infective causes and seem to be the main causative factor in cases in
renal transplants or in countries with high prevalence of tuberculosis. In the largest
collection of data so far on this disease, Joss et al noted 18 cases of GIN from of etiologies
such as sarcoidosis (n=5), drug induced (n=2), idiopathic (n=9) and tubulointerstitial
nephritis with uveitis (n=2). The most common presentation of GIN was advanced renal
failure with minimal proteinuria. (Joss 2007)
Sarcoidosis and Kidney Disease
95
Despite great clinical variability, the most common clinical syndrome associated with
sarcoidosis and GIN is chronic kidney disease with decline in renal function usually over
weeks to months
(Jean-Philllipe 2005). Acute renal failure as an initial presentation is also
well known (ORiordan et al 2001). Renal dysfunction may progress at variable rates but can
irreversibly progress to end stage renal disease despite high dose glucocorticoid treatment.
(Tsiouris et al 1999) Consistent with a pattern of tubulointerstitial disease, proteinuria is
either absent or mild. Urine analysis shows leucocytes and granular casts. Rarely, patient
may present as frank hematuria lasting several weeks. (Mills et al 1994) Functional tubular
abnormalities can occur in as much as 50% of cases of sarcoidosis when aggressively
investigated which include renal glycosuria, urinary sodium and potassium wasting,
Fanconis Syndrome, decreased urinary concentration ability, proximal or distal tubular
acidosis. (Muther et al 1981) It is uncertain whether the presence of interstitial lesions solely
contributes to these abnormalities but hypercalcemia and hypergammaglobulinemia also
play a pathogenic role. (Mery, 2005)
GIN is usually associated with enlarged kidneys mimicking polycystic kidney disease or
renal carcinoma. (Mery, 2005)
Renal sonogram shows bilateral renal masses which are either
hyper- or hypoechoic in comparison to adjacent renal parenchyma. Computer tomogram
shows the renal masses to be low intensity. A Gallium-76 citrate scan commonly reveals
increased uptake suggesting active granulomatous inflammation. (Mery and Kenouch, 1988)
Serum ACE concentration is a poor marker of active renal lesion and may even be normal in
active GIN with severe renal failure. (Hannedouche et al 1990)
In most cases, a diagnosis of GIN is made in the context of typical extra-renal manifestations
of sarcoidosis and/or hyperkalemia. Rarely renal involvement may be isolated and
preceding other sites of the disease for months to years. Some have even considered isolated
GIN as a localized form of sarcoidosis. In such isolated cases, it is important to rule out drug
induced interstitial nephritis which is far more easily treatable cause of GIN that sarcoidosis
itself. (Muther et al 1981) Another syndrome commonly associated with Sjogrens Syndrome
but also reported with sarcoidosis is the TINU syndrome or the Dobrin Syndrome
(Sinnamon et al 2008) which is characterized by acute interstitial nephritis, anterior uveitis
and epitheliod granulomas in bone marrow and lymph nodes. The renal lesion consists of
interstitial infiltrates mainly composed of mononuclear cells and few eosinophils. Although
no interstitial granulomas are seen in TINU, the interstitial cell infiltrate is the same as a
sarcoid granuloma. Therefore it is possible that some cases described as Dobrin syndrome
may be atypical forms of sarcoidosis.
Analyzing all cases of GIN, Joss et al, noted that the background diagnosis of sarcoidosis
was known in only 1 of 5 patients of GIN who eventually were categorized as sarcoid GIN.
Mean age of presentation was 56. 8 years. ACE levels were elevated in a minority of patients
(1 out of 5) and hypercalcemia was seen in only 2 patients. Pulmonary findings of hilar
lymphadenopathy was seen in only 1 patient and one had the TINU syndrome.
(Joss et al 2007)
Renal pathology of GIN consists of the typical non-caseating granuloma widely distributed
throughout the cortex and the medulla, although the density of these lesions may differ
from patient to patient. (Mery 2005) The sarcoid granuloma consists of lymphocytes,
mononuclear, cells and plasma cells. The center of the granuloma consists of epitheliod and
Chronic Kidney Disease
96
multinucleate giant cells both of which are derived from activated macrophages.
Multinucleate giant cells are formed by the coalescence of epitheliod cells. Lymphocytes
largely consist of T-helper cells (CD 4
+
) in the center and CD 8
+
lymphocytes in the
periphery. Some granulomas have small arteries in their center. Although granulomas may
also form in drug induced interstitial nephritis it is less well formed than in sarcoidosis.
Varying degrees of fibrosis may also be present. The severity of fibrosis correlates with
tubular atrophy and degeneration. In the absence of any predominant glomerular
pathology, the glomeruli are either normal or show mesangial hypertrophy and thickening
of the basement membrane. Electron microscopy may show fusion of epithelial foot
processes (Farge et al 1986). However, there are no significant immune deposits in either the
glomeruli or tubules as seen by immunoflourescent microscopy. In a significant number of
cases, immunoflourescence with anti-ACE serum showed localization in the sarcoid
granuloma in addition to normal staining of the brush border of the proximal tubules. (Mery
et al 1988) See Figure 4.
Fig. 4. Renal biopsy showed a granulomatous interstitial nephritis with a broadened
interstitial, cellular infiltrates and granuloma with typical multinucleated giant cells
(arrowheads). Kettritz R et al. Nephrol. Dial. Transplant. 2006; 21:2690-2694
Sarcoidosis and Kidney Disease
97
In contrast to conventional pathological dogma, Joss et al showed that asteroid bodies and
calcification were not common in sarcoid GIN. (Joss et al 2007) Interestingly, asteroid bodies
were seen in 1 case of drug induced AIN. However, lymphocyte cuffing and giant cell
infiltration were prominent in sarcoid granulomas in the kidney. Necrosis and eosinophil
infiltration of the interstitum was more common in drug induced GIN as compared to
sarcoidosis. It is now believed that idiopathic GIN, TINU and sarcoidosis represents a
clinicopathological spectrum and that idiopathic GIN or TINU may subsequently develop
typical extra-renal manifestations of sarcoidosis.
6.1 Treatment
The mainstay of treatment of sarcoid GIN is glucocorticoids. Initial treatment requires a
daily dose of prednisone or prednisolone preferably 1-1. 5 mg/kg. Response to treatment
can often be dramatic in terms of improvement of renal insufficiency. The best response to
glucocorticoids was noted in a study by Mahevas et al. in which 47 patients with renal
sarcoid received prednisolone while 10 also received pulse methylprednisolone. (Mahevas
et al 2009) The authors concluded that at 24 months, a complete and partial remission
occurred in 30 and 5 patients respectively. But no response was noted in patients with
severe interstitial fibrosis of greater than 50%. Underlying functional tubular dysfunction
improves with progressive drop in serum creatinine. An important point to realize here is
that steroid treatment has to be prolonged and must exceed at least 6 months as
nephropathy relapses very frequently with short term therapy (Gene and Cheviot 1988). A
commonly followed strategy is to give the initial dose for 2 months followed by progressive
taper and switching to an alternate day therapy. A maintenance therapy period for 1 year
at least is recommended. Serial renal biopsies have shown a regression of granuloma in
conjunction with improvement of renal function
(
Farge et al 1986) although given the
variability in results
(Gene and Cheviot 1988) routine biopsies after starting steroids is not
recommended. Treatment in advanced disease is often associated with interstitial fibrosis
along with focal segmental glomerulosclerosis and vascular lesions. However, vascular
lesions are more common with long term corticosteroid therapy and are associated with
delayed development of hypertension which is a major contributor to progression of renal
failure. (Mery and Kenouch 1988)
While analyzing outcomes of steroid treatment in a heterogeneous population of GIN, Joss
et al, presented data of 16 patients of which 5 were labeled as sarcoidosis. Patients were
treated with prednisolone (starting dose of 0. 55mg/kg)
(Joss et al 2007) for a mean period of
25 months and then followed up for a period of 45 months. Overall, renal function stabilized
or improved at the end of the study with mean GFR improving from 21 to 56 ml/min. One
patient who was on dialysis at the beginning of therapy was able to discontinue dialysis
within 3 months. Six patients relapsed on dose reduction of which 4 were sarcoid GIN who
required azathioprine to break steroid dependence. Sarcoid patients required longer
treatment (36 months) as compared to idiopathic or TINU patients. The greatest renal
recovery occurred in the first year of treatment. There was no difference in renal outcome
when analyzing the degree of interstitial fibrosis. Age less than 60 years was associated with
a better outcome. Table 1 summarizes data on treatment of GIN in some important studies
so far.
Long term results with steroid therapy in sarcoid GIN have not been rigorously tested in
randomized controlled trials. In a large case series of 39 patients with sarcoid renal disease,
Chronic Kidney Disease
98
Parameter Joss et al Robson et al.
ORiordan
et al.
Hannedouche
et al.
Brause
et al.
n 18 16
1
7 5 6 5
Cause Mixed
Idiopathic/
TINU/sarcoid
Idiopathic
Isolated
sarcoid
Sarcoid Sarcoid
Age (yr) 55 56 69 48 to 71 62 61
Male gender (%) 61 56 71 60 50 60
Renal function at
baseline
BaselineCC
(ml/min)
21 24 14 6 NA NA
Baseline
creatinine
(mol/L)
373 357 420 NA 566 396
Hypercalcemia 3/18 3/16 2/7 0/5 2/6 1/5
Raised serum
ACE
4/17 4/15 3/7 1/5 4/4 1/5
Improved renal
function
17/18 15/16 5/7 4/5 6/6 5/5
Long-term RRT 0/18 0/16 2/7 0/5 0/6 0/5
Prednisolone (%) 89 88 100 100 100 100
Mean follow-up
(mo)
45 48 25 35 75 NA
Renal function at
last visit
ECC (ml/min) at
end of therapy
56 53 22 20 NA NA
Creatinine
(mol/L) at end
of study
159 159 296 NA 192 225
1
Data excluding the two cases of drug-induced GIN.
Table 1. Comparison on treatment of GIN in literature.
17 patients with biopsy-proven tubulo-interstitial nephritis with significant renal
impairment were analyzed over a one year period of corticosteroid therapy. (Robson et al
2003). All patients were initially started on prednisolone at 0. 5 mg/kg body weight at a
daily dose of 3060 mg which was tapered by 5 mg each week once the renal function has
improved and/or stabilized. Thereafter, patients were maintained on 57. 5 mg daily
indefinitely. Mean duration of study was 84 months. Estimated glomerular filtration rate
(eGFR) at baseline was 26. 814 ml/min which improved to 49. 65. 2 ml/min (P<0. 01) at 1
year, and 47. 96. 8 ml/min (P<0. 05) at last review. Interestingly, the response to treatment
was similar regardless of the degree of renal impairment at baseline, race and the degree of
tubulo-interstitial scarring on renal biopsy. Three patients developed side effects that could
Sarcoidosis and Kidney Disease
99
be attributed to steroids which included acute psychosis and type 2 diabetes. Long term use
of corticosteroids, especially in adolescents, can cause substantial side effects including
diabetes, growth retardation and cataract. Alternative agents that have been attempted in
treating sarcoid GIN include mycophenolate
(Moudgil 2002) and mizoribine (Rajakariar et al
2006 and Ito et al 2009) which are limited to case reports and have been primarily used in
pediatric patients to break steroid dependence or ameliorate significant side effects. Other
agents which have been tried in systemic extra-renal sarcoidosis include mycophenolate
mofetil, methotrexate, azathioprine, antimalarials, and phosphodiesterase inhibitors such as
pentoxifylline and thalidomide although no data on treating renal sarcoidosis exists.
(Baughman 2003) There has been great interest in the use of TNF-antagonists as another
modality to treat sarcoid GIN in order to avoid use of steroids. TNF-alpha, which is
expressed by monocytes, is critical in the development of these noncaseating granulomas.
TNF-alpha receptor antagonists have also been shown to prevent the initiation and
perpetuation of inflammation and subsequent interstitial fibrosis. Etanercept is a soluble
TNF-alpha receptor fusion protein that binds TNF-alpha. Infliximab and adalimumab are
monoclonal antibodies that bind specifically to and neutralize TNF-alpha. While etanercept
is an ineffective agent in the treatment of systemic sarcoidosis,
(
Ulz et al 2003) infliximab has
been shown to be effective in a case of renal sarcoid. Thumfart et al, described the case of a
boy presenting with severe arterial hypertension and acute renal failure caused by an
isolated sarcoid granulomatous interstitial nephritis. Renal function improved initially with
prednisone treatment but later, the patient showed signs of severe steroid toxicity and
progressive renal failure. Monthly treatment with infliximab was initiated resulting in a
steady improvement in renal function and resolution of renal granulomata, as well as
reduction in antihypertensive medication. (Thumfart 2005) Ahmed et al presented a patient
with acute renal failure due to isolated granulomatous infiltration of the renal parenchyma.
(Ahmed et al 2007) Renal biopsy showed granulomatous interstitial nephritis with
noncaseating granulomas. There was no evidence of extrarenal sarcoid involvement.
Prednisone 60mg daily resulted in significant improvement in renal function. Due to
recurrent flares while tapering the prednisone and steroid toxicity, treatment with
infliximab was instituted and resulted in stabilization of renal function. This case
demonstrated that steroid-dependant or refractory renal sarcoidosis cases may respond to
infliximab. We recently reported the case of a 46-year-old woman with multi-organ
sarcoidosis, type 2 diabetes, subnephrotic-range proteinuria, hypertension and recurrent
episodes of hypercalcemia-induced acute kidney injury who was referred for evaluation of
worsening renal function and nephrotic range proteinuria. (Gupta et al 2008) A kidney
biopsy showed sarcoid GIN with moderate-to-severe chronic tubulointerstitial disease,
hypertensive vasculopathy, and diabetic glomerulosclerosis. Because steroids had caused
multiple side effects including diabetes, hypertension and obesity and attempts to wean
steroids had caused hypercalcemia and acute renal failure, Adalimumab (Humira
TM
) 40
mg/0. 8 cc weekly for 6 months was initiated. After 6 months of treatment with
adalimumab, serum creatinine improved from 345 mol/L (3. 9 mg/dL) to 1. 8 mg/dl (her
baseline for years) and proteinuria improved from 10 g/day to 3. 5 g in 24 hours
respectively. A repeat biopsy showed persistent diabetic glomerulosclerosis, moderate
chronic tubulointerstitial inflammation with complete resolution of interstitial epitheliod
granulomas. Although adalimumab and infliximab are generally safe, some side effects
Chronic Kidney Disease
100
include risk of lymphoma and reactivation of latent tuberculosis
(
Denys et al 2007). These
agents may hold promise for the future once large scale randomized studies are available to
show consistent benefits with minimal side effects.
7. Renovascular diseases associated with sarcoidosis
Renovascular diseases secondary to sarcoidosis are distinctly rare and attributed to a form
of secondary vasculitides. Systemic vasculitis associated with sarcoidosis has been
reported as an isolated entity in the literature after excluding other common causes of
vasculitis. It is predominantly large vessel vasculitis although few instances of small
vessel vasculitis have been reported. In a large case series and review of literature on
sarcoid vasculitis, Fernandes et al, noted that most cases were children and clinical
presentation resembled hypersensitivity vasculitis, Takayasus arteritis, polyarteritis
nodosa or microscopic polyangitis. (Fernandes 2000) Clinical features included fever,
peripheral adenopathy, hilar adenopathy, rash, pulmonary parenchymal disease,
musculoskeletal symptoms, and scleritis or iridocyclitis with biopsy showing necrotizing
sarcoid granulomata. Interestingly, no renal involvement was noted. Notably the authors
found large vessel vasculitis largely in the African American population while small
vessel vasculitis predominantly affected white races. Godin et al described a known case
of pulmonary sarcoidosis with persistent hypertension. (Godin et al 1980) Diagnostic
evaluation for renovascular hypertension included aortography which showed severe
stenosis of right renal artery. Surgical exploration showed extensive periaortic and
perirenal fibrosis with extrinsic compression of renal artery. Pathological examination of
the kidney revealed epitheloid infiltration of the adventia of renal artery suggestive of
sarcoid angitis. Surgical biopsy was performed on both kidneys. The right kidney,
protected by arterial stenosis, was slightly altered, while the left kidney showed extensive
interstitial, tubular, and glomerular lesions which included focal and segmental
hyalinosis. Marcussen et al, reported an autopsy case of a middle aged man who died of
myocardial infarction secondary to fulminent vasculitis. (Marcussen and Lund 1989)
Pathology showed widespread giant cell vasculitis with simultaneous involvement of the
renal arteries, veins, and arterioles along with typical interstitial sarcoid granuloma.
Shintaku et al, showed granulomatous inflammation of small renal vessels and crescentric
GN on the autopsy of a patient with pulmonary hemorrhage and rapidly progressive
renal failure. (Shintaku et al 1989) Thus, sarcoid angitis, especially causing small vessel
vasculitis in the kidney may represent a very severe form of sarcoidosis. In their review,
Fernandes et al, noted that four out of six patients responded well to steroid treatment
alone but had relapses when attempts were made to taper or withdraw steroids
.
(Fernandes 2000) Frequently, there is an overlap between sarcoidosis and well known
causes of granulomatous vasculitis. For instance, Watson et al described a case of
longstanding pulmonary sarcoidosis presenting with rapidly progressive renal failure
with p-ANCA positivity. (Watson 1996) Renal biopsy demonstrated focal and segmental
fibrinoid necrosis with crescentric GN and focal fibrinoid necrosis in arterial wall, but no
granulomata and pauci-immune deposits on immunofluorescence. Unlike patients with
ANCA positive vasculitis, the index case responded poorly to pulse steroids and
cyclophosphamide and progressed rapidly to end stage renal disease.
Sarcoidosis and Kidney Disease
101
8. Kidney transplantation in patients with sarcoidosis
The usual cause of end stage renal disease in sarcoidosis requiring renal replacement
therapy is usually due to hypercalcemic nephropathy rather than granulomatous interstitial
nephritis or a glomerular disease. The outcome in renal transplantation in patients with
sarcoidosis has been described in the literature. The first recurrence of sarcoid GIN in renal
allograft was diagnosed 6 years after deceased donor kidney transplantation in a patient
that was diagnosed with GIN before transplantation
(
Shen et al 1986). A recent French study
aimed to describe a multicenter experience with kidney transplantation in patients with
sarcoidosis. (Aouizerate et al 2010) In this study, the authors retrospectively identified 18
patients who underwent renal transplantation. Patient medical charts, demographics were
reviewed. The median time between the last sarcoidosis episode and renal transplantation
was 78 (8 to 900) months. Only 3 out of 18 patients had been on immunosuppression prior to
transplantation. Vast majority of the patients had in the past received steroids and other
immunosuppression for their sarcoid before transplantation. Renal disease was attributable
to biopsy proven renal sarcoid in 10 out of the 18 patients and was attributed to other causes
in 8 patients. Mean age of transplantation was 43. 5 +/- 11 years. 17 out of 18 patients had a
deceased donor transplant. Mean donor age was 36. 5 +/ 15 years. Mean cold ischemia time
was 16. 6 +/- 8 hours. 11 patients received induction therapy with anti-thymocyte globulin
or Il-2 receptor antagonists. Maintenance immunosuppression included calcineurin inhibitor
(CNI) for all patients, mycophenolate mofetil or azathiporine, sirolimus and corticosteroids
for 16 out of the 18 patients. At the end of the 42 month follow up period, patient and death
censored graft survival was 94. 4% and mean GFR was 60 cc/min per 1. 73 m2. Recurrence
of sarcoidosis after renal transplantation was observed in 5 (27%) of patients. The median
period between renal transplantation and recurrence was 13 months and four of five
patients exhibited recurrence in the first 18 months after renal transplantation. Recurrences
involved in the same organ in four of five patients and included renal involvement in three
patients and lung and liver involvement in one patient. Mean GFR at end of follow-up was
significantly lower in the three patients with recurrence than that for the entire cohort. (31
versus 60 cc/min per 1. 73 m2). Analysis of the recurrences showed that they occur in the
first 18 months after transplantation. Primary disease related to sarcoidosis was strongly
associated with recurrence (40% in the group with renal sarcoidosis versus 12. 5% in a group
with a primary nephropathy, and median period between last episode of sarcoidosis and
renal transplantation was shorter in the case of sarcoidosis recurrence (42 versus 78 months
respectively). This study showed that patients with initial renal involvement display
sensitivity to disease recurrence in allograft. The incidence of recurrence was significant as
all patients were maintained on triple immunosuppressive therapy including steroids and
mycophenolate mofetil. This study showed that renal transplant can be conducted safely in
transplant patients with sarcoidosis, but recurrences do occur and affect overall graft
outcome.
Kukura reported a case of recurrence of sarcoidosis in the renal allograft during pregnancy.
(Kukura et al 2004) This was a 27 yr old female diagnosed with sarcoidosis at age 14 by
lacrimal and parotid gland biopsy. 4 years after presentation, she developed hypertension
and renal insufficiency. Kidney biopsy showed interstitial nephritis and nephrosclerosis, but
no granulomas. Patient was eventually started in hemodialysis and underwent kidney
Chronic Kidney Disease
102
transplantation with excellent graft function with a creatinine of 1. 32 mg/dl and a negative
urinalysis. Patient was maintained on cyclosporine, azathioprine and prednisone 25 mg by
mouth daily. 2 years after transplantation once the steroids were withdrawn, patient
continued to have good kidney function with an allograft biopsy showing mild chronic
allograft nephropathy only. Immunosuppression consisted of azathioprine and
cyclosporine. At 3 years after kidney transplantation, patient became pregnant. 29 weeks
into pregnancy, renal function worsened. Biopsy showed numerous noncaseating
granulomas bound to the arteries, initial arteritis in one artery, mild interstitial mononuclear
inflammation and tubulitis. Graft function improved with pulse methylprednisolone and
tapered steroids were used. After delivery, renal allograft biopsy was performed 6 months
which showed baseline disease of mild chronic allograft nephropathy and sporadic
granulomas. This case demonstrates that steroid withdrawal after kidney transplantation
may lead to sarcoidosis recurrence.
The implication that sarcoid reflects a disease phenomena related to the immunologic
stimulus makes sarcoidosis an unlikely diagnosis to be made in an immunosuppressed
patient such as an organ transplant recipient. However, Schmidt et al showed that after
kidney transplantation, sarcoidosis can occur in the lung and pleura. (Schmidt et al 1999) In
this case, a 41 yr old with history of IgA nephropathy and no past medical history received a
living related kidney transplant and had been receiving tacrolimus therapy. He was found
to have a large pleural effusion 17 months after kidney transplant. Diagnosis of sarcoidosis
was established by identifying noncaseating granulomas, some with multinucleated giant
cells in the pleural and lung tissue. All viral and bacterial workup was negative. The
effusion resolved after initiating corticosteroid therapy. One month into therapy, the
effusion resolved and patient continued to be asymptomatic twenty months after therapy.
The authors did not speculate on the pathogenesis of granuloma formation since both
tacrolimus and corticosteroids interfere with T lymphocyte function and granuloma
formation. They speculated that activation of tissue chemokines of the IP-10 type during the
posttransplant period, along with subsequent recruitment of lymphocytes and macrophages
may have resulted in the sarcoidosis.
9. Conclusion
Sarcoidosis is a disease that primarily affects the reticuloendothelial system but can affect all
tissues and organ systems. In this chapter, we described the effects of sarcoidosis on the
kidneys. This disease affects patients worldwide and is defined pathologically by the
presence of noncaseating granulomas in the involved tissue. The etiology of sarcoidosis has
yet to be determined but some have proposed a possible infectious etiology. Commonly
sarcoid patients present with hypercalcemia, hypercalcuria, and nephrolithiasis due to the
overproduction of calcitriol from the epitheliod granulomas. We also described the rare
glomerular and renovascular manifestations of sarcoidosis. Granulomatous interstitial
nephritis is most commonly associated with sarcoidosis. It is a histological diagnosis and
can be treated with both steroids and TNF-alpha antagonists. Kidney transplantation is safe
in patients with sarcoidosis but we must keep in mind the disease can recur in the allograft.
In conclusion, sarcoidosis is a complex disease and presents both a diagnostic and
management challenge to the physician.
Sarcoidosis and Kidney Disease
103
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7
Origins of Cardiorenal Syndrome and
the Cardiorenal Connection
L. G. Bongartz
1,2
, M. J. Cramer
1
and J. A. Joles
2
1
Dept. of Cardiology, University Medical Center Utrecht, Utrecht
2
Dept. of Nephrology, University Medical Center Utrecht, Utrecht
The Netherlands
1. Introduction
In recent years, the relationship between the heart and the kidneys in disease has received
increasing attention from the clinical and scientific medical community. This was initiated
by epidemiological observations in the late 1990s of increasing patient numbers with
concurrent heart and kidney problems, and the association with a significantly higher
mortality ratio. This has led to intense discussions about the value of the recognition of
cardiorenal disease on the one hand, and the existence of a specific cardiorenal syndrome
on the other hand.
1-10
The idea of specific interaction between heart and kidneys is not new. There are numerous
examples and anecdotes that show that people in the past from various societies considered
the heart and the kidneys to have a special relationship.
1.1 Heart and kidneys in ancient times
The Egyptian Book of the Dead (1600-1240 B.C.), which served as a reference work to
assist the deceased in the afterlife, is one of the first known texts that mentions the heart and
kidneys in parallel:
"Homage to thee, O my heart!
Homage to you, O my kidneys!".
11
The heart and the kidneys were the only organs left inside the body during the process of
mummification. The heart was weighed against the feather of truth by the jackal-headed
Anubis (Figure 1), but the exact role of the kidneys for the passage into afterlife is uncertain.
Blood vessels are well preserved in mummies, and there is evidence that cardiovascular
disease affecting both the heart and the kidneys were not uncommon.
12
Eknoyan
13
researched the Bible and found that:
[T]he kidneys are mentioned five times in the Bible as the organs examined by God to
pass judgment on a person. They are mentioned either before or after but always in
parallel with the heart,
as for example, "I, the Lord, search the heart, I try the reins,
even to
give every man according to his ways, and according
to the fruit of his doings" (Jer. 17:10), and,
"Examine me,
O Lord, and prove me; try my reins and my heart" (Psalms 26:2).
Chronic Kidney Disease
108
Fig. 1. The weighing of the heart against the feather of truth. This papyrus was found in the
tomb of the scribe Hunefer in Thebes. It dates from the 19th Dynasty, about 1285 BC. It can
be seen in the British Museum.
In Hebrew lore the kidneys owned the status as the organs which give the heart advice and
counsel, and which symbolize the innermost sources of thought and desire, those hardly
accessible to man but tested by God.
14
1.2 Heart and kidneys in Traditional Chinese Medicine
No less lyrical, albeit more clinical descriptions are found in China, where the heart and the
kidneys are described in various medical texts. In Traditional Chinese Medicine (TCM), the
kidney represents water and is considered a yin organ whereas the heart represents fire
and is a yang organ.
15
In TCM, the kidney not only regulates the urinary system, but also
controls the reproductive, endocrine and nervous system. It stores Jing, which is considered
a vital life force responsible for development and reproduction. The heart rules the blood
vessels and blood supply to the organs, but also stores the spirit, reflected in a persons
mental, cognitive and intellectual abilities.
Dr. Shen Jin'ao writes in his book Dr. Shen's Compendium of Honoring Life (Shen Shi
Zunsheng Shu) from 1773:
The heart resides in the vessels. It rules the kidney network, not via a controlling
position in the restraining circle of relationship between the organ networks [where the
kidney actually restrains the heart], but simply because it is the general master of all
Origins of Cardiorenal Syndrome and the Cardiorenal Connection
109
organ networks. Before the heart fire can harmoniously blend with the kidney water,
however, the kidney water must be sufficient. Otherwise the heart fire will flare out of
control, and all kinds of heart and kidney ailments will arise.
In the 5 Elements network of Chinese medicine (Figure 2) a disorder called heart and
kidney failing to link (xin shen bu jiao) is presented, resulting in a variety of symptoms
ranging from restlessness and palpitations to dizziness, and dark, scanty urination or
nocturia.
16
If both kidneys and heart are weakened, there may be palpitations, shortness of
breath, dizziness, darken complexion, purple lips and nails, sensitivity to low temperatures,
urinary difficulty, edema that is more apparent in the lower limbs, and a bulky tongue. If
the kidneys and heart are in disharmony, there may be palpitations, dream-disturbed sleep,
forgetfulness, dizziness, thirst, red cheeks, night sweats, lumbar and knee soreness,
nocturnal emission, and a red tongue.
Fig. 2. The Five Elements theory of TCM and the relationships between the organs, with
generation (solid arrows) and restriction cycles (dashed arrows).
Another piece of traditional Chinese Medicine text gives a pretty accurate description of the
symptoms of cardiorenal failure:
When the kidney fails to evaporate fluid which then floods and ascends to depress the
function of heart yang there may be clinical manifestations such as oedema, chills and
cold limbs, accompanied by palpitations, shortness of breath and stuffiness in the chest,
indicating retained water affecting the heart.
17
1.3 Cardiorenal disease in the European Middle Ages
In Western society, during the Middle Ages, heart disease per s was not very well
described in medical doctrines, although the heart was considered the source of the spiritus
vitalis. Medieval doctors viewed the outward appearance and excretions of the whole body
Chronic Kidney Disease
110
or body parts as a reflection of ones state of health, and as such the symptoms of congestive
heart failure were approached as separate clinical entities.
18
The examination of urine was
however a widely used diagnostic tool. As one of the first Western cardio-nephrologists,
Gentile da Foligno (Gentilis de Fulgineo; 1272? 1348) considered heart disease as one of the
major inflictions modulating the color and output of urine in his commentary on De pulsibus
(About Pulses) composed by Aegidius Corboliensis (Figure 3).
19
Fig. 3. First page of De pulsibus. Town Library, Foligno. Reproduced from ref.
19
.
1.4 Heart-kidney interactions in the late 19
th
and early 20
th
century
During the Industrial Revolution the medical sciences expanded and scientific methods
became more and more reliant on experiments and observation. Richard Bright (17891858)
and Ludwig Traube (18181876) both documented that cardiac hypertrophy was a common
anomaly resulting from chronic renal disease.
20, 21
Traube refers in his writings to William
Senhouse Kirkes (1822 1864) who reviewed 14 autopsy cases of with apoplexy and diseased
kidneys, of which only one did not have an enlarged heart (Figure 4).
22
He concludes that:
"... I believe that the affection of the kidneys is the primary disease... [it] has among its
most frequent and permanent accompaniments an hypertrophied condition of the left
Origins of Cardiorenal Syndrome and the Cardiorenal Connection
111
ventricle ... of the various explanations of this pathological fact the most probable
perhaps is that which regards the blood as so far altered from its normal constitution ...
as to move with less facility through the systemic capillaries, and thus to require
increased pressure, and consequently increased growth of the left ventricle, to effect its
transmission."
Fig. 4. Beginning of Kirkes publication in the Medical Times & Gazette, 1855.
In a lecture delivered at the University College in London in 1913, Thomas Lewis
23
speaks
about paroxysmal dyspnoea in cardio-renal patients and after a very interesting review of
the clinical and pathological findings of multiple cases, he concludes:
We come to this standpoint-that the clinical or anatomical distinction between cardiac
and renal asthma, is no certain one. Asthma occurring, in patients who show on the one
hand prominent cardiac lesions, on the other hand prominent renal lesions, may or may
not be due to a single cause.
Alfred Stengel
24
proposed a definition of cardio-renal disease (Figure 5) when he wrote in
1914:
The clinician encounters many cases, mainly in persons of middle age or older, in
which evidences of cardiac weakness and other circulatory disturbances, such as high
pressure, are associated with signs of failure of renal function or urinary indications of
renal disease. When this combination of symptoms is of such character that the observer
cannot readily assign to either the cardiovascular system or to the kidneys the
preponderance of responsibility, the term "cardio-renal disease" is often employed. The
term, therefore, comprises cases of combined cardiovascular and renal disease without
such manifest predominance of either as to justify a prompt determination of the one
element as primary and important and the other as secondary and unimportant.
Chronic Kidney Disease
112
Fig. 5. Example from a 1915 Death Certificate from Massachusetts. From Rudys List of
Archaic Medical Terms at http://www.antiquusmorbus.com/English/Heart Stroke.htm
The observations on the cardiac consequences of chronic kidney disease were later
expanded, and Gouley
25
was coined the term uremic myocardiopathy in 1940 and in 1944
Raab
26
proposed that cardiotoxic substances accumulate in uremia. Rssle,
27
and
Langendorf and Pirani
28
later showed that interstitial widening and fibrosis were common
in hearts of patients dying from uremia.
1.5 The Cardiorenal Syndrome in modern times
The advent of the Cimino-shunt and the development of hemodialysis (HD) as the mainstay
treatment for end-stage renal disease (ESRD) resulted in further increasing interest in the
structural and functional cardiac status of HD patients.
29-32
The full extent of the problem of
cardiovascular disease in chronic kidney disease (CKD) and ESRD patients was then charted
in the 1990s, showing that a large proportion of patients starting dialysis already suffers
from cardiac abnormalities and dysfunction and that survival of these patients after a
myocardial infarction (MI) was dismal.
33-35
In 2003, a statement from several councils from
the American Heart Association (AHA) was published in Hypertension and Circulation
underscoring the problem of increased cardiovascular risk in CKD, and the lack of
knowledge on pathophysiology.
36
This was followed by two seminal papers published in
the New England Journal of Medicine showing the exponentially increased risk for adverse
outcome with decreasing kidney function, in normal patients but even more so after they
had experienced a myocardial infarction.
37, 38
At the same time, the scientific and clinical
community became increasingly aware of the effect of decreased kidney function or kidney
damage on the prognosis of patients with heart failure.
39-41
Interestingly, in a study on the
predictive value of 10 different biomarkers in over 3000 patients from the Framingham
Heart Study, levels of brain natriuretic peptide and urinary albumin-to-creatinine ratio most
strongly predicted major cardiovascular events.
42
One patient study even suggested that the
decline of renal function is accelerated after an acute MI.
43
These epidemiological
associations resulted in a strong clinical suspicion that the combination of heart and kidney
disease is associated with accelerated disease progression and adverse outcome.
2. The Severe Cardiorenal Syndrome and the Cardiorenal Connection
The epidemiological data, the AHA statement, and our own clinical observations of
cardiorenal failure in patients led us to propose the Severe Cardiorenal Syndrome (SCRS)
Origins of Cardiorenal Syndrome and the Cardiorenal Connection
113
as a separate disease entity with the Cardiorenal Connection (CRC) as the putative
pathophysiological model.
44
We defined the SCRS as a condition in which combined cardiac
and renal dysfunction amplifies progression of failure of the individual organ, leading to
grossly increased cardiovascular morbidity and mortality. The CRC works in conjunction
with the hemodynamic control model of heart-kidney interactions as stipulated by the late
professor Guyton (Figure 6).
Fig. 6. The cardiorenal connection works extensive to Guytons model to drive accelerated
cardiovascular damage in combined renal and heart failure.
The cardiorenal connectors that we put forward were:
the balance between nitric oxide (NO) and reactive oxygen species (ROS),
the sympathetic nervous system (SNS)
the renin-angiotensin system (RAS), and
inflammation.
We envisioned that both heart and renal failure lead to derangement of the Guytonian
model of hemodynamic control, but also results in activation/disturbance of the connectors
Chronic Kidney Disease
114
of the CRC. The connectors have a modulating effect on hemodynamic control but can also
induce cardiovascular damage, thereby mediating further functional deterioration.
44
We
proposed that activation of the CRC leads to a vicious cycle in which all the connectors
become disturbed, synergize and further activate each other. This ultimately results in
worsening of both cardiac and renal damage and failure.
2.1 Summary of the Cardiorenal Connection
A shift in the balance between NO and ROS towards ROS is a central event in many
cardiovascular diseases.
45
In the SCRS,
the balance between NO and the ROS is skewed
towards the latter
by increased production of ROS, a low anti-oxidant status, and
lower
availability of NO.
46
In the cardiorenal connection, this imbalance may influence
sympathetic nervous activity,
47
release of renin and angiotensin,
48
and promote
inflammation by oxidative modification of substances.
49
Sympathetic nervous activity is also increased in both renal and heart failure. By affecting
the other cardiorenal connectors it can play a significant role in the SCRS. It stimulates renin
release from the kidneys,
50
generates ROS which induces vascular wall growth,
51
and
induces inflammation.
52
The RAS is activated in both renal and heart failure
53, 54
and angiotensin II affects the other
cardiorenal connectors in different ways. It activates the SNS in both heart and kidney
failure,
55, 56
it generates ROS via nicotinamide adenine dinucleotide phosphate (NADPH)-
oxidase,
57
and activates pro-inflammatory gene expression via nuclear factor-B.
58
Persistent inflammation has been found in both renal and heart failure. By altering the
functioning of the RAS,
59
and promoting ROS
60
and noradrenaline formation,
61
inflammation can contribute to the positive feedback loops in the cardiorenal connection.
The Severe Cardiorenal Syndrome is thus not a syndrome in which cardiac and renal failure
simply co-exist side-by-side. Cardiac and renal failure are intimately linked by the
cardiorenal connectors, because failure of either organ can excite the cardiorenal connectors,
but the connectors themselves also affect the structure and function of both organs.
Logically, the cardiorenal connectors become more pronounced in combined failure.
6
3. Previous research on the cardiorenal interactions
In a recent comprehensive review in Circulation, Bock and Gottlieb
10
state that:
each dysfunctional organ has the ability to initiate and perpetuate disease in the
other organ through common hemodynamic, neurohormonal, and immunological/
biochemical pathways. They also write: our understanding of the complex
physiological, biochemical, and hormonal derangements that encompass the CRS is
woefully deficient.
Despite general acknowledgement of the adverse prognosis of concurrent cardiac and renal
disease, many clinicians and researchers are skeptical about the true existence of a specific
heart-kidney interaction that goes beyond known physiological interactions. Thus the
question was raised whether kidney disease and heart disease simply co-exist or that they
indeed worsen each others progression. Clinical studies can not provide the answer to this
Origins of Cardiorenal Syndrome and the Cardiorenal Connection
115
question because they are observational, lack histological end-points, and are confounded
by selection bias, inconsistent definition of end-points, and medication use. Therefore,
further exploration of the mechanisms of cardiorenal interactions must rely on animal
studies, in which timing and severity of the disease are controlled, progression of disease
can be followed, and histological end-points are assessed.
Much of what we know today on the structural cardiac consequences of chronic kidney
disease results from the extensive research in rats with CKD by the group of Kerstin Amann
and Eberhard Ritz in the late 80s and early 90s.
62
Despite numerous cardiac changes, in the
rat CKD model of subtotal (5/6
th
) nephrectomy (SNX) cardiac systolic function is generally
maintained.
63, 64
Conversely, after MI by ligation of the left coronary artery in rats, renal
histological damage or proteinuria is absent although glomerular filtration rate (GFR) may
be decreased.
65, 66
Thus, it appears that both organs need to be affected to cause acceleration
of damage and failure typical for the CRS. Only two animal studies investigated the effect
of dual damage to heart and kidneys, with MI following shortly after a renal insult in rats,
with conflicting results.
65, 67
Different models of nephrectomy exist in mice, but these are not
as robust as those in rats, with variable changes in renal function and cardiac
abnormalities.
68-72
The renal hemodynamic response to heart failure (HF) induced by pacing in dogs has also
been investigated,
73-75
but whether there is histological damage is unknown. Furthermore,
there is no proven model of CKD in dogs. Taken together, there is still a paucity of models
that investigate the interaction between kidney and heart failure in a chronic set-up with
integrated physiological and histological assessment. From the available data, combining
the SNX model of CKD and the coronary ligation model of HF appears to be the most robust
option to investigate the SCRS.
3.1 The role of nitric oxide in the Severe Cardiorenal Syndrome
We developed a model of the SCRS based on CKD and depletion of NO availability. The
rationale for these investigations was that the pathogenesis of CKD (in the presence of
hypertension, diabetes or aging) is associated with low NO availability,
76,77
while
experimental SNX induces nephron number reduction in a healthy animal. Furthermore, in
SNX, cardiac systolic function generally remains preserved, while in patients left ventricular
dysfunction (LVSD) develops during the course of CKD progression.
78
Reduced NO availability is considered a hallmark of CKD.
46, 79
NO can function as an
effector of the CRC by way of its vasodilatory action. It also modulates GFR and tubulo-
glomerular feedback.
80
Reduced NO availability will result in tissue damage by oxidative
stress. In extension to our proposal of the Cardiorenal Connection,
44
we postulated that the
balance between NO and ROS is a key modulator of the other cardiorenal connectors.
81
Many effects of the other CRCs may be mediated by changes in the redox-balance and NO
availability.
82, 83
Also, it has been shown that constitutive NO production supports basal cardiac function.
84
Apart from its role in endothelial dysfunction, NO availability also modulates cardiac
contractility, as NO synthase (NOS) inhibition reduces cardiac output, and causes cardiac
damage in high doses.
85, 86
Chronic Kidney Disease
116
We thus hypothesized that a reduction in NO availability would accelerate the development
of cardiac dysfunction. Indeed, treatment with an oral NO synthase (NOS) inhibitor (L-
NNA), at a very low dose, induced NO depletion and severe cardiac dysfunction.
87
Furthermore, proteinuria, severe glomerulosclerosis and cardiac interstitial fibrosis were
worsened compared to rats with CKD without NOS inhibition. Another remarkable finding
was that the effects on cardiorenal dysfunction but also on systemic NO production were
irreversible after cessation of the NOS inhibitor, during a 7 week follow-up. A five times
higher dose of NOS inhibition in control rats, which caused a similar level of hypertension
and NO depletion, induced LVSD that was not as severe as in the CRS rats. Furthermore, all
effects on blood pressure, cardiac function and NO availability were completely reversible,
and had no effect on kidney structure and function. Combining NOS inhibition with SNX
also, worsened kidney injury. The more severe hypertension and direct effects of NOS
inhibition may have played a role in this.
We concluded that during CKD development the heart is very sensitive to depression of
systemic NO availability. Compared to the normal kidney, the damaged kidney is more
sensitive to alterations of NO availability as well, possibly because of a loss of
autoregulation.
88
Thus, maintaining adequate NO availability appears to be very important
for progression of cardiorenal failure during progression of CKD, and the combination of
CKD and NO depletion appears to produce a functional model of the SCRS in which cardiac
function is further compromised.
That supplementation of NO is useful as a rescue therapy was shown in a subsequent study,
where treatment with the oral tolerance-free NO donor molsidomine (MOLS) significantly
improved cardiac diastolic and systolic function, abrogated mortality, and also slightly
improved kidney function and injury.
89
The cardiac effect of MOLS appeared to be a
combination of reduced cardiac loading and improved contractility and relaxation. Systolic
blood pressure was only mildly reduced and GFR was even slightly improved. Thus, MOLS
appears to be an attractive and safe therapeutic option for CKD patients suffering from
cardiac dysfunction of non-ischemic origin. The pathophysiology of the continuing low NO
production in this model is likely very complex and may include low NOS expression or
activity, substrate deficiency, high oxidative stress levels, and increased amounts of
endogenous NOS inhibitors.
79
In conclusion, the cardiorenal connection has intrigued scientists and physicians for
centuries. The existence of a specific cardiorenal syndrome has been suggested since the
start of the 20
th
century, but has recently gained widespread attention in the scientific
literature. We proposed the Severe Cardiorenal Syndrome, in which CKD and HF induce
derangements to cause a vicious cycle of cardiovascular damage and progression of failure
of both organs. Understanding of pathophysiological mechanisms is expanding and animal
models provide an invaluable tool to investigate the bidirectional nature of cardiorenal
interactions.
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8
Sub-Types and Therapeutic
Management of the Cardiorenal Syndrome
Margot Davis and Sean A. Virani
University of British Columbia
Canada
1. Introduction
Cardiorenal syndrome (CRS) describes the inter-relationship and complex
pathophysiological processes by which dysfunction of either the heart or the kidneys is
related to dysfunction in the other organ system. Historical definitions may have been
overly simplistic; newer definitions have tried to capture the complex interactions and
feedback processes which exist between the two organs. These definitions classify the CRS
into five discrete categories, based on both the organ system in which the primary
dysfunction occurs and the time course of disease development/progression.
The CRS is more common than many clinicians realize. Over one third of patients in heart
failure (HF) registries have evidence of renal dysfunction, and a similar proportion of
dialysis patients have symptoms of congestive HF or clinical evidence of left ventricular
dysfunction (Adams et al., 2005; Stack & Bloembergen, 2001). Importantly, the presence of
the CRS is a strong adverse prognostic marker in patients with either primary cardiac
disease or primary renal disease.
While originally thought to reflect renal hypoperfusion secondary to low cardiac output, it
is now understood that the CRS is underpinned by far more complex processes. From a
hemodynamic standpoint, it seems likely that venous congestion is at least as important to
the pathophysiology of disease progression as is low forward flow. Other contributing
factors include activation of neurohormonal axes, including the sympathetic nervous system
and the renin-angiotensin-aldosterone system, as well as oxidative injury and endothelial
dysfunction (Bock & Gottlieb, 2010). More recently, it has become recognized that anemia
may also be intimately involved in the process, both as a consequence and as a causative
agent of the CRS. Finally, it is well recognized that many common risk factors for
cardiovascular disease and for chronic kidney disease (CKD) co-exist in these patient
cohorts.
Management of the CRS is challenging. Therapies for HF often cause worsening of renal
function, while treatment of renal failure commonly involves fluid administration, which
may precipitate disease decompensation among those with HF. Unfortunately, most large
randomized trials in the HF population have excluded patients with elevated serum
creatinine levels, and there is little evidence to guide therapy in this group of patients.
Observational studies suggest that there may be a mortality benefit associated with the use
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of standard HF medications, such as angiotensin converting enzyme inhibitors, angiotensin
receptor blockers and beta blockers in patients with HF and CKD, regardless of glomerular
filtration rate (GFR) (Berger et al., 2007; Cice et al., 2003).
Many novel therapies for HF have been introduced over recent years, several of which were
appealing for treatment of the CRS, given the pathophysiological processes towards which
they were directed. Unfortunately, natriuretic peptides, vasopressin antagonists, and
adenosine antagonists have all failed to show meaningful clinical benefits in patients with
HF (Hernandez, 2010; Konstam et al., 2007; Massie et al., 2010). Other approaches,
particularly peripheral ultrafiltration, have shown more promise in this patient population
(Costanzo et al., 2005).
2. Definitions and sub-types of the cardiorenal syndrome
Historically, the CRS is thought to have been due to impaired renal perfusion secondary to
low cardiac output states or the result of HF therapies negatively impacting renal function.
In 2004, the National Heart, Lung and Blood Institute defined CRS as a state in which
therapy to relieve heart failure symptoms is limited by further worsening in renal function
(National Heart, Lung and Blood Institute, 2004). By this paradigm, the heart was
considered to be the central driving force behind impaired renal function in patients with
HF.
Our understanding of the pathophysiology behind the CRS has evolved in the last number
of years and there is increasing recognition of the complexity of interactions which exist
between the heart and the kidneys, particularly when either or both organs are diseased.
This organ cross talk is bidirectional in nature and the resultant dialogue is dependent on
whether the heart or the kidney is the primary affected organ as well as the time course over
which the associated pathophysiological changes may occur.
It is within this context, that newer definitions for the CRS have been proposed which
recognize that either the heart or kidney may be the primary site of organ injury. A more
comprehensive definition and classification schema for the CRS has the advantage of
allowing clinicians to make a more accurate diagnosis which in turn informs our
understanding of a given patients natural history, prognosis and optimal treatment
strategy.
The definition and classification system for CRS introduced by Ronco and colleagues in 2008
(Ronco et al., 2008) is now widely considered to be the preferred mechanism for describing
patients and the pathophysiological processes associated with CRS. Ronco and colleagues
broadly define CRS as a pathophysiological disorder of the heart and kidneys whereby
acute or chronic dysfunction of one organ may induce acute or chronic dysfunction of the
other. Additionally, they characterize five sub-types of the CRS based on this definition.
These are described and discussed below. It should be noted that CRS types 1-5 may
frequently co-exist in a given patient, underscoring the complexity of interaction between
the heart and kidney and the importance of appointing chronology to these processes.
2.1 Cardiorenal syndrome type 1 (acute cardiorenal syndrome)
Type 1 CRS is distinguished by an acute deterioration in cardiac function or acute cardiac
injury, from any cause, that secondarily results in acute kidney injury (AKI).
Sub-Types and Therapeutic Management of the Cardiorenal Syndrome
125
Pathophysiologically, Type 1 CRS is characterized by decreased cardiac output with
impaired renal perfusion as well as elevated central venous pressures and acute renal
edema. Renal ischemia may be mediated by decreased oxygen delivery due to impaired
myocardial contractile performance, elevated interstitial pressures in the renal medulla and
by peripheral/systemic vasoconstriction which occurs as a compensatory mechanism in the
face of low cardiac output.
Historically, decreased forward cardiac flow was thought to be the primary determinant for
AKI in this context, however recent clinical trials have suggested this mechanism may not
be as important in the development of CRS Type I as previously hypothesized. Specifically,
data from ADHERE (Acute Decompensated Heart Failure National Registry) which
included over 100,000 patients admitted to hospital in the United States with acute
decompensated heart failure (ADHF) showed that <2% of patients had systemic
hypotension, a surrogate for low cardiac output, while the vast majority of patients had
symptoms/signs of volume overload (Adams et al., 2005). This is corroborated by the
findings of the ESCAPE (Evaluation Study of Congestive Heart Failure and Pulmonary
Artery Catheterization Effectiveness) Trial in which 433 patients admitted to hospital with
ADHF were randomized to pulmonary artery catheterization versus standard care to assess
the efficacy of tailored haemodynamic therapy (Binanay et al., 2005). In the ESCAPE Trial,
cardiac index was not associated with baseline renal function or deterioration in renal
function, however right atrial pressure was weakly correlated with baseline creatinine and
GFR (Nohria et al., 2008).
The impact of central venous pressures (CVP) on worsening renal function in the setting of
ADHF has been receiving greater attention in recent years. Elevated CVP is more predictive
of a decline in renal function than other relevant haemodynamic variables such as cardiac
index, blood pressure and pulmonary capillary wedge pressure (Mullens et al., 2009).
Moreover, elevated CVP predicts risk of re-hospitalization for HF and death suggesting that
it is a potent prognosticator for poor outcomes and a potential target for therapy (Uthoff et
al., 2011). Elevated intra-abdominal venous pressures have also been shown to have a
similar relationship with GFR at baseline and changes in GFR with therapy (Bock &
Gottlieb, 2010; S. E. Bradley & G. P. Bradley). This may be the result of a direct mechanical
effect on renal blood flow or simply a reflection of elevated CVP.
Among patients with ADHF, activation of the sympathetic nervous system (SNS) and the
renin-angiotensin-aldosterone system (RAAS) is a homeostatic mechanism intended to
maintain intraglomerular perfusion pressures and preserve GFR. Paradoxically however,
systemic vasoconstriction by these mechanisms increases cardiac afterload leading to further
decline in cardiac output and renal blood flow. Additionally, these neurohormones have a
maladaptive effect on the myocardium resulting in fibrosis and ventricular remodeling.
Treatment with -blockers is relatively contra-indicated in the face of an acute
decompensation due to their negative inotropic effects and the relative dependence of
cardiac output on heart rate in this patient population; therefore, SNS activation in CRS
Type 1 may remain unchecked leading to ischemia of both renal and cardiac tissue beds.
Acute administration of RAAS inhibition may exacerbate renal injury in CRS Type 1 by
reducing pressure in Bowmans capsule; this effect may be magnified in the presence of
volume shifts associated with diuretics, which remains the mainstay of therapy. Moreover,
diuretics may directly result in additional neurohormonal activation and there is now an
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increasing body of literature suggesting that diuretics, in and of themselves, may be
associated with worse outcomes in patients with ADHF independent of other relevant
clinical variables. In a single centre retrospective analysis of 1354 patients admitted with
ADHF, Eshaghian and colleagues (Eshaghian et al., 2006) demonstrated that patients
requiring the highest doses of diuretics, stratified by quartiles, had higher rates of sudden
death, death due to progressive pump failure and all cause mortality compared to patients
in the lowest quartile of diuretic dose. This type of observation has fueled a growing interest
in identifying alternate strategies for fluid management in the acute setting, independent of
diuretic administration (see section 3.8).
Of particular concern among patients who present with the features of CRS Type 1 is the
impact of diagnostic imaging and invasive cardiac procedures which may have an
additional and direct toxic effect on the kidneys through a variety of mechanisms.
Individuals who present with an acute deterioration in cardiac function will frequently
require imaging or investigation to identify a precipitant or cause for their symptoms.
Independently, percutaneous interventions and cardiac surgery impart a risk of AKI which
is higher in patients who have pre-existing or concomitant acute renal insufficiency
(Anderson et al., 1999; Best et al., 2002).
Upwards of 70% of patients admitted to hospital with ADHF will experience a rise in serum
creatinine over the course of their admission (Gottleib et al., 2002); this may be the result of
therapies administered, either medical or invasive, or a consequence of the various
pathophysiological processes which characterize CRS Type 1. Regardless of mechanism,
worsening renal function portends a poor prognosis and is associated with higher mortality
rates. (Gottleib et al., 2002; Damman et al., 2007).
2.2 Cardiorenal syndrome type 2 (chronic cardiorenal syndrome)
Chronic HF leading to chronic kidney disease is the hallmark of CRS Type 2. The prevalence
of CKD in HF cohorts has been variably reported depending on the patient population
examined e.g. hospitalized versus ambulatory patients. Further complicating our
understanding of disease prevalence is the fact that early clinical trials of chronic HF
excluded patients with established renal insufficiency and most did not determine
glomerular filtration rate (GFR) which is of particular clinical importance given that HF is a
disease of the elderly.
For example, the SOLVD (Studies of Left Ventricular Dysfunction) trials examined the impact
of the angiotensin converting enzyme (ACE) inhibitor Enalapril on mortality and symptom
development in patients with left ventricular dysfunction (The SOLVD Investigators, 1991; The
SOLVD Investigators, 1992). While those with serum creatinine levels >2.0 mg/dL were
excluded from the original trial, a retrospective analysis of study patients revealed at least
moderate renal impairment (GFR < 60 ml/min) was present in 26% and 56% of participants in
the prevention and treatment arms of the trial, respectively (Dries et al., 2000). Across the
series of trials which composed the CHARM (The Candesartan in Heart Failure: Assessment of
Reduction in Mortality and Morbidity) Program, moderate renal impairment was detected in
36% of the 2680 study participants at baseline (Hillege et al., 2006).
Determining the prevalence of pre-existing CKD is particularly challenging among
hospitalized HF patients. Some clinicians may attribute AKI at the time of HF
Sub-Types and Therapeutic Management of the Cardiorenal Syndrome
127
hospitalization solely to CRS Type 1 thereby underestimating the presence of concomitant
CRS Type 2 in this cohort of patients. Novel biomarkers of AKI may help clinicians to
decipher the relative contributions of CRS Type 1 versus CRS Type 2 in patients hospitalized
for HF who have poor renal function upon presentation (Siew et al., 2011; Coca et al., 2008).
Regardless of cause, renal insufficiency in hospitalized HF patients appears to be relatively
common; among those enrolled in ADHERE, the prevalence of at least moderate renal
impairment, as determined by GFR, was greater than 60% at baseline (Heywood et al., 2007).
This is in sharp contrast to initial reports from the same registry which suggested a
prevalence rate of only 20% when a serum creatinine of 2.0 mg/dL was employed as a cut
off (Adams et al., 2005). Calculation of GFR, therefore, is paramount to accurately
identifying the burden of renal disease in all forms of CRS.
The true burden of pre-existing renal dysfunction among patients with HF was best
characterized in a meta-analysis performed by Smith and colleagues. In their systematic
review of the literature, approximately 80,000 hospitalized and non-hospitalized patients
with HF were identified across 16 clinical trials. While 29% of patients were found to have
moderate to severe renal impairment (GFR <53 mL/min or cystatin C of >1.56 mg/dL), 63%
were found to have at least some degree of impaired kidney function. Moreover, these
findings are likely to underestimate the true prevalence of renal insufficiency in HF
populations given that 8 of the clinical trials included in the meta-analysis excluded patients
on the basis of age or an elevated serum creatinine at baseline (Smith et al., 2006).
In the meta-analysis performed by Smith and colleagues, renal impairment at baseline
conferred an increased risk of mortality at one year follow-up compared to patients with
normal kidney function (Smith et al., 2006). The adjusted hazard ratio for patients with any
renal impairment or moderate to severe renal impairment was 1.56 and 2.31 respectively.
Excess risk was conferred in an incremental fashion with each 10 mL/min reduction in GFR
correlating to a 7% increase in the risk of death. This observation is strengthened by similar
findings across a spectrum of clinical trials in both hospitalized and ambulatory HF
populations (Adams et al., 2005; Dries et al., 2000; Fonarow et al., 2005; Heywood et al., 2007;
Hillege et al., 2006).
Many of the pathophysiological mechanisms which characterize CRS Type 1 are also
implicated in the development of CRS Type 2, although many of these processes may occur
slowly and over longer periods of time. For example, elevated central venous pressure is
strongly associated with a decline in eGFR among patients with chronic HF (Damman et al.,
2009; Firth et al., 1988); as described above, the same is true for patients with ADHF and
CRS Type 1. Elevated CVP and secondarily an elevation in renal venous pressure may
trigger a number of downstream events, including interstitial ischemia, neurohormonal
activation and decreased responsiveness to natriuretic peptides which all combine to reduce
GFR directly or indirectly (Damman et al., 2007; Bock & Gottlieb, 2010) in the setting of
chronic HF. Chronically low cardiac output, particularly in combination with micro and
macrovascular renal disease, may also contribute to fibrosis and structural changes in the
kidney which result in impaired renal function.
RAAS activation occurs in both HF and CKD with an associated increase in Angiotensin II
levels (AII). AII mediates oxidative injury and endothelial dysfunction through both the
formation of reactive oxygen species and a decrease in nitric oxide bioavailability. Each of
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128
these processes, in turn, can result in haemodynamic abnormalities at the level of the heart
and kidney contributing to a decline in GFR (Bock & Gottlieb, 2010).
While neurohormonal inhibition and diuretic therapy are the mainstay of pharmacological
HF management, these agents are also implicated in the worsening of GFR associated with
CRS Type 2. ACE inhibitors and angiotensin receptor blockers (ARBs) result in systemic
hypotension as well as efferent arteriolar vasodilatation with an associated decline in
intraglomerular pressure and GFR. These effects may be magnified in the presence of
concomitant diuretic use and relative intra-vascular volume depletion. The treatment of CRS
is discussed in detail below.
The presence of anemia is common in patients with HF, an observation which is consistent
across a number of clinical trials in the HF arena. A review of the literature suggests a
prevalence rate of between 9-25% depending on the HF patient population studied and the
cut-off criteria used to diagnose anemia (Virani et al., 2008; Al-Ahmad et al., 2001; Sharma et
al., 2004; Anand et al., 2005; Horwich et al., 2002). Regrettably, many of these studies
excluded patients based on renal function and therefore the relative contribution of low GFR
to the development of anemia in these patient cohorts is lacking. Anemia in the presence of
HF portends a poor prognosis with absolute haemoglobin (Hgb) levels correlating with 1
year survival; a precipitous increase in mortality is observed when Hgb drops below 120
g/L (Horwich et al., 2002; Ezekowitz et al., 2003).
The development of anemia in CRS Type 2 is likely multifactorial and underpinned by a
number of processes occurring simultaneously; malnutrition, the formation of reactive
oxygen species, cytokine release and erythropoietin (EPO) deficiency/resistance have all
been implicated. When present, anemia may lead to further cardiac and renal dysfunction
through impaired oxygen delivery and tissue hypoxia, neurohormonal activation, decreased
renal blood flow and expansion of plasma volume with resultant cardiac remodeling
(McCullough & Lepor, 2005). These mechanisms establish and propagate a vicious cycle of
maladaptive processes which lead to worsening anemia, HF and kidney function as a net
result.
2.3 Cardiorenal syndrome type 3 (acute renocardiac syndrome)
The RIFLE Criteria define acute kidney injury as a twofold increase in serum creatinine or a
GFR decrease by 50 percent or urine output of <0.5 mL/kg per hour for 12 hours (Bellomo et
al., 2004). By this definition, AKI is prevalent in nearly 9% of hospitalized patients (Uchino
et al., 2006) with an associated 4-fold increased risk of mortality compared to patients
without evidence of renal injury (Ricci et al., 2008). Much of that excess risk may be
attributable to cardiac sequelae of AKI. CRS Type 3 characterizes this interaction and may be
defined broadly as primary acute kidney injury, due to any number of causes, which
secondarily leads to acute cardiac dysfunction.
A number of pathophysiological processes may be initiated as a consequence of AKI which
have significant downstream cardiac effects. Biochemical abnormalities including
hyperkalemia may pre-dispose to malignant cardiac arrhythmias and an increased risk of
sudden cardiac death. Acidemia and uremia have direct myocardial depressant effects and
may precipitate acute biventricular cardiomyopathy; these effects are exacerbated in the face
of volume expansion.
Sub-Types and Therapeutic Management of the Cardiorenal Syndrome
129
Volume overload due to impaired solute and fluid clearance may also result in hypertension
and pulmonary edema. Moreover, the resultant elevations in intra-cardiac filling pressures
reduce the transmyocardial perfusion gradient during diastole leading to sub-endocardial
ischemia and overall worsening of ventricular function. Release of pro-inflammatory
cytokines and reactive oxygen species in response to renal injury may result in endothelial
dysfunction in addition to having direct toxic effects on the myocardium with resultant
apoptosis and myocardial fibrosis.
Activation of the SNS and RAAS as a result of AKI may also lead to deleterious
haemodynamic consequences including increased systemic vascular resistance and
increased myocardial oxygen consumption, both of which lead to decreased cardiac output.
While AII also causes left ventricular hypertrophy, ventricular remodeling and accelerates
the development of atherosclerosis, these effects are likely of greater relevance in the setting
of Chronic Renocardiac Syndrome (CRS Type 4).
2.4 Cardiorenal syndrome type 4 (chronic renocardiac syndrome)
CRS Type 4 describes a clinical scenario where primary CKD leads to structural and/or
functional cardiac abnormalities which may be associated with clinically significant adverse
cardiac events. Indeed, the presence of CKD portends a poor cardiac prognosis with the
attributable risk of adverse events correlating in a step-wise manner to reduction in GFR (Go
et al., 2004). Moreover, individuals with CKD have an accelerated natural history of their
cardiac disease and are more likely to die from cardiac causes rather than progress to renal
replacement therapy (Collins et al., 2008; Foley et al., 2005; Keith et al., 2004).
For example, in ALLHAT (The Antihypertensive and Lipid-Lowering Treatment to Prevent
Heart Attack Trial) the risk of myocardial infarction (MI)/stroke, revascularization, death
due to coronary disease and all forms of atherosclerotic vascular disease was increased as
GFR decreased (Wali & Henrich, 2005). Among patients with CKD who experience an acute
coronary syndrome, prognosis may also be stratified according to GFR. Shlipak and
colleagues reviewed approximately 130,000 elderly patients hospitalized with an acute
coronary syndrome and found a 2.5 fold increased risk of death between patients in the
highest (CrCl > 0.92 mL/sec) and lowest (CrCl 0.17-0.54 mL/sec) tertile of creatinine
clearance (Shlipak et al., 2002). Moreover, an analysis of nearly 120,000 patients from the
Cooperative Cardiovascular Project suggested that renal function was a more accurate
predictor of long term mortality post-MI than left ventricular systolic function, the presence
of heart failure or prior MI (Smith et al., 2008). This relationship has been demonstrated in a
multitude of clinical trials across a variety of cardiac cohorts and the observation between
CKD and poor cardiac outcomes remains robust (Ronco et al., 2008).
There are many postulates as to the mechanisms underlying poor cardiac outcomes in
patients with chronic renal dysfunction. It would appear that the burden of coronary artery
disease and myocardial ischemia is greater in patients with CKD than those without (Ix et
al., 2003). This may be due to a higher preponderance of traditional risk factors for coronary
artery disease in this patient population (Muntner et al., 2005; Parikh et al., 2006) or simply
that CKD, in and of itself, imparts increased risk of adverse cardiac events (Levey et al.,
2003). In the Framingham Offspring Cohort, two or more traditional cardiovascular risk
factors were identified in 73% of patients with CKD (GFR <60 mL/min) compared to 51.4 %
Chronic Kidney Disease
130
of participants without CKD. A statistically significant increase in hypertension and diabetes
along with a trend towards increased dyslipidemia were more prevalent in the CKD cohort
(Parikh et al., 2006). Existing data would suggest that CKD is independently associated with
a higher risk for cardiovascular endpoints in affected patients; the magnitude of this excess
risk, however, does not support elevating CKD to the level of a cardiovascular disease
equivalent as is the case with diabetes or prior MI (Wattanakit et al., 2006).
Other potential pathophysiological processes involved in the development and acceleration
of coronary atherosclerosis in patients with CKD include abnormalities of mineral
metabolism leading to vascular calcification and endothelial dysfunction secondary to both
chronic inflammation and EPO deficiency. Uremia, hypertension and increased vascular
stiffness contribute to progressive left ventricular hypertrophy and diastolic dysfunction,
which in time may progress to systolic dysfunction. Neurohormonal activation results in
myocardial fibrosis and maladaptive ventricular remodelling which may hasten this
process. In the presence of volume expansion, patients with either systolic or diastolic
dysfunction remain at high risk for developing decompensated heart failure.
Observational trials very clearly demonstrate that those with CKD, as a result of actual or
perceived contraindications, are less likely to receive efficacious and evidence based
therapies compared to cohorts of patients with normal renal function (Al-Suwaidi et al.,
2002; Parikh et al., 2006). An even more important observation is that those patients with
CKD who do receive appropriate guideline based interventions have better outcomes
(Shlipak et al., 2002); therapeutic prejudice of healthcare teams and providers in relation to
patients with renal dysfunction is most certainly misplaced, particularly since this group of
patients have a high burden of disease and therefore may receive the greatest degree of
benefit from aggressive intervention.
2.5 Cardiorenal syndrome type 5 (secondary cardiorenal syndrome)
Secondary cardiorenal syndrome is the result of a systemic disorder leading to simultaneous
cardiac and renal injury; each of these processes may be acute or chronic in nature and CRS
Type 5 does not preclude involvement of other organs and tissue beds. Moreover, other sub-
types of the CRS may exist concomitantly due to pre-existing co-morbidities.
The prevalence of CRS Type 5 overall has not been well described, primarily due to a
paucity of data in this arena, however the frequency of cardiac and renal involvement for
specific systemic disease states may be described in the literature. For example, myocardial
injury in the absence of an acute coronary syndrome, as manifested by a positive troponin
assay, is present in up to one-half of patients with sepsis admitted to a critical care unit
(Amman et al., 2003). Similarly, AKI may occur in 70% of this patient population (Kim et al.,
2011). Dysfunction of either or both organ systems portends a poor prognosis.
Connective tissue disease, sarcoidosis, amyloidosis, diabetes and sepsis are the most
commonly referred to systemic process that may predispose to secondary CRS (Ronco et al.,
2008). While a discussion of cardiac and renal involvement in each of these disease states is
beyond the scope of this chapter, it is clear that definitive treatment must be focused at
correcting the underlying pathophysiological process while providing supportive care for
the heart and kidneys in the interim.
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131
3. Management of the cardiorenal syndrome
Management of the CRS presents a challenge to the clinician. Treatment of HF with
standard therapies often results in worsening of renal function. Moreover, most randomized
clinical trials of HF therapies, including -blockers, ACE inhibitors, ARBs and aldosterone
antagonists, have excluded patients with significant renal dysfunction. Therefore, the results
of these trials, most showing significant reductions in morbidity and mortality in the general
HF population, may not be applicable to the CRS population. Observational studies and
small randomized studies, however, have suggested that many of these drug classes may
have similar benefit in patients with renal dysfunction (Berger et al., 2007; Cice et al., 2003).
A number of novel strategies have been described that may offer specific benefit in the CRS
population, although data from clinical trials have not always been encouraging.
Management of chronic CRS is overall similar to the management of HF in general,
employing a combination of diuretics, inhibitors of the RAAS, and -blockers. In the
hospitalized patient with CRS and ADHF, diuretics remain a mainstay of therapy, but may
be supplemented by additional therapies including novel pharmacologic agents, inotropic
support, and ultrafiltration.
3.1 Diuretics
While fluid removal with diuretics is a cornerstone of HF management, diuretic resistance is
highly prevalent in patients with decreased renal function, making this aspect of care for the
patient with CRS particularly challenging. Furthermore, effective diuresis can result in
further deterioration in renal function, particularly when the rate of fluid removal exceeds
the rate of fluid movement from the extravascular space to the intravascular space, resulting
in low effective circulating volume. Thus, two of the greatest obstacles in treating patients
with CRS are overcoming diuretic resistance and effectively removing fluid without
compromising renal function.
Loop diuretics (LD) such as furosemide act at the thick ascending limb of the loop of Henle,
inhibiting the Na
+
/K
+
/2Cl
-
cotransporter. LD are protein bound, preventing filtration at the
glomerulus, but are actively secreted in the proximal tubule. Effective delivery to the loop of
Henle requires effective delivery to the bloodstream (through intestinal absorption or direct
intravenous administration), adequate renal blood flow, intact proximal tubule secretion,
and delivery of tubular contents to the more distal nephron. There are therefore a number of
mechanisms by which diuretic resistance may occur (Jentzer et al., 2010).
Delayed intestinal absorption is common in patients with HF, owing to intestinal wall
edema. This can be most effectively overcome by using intravenous LD in patients who are
markedly volume overloaded, and transitioning to oral administration once signs of
congestion elsewhere (i.e. peripheral edema, venous congestion on chest X-ray) have
resolved. Reduced renal blood flow (RBF) and GFR are also prevalent in patients with HF
and CRS as a result of intrinsic renal dysfunction, decreased cardiac output, and alteration
in glomerular haemodynamics by agents such as non-steroidal anti-inflammatory drugs
(NSAIDs), ACE inhibitors, and ARBs. Avoiding agents such as NSAIDs, optimizing
systemic hemodynamics, and increasing LD dose can help to overcome this aspect of
resistance to LD. Similarly, proximal tubular secretion of LD is reduced in patients with CRS
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because organic acids that accumulate in the uremic state compete for the same transporters;
increased doses of LD may be required to overcome this problem.
Through intravascular volume depletion, LD may result in activation of the RAAS. This
leads to increased sodium absorption by the proximal and distal tubule. This issue is
compounded by the fact that post-diuretic rebound sodium avidity occurs between bolus
doses of LD, negating much of the natriuretic benefit achieved. Strict dietary sodium
restriction and administration of RAAS antagonists (i.e. ACE inhibitors and ARBs) may help
to prevent this. Historically, it has been believed that continuous infusions of diuretics may
also be effective in minimizing rebound sodium absorption; the recent DOSE (Diuretic
Strategies in Patients with Acute Decompensated Heart Failure) trial suggests that there
may be no difference in diuretic efficacy between intermittent intravenous bolus dosing and
continuous infusions (Felker et al., 2011).
The braking phenomenon is a short-term effect, whereby the nephron becomes less
sensitive to LD after an initial dose. This is thought to result from upregulation of the
Na
+
/K
+
/2Cl
-
cotransporter in the thick ascending loop of Henle. Higher doses of LD may be
necessary to overcome this. With chronic LD administration, distal tubule hypertrophy
occurs. This allows increased distal sodium reabsorption, tending to negate the inhibition of
sodium reabsorption that has occurred in the loop of Henle.
A strategy of combination diuretic administration, with the addition of a thiazide diuretic
such as metolazone 5-10 mg 30 minutes prior to LD administration can help to prevent
sodium retention by this mechanism. Thiazides inhibit the NaCl cotransporter in the distal
convoluted tubule. Caution is needed, however, as combination diuretic therapy can result
in profound electrolyte abnormalities. Serum levels of potassium and magnesium must be
closely monitored and infrequent metolazone dosing (i.e. three times per week) or co-
administration of a potassium-sparing diuretic may be necessary to prevent life-threatening
hypokalemia.
Finally, sodium and water retention may be upregulated in the distal nephron in patients
with CRS, mediated by elevated levels of aldosterone and vasopressin, respectively.
Administration of aldosterone antagonists or other potassium-sparing diuretics will
minimize sodium retention in this situation; the new vasopressin antagonists have a role in
preventing excessive absorption of free water (see section 3.6). Free water restriction may
also be necessary in patients with refractory fluid overload or significant hyponatremia. An
important caveat to the use of aldosterone antagonists in CRS is the risk of hyperkalemia in
patients with renal impairment; these agents should generally be avoided in patients with
GFR <30 mL/min.
Major drawbacks to the use of LD include neurohormonal activation, ototoxicity, electrolyte
abnormalities (particularly hypokalemia and hypomagnesemia), dysrhythmias, and
intravascular volume depletion with resultant worsening renal function and/or
hypotension in patients who are preload-dependent or receiving concomitant vasodilator
therapy.
A novel approach to diuretic use involves the co-administration of loop diuretics and
hypertonic saline solution (HSS). Small studies in patients with ADHF have demonstrated
that, compared to intravenous bolus loop diuretics with a low sodium diet, administration
Sub-Types and Therapeutic Management of the Cardiorenal Syndrome
133
of intermittent boluses of HSS with loop diuretics and moderate dietary sodium restriction
resulted in more rapid diuresis, normalization of neurohormonal activity, shorter
hospitalizations, and less renal dysfunction (Licata et al., 2003; Paterna et al., 2000). After
discharge, these results were maintained by continuing moderate sodium restriction (<2.8
g/day) with strict fluid restriction (<1 L/day), resulting in fewer readmissions and
improved survival compared to continued strict sodium (<2 g/day) and similar fluid
restriction. The mechanism by which HSS provides these benefits is unclear, but may be
related to the osmotic load drawing interstitial fluid into the intravascular space, leading to
neurohormonal blockade, reduced vascular resistance, improved cardiac output, and
reduced interstitial edema. In addition, the sodium load in the kidney may induce a sort of
transient diabetes insipidus, resulting in rapid diuresis (Di Pasquale et al., 2007). Further
research and larger scale studies are required to confirm the benefits of HSS in patients with
CRS.
3.2 Renin-angiotensin-aldosterone system antagonists
Inhibitors of the renin-angiotensin system, including ACE inhibitors and ARBs have proven
survival benefit in patients with left ventricular dysfunction (The SOLVD Investigators,
1991; The SOLVD Investigators, 1992), and have also been shown to slow the rate of decline
of renal function in patients with diabetic chronic kidney disease (Lewis et al., 1993). It
stands to reason, therefore, that these agents would be beneficial in the CRS, although large-
scale clinical trials in the HF population have typically excluded patients with significant
renal dysfunction.
The CHARM studies investigated the effects of candesartan compared with placebo in a
broad population of patients with HF. Patients with serum creatinine >3.0 mg/dL were
excluded, but among the study population, there was no statistically significant interaction
between eGFR and treatment effect, suggesting a mortality benefit of ARBs in patients with
HF and mild-to-moderate renal dysfunction that is equivalent to that seen in patients with
HF and preserved renal function (Hillege et al., 2006). An analysis of CONSENSUS
(Cooperative North Scandinavian Enalapril Survival Study) which demonstrated a mortality
benefit of enalapril compared to placebo in patients with HF, found a greater benefit in
patients with baseline serum creatinine above the median (123 umol/L) than in those with
serum creatinine below the median (Swedberg et al., 1990). A retrospective analysis of the
Minnesota Heart Survey stratified 4573 patients hospitalized with HF by GFR, and revealed
that patients at all stages of CKD had reduced in-hospital mortality when an ACE inhibitor
or ARB was used in hospital, and reduced one-year mortality when discharged on an ACE
inhibitor or ARB (Berger et al., 2007). This same analysis, however, demonstrated that
patients with severe renal dysfunction were far less likely to receive either agent than those
with normal renal function.
In HF, elevated angiotensin II levels cause efferent arteriolar vasoconstriction, elevating
glomerular filtration pressure and preserving GFR. Inhibition of this process with ACE
inhibitors or ARBs may result in an initial decline in GFR, but in the long term protects the
glomerulus from high filtration pressures and may help to preserve long-term renal
function (Heywood, 2004). Although there appear to be benefits of using these agents in the
CRS population, caution must be taken when initiating ACE inhibitors and ARBs in patients
with renal dysfunction, particularly with regard to volume status and avoidance of NSAIDs.
Chronic Kidney Disease
134
Volume depletion increases the risk of significant renal dysfunction associated with ACE
inhibitors and ARBs. Increases in creatinine of up to 30% are acceptable, and may identify a
group of patients most likely to benefit from angiotensin inhibition (Koniari et al., 2010). HF
patients who are unable to tolerate ACE inhibitor therapy because of hypotension, renal
dysfunction, or hyperkalemia have a particularly high one-year mortality rate, in excess of
50% (Kittleson et al., 2003).
3.3 -adrenergic receptor blockers
-blockers are considered standard therapy in patients with HF and systolic dysfunction.
They exert a number of beneficial effects, including prevention of ventricular arrhythmias,
prevention of ventricular remodeling, reduction in myocardial oxygen demand, increased
myocardial oxygen supply, and inhibition of other deleterious neurohormonal pathways.
Their significant mortality benefit in patients with HF is well established through large
clinical trials. Unfortunately, the majority of these studies excluded patients with significant
renal dysfunction, but retrospective analyses of trials data have offered insight into the
benefits in patients with mild-to-moderate renal impairment. COPERNICUS (Carvedilol
Prospective Randomized Cumulative Survival Study), for example, demonstrated a 35%
reduction in the risk of death in patients with severe HF treated with carvedilol compared to
placebo, but excluded patients with a serum creatinine greater than 2.8 mg/dL. Similarly,
the CAPRICORN (Carvedilol Post-Infarct Survival Control in Left Ventricular Dysfunction)
trial showed a 23% reduction in all-cause mortality in patients with EF 40% after
myocardial infarction treated with carvedilol compared with placebo, but excluded patients
with significant renal impairment (Dargie, 2001). A post-hoc analysis of individual patient
data from these two trials, however, demonstrated that in patients with HF and mild-to-
moderate CKD, carvedilol was safe and efficacious, associated with reductions in all-cause
mortality, cardiovascular mortality, and HF hospitalization (Wali et al., 2011). CIBIS-II (The
Cardiac Insufficiency Bisoprolol Study II) demonstrated a 34% reduction in mortality in
patients with HF treated with bisoprolol compared to placebo, and excluded patients with
serum creatinine 300 umol/L (3.4 mg/dL) (CIBIS-II Investigators and Committees, 1999). A
post-hoc analysis of this trial showed that although patients with GFR <60 mL/min had
higher overall mortality than those with GFR 60 mL/min, the benefit of bisoprolol was
similar in both groups (Erdmann et al., 2001). The relative risk of mortality in the group with
GFR <60 mL/min treated with bisoprolol compared to placebo was 0.66, and there was a
non-significant trend towards an even greater benefit in the small number of patients with
GFR <30 mL/min.
An analysis of MADIT-II (Multicenter Automatic Defibrillator Implantation Trial II), which
demonstrated a 31% reduction in the risk of all-cause mortality with the addition of an
implantable cardioverter-defibrillator to medical therapy in patients with ischemic
cardiomyopathy and EF 30%, examined the predictors of sudden cardiac death (SCD) in
the subset of patients in the medical arm of the study with impaired renal function, defined
as GFR 75 mL/min. -blocker therapy was a negative predictor of SCD, with a hazard ratio
of 0.61 (Chonchol et al., 2007).
Smaller studies have examined the benefits of -blocker therapy in patients with end-stage
renal failure. In a non-randomized study of 134 patients with HF and either chronic renal
impairment, anemia, or both, treatment with -blockers for 12 months was associated with
Sub-Types and Therapeutic Management of the Cardiorenal Syndrome
135
improvement in both creatinine clearance and hemoglobin levels, while those patients who
did not receive -blockers had worsening renal function and anemia over the same time
period (Khan et al., 2006). In patients with HF and normal renal function at baseline, lack of
treatment with a -blocker was associated with increased risk of developing renal failure
over 20 years of follow-up (Tanaka et al., 2007). In hemodialysis patients with dilated left
ventricles, treatment with metoprolol resulted in reduced ventricular dimensions, increased
fractional shortening, and reduced levels of natriuretic peptides (Hara et al., 2001). A
randomized trial of 114 hemodialysis patients with dilated cardiomyopathy showed that
carvedilol, compared to placebo, was associated with improved ejection fraction, improved
survival, and fewer HF hospitalizations (Cice et al., 2003). Although large-scale clinical trials
in this population are lacking, the weight of evidence suggests that treatment with -
blockers in the CRS population is likely to be associated with reductions in mortality and
morbidity.
3.4 Inotropic agents
Inotropic medications such as dobutamine and milrinone are frequently used in patients
with ADHF, particularly in the setting of the CRS where low cardiac output is felt to be a
major contributor to rapidly declining renal function. Both agents are vasodilating
inotropes, but they have different mechanisms of action. Dobutamine is an adrenergic
agonist that affects inotropy and chronotropy via -1 activity and peripheral vasodilation
via -2 activity. Milrinone is an inhibitor of type III phosphodiesterase and results in
increased intracellular cyclic adenosine monophosphate (cAMP). This, in turn, results in
increased inotropy (without chronotropy) as well as peripheral vasodilation. Although both
agents have attractive hemodynamic profiles in the treatment of CRS, evidence suggests that
they should not be part of standard therapy in this condition. OPTIME-CHF (Outcomes of a
Prospective Trial of Intravenous Milrinone for Exacerbations of Chronic Heart Failure)
compared intravenous milrinone to placebo in patients with ADHF not requiring inotropic
therapy for shock or other indications. There was no difference between the two groups in
the primary endpoint of total number of days in hospital by 60 days after randomization.
There was also no difference in the rate of progression of HF, but the patients treated with
milrinone had higher rates of treatment failure, largely driven by higher rates of
hypotension and atrial arrhythmias.
The ADHERE registry compared outcomes of patients with ADHF treated with vasodilating
medications (nitroglycerin, nesiritide) and inotropic agents (dobutamine, milrinone). Even
after adjustment for baseline variables including age, gender, blood pressure, BUN,
creatinine, sodium, heart rate, and symptom severity, odds ratios for mortality between
individual inotropes and individual vasodilators ranged from 1.45 to 2.17. Inotropic agents,
therefore, are recommended by major society guidelines only for short-term use in patients
with cardiogenic shock or refractory volume overload with diuretic resistance, and not
recommended for routine use in hospitalized patients with ADHF. In addition, patients
receiving these agents must be carefully monitored for hypotension and arrhythmias, and it
should be recognized that the use of these agents is associated with a worse prognosis.
Dopamine is an endogenous catecholamine that binds dopamine receptors (D1-D5) as well
as and adrenergic receptors with varying affinity depending on the dose administered.
At low doses (2-5 mcg/kg/min), it primarily binds dopaminergic receptors and causes
Chronic Kidney Disease
136
vasodilation of renal, splanchnic, cerebral, and coronary vessels. At higher doses,
adrenergic effects dominate, resulting in positive inotropy and chronotropy as well as
adrenergic-mediated vasodilation, with progressively increasing adrenergic activity at still
higher doses resulting in vasoconstriction.
For many years the use of renal-dose dopamine was advocated in acute renal failure, the
rationale being that dopamine in doses up to 5 mcg/kg/min in animals and healthy
volunteers resulted in increased renal blood flow and natriuresis via selective dopamine
receptor binding. In recent years this approach has fallen out of favor, as multiple
retrospective and small prospective studies failed to convincingly demonstrate any benefit
in terms of renal function or survival. A meta-analysis of 61 trials comparing low-dose
dopamine to placebo or no treatment found that dopamine was associated with a 24%
increase in urine output on day 1 but was not associated with reductions in mortality, need
for renal replacement therapy, or adverse events (Friedrich et al., 2005). Only one of the 61
studies included patients with HF, and this study did not assess mortality; only three of the
studies included patients who were receiving diuretics. More recently, data from the DAD-
HF (Dopamine in Acute Decompensated Heart Failure) trial has been presented, comparing
low-dose dopamine plus low-dose furosemide to high-dose furosemide alone in patients
with ADHF. The two regimens were not associated with statistically significant rates of
diuresis, but the patients receiving dopamine plus low-dose furosemide were less likely to
develop worsening renal function (36% and 4% of patients in dopamine/furosemide and
furosemide only groups, respectively, had >25% increase in serum creatinine). As more data
become available regarding outcomes with low-dose dopamine in this specific population,
renal-dose dopamine may turn out to be useful after all.
3.5 Vasodilators
Nesiritide, a synthetic B-type natriuretic peptide (BNP), has been used in the management of
ADHF, particularly in patients at risk for worsening renal function with standard therapies.
Like naturally occurring BNP, released from ventricular myocardium under conditions of
increased wall stress, nesiritide is a vasodilator, causing both arterial and venous dilatation
as well as mild diuresis. Its rapid onset of action, apparent neurohormonal benefits, and lack
of need for invasive hemodynamic monitoring led to much initial enthusiasm for its use in
ADHF, as well as FDA approval for this indication (Publication Committee for the VMAC
Investigators, 2002). Use of this agent took a sharp decline, however, after meta-analyses
suggested increased 30-day mortality and increased risk of renal failure with nesiritide
(Hauptman et al., 2006; Sackner-Bernstein et al., 2005a; 2005b). The definitive randomized
clinical trial, ASCEND-HF (Acute Study of Clinical Effectiveness of Nesiritide in
Decompensated Heart Failure), recently demonstrated that while nesiritide is safe with no
increased risk of 30-day death or hospitalization or increased risk of renal failure, it offers no
significant clinical benefit when added to standard therapy in patients with ADHF
(Hernandez, 2010).
3.6 Vasopressin antagonists
Arginine vasopressin (AVP), a nonapeptide synthesized by the hypothalamus and released
by the posterior pituitary gland in response to increased plasma osmolality or decreased
plasma volume, binds to 3 distinct receptor subtypes (V1a, V1b, and V2). V1 receptors
Sub-Types and Therapeutic Management of the Cardiorenal Syndrome
137
mediate cardiac myocyte hypertrophy, vasoconstriction, and platelet aggregation. When
AVP binds V2 receptors expressed in the renal collecting duct, the short-term result is
increased translocation of vesicles containing aquaporin-2 (AQP2) water channels to the
apical membrane of principal cells; in the long-term, AVP-V2 receptor binding results in the
up-regulation of AQP2 protein expression. AQP2 mediates water transport across the apical
membrane of the principal cell, resulting in urinary concentration and increased solute-free
water retention (Schrier et al., 2009). AVP also stimulates urea reabsorption, resulting in an
augmented medullary concentrating gradient and increased levels of blood urea nitrogen
(Sands, 2003).
In HF and CRS, low cardiac output causes nonosmotic AVP release, leading to
inappropriate water retention. Low serum sodium and elevated blood urea nitrogen are
strong predictors of mortality in HF, and both are mediated, at least in part, by AVP activity
in the kidney. Augmentation of cardiac output with vasodilator medications is associated
with reductions in plasma AVP (Bichet et al., 1986). Early studies demonstrated effective
water removal without worsening renal function (Gheorghiade et al., 2007). Thus, the use of
agents that interfere with AVP-mediated water retention has been an attractive concept in
CRS. The SALT-1 and SALT-2 trials showed that tolvaptan, a selective oral V2 receptor
antagonist, caused increases in serum sodium levels in patients with HF, cirrhosis, and the
syndrome of inappropriate antidiuretic hormone (Schrier et al., 2006). Unfortunately, the
randomized EVEREST (Efficacy of Vasopressin Antagonism in Heart Failure Outcome
Study with Tolvaptan) trial subsequently failed to demonstrate a mortality benefit or
reduction in HF morbidity in patients hospitalized with HF treated with tolvaptan, despite
sustained reductions in body weight with preserved renal function (Konstam et al., 2007). It
seems, therefore, that vasopressin antagonists have little role in influencing clinical
outcomes in patients hospitalized with HF and the CRS, although they may be useful in
patients with hyponatremia that is difficult to manage with standard therapies. Additional
studies are needed to further define the role of tolvaptan and other vasopressin antagonists
in the outpatient setting.
3.7 Adenosine antagonists
Adenosine is a purine nucleoside breakdown product of adenosine triphosphate. It interacts
with four main receptor subtypes: A1, A2a, A2b, and A3. With the exception of coronary
vasodilatation and increased renal medullary blood flow, its cardiovascular and renal effects
are largely mediated via the A1 receptor. Binding of adenosine to A1 receptors in the heart
results in slowing of the heart rate and decreased atrial contraction. In the kidney, adenosine
is released from the macula densa in response to sodium delivery to the distal nephron via
tubuloglomerular feedback (TGF). Adenosine released through TGF acts on local A1
receptors, causing afferent arteriolar vasoconstriction and reduction in GFR as well as
increased proximal tubular sodium reabsorption. Blockade of these receptors should,
therefore, result in improved renal blood flow and GFR and decreased sodium and water
reabsorption.
In the setting of CRS, loop diuretics cause increased sodium delivery to the distal tubule,
making the role of adenosine particularly relevant in this population. Animal studies
showed that rolofylline, a selective A1 receptor antagonist, caused increased urine flow and
urinary sodium excretion without increasing potassium excretion and without affecting
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138
either blood pressure or renal function, and protected against nephrotoxic medication-
induced acute renal failure (Nagashima & Karasawa, 1996; Yao et al., 1994). A small clinical
study supported this, demonstrating that the addition of rolofylline to diuretics in patients
with volume overload and renal impairment resulted in an improvement in renal function
and increased diuresis with reduced diuretic requirements (Givertz et al., 2007).
Unfortunately, the PROTECT (Placebo-Controlled Randomized Study of the Selective A
Adenosine Receptor Antagonist Rolofylline for Patients Hospitalized With Acute
Decompensated Heart Failure and Volume Overload to Assess Treatment Effect on
Congestion and Renal Function) study, which randomized 2033 patients with ADHF to
intravenous rolofylline or placebo, failed to demonstrated any difference between groups in
the primary endpoint of treatment success (moderate or marked improvement in dyspnea at
24 and 48 hours without treatment failure), treatment failure (death or readmission for HF
by 7 days, persistent worsening renal failure, or worsening HF), or no change (Massie et al.,
2010). There were no differences in the number of patients who developed renal impairment
or in the secondary endpoint of death or rehospitalization for cardiac or renal causes at 60
days. The overall adverse event rates were similar between groups, although more patients
in the rolofylline group had seizures, a known side effect of A1 antagonists mediated via
central nervous system A1 receptors that regulate electrical excitability. Based on the lack of
clinical efficacy, coupled with the increased risk of seizures, rolofylline is not recommended
for the treatment of CRS.
Another intravenous selective A1 antagonist, tonapofylline, was also investigated in Phase II
clinical trials after preclinical studies and small human studies suggested effective
natriuresis. The TRIDENT-1 (Safety and Tolerability of IV Tonapofylline in Subjects With
Acute Decompensated Heart Failure and Renal Insufficiency) and POSEIDON (Oral BG9928
in Patients with Heart Failure and Renal Insufficiency) trials were both terminated early
after review of interim safety data from TRIDENT-1 revealed that two patients in the
tonapofylline group had had seizures (Ensor & Russell, 2010). Of note, seizures were not
reported in studies of oral tonapofylline, and in rat studies, tonapofylline did not cross the
blood-brain barrier (Ensor & Russell, 2010). There is insufficient data to determine whether
oral formulations of A1 antagonists are safe or clinically useful.
3.8 Ultrafiltration
Extracorporeal fluid removal has been used for decades in ADHF, typically reserved for
patients with fluid overload states that are refractory to diuretics and other medical
therapies. Small studies of ultrafiltration in HF have previously demonstrated effective fluid
removal, rapid symptom improvement, attenuated neurohormonal activity, and
hemodynamic improvements including reduced LV filling pressures and reduced
pulmonary arterial pressures without reductions in systemic blood pressure or cardiac
index (Marenzi et al., 1993; Rimondini et al., 1987). The landmark UNLOAD (Ultrafiltration
versus intravenous diuretics for patients hospitalized for acute decompensated heart failure)
trial randomized 200 patients with ADHF and volume overload to veno-venous
ultrafiltration or intravenous diuretics (Costanzo et al., 2005). Patients in both groups had
similar improvements in dyspnea scores, but the patients in the ultrafiltration group had
greater weight loss and net fluid loss at 48 hours. Importantly, there were fewer
rehospitalizations, rehospitalization days, and unscheduled clinic visits at 90 days in the
Sub-Types and Therapeutic Management of the Cardiorenal Syndrome
139
ultrafiltration group than in the IV diuretic group. No differences in renal outcomes were
seen.
Ultrafiltration can be performed via peripheral or central veins, with rates of fluid removal
regulated by a hematocrit sensor and ranging from 10 to 500 mL per hour. Blood flow rates
range from 10 to 40 mL per minute, and total extracorporeal blood volume can be as low as
33 mL. Maintenance of a constant hematocrit ensures that the rate of fluid removal from the
intravascular compartment is equivalent to the rate of fluid shift from the extravascular to
intravascular compartments. Low extracorporeal blood volume and slow fluid removal
minimize neurohormonal activation and prevent hypotension. In contrast to the hypotonic
fluid removal that occurs with diuresis, ultrafiltration removes isotonic fluid, potentially
resulting in greater total sodium removal. The mechanism of the sustained benefit seen in
the UNLOAD trial is thought to be related to the attenuation of neurohormonal activity and
to the removal of isotonic fluid.
The major limitation to the widespread use of ultrafiltration in HF and the CRS is likely to
be the cost of the filters used. In addition, questions remain about patient selection, optimal
timing of initiation of therapy, and determination of total fluid volume to be removed. The
specific role of ultrafiltration in patients who develop worsening renal function with diuretic
therapy is being investigated in CARESS-HF (Cardiorenal Rescue Study in Acute
Decompensated Heart Failure), and will help to define the role of this therapy specifically in
the CRS population.
3.9 Erythropoietin and correction of anemia
Anemia is common in both HF and CKD, and the term cardiorenal-anemia syndrome
refers to the coexistence of anemia and the CRS. EPO is widely used in the CKD population
to correct anemia to a moderate degree. Studies in this population have shown improved
parameters of cardiac performance with EPO therapy, including reduction of left ventricular
hypertrophy and dilatation, improved left ventricular ejection fraction, and increased
cardiac output (Linde et al., 1996; Low et al., 1989; Low-Friedrich et al., 1991). Studies of
EPO and iron administration to patients with HF with or without CKD have shown
inconsistent results, but some studies have demonstrated modest improvements in
symptoms and functional capacity as well as renal function, ejection fraction, and left
ventricular dimensions (Bolger et al., 2006; Palazzuoli et al., 2006; Silverberg et al., 2000;
Toblli et al., 2007). The FAIR-HF (Ferric Carboxymaltose in Patients with Heart Failure and
Iron Deficiency) study demonstrated improved symptoms and functional capacity in
patients with HF and iron deficiency, even in the absence of overt anemia, treated with
intravenous iron as compared to placebo (Anker et al., 2009). The ongoing IRON-HF (Iron
Supplementation in Heart Failure Patients With Anemia) and RED-HF (Reduction of Events
With Darbepoetin Alfa in Heart Failure ) studies will likely further clarify the role of iron
and EPO therapies in patients with HF and provide additional insights into the
management of the CRS.
4. Conclusions and future directions
The Cardiorenal Syndrome is a pathophysiologic state involving complex feedback
processes between the failing heart and failing kidneys, and is associated with a
Chronic Kidney Disease
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significantly increased risk of morbidity and mortality compared to either disease process
alone. New classification schemes add to our understanding of the processes involved, and
help to guide therapy. As the pathophysiology of the CRS becomes better understood, there
is potential for the development of novel and rational treatment strategies. Although many
promising agents introduced in recent years have produced disappointing results in clinical
trials, other strategies, including HSS, ultrafiltration, and low-dose dopamine still hold
potential. Larger scale trials of these and other agents are required before their use can be
widely adopted. Fortunately, such trials are already underway for ultrafiltration, EPO, and
dopamine and the results of these studies are eagerly anticipated. Similarly, established
therapies such as -blockers, ACE inhibitors, and ARBs must be rigorously tested in patients
with concomitant cardiac and renal dysfunction to ensure they provide clinical benefit
across the spectrum of disease states which characterize the cardiorenal syndrome.
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9
Atherosclerotic Renovascular Disease
Gen-Min Lin, Chih-Lu Han, Chung-Chi Yang
and Cheng-Chung Cheng
National Defense Medical Center
Taiwan
1. Introduction
Atherosclerotic renovascular disease (ARVD), also known as atherosclerotic renal artery
stenosis is increasingly recognized to be a cause of chronic renal failure. According to a
recent administrative data regarding general population of the elderly greater than 65 years
of age in the United States, the prevalence and incidence rates of ARVD were estimated
0.5% and 3.7 per each 1000 person-years respectively (Kalra et al., 2005). In addition, some
epidemiological researches demonstrated that the prevalence among those with end-stage
renal disease beginning renal replacement therapy was estimated from 5% to 22% (Rimmer
& Gennari, 1993; Mailloux et al., 1994; Appel et al., 1995; van Ampting et al., 2003). Of note,
ARVD is not only responsible to impaired kidney function but also reflects a status of
patients at risk for systemic cardiovascular diseases (Kalra et al., 2005). It has been well
known that a variety of risk factors for atherosclerosis share common pathway underlying
atherosclerotic renal artery stenosis, coronary artery disease, and peripheral vascular
disease. On the contrary, significant high-grade bilateral or isolated renal artery stenosis
may cause renovascular hypertension estimating over 50% of ARVD populations by
activation of renin-angiotensin-aldosterone system and lipoxygenase pathway that further
deteriorate the kidney function (Romero 1997). A previous report uncovered that ARVD
was estimated from 1% to 6% in patients with hypertension (Simon et al., 1972). In this
regard, a vicious cycle will be established in the progression of renal arterial atherosclerosis,
which is characterized by refractory hypertension, acute cardiac events (ie, heart failure,
cardiogenic pulmonary edema or acute coronary syndrome), and hence leads to acute or
chronic renal failure due to hypertensive or ischemic nephropathy (Buller et al., 2004).
Therefore, an early alert of patients at risk for ARVD is critical in slowing down the rate of
kidney function loss and providing treatment for underlying cardiovascular disease as well.
In this chapter, we will fuel the readers with the classic knowledge in this field and propose
the latest evidence-based medicine to manage patients with this disease.
2. The pathogenesis of atherosclerosis
Atherosclerosis is affected by the traditional risk factors including hypertension, smoking,
hyperlipidemia, diabetes mellitus and family history of premature coronary artery disease
systemically. Regionally, blood flow disturbances near arterial branches, bifurcations and
curvatures result in complex spatiotemporal shear stresses that are associated with
Chronic Kidney Disease
150
atherosclerosis susceptibility (Davies, 2009). In these predisposed areas, hemodynamic shear
stress, the frictional force acting on the endothelial cell surface is weaker than in protected
regions. Studies have identified shear stress to be an important determinant of endothelial
function and phenotype. High shear stress (>15 dyne/cm
2
) induces endothelial quiescence
and an atheroprotective gene expression profile, while low shear stress (<4 dyne/cm
2
),
which is prevalent at atherosclerosis-prone sites, stimulates an atherogenic phenotype
(Malek et al, 1999). As we know, thrombosis formation in situ and distal embolic dislodge
from great vessels, determined by the burden and the stability of atherosclerosis, are the two
major mechanisms leading to target organ infarction. With recent substantial evidence,
systemic inflammation caused by either external stimulus such as microbial infection or
internal immunologic response may trigger acute vascular events via pathogenic athroma
plaque rupture. Therefore, when and how to stablize and regress the process of
atherosclerosis becomes a cirtical step to prevent target organ damage.
2.1 Systemic arterial atherosclerosis: the evidence from angiography and autopsy
Advanced atherosclerosis is highly prevalent among patients with ARVD characterized by
coexistence with abdominal aortic aneurysm, severe coronary artery disease, ischemic stroke
and peripheral vascular disease in post-mortem and angiographic studies (Table 1).
ANG
(Patient: n)
Age
(year)
CAD
(%)
PAD
(%)
ARVD
(%)
Predictors
Crowley,
1998
CAD: 14,152 61+12 63% NA
6.3%
(bil: 1.3%)
Predictors for ARVD
progression:
-Female gender, OR: 1.8
-PAD, OR: 1.8
-Hypertension, OR: 1.5
-significant CAD, OR: 1.2
Conlon,
2001
CAD: 3,987 61+9 100% NA
9.1%
(4.8%)*
-CAD: 2VD vs.1VD, OR: 1.9
-CAD: 3VD vs 1VD, OR: 2.5
Liu,
2004
CAD: 141 59+10 31% NA 18.4% -CAD vs. non-CAD, HR: 2.8
Leandri,
2004
CAD: 467 64+11 69% NA 9.0%
-CAD: 2VD vs.1VD, OR: 2.8
-CAD: 3VD vs 1VD, OR: 3.0
Buller,
2004
ARVD: 837 67+10 68%
-Carotid: 12%
-A.A.A or lower limb
PAD: 12%
14.4%
(bil: 3.1%)
-Age per 10 year, OR:1.7
-Female gender. OR:1.9
-A.A.A or lower limb PAD,
OR: 2.1
-Carotid, OR: 3.0
Zhang,
2006
CAD: 1,200 62+10 51% NA
9.7%
(bil: 1.7%)
Age, hypertension, renal
insufficiency, CAD
Ozkan,
2009
PAD: 629 62+11 43%
Aortoiliac, crural,
femoropopliteal: 83%
9.6%
Age, hypertension and
aortoiliac stenosis
Atherosclerotic Renovascular Disease
151
ANG
(Patient: n)
Age
(year)
CAD
(%)
PAD
(%)
ARVD
(%)
Predictors
Kuroda,
2000
Stroke: 346 69+11 33%
-Carotid: 29.2%
-A.A.A: 13.3%
10.4%
*
-Renal insufficiency, OR: 6.6
-Hypertension:, OR: 4.1
-Carotid >50%, OR: 4.8
-Female gender, OR: 3.4
Fujii,
2006
Stroke: 346 71+11 39% NA
26.1%
*
(bil: 19%)
Hypertension, renal
insufficiency, PAD
Table 1. Associations between systemic atherosclerosis and ARVD: n: number; ANG: renal
angiography; CAD: coronary artery stenosis>50%; ARVD: renal artery stenosis>50%; PAD:
peripheral artery stenosis>50%; NA: not available; A.A.A: abdominal aortic aneurysm;;
carotid: carotid artery stenosis>50%; HR: hazard ratio; OR: odds ratio; VD: number of
diseased coronary artery; renal artery stenosis>60%; * renal artery stenosis>75%.
2.1.1 The nature course of ARVD
According to the shear stress rule, ostial and proximal lesions are mostly encountered and
20%-50% of cases are bilateral sites in ARVD (Safian & Textor, 2001). A significant
progression of ARVD was observed in 11.1% of 14,152 subjects with high cardiovascular
risks within a 2.6-year period in an angiographic study (Crowley et al, 1995) and in 35%-51%
from 3 to 5 years in a doplex unltrasonography study (Caps et al, 1998). From these reports,
the predictors to disease progression include old age, female gender, hypertension, diabetes
and the presence of significant coronary artery disease or peripheral vascular disease in
which the odds ratios range from 1.2 to 2.1. On the other hand, patients with ARVD are
associated with approximately 2-times risk of the occurrence of adverse coronary events and
mortality as compared to those without ARVD in a long-term follow-up (Conlon et al, 2001;
Edwards et al, 2005).
2.2 How to select patients at risk for prompt screening
As prescribed previously, patients at higher risk for atherosclerosis should receive an
advanced step for screening the presence of ARVD (table 2)
Among these clinical features, the only statistically significant predictor to ARVD is the
presence of abdominal bruit. The prevalence ranges from 6.5% to 31% in the healthy
population (Watson & William, 1973), and 28% in hypertensive patients (Julius and Steward,
1967). However, in patients with angiographically proven ARVD, the prevalence increases
up to 80% (Turnbull, 1995). Besides, the sensitivity of a systolic-diastolic abdominal bruit in
the diagnosis of RAS has been reported from 39% to 63% and the specificity of 90% to 99%
((Turnbull, 1995). Thus, the presence of a systolic-diastolic bruit is highly suggestive of RAS
and should be screened for, while the absence of a bruit does not exclude RAS (Rosener
2001).
2.2.1 Differential diagnosis
Some clinical situations have to been addressed in the diffential diagnosis of ARVD (table 3).
Chronic Kidney Disease
152
Categories Criteria
Physical findings -Abdominal or flank bruit
Metabolic
syndorme
International Diabetes Federation:
- Central obesity is defined as waist circumference with ethnicity specific
values or if BMI is >30 kg/m, central obesity can be assumed and waist
circumference does not need to be measured.
And and any two of the following:
-Triglycerides: > 150 mg/dl, or specific treatment for this lipid
abnormality.
- HDL cholesterol: < 40 mg/dl in males, < 50 mg/dl in females, or
specific treatment for this lipid abnormality
- BP: systolic BP > 130 mmHg or diastolic BP >85 mm Hg, or treatment
of previously diagnosed hypertension.
- FPG >100 mg/dl, or previously diagnosed type 2 diabetes. If
FPG>100 mg/dl, OGTT glucose tolerance test is strongly
recommanded but is not necessary to define presense of the syndrome
Hypertension
-Refractory hypertension (BP >160/95 mm Hg while receiving three or
more antihypertensive agents) or associataed acute pulmonary edema
-Accelerated hypertension (increase in BP>15% in 6 months)
-Severe hypertension (DBP >115 mm Hg or Grade III or IV retinopathy)
-Recent-onset (within the last 2 years) hypertension
-Onset of hypertension after age 60
Renal insufficiency
-Elevated serum blood urea nitrogen > 20mg/dl or creatinine > 1.4 mg/dl
-Cockcroft-Gault CrCl < 50 ml/min without clear etiology
-Acute renal failure attributable to ACEI or ARB therapy
Atheroscelrosis
-Abdominal aortic atherosclerosis or lower extremity artery stenosis
-Peripheral artery disease or carotid artery stenosis / ischemic stroke
-Coronary artery disease > 2 vessel disease
Table 2. Patients at risk for further ARVD screening. BP: blood pressure; DBP: diastolic BP;
FPG: Fasting plasma glucose; OGTT: oral glucose tolerance test; CrCl: creatinine clearance;
ACEI: ACE inhibitors; ARB: angiotensin II receptor blockers.
Clinical situations Differential diagnosis
Abdominal bruit
Splenic arteriovenous fistula, hepatic cirrhosis,
hepatoma, abdominal aortic aneurysm and coarctation,
celiac artery compression syndrome, intestinal ischemia,
and pancreatic carcinoma
Rrogressive renal insufficiency
or renovascular hypertension
-Benign hypertensive nephrosclerosis
-Atheroembolic renal disease
Renal artery stenosis
-Renal artery dissection
-Fibromuscular dysplasia
Table 3. Differential diagnosis of ARVD.
Atherosclerotic Renovascular Disease
153
Most of these clinical situations could be differentiataed correctly by the image study such
as computed tomography and renal angiography. They are not neccessry mutually exclusive
and may be coexisted. For instance, benign hypertensive nephrosclerosis, a renal
parenchymal disease can be present together with ARVD. Atheroembolic renal disease is
associated with aortic manupulation or occurs spontaneously. The clinical features include
abrupt decline of renal functions and evidence of atrial fibrillation with extrarenal embolism
(Hazanov, 2004). Fibromuscular dysplasia (FMD) characterized by fibrous thickening in
arterial wall usually involves 60%-75% of renal and 25%-30% of carotid artery stenosis
(Luscher et al, 1981; Gray et al 1996) and is respoisble for 25 % cases of renovascular
hypertension (Pickering, 1989). In angiographic findings, FMD demostrates classic images of
string-of-beads appearance, aneurysm, and focal or tubular stenosis. In conrast to ARVD,
FMD occurs predominantly in young women of childbearing age and involves the middle
and distal portion of main renal artery (Das et al, 2007).
2.3 The screening and diagnostic modality
With the progression of the technology, a variety of modalities emerge for screening and
diagnosis of ARVD (table 4). In addition to renogram and nuclear scintigraphic captopril
renogram, doplex ultrasonography has been used successfully to detect the presence of
renal artery stenosis due to the non-invasive and contrast-free characteristics. However, it is
usually limited by a wide operator-dependent variation, obesity of patient and time
consuming. Magnetic resonance (MR) angiography increases the comparability between
examinations (Fig. 1A). Both of the sensitivity and specificity are estimated within 90-95%.
Till now, multi-detector computed tomography (MDCT) angiography (Fig. 1B) almost
replaces the role of catheter angiography as the first diagnostic tool for ARVD because of its
high utility and detection rate in evaluation of other abdominal problems.
Categories Sensitivity/specificity PPV/NPV Limitations
Renogram 75% /75% 50-75% /60% Almost for screening only
Captopril renogram 83-90% / 80-93% 70-92% /60-100% Hypotension
Doplex
ultrasonography
75-90% /62-90% 60% /95%
Wide operator-dependent
variation, time consuming
MR angiography 88-95% /90-95% 60-75% /90-98%
Gadolinium-induced
renopathy
MDCT angiography 94-100% / 93-100% 71-100% /95-100% Contrast-induced renopathy
Catheter
angiography
100% 100%
Contrast-induced renopathy,
bleeding, arterial dissection,
distal embolism
Table 4. Diagnostic modalities for ARVD. PPV: positive predictive value; NPV: negative PV.
2.4 Therapeutic indications
As we know, ARVD is highly associated with systemic atherosclerosis and occurs after the
occurrence of coronary artery disease and peripheral artery disease. Accordingly, an early
medical intervention and risk factors reductions to prevent the development of ARVD in the
Chronic Kidney Disease
154
Fig. 1. A: MR angiography demonstrates a right ostial renal artery stenosis (an arrowhead);
B: MDCT angiography demonstrates diffuse atherosclerosis of left renal artery (an
arrowhead indicate the proximal lesion of left renal artery).
presence of systemic atherosclerosis and many risk factors is important. On the other hand,
a critically unilateral or bilateral stenosis of renal artery disease may need further
mechanical manipulations such as renal angioplasty, stenting and bypass surgery. We will
describe the two parts of therapy in detail in the following paragraphs.
2.4.1 Medical treatment
Life style modification and a control of established risk factors is the golden rule for most
atherosclerotic vascular disease including diabetes, obesity, hypertension, low density
lipoprotein cholesterol (LDL-C), inflammation and smoking. However, no reports prove the
effect of medical control to reduce the occurrence of ARVD or prevent disease progression.
It is reasonable that medical treatment should be started in middle-aged persons at risk to
prevent ARVD. The choice of pharmacological agents and the goal aimed to achieve with or
without vascular events will be listed in table 5.
Among the antihypertensive agents, ACE inhibitors or angiotensin II receptor blockers
(ARBs) are observed with the most effectiveness in control of the blood pressure for patients
with ARVD (Dworkin & Jamerson, 2007). Surgical intervention should be considered if
refractory hypertension persisits. However, adequate control of blood pressure by chronic
administration of antihypertensive drugs can not be guarantteed the prevention of stenotic
lesions progression and post-stenotic renal.atrophy.
2.4.2 Interventional treatment
Renal artery revascularization for bilateral or unilateral disease in a single viable kidney is
indicated in the following situations (Greco & Breyer, 1997; Textor, 2004).
1. Severe or refractory hypertension
2. Recurrent episodes of acute pulmonary edema
3. Unexplained progressive renal insufficiency
4. Progressive renal function impairment with optimal blood pressure control.
Beyond these criteria mentioned above, the procedure of revascularization should be
performed after weighing the benefits against the hazards. Therefore revascularization
Atherosclerotic Renovascular Disease
155
Risk factors Medications (non) CVD / Goal Precautious
Diabetes
mellitus
Insulin, secretagogues,
sensitizers,
-glucosidase
inhibitors,
peptide analogs
Non-CVD/ HbA1c< 6.5 %
CVD/ LDL-C < 7.0 %
Adverse cadiovascualr
effects and metabolic
abnormalities of anti-
diabetic agents
Hypertension
ACEI/ ARB,
BB, CB,
diuretics
Non-CVD/ BP< 120/85
mg/L
CVD/ BP< 140/90mg/dl
-A J-curve relationship
between hypertension and
cardiovascular mortality
-ACEI/ARB should be used
carefully in bilateral ARVD
LDL-C
Statin, fibrates, resins,
niacin, ezetimibe,
Non-CVD/ LDL-C< 100
mg/L
CVD/ LDL-C < 70mg/dl
Multi-drug interaction and
dose effect related
rhabdomyolysis
Inflammation
-Antiplatelet agents
-Statin
-Investagated drugs
Non-CVD/ hs-CRP< 2mg/L
CVD/ not established
According to the JUPITER
trial only (Ridker et al, 2008)
Table 5. Modifiable risk factors for ARVD. CVD: cardiovascular disease including
myocardial infarction and ischemic stroke; ACEI: ACE inhibitors; ARB: angiotensin II
receptor blockers; BP: blood pressure; BB: beta-blocker; CB: calcium receptor blocker;
JUPITER: Justification for the Use of Statins in Primary Prevention: An Intervention Trial
Evaluating Rosuvastatin trial; CRP: C-reactive protein.
should be aimed for patients with a reversible status of chronic renal insufficiency and
resistant hypertension instead of reduing their mortality. A review literature has
demonstrated that half of patients with ARVD have no change in renal function, while one
fourth improve and one fifth deterioate their renal function after renal stenting (Fig.1A & B)
(Leertouwer et al, 2000).
Fig. 2. An atherosclerotic ostial lesion at right renal artery. Panel A: catheter renal
angiography (An arrowhead indicates a lesion from ostial to proximal right renal artery;
Panel B: post-percutaneous transluminal angioplasty with stenting (An arrowhead indicates
stenting site of right renal artery).
Chronic Kidney Disease
156
Accordingly, only 20-25% of patients may be eligible for elective renal revascularization.
There have some image, histology and clinical evidence to select patients with ARVD
having benefits to undergo renal artery revascularization which is described as follow
(Novick et al, 1987; Muray et al, 2002).
1. Visualization of the collecting system either on an intravenous pyelogram or during the
pyelogram phase after renal arteriography
2. Kidney length 9 cm.
3. The presence of intact glomeruli on frozen section biopsy at the time of surgery.
4. Rapid decline of renal functions after ACEI/ARB administrations.
There are three methods for renal artery revascularization (table 6).
Revascularization Indications Comments
PTA without stenting
(Connolly et al, 1994)
Non-ostial lesions
-65-70% success rate for lesions
-35-50% improvement of renal
functions
PTA with stenting
(ASTRAL investigators,
2009; Stone et al, 2011;
White, 2010; Davies et al,
2009)
Ostial and non-ostial lesions
-Significantly lower restenosis
rate than PTA alone
-98.8% success rate for lesions
-10.6-19% TVR rate within 5-10
years period
-Inconclusive results of the
improvement of renal functions
-Complication rate: 9% in 24
hours; 20% in 1 month;
mortality rate <1%
Bypass surgery
(ACC/AHA 2005
guidelines; Hansen et al,
1992)
-Multiple small renal arteries
-Early primary branching of
the main renal artery
-Aortic reconstruction near
the renal arteries
-85-90% success rate for lesions
-55-65% improvement of
lesions of renal functions
-In-hospital mortality rate: 3-
10%
Table 6. Comparison of three types of revascularization intervention. PTA: percutaneous
transluminal angioplasty; TVR: target vessel revascularization; ASTRAL: Angioplasty and
Stenting for Renal Artery Lesions; ACC/AHA: American College of Cardiology
Foundation/American Heart Association.
3. Conclusion
ARVD reflecting a status of systemic atherosclerosis is associated with chronic renal disease.
Life style modification and risk factors reduction are important for the primary prevention
of ongoing renal dysfunction and secondary prevention of subsequent cardiovascular
events. Some clinical features of patients at risk for ARVD should be highlighted and both
medical treatment and mechanical procedures should be taken as early as possible if
uncontrolled hypertension leading to end-organ damage or progressive renal insufficiency
develops.
Atherosclerotic Renovascular Disease
157
4. Acknowledgement
We thank Dr. Yu-Guang Chen, Tri-Service General Hospital for his kindly providing the
image of MDCT angiography.
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10
Pharmacologic Adjuvants to Reduce
Erythropoietin Therapy Dose in Anemia of
Chronic Kidney Disease and
End Stage Renal Disease
Adeel Siddiqui, Aqeel Siddiqui and Robert Benz
Lankenau Medical Center and Lankenau Institute for Medical Research
Wynnewood, Pennsylvania
USA
1. Introduction
Anemia is one of the leading causes of morbidity in chronic renal failure.
1
Chronic kidney
disease (CKD) associated anemia is largely due to reduced erythropoietin (EPO) release and,
to a lesser degree, to shortened red cell survival.
2
To overcome EPO deficiency in this
population, the development and administration of erythropoiesis-stimulating agents
(ESAs) such as recombinant human EPO and darbepoetin alfa (DPO) has resulted in
substantial health benefits, including improved quality of life, reduced blood transfusion
requirements, decreased left ventricular mass, diminished sleep disturbance and enhanced
exercise capacity.
1-7
Unfortunately in recent clinical trials, a proportion of patients exhibited
complications such as fatal or nonfatal stroke, access thrombosis, increase in thrombotic
events and exacerbation of malignancy associated with overly aggressive correction of
anemia.
8-10
It is not established whether these complications are related to higher dose of
EPO, underlying EPO resistance factors (i.e. inflammation) or achieving higher hematocrit
(HCT). A multifactorial combination of predisposing circumstances is possible. A number of
pharmacologic agents have been evaluated as adjuvant to ESAs therapy. These agents
include iron, L-carnitine, ascorbic acid, androgens, statins, pentoxifylline and N-
acetylcysteine. In this review article we will focus on the agents that have been used in
conjunction with EPO to correct anemia in patient with chronic kidney disease and end-
stage renal disease in an effort to reduce the dose requirement of EPO.
2. Iron
Iron is one of the most integral components of hematopoiesis in the anemia of kidney
disease. Trapped iron storage or decreased availability of iron is the most common factor
for the resistance to the effect of ESAs. Absolute iron deficiency is likely to be present in
patients with CKD when: the percent transferrin saturation (plasma iron divided by total
iron binding capacity x 100) falls below 20; the serum ferritin concentration is less than 100
ng/mL among advance CKD(predialysis) and peritoneal dialysis patients and less than
Chronic Kidney Disease
162
200 ng/mL among home hemodialysis patients.
11
However, functional iron deficiency is
associated with transferrin saturation (TSAT) 20 percent and elevated ferritin levels
(between approximately 200 to 800 ng/mL) or higher. An elevated ferritin level in this
condition is likely secondary to the acute phase reaction of underlying inflammation. The
2006 K/DOQI guidelines recommend goals of iron therapy during administration of ESAs.
For predialysis and peritoneal dialysis patients: TSAT >20 percent or content of hemoglobin
(Hb) in reticulocytes >29 percent and serum ferritin concentration >100 ng/mL. For patients
undergoing hemodialysis: transferrin saturation >20 percent or content of Hb in
reticulocytes >29 percent and serum ferritin concentration >200 ng/mL.
12
A number of clinical trials have compared which route of iron administration Intravenous
(IV) vs Oral (PO) is superior in treating anemia of CKD.
13-22
First we will discuss this issue in the Chronic Kidney Disease (CKD) population.
3. Anemia in chronic kidney disease
In a prospective trial by Stoves et.al, PO vs IV route of iron administration was studied.
Forty five anemic patients with CKD, not on dialysis, were randomized to receive oral
(ferrous sulfate 200 mg tid) or intravenous (300 mg iron sucrose monthly) iron therapy. EPO
was started at the same time and the dose adjusted according to a pre-established protocol.
After an average follow up of 5.2 months, there were no significant differences in Hb
response and EPO dose between the two groups.
13
A prospective study by Charytan et. al.
in 96 CKD anemic patients on EPO compared the efficacy of IV iron (5 doses of 200 mg iron
sucrose weekly) to oral iron (ferrous sulfate 325 mg tid). They found an increase in Hb and
ferritin following IV iron, whereas the oral iron group demonstrated an increase in Hb
without increase in iron stores.
14
Both of the above studies failed to show IV iron superior to
PO in either selected group of CKD patients. Van Wyck et.al. conducted a larger study of
182 non dialysis-dependent CKD (stages 3 to 5) patients to compare oral iron vs. IV iron.
That randomized, controlled, multicenter trial tested IV iron as sucrose 1 g in divided doses
over 14 days vs oral ferrous sulfate 325 mg three times a day for 56 days. Inclusion criteria
for the group were Hb 11 g/dL, TSAT 25%, and ferritin 300ng/mL. EPO/DPO dose was
not changed for eight weeks prior to or during the study. The proportion of patients
achieving the primary outcome (Hb increase 1 g/dL) was greater in the IV iron treatment
group than in the oral iron treatment group (44.3% vs. 28.0%, P = 0.0344), as was the mean
increase in Hb by day 42 (0.7 vs. 0.4 g/dL, P = 0.0298).
15
Agarwal and colleagues conducted
a randomized, multicenter, controlled trial in 75 adult, anemic, iron-deficient, non-dialysis
CKD patients not receiving ESAs. The patients were randomly assigned to receive either IV
ferric gluconate 250 mg weekly for 4 weeks or oral ferrous sulfate 325 mg three times a day
for 42 days. Both oral and IV iron similarly increased Hb in anemic CKD patients not
receiving ESAs.
16
A new IV iron preparation, ferumoxytol has been approved in the United States.
It appears to be safe and effective when given as a rapid infusion of up to 510 mg in
patients with CKD and patients on dialysis. A Phase III trial randomly assigned 304
patients with CKD in a 3:1 ratio to two 510-mg doses of intravenous ferumoxytol within 5
3 days or 200 mg of elemental oral iron daily for 21 days. Among patients who were not
receiving ESAs, Hb increased 0.62 1.02 g/dL with ferumoxytol vs. 0.130.93
Pharmacologic Adjuvants to Reduce Erythropoietin Therapy Dose
in Anemia of Chronic Kidney Disease and End Stage Renal Disease
163
g/dL with oral iron. Among patients who were receiving ESAs, Hb increased 1.161.49
g/dL with ferumoxytol vs. 0.191.14 g/dL with oral iron. The increase in Hb at day 35, the
primary efficacy end point, was 0.82+/-1.24 g/dL with ferumoxytol and 0.16+/-1.02 g/dL
with oral iron (P<0.0001).
17
The authors concluded that a regimen of two doses of 510 mg of
intravenous ferumoxytol administered rapidly within 53 days was well tolerated and had
the intended therapeutic effect. The side effects associated with IV iron in the above-
mentioned studies were headache, myalgia, and hypotension (particularly in thin, older
women<65 kg). Intravenous iron sucrose has shown better tolerability. Oral iron has more
GI associated side effects including constipation, diarrhea, nausea and vomiting.
13-17
As a result of these studies the K/DOQI guidelines have recommended that either oral iron
therapy or intravenous iron therapy can be given in CKD patients.
4. Anemia in end stage renal disease
Among hemodialysis patients, studies show that transferrin saturation and serum ferritin
levels usually continue to fall and anemia fails to correct despite ongoing oral iron therapy.
MacDougall et.al. studied 37 iron-replete hemodialysis patients beginning EPO therapy
randomized into three groups with different iron supplementation: Group1, IV iron dextran
5 ml (equivalent to 250 mg of elemental iron) every 2 weeks; Group 2, oral ferrous sulfate
200 mg tid; Group 3, no iron. Subjects were treated with 25 U/kg of EPO thrice weekly
subcutaneously. After a period of 16 weeks, the Hb response in the group receiving IV iron
(7.3+/-0.8 to 11.9+/-1.2 g/dL) was significantly greater than that for the other two groups
(7.2 +/-1.1 to 10.2 +/-1.4 g/dL and 7.3+/-0.8 to 9.9+/-1.6 g/dL for Groups 2 and 3,
respectively; p < 0.005 for both groups vs. Group 1 at 16 weeks). Serum ferritin levels
remained constant in those receiving IV iron (345 +/-273 to 359+/-140 mcg/L) in contrast to
the other two groups in which ferritin levels fell significantly (309+/-218 to 116+/- 87
mcg/L and 458+/-206 to 131+/- 121 mcg/L for Groups 2 and 3, respectively; p < 0.0005 for
Group 1 vs. Group 2, and p < 0.005 for Group 1 vs. Group 3 at 16 weeks). Dosage
requirements of EPO were also less in Group1.These results suggested that even in iron-
replete patients, those supplemented with IV iron have an enhanced Hb response to EPO
with better maintenance of iron stores and lower dosage requirements of EPO.
18
Wingard et.al. conducted a prospective study on 46 EPO treated hemodialysis patients and
randomized them into four different oral iron preparations. These four preparations
included Chromagen (ferrous fumarate from Savage Laboratories), Feosol (ferrous sulphate
from Smith Kline Beecham), Niferex (polysaccharide, Central Pharmaceutical) or Tabron
(ferrous fumarate; Parke-Davis). All patients were prescribed approx 200 mg of elemental
iron daily with at least 100 mg of ascorbic acid for six months. The study concluded that
with emphasis on compliance, oral iron supplementation at the dose used for this study was
able to maintain adequate iron status in the short term (less than 6 months) without the need
for IV iron dextran. However, IV iron dextran eventually (after 6 months) would be
necessary because of the downward trend in iron stores.
19
Ferumoxytol was studied in a randomized, open-label, controlled, multicenter Phase 3 trial
by Provenzano et.al. to evaluate the safety and efficacy of IV ferumoxytol compared with
oral iron.
20
Anemic patients on HD and on a stable ESA regimen received either two
injections of 510 mg of ferumoxytol within 7 days (n = 114) or 200 mg elemental oral iron
Chronic Kidney Disease
164
daily for 21 days (n = 116). Ferumoxytol resulted in a mean increase in Hb of 1.02+/-1.13
g/dL at day 35 compared with 0.46+/-1.06 g/dL with oral iron (p = 0.0002). There was a
greater mean increase in TSAT with ferumoxytol compared with oral iron at day 35 (p <
0.0001).
5. Conclusion
For patients with chronic kidney disease who are not on dialysis, oral iron or IV iron can be
used for iron supplementation. This conclusion is consistent with the opinion of the Work
Group from K DOQI guidelines.
The preferred route of administration of iron in patients with chronic kidney disease on
hemodialysis is intravenous as supported by K DOQI guidelines as of 2006.
6. Ascorbic acid
Vitamin C or ascorbic acid has been studied in the metabolism of iron and anemia
management. The first studies were performed in guinea-pigs. It was found that ascorbic
acid deprivation increased the total non-haem iron concentration in the spleen and reduced
it in the liver, and in both organs ferritin was diminished and haemosiderin increased.
Repleting the ascorbic acid restored the normal distribution of iron between the two storage
compounds, and in the spleen the total storage iron concentration returned to control levels
within 24 hours.
21
Another important property of ascorbic acid is its ability to increase the
availability of storage iron to chelators.
22
In hemodialysis patients this role of ascorbic acid
was investigated by Deicher who conducted a cross-sectional, single-centre observational
study. Pre-dialysis plasma Vitamin C concentrations were measured and response to EPO
(Hb concentration/ international units EPO/kg/week) was recorded. Univariate analysis
yielded a significant correlation between Vitamin C plasma levels and response to EPO. It
was found that in unselected hemodialysis patients Vitamin C plasma levels account, at
least partially for the response to EPO.
23
That work led to ascorbic acid investigations for use
in EPO-treated hemodialysis patients, particularly those with EPO- hypo responsiveness,
elevated serum ferritin levels, and functional iron deficiency (transferrin saturation 20
percent and elevated ferritin level between 200 to 800 ng/ml or higher). Studies evaluated
the role of IV Vitamin C in hemodialysis patients and showed that in those patients who
develop resistance to EPO with functional iron deficiency, the resistance can be overcome
by giving Vitamin C instead of iron,thus avoiding hemosiderosis.
24
In another comparative
larger study, Tarng et.al. were able to show similar results in a prospective trial of dialysis
patients. Sixty-five HD patients with serum ferritin levels greater than 500 mcg/L were
recruited and divided into the control (N = 19) and intravenous ascorbic acid IVAA (N = 46)
groups. IVAA patients with a hematocrit (HCT) of less than 30% received 300 mg of ascorbic
acid three times per week for eight weeks. Controls had a HCT of more than 30% and did
not receive the adjuvant therapy. Red blood cell and reticulocyte counts, iron metabolism
indices, erythrocyte zinc protoporphyrin (E-ZPP), and the concentrations of plasma
ascorbate and oxalate were examined before and following the therapy. Thirteen patients
(four controls and nine IVAA patients) withdrew by the end of the study. Eighteen patients
had a dramatic response to IVAA with a significant increase in Hb and reticulocyte index
and a concomitant 24% reduction in EPO dose after eight weeks. This paralleled a
Pharmacologic Adjuvants to Reduce Erythropoietin Therapy Dose
in Anemia of Chronic Kidney Disease and End Stage Renal Disease
165
significant rise in serum iron and TSAT and a fall in E-ZPP and serum ferritin (baselines vs.
8 weeks, serum iron 68+/-37 vs. 124 +/-64 mcg/dL, TSAT 27+/-10 vs. 48+/-19%, E-ZPP
123+/-44 vs. 70+/-13 micromol/mol heme, and serum ferritin 816+/-435 vs. 587+/-323
mcg/L, p<0.05). Compared with responders, mean values of Hb, EPO dose, iron
metabolism parameters, and E-ZPP showed no significant changes in controls (N = 15) and
in non-responders (N = 19).
25
A single PO study by Benz et. al. was conducted in 21 EPO resistant anemic hemodialysis
patients with ferritin levels greater than 350 ng/mL had received oral daily ascorbic acid at
a dose of 500 mg/day and were retrospectively studied. Hemoglobin, HCT, EPO dose,
ferritin, and transferrin saturation were recorded at baseline and after three months of
treatment. EPO dose/HCT was calculated. Serum oxalate levels were also measured. In this
study, daily oral ascorbic therapy decreased ferritin levels and EPO dose requirements
while raising Hb and HCT level. Hb increased 9% from 11.4 to 12.2 gm/dl (p = 0.05), HCT
increased 10% from 33.3 to 36.7% (p = 0.05), and EPO dose requirement decreased 33% from
26,229 to 17,559 U/week (p = 0.03). Ferritin levels decreased 21% from 873 to 691 ng/mL (p =
0.004) Patients with oxalate levels >27 micromol/L were instructed to stop ascorbic acid
treatment, and mean levels decreased from 107 to 19 micromol/L (p = 0.01) over a mean
time of 71 days. This beneficial profile of the effects of ascorbic acid therapy is consistent
with improvement of EPO resistance and cost savings in this population.
26
The primary concern for using Vitamin C in dialysis patients is secondary oxalosis because
of the impairment in renal excretion and inadequate removal by dialysis procedures.
27-28
Tarng et.al. showed that oxalate levels increase modestly after 8 weeks of IV Vitamin C but
information on longer courses of treatment is limited.
25
Canavese prospectively studied the
dose of Vitamin C and effect on oxalate levels in 30 dialysis patients. Eighteen patients were
administered intravenous ascorbate during 18 months (250 mg/wk, subsequently increased
to 500 mg), and 12 patients were taken as reference untreated cases. The study found that
plasma oxalate levels progressively increased as the dose of IV Vitamin C was increased
from 250 to 500 mg/week. After six months at a dose of 500 mg per week, 7 of 18 patients
(40 percent) attained plasma oxalate levels that exceeded the range that would be associated
with calcium oxalate super saturation at usual calcium concentrations.
29
The 2006 K/DOQI guidelines for anemia in CKD stated that there was insufficient evidence
to recommend Vitamin C as an adjuvant to EPO therapy.
30
However, several of the clinical
studies were published subsequent to the development of those guidelines.
7. Pentoxifylline
Pentoxifylline (PTX) is a methyl xanthine derivative, which is approved for use in
peripheral vascular disease and may also have anti-inflammatory effects according to
studies. Benbernou et. al. studied pentoxifyline and examined its regulatory effect on T
helper (TH1-and TH2) cell-derived cytokines in human whole blood and peripheral blood
mononuclear cells stimulated with phytohemagglutinin and phorbol myristate acetate.
The results showed that PTX at the appropriate concentrations (5 x 10
(-4)
M ) could induce
selective suppression of interleukin (IL) -2 and interferon (INF) -gamma, whereas at high
concentrations this drug could act as a suppressive agent of both TH1- and TH2-derived
cytokines.
31
Bienvenu showed similar results that PTX possesses a much broader spectrum
Chronic Kidney Disease
166
of activity on cytokine production than was initially described, and it appears to be a
potential and promising immunotherapeutic agent.
32
These studies led to PTXs possible
role in treating EPO resistant anemia. Navarro et. al. conducted a prospective small study
of 7 anemic patients with CKD, who were treated with pentoxifylline (400 mg orally
daily) for 6 months with the goal of defining the effects of pentoxifylline, as an agent with
anti-tumor necrosis factor (TNF)-alpha properties. The results showed Hb significantly
increased in the pentoxifylline-treated patients at the 6th month (9.9+/-0.5 g/dL at
baseline;10.6+/-0.6 g/dL at the 6th month, respectively, p < 0.01), whereas no increase
was seen in the control group. Serum EPO levels remained stable in all patients. However,
the serum TNF-alpha concentration decreased significantly in patients receiving
pentoxifylline. The study suggested that the inhibition of erythropoiesis by cytokines may
play a significant role in renal anemia. The administration of agents with anti-cytokine
properties, such as pentoxifylline, can improve the hematologic status in this
population.
33
Another small study was conducted by Cooper and colleagues on 16
dialysis EPO resistant anemic patients. The patients were treated with oral pentoxifylline
400 mg daily for 4 months. Ex-vivo T cell generation of TNF-alpha and IFN-gamma from
the patients was assessed before and 6 to 8 weeks after the therapy. A total of 12 of 16
patients completed the study. Before therapy, mean Hb concentration was 9.5+/-0.9 g/dL.
After 4 months, the mean Hb concentration increased to 11.7+/-1.0 g/dL (p = 0.0001).
Baseline ex vivo T cell expression of TNF-alpha decreased from 58% +/-11% to 31%+/-
23% (p= 0.0007) after therapy. Likewise, IFN-gamma expression decreased from 31%+/-
10% to 13%+/-10% (p = 0.0002). EPO doses remained unchanged in all but one patient in
whom the dose was reduced in response to a higher Hb. One patient who was previously
transfusion dependent was able to stop receiving monthly transfusions. Pentoxifylline
therapy may significantly improve Hb response in patients with EPOresistant anemia in
renal failure.
34
This small, open-label, uncontrolled study suggests the need for a larger, controlled trial
with this agent. Until such a trial is conducted, pentoxifylline is not recommended as an
EPO-adjuvant except in the experimental setting.
8. Statins
Statins (HMG-CoA reductase inhibitors) are a class of drugs used to lower cholesterol levels
by inhibiting the enzyme HMG-CoA reductase, which plays a central role in the production
of cholesterol in the liver. As mentioned above, cytokines play a role in inhibition of
erythropoiesis. Statins have been evaluated as an adjuvant to EPO with the thought that
they have anti-oxidant and anti-inflammatory properties. In one retrospective study, 70 HD
patients were treated with statins for a period of 4.7 months and were found to have the
mean Hb level rise from 10.6 to 12.5 g/dL (p < 0.0005) with an associated 25 percent
decrease in EPO requirements.
35
Another study investigated whether the anti-inflammatory
effect of statins improved EPO responsiveness in hemodialysis patients. It examined
patients with Type 2 diabetes mellitus, who had been shown to have EPO resistance. One
hundred and three patients were stratified into statin and non-statin groups.The outcome of
interest was EPO dose. The mean EPO dose (units/kg per week) was significantly lower in
the statin group (275.6 273.2, vs. 449.5 555.9, p < 0.05). Twenty percent of patients in the
Pharmacologic Adjuvants to Reduce Erythropoietin Therapy Dose
in Anemia of Chronic Kidney Disease and End Stage Renal Disease
167
statin group required EPO dose in excess of an EPO equivalent of 500 units/kg per week,
compared to 30.88% in the non-statin group. The two-way analysis of variance showed no
interaction between the use of statins and the presence of Type 2 diabetes mellitus on EPO
dose. This study demonstrated that hemodialysis patients who were on statins had a
significantly lower EPO requirement. This association is possibly due to the pleiotropic
effect of statins.
36
A prospective study tested the effect of statin therapy on ESA hypo- responsiveness, and
emphasized its anti-inflammatory benefits in maintenance hemodialysis patients. This study
enrolled 30 patients with baseline cholesterol >220 mg/dL. Low-dose atorvastatin (10
mg/day) was prescribed for 12 weeks. They prospectively recorded biochemistry and
hematological profiles, ESAs prescription and inflammatory markers at baseline, 4 weeks
and 12 weeks. Statistically significant changes were noted after 4 and 12 weeks of statin
therapy for cholesterol (272.5 to 184.4 and 196.4 mg/dL, p < 0.05) and ESA hypo-
responsiveness, reported as EPO to HCT ratio (EHR) (129.3+/-58.2 to 122.3+/-53.5 and
121.0+/-53.3 EPO U/HCT/week, p < 0.05). Mean values for proinflammatory cytokines
included interleukin-6 and TNF-alpha levels decreased by 30.8 and 10.6%, respectively.
These data suggest that statin therapy may benefit patients with ESA hypo-responsiveness.
This benefit in ESA hypo-responsiveness is associated with the effects of statins on
inflammation.
37
These preliminary studies may justify future studies to use statins as an EPO dose reducing
adjuvant in patients with inflammation-mediated EPO resistant anemia of CKD.
9. Carnitine
L-carnitine is a small molecule (molecular weight: 161.2) that is derived from dietary
products, mainly red meat and milk. Endogenous carnitine production takes place in the
liver from lysine, methionine, ascorbate, niacin and pyridoxine. L-carnitine is required for
the transport of long-chain fatty acids into the mitochondria and is an integral part of energy
metabolism via ATP formation.
L-carnitine has been shown to improve anemia in uremic patients by stabilizing erythrocyte
membrane function or erythropoiesis. End-stage renal disease patients are known to have
carnitine deficiency.
38
This could be a contributing factor of anemia requiring higher dose of
EPO. Thus, it has been used therapeutically in dialysis patients with and without
concomitant EPO. Carnitines role as an adjuvant to EPO in kidney disease is unclear. Most
studies have involved HD patients with IV carnitine administration.
A 2002 meta-analysis evaluated the efficacy of IV carnitine supplementation in lowering the
required dose of EPO using data from six randomized trials. The EPO dose was found to be
significantly lower among those administered carnitine, with a beneficial response reported
in four of the six studies.
38
Two studies showed improvement in Hb and HCT with PO
carnitine but they were published before EPO was available.
39,40
In one study, 24 dialysis
anemic patients were divided into two groups, controls (inert placebo), treated patients (L-
carnitine 1.6 g PO daily) for one year. A significant increase in HCT, Hb, red cell count and
mean corpuscular Hb concentration was observed. In comparison with the control group, an
Chronic Kidney Disease
168
early improvement could be detected by the 3
rd
month, with further increases in the
successive months of treatment in the L-carnitine cohort.
There is some evidence in the literature suggesting that accumulation of metabolites
(trimethyleamine and trimethylamines-N-oxide)of oral carnitine, may have potential
toxicity
41
. Marcus et.al. conducted a study using oral carnitine and showed that a small dose
of L-carnitine is sufficient to increase the blood concentration of carnitine.
41
The concern
remains about the accumulation of trimethylamines-N-oxide and its potential toxicologic
effects include neurological toxicity and uremic breath.
The 2006 K/DOQI guidelines for anemia in CKD stated that there was insufficient evidence
to recommend L-carnitine.
42
10. Androgens
There is no literature available in CKD patients not on dialysis. Before EPO was available,
androgens (which may increase endogenous EPO production, sensitivity of erythroid
progenitors to the effects of EPO, and red blood cell survival) were used regularly in the
treatment of anemia in dialysis patients.
43-46
Their use for anemia in dialysis patients has
declined markedly since EPO was approved.
EPO and androgens combination in hemodialysis patients has been studied:
Ballal et.al. performed a study in a group of 15 adult male hemodialysis patients.
47
Seven
patients were treated with EPO alone at a dose of 2,000 U intravenously (IV) three times a
week. An additional group of eight patients was treated with 2,000 U of EPO three times a
week and also received 100 mg of nandrolone decanoate intramuscularly (IM) each week.
After 12 weeks of therapy, HCT values increased slightly in the group receiving EPO alone,
from 25.3+/-0.8 to 27.4+/-1.5. In contrast, EPO in combination with nandrolone decanoate
resulted in a greater increase in HCT values, from 24.4+/-1.4 to 32.9 +/-1.8 (p < 0.001). The
results showed that the groups receiving low-dose EPO alone had a poor erythropoietic
response. In contrast, patients receiving androgen in addition to EPO had a significantly
greater increase in HCT values with treatment. These data show that androgen therapy
significantly augments the action of exogenous EPO such that lower doses of EPO may be
sufficient for an adequate hematopoietic response.
In a prospective, randomized study by Berns et al. in a chronic hemodialysis population,
patients received EPO 40 U/kg intravenously three times weekly either alone (Group 1, n =
6) or with weekly intramuscular injection of 2 mg/kg nandrolone decanoate (Group 2, n = 6)
for up to 16 weeks. Baseline HCT, ferritin, N-terminal parathyroid hormone, and aluminum
levels were similar. The mean weekly rate of rise in HCT was 0.32+/-0.13% in Group 1 and
0.37+/-0.11% in Group 2, (p = NS). Three of 6 patients in Group 1, but only 1 of 6 patients in
Group 2, reached the target HCT of 30% within 16 weeks. Two patients in Group 2
requested that the nandrolone decanoate be stopped prior to reaching target HCT because
of unacceptable side effects (acne).
48
Nandrolone decanoate did not enhance the response
rate to this EPO dose and is associated with significant side effects.
In a longer open-label study with low-dose EPO therapy, 19 chronic hemodialysis patients
were randomly assigned to receive EPO (1,500 units IV at each HD treatment) either alone
Pharmacologic Adjuvants to Reduce Erythropoietin Therapy Dose
in Anemia of Chronic Kidney Disease and End Stage Renal Disease
169
or with nandrolone decanoate (100 mg intramuscularly weekly) for 26 weeks.
49
The mean
increase in HCT and the final achieved HCT were greater in the nandrolone decanoate
treated group (8.2 and 33.2 percent, respectively) than in the group treated with EPO alone
(3.5 percent and 28.3 percent, respectively). No serious side effects were reported.
Thirty two hemodialysis patients were randomly assigned to receive low dose EPO therapy
(1,000 units SC at each HD treatment) either alone or with nandrolone decanoate 50 mg
intramuscularly twice weekly for six months.
50
The increase in Hb in the
nandrolone decanoate treated group (from 7.5 to 10.4 g/dL) was not statistically different
from the control group (7.3 to 10.0 g/dL). Side effects, including gynecomastia, hirsutism,
menstrual irregularity, and increases in liver enzymes and triglyceride levels, were
common.
The limiting factor in these studies was small size and relatively short follow ups, and none
attempted to maintain currently recommended Hb levels. The 2006 K DOQI guidelines for
anemia in CKD stated that androgens should not be used as an adjuvant to EPO.
11. N-acetylcysteine
Nacetylcysteine (NAC) is a drug and nutritional supplement used primarily as a mucolytic
agent and also in the management of acetaminophen overdose. To explore the efficacy of
oral NAC supplementation for anemia and oxidative stress in hemodialysis patients, Chien
et al studied 325 dialysis patients. In this study, 49 pateints received NAC 200 mg orally
three times a day during the first 3 months of dialysis, while the other 276 patients not
receiving NAC were observed. During the 4-month study, 11 patients receiving NAC
withdrew but had no severe adverse effects, while 49 patients not receiving NAC had
negative confounding events. Thus only the data of the remaining patients, 38 taking NAC
and 227 not taking NAC, were analyzed for efficacy.
When the EPO dosage was stable, only the NAC group had a significant increase in HCT,
accompanied with a decrease in plasma levels of 8-isoprostane and oxidized low-density
lipoprotein. Analyzed as a nested case-control study, NAC supplementation was also found
to be a significant predictor of positive outcomes in uremic anemia.
51
To determine the
contribution of injectable iron administered to hemodialysis patients in causing oxidative
stress and the beneficial effect of NAC in reducing it, Swarnalatha et al conducted a
prospective, double blinded, controlled, cross over trial on 14 adult hemodialysis patients
who were randomized into two groups; one group received NAC in a dose of 600 mgs by
mouth twice daily for 10 days prior to intravenous iron therapy and the other group
received placebo. Both groups received intravenous iron therapy, 100 mg of iron sucrose in
100 mL of normal saline given over a period of one hour. Blood samples for the markers of
oxidative stress were taken before and after the iron therapy. After a week of wash-out
period for the effect of NAC, subjects crossed over to the opposite regimen. They measured
the lipid peroxidation marker, malondiaaldehyde (MDA), to evaluate the oxidative stress
and total anti-oxidant capacity (TAC) for the antioxidant level in addition to the highly
sensitive C-reactive protein (HsCRP). Non-invasive assessment of endothelial dysfunction
was measured by digital plethysmography before and after intravenous iron therapy. There
was an increase of MDA (21.97 + 3.65% vs 7.06 + 3.65%) and highly sensitive C-reactive
protein (HsCRP) (11.19 + 24.63% vs 13.19 + 7.7%) after iron administration both in the
Chronic Kidney Disease
170
placebo and the NAC groups. NAC reduced the baseline acute systemic generation of
oxidative stress when compared to placebo, which was statistically significant with MDA
(12.76 +/- 4.4% vs 9.7 +/- 4.4%) but not with HsCRP. Pre-treatment with NAC reduced the
endothelial dysfunction when compared to placebo, but it was not statistically significant.
The author concluded that in those HD patients, NAC reduced the oxidative stress before
and after the administration of intravenous iron therapy in addition to the endothelial
dysfunction induced by this treatment.
52
Finnigan and Benz reported the results of treating 12 ESRD EPO resistant hemodialysis
subjects with oral NAC 600 mg by mouth twice daily for 6 months. In that small pilot study,
NAC therapy was associated with a 53% reduction in the EPO Resistance Index (weekly
EPO dose/weight in Kg/Hb).
53
These preliminary studies suggest the need for a larger, controlled trial with NAC. Until
then, routine use of NAC as an EPO- adjuvant cannot be recommended.
12. Discussion
Anemia of CKD/ESRD has multiple etiologies, although the decrease in EPO production by
the diseased kidneys is the major contributor. Recently, studies targeting higher Hb levels or
using higher EPO dosing regimens in the correction of anemia have shown detrimental
effects including increased all cause mortality, cardiac and cerebral vascular events and
vascular access thrombosis
8-10,54
It is not clear whether this is due to higher HCT or EPO the
molecule itself at higher concentration. This review article focused on adjuvant oral and
parenteral agents that have been used along with EPO to reduce its dose and give
foundation to research in randomized control trials. There may also be a potential benefit of
these agents to use along with EPO in reducing cost and expenditures especially when the
bundling method of dialysis payment is in effect.
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11
Molecular Mechanisms of
Nephro-Protective Action of HE-86 Liquid
Extract in Experimental Chronic Renal Failure
Li-qun He
1
, Dong Feixia
2
, Qiang Fu
3
and Jun Li
4
1
Department of Nephrology, Shuguang Hospital Affiliated with Shanghai University of
Traditional Chinese Medicine, Ministry of Education Key Laboratory of Liver and Kidney
Disease Syndrome, E-Institutes of Shanghai Municipal Education Commission, Innovative
Research Team in Universities of Shanghai Municipal Education Commission
2
Department of Nephrology, Wenzhou TCM Hospital Affiliated to Zhejiang University of
Traditional Chinese Medicine, Wenzhou
3
Heilongjiang University of Traditional Chinese Medicine, HeiLongJiang
4
Zhuhai City NO 5 Hospital, GuangDong
China
1. Introduction
Chronic renal injury can be mediated by angiotensin II (Ang II) through hemodynamic and
inflammatory mechanisms and attenuated by individual suppression of these mediators.
Hypertension is usually associated with the development of vascular and renal fibrosis [3].
This pathophysiological process is characterized by structural changes in vasculature caused
by increased synthesis and rearrangement of extracellular matrix proteins, such as the
collagen type I [4]. Several studies support a major role for the renin-angiotensin system in
the development of fibrosis [5, 6].
Hypertension injures blood vessels and thereby causes end-organ damage. The mechanisms
are complicated and although they have been studied for decades in experimental animal
models [7], they are only currently being elucidated. From the efforts of many investigators,
we are now in the position of constructing a chain of events from the endothelium to the
underlying matrix, to the vascular smooth muscle cells, and beyond to the adventitia, and
surrounding tissues. The endothelial layer acts as a signal transduction interface for
hemodynamic forces in the regulation of vascular tone and chronic structural remodeling of
arteries [8]. Infiltration of the permeabilized endothelium by leukocytes sets the stage for an
inflammatory cascade, involving cytokines, chemokines, growth factors, and matrix
metalloproteinases. Altered integrin signaling, the production of tenacin, epidermal growth
factor signaling, tyrosine phosphorylation, and activation of downstream pathways
culminate in vascular smooth muscle cell proliferation [9]. Evidence is accumulating that
matrix molecules provide an environment which decreases the rate of programmed cell
death [10].
Chronic Kidney Disease
176
Hypertension is a major risk factor for renal and cardiac damage, however, the mechanisms
are incompletely understood. Angiotensin (Ang) II, the key effector of the local and
circulating renin-angiotensin system (RAS), plays a central role [11-12]. In addition to its
vasoactive and growth-promoting action, Ang II stimulates circulating leukocytes and
endothelial cells, thereby promoting inflammation and interstitial extracellular matrix
accumulation [13-17]. Many inflammation-mediating genes are activated by the
transcription nuclear factor-B (NF-B), which resides inactive and bound to the inhibitory
protein I-B in the cytoplasm of T lymphocytes, monocytes, macrophages, endothelial cells,
and smooth muscle cells [18-19]. Ang II stimulates NADPH oxidase, which generates
reactive oxygen species (ROS) [20]. ROS may act as signal transduction messengers for
several important transcription factors, including NF-B and AP-1 (activator protein-1) [21].
Recently, Ozes et al [22]showed that Akt/protein kinase B (Akt) is essential in tumor
necrosis factor- (TNF-)induced activation of NF-B. Takahashi et al, [23] as well as
Ushio-Fukai et al, [24] have demonstrated Akt activation by Ang II, which may involve
ROS. Akt-induced activation of NF-kB upregulates numerous genes, including interleukin
(IL)-1, IL-6, IL-8, interferon-, TNF-, intercellular adhesion molecule-1 (ICAM-1), vascular
cell adhesion molecule-1 (VCAM-1), and the chemokine MCP-1 (monocyte chemoattractant
protein-1). Several reports [25-27] indicated that angiotensin converting enzyme (ACE)
inhibition decreased NF-B in renal disease.
We have previously demonstrated that traditional Chinese medicine prescription
documented in the ancient Chinese pharmacopoeia or monographs promoted blood
circulation, decreased blood stasis, and improved renal function. They decreased urinary
protein excretion ,balanced lipid metabolism and enhanced the effects of antioxidant in the
treatment of patients with early and middle stage chronic renal failure [28-32].
It has been showen broad foreground to postpone progression of chronic renal dysfunction.
But it is unclear that effective composition and mechanism of renal protection. Therefore,
the study presented here was designed to test the hypothesis that HE-86 liquid extract,
which is effective unite refined from above Chinese prescription, would prevent chronic
renal failure rats induced by nephrectomized, in association with decreased expression of
angiotensin II and AT- II receptors, further to suppress high expression of inflammatory and
growth factors. In an attempt to obtain more effective renal protection, research design
consisted of a group of Nx rats receiving a HE-86 liquid extract treatment comparing with
chronic renal failure rats induced by subtotal nephretomized without treatment. At same
time, in the present study, we also assess the influence of renal mass reduction (RMR)
caused by subtotal (5/6) nephrectomy on gene expression for NF-B, TNF- and TGF-beta1
and evaluate the correlation between expression of these genes and activity of the intrarenal
renin-angiotensin systems. The research result showed HE-86 played a critical role in
improving renal disease and was a key mediator in delay process of vascular fibrosis,
characterized by reduced lumen diameter and arterial wall thickening attributable to
excessive deposition of extracellular matrix (ECM) through by the model study.
2. Materials and methods
2.1 Experimental design
Thirty-six of the normal kidney mass were removed from adult male Munich-Wistar rats
(BiKai, Shanghai, China) weighing 200210 g to make animal models of CRF. In a first
Molecular Mechanisms of Nephro-Protective
Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure
177
session, two thirds of the left kidney were removed. One week after the first operation, the
right kidney was removed. These procedures were performed under anaesthesia with
sodium pentobarbital (The ShuGuang pharmaceutical factory in Shanghai). Two weeks after
5/6-nephrectomy, 24 rats were divided into pairs such that both rats in each pair exhibited
almost the same levels of serum creatinine, blood urea nitrogen (BUN) (Table 1). One rat
from each pair was assigned to (i) control uraemic group (n=12), the other to (ii) treatment
uraemic group (n=12) which received HE-86, extract liquid which is effective composition
isolated from Chinese medicine prescription, everyday at a dose of 0.75 g/100 g body
weight for 8 weeks. For normal controls, rats underwent a sham operation consisting of
laparotomy and manipulation of the renal pedicles but without damage to the kidney(n=12).
The treatment group were administered by HE-86 infuse the stomach as pair-fed with the
control uraemic rats, and the normal rats were fed ad libitum with standard solid chow
(BiKai Animal Lab. Company, Shanghai, China) containing 24.5% protein.
N BUN(mmol/L) Scr(mol/L)
sham 12 7.510.75 19.004.00
control 12 16.170.99* 49.506.53*
treatment 12 16.182.42* 49.239.36*
Table 1. The variation of serum creatinine and blood urea nitrogen before treatment.
Blood pressure was measured before treatment and every two weeks after surgery. The
levels of serum creatinine (Scr), Blood urea nitrogen (BUN), 24h urine protein excreation
and urine TGF were determined at 4 or 8 weeks after starting the administration of HE-86,
respectively. The remnant kidneys were removed after perfusion at the end of experiment
for histopathological and gene expression studies.
2.2 Analytical procedures
Renal Function Assessment and Blood Pressure Measurement
Serum creatinine (Scr) and Blood urea nitrogen (BUN) were measured using a Beckman Cx4
analyser (Fullerton, CA, USA), respectively.
24h Urinary protein concentrations were determined by the Bradford method, adapted to a
microtiter plate assay. Coomassie reagent (USB, Cleveland, OH) was added to the diluted
urine samples. After 10 minutes, the absorbance at 595-nm wavelength was read on ELX800
microplate reader (Bio-Tek Instruments, VT). The protein concentrations were calculated by
reference to bovine serum albumin (Sigma) standards.
Systolic blood pressure was recorded by tail plethysmography using the BP2000 blood
pressure analysis system (Visitech Systems, Inc., Apex, NC) in conscious rats at baseline and
every 2 weeks throughout the experimental time course.
Chronic Kidney Disease
178
2.3 Immunohistochemical analysis
Immunostaining of NF-b (Sigma) in renal tissue sections was performed using the
streptavidinbiotinylated peroxidase complex (SABC) method. The tissue specimens were
divided into thin sections (4-m thick) that were then deparaffinized. The sections were
washed three times with distilled water for 5 min. The sections were treated with Protease K
(Try box produced by BSD living creature technique company of Wuhan) in distilled water
at 37C for 15 min, and washed three times with PBS for 10 min. Endogenous peroxidase
activity was blocked by incubating the sections with 0.3% H2O2 in methanol for 20 min at
room temperature. The sections were washed three times with PBS for 5 min. The sections
were incubated with 10% rabbit serum at 37C for 60 min to reduce the non-specific
background staining, and washed three times with PBS for 5 min. Then, the sections were
incubated with a monoclonal anti- NF-b antibody (7 g/ml) dissolved in PBS containing
3% BSA and 0.1% NaN3 at 4C overnight, and washed three times with PBS for 10 min;
followed by incubation with a biotinylated rabbit antibody against mouse IgG+IgA+IgM (10
g/ml) at 37C for 40 min. The sections were washed three times with PBS for 5 min, and
then incubated with peroxidase-labelled streptavidin at 37C for 30 min. After washing
three times with PBS for 10 min, the reaction was completed by the addition of
diaminobenzidineH2O2 solution for 15 min, and washed three times with distilled water
for 5 min, then the slides were counter-stained with methylgreen.
The primary anti- NF-b antibody (1 : 100) was incubated with NF-b (10 mg/ml) at 4C
overnight. After centrifuging the mixture at 10,000xg for 30 min, the supernatant was used
as negative control for the primary antibody solution followed by the usual SABC method.
There was no positive staining in the renal cortex when the primary antibody was pre-
incubated with NF-b.
The immunostaining of NF-b was quantified using an image analyser IMS (FUDAN
university of medical science portrait examination center) by evaluating the positively
stained area of the sections under the same light intensity for microscopy. The intensity of
colour component for red, green or blue was graded from 0 to 256. Areas which showed
intense brown color were extracted from the microscopic fields (number of fields for each
tissue sample, six fields; magnification on the display: x300) under the following conditions;
red component ranging from 104 to 158, green component from 81 to 129, and blue
component from 70 to 123.
3. Real-time quantitative Polymerase Chain Reaction (PCR) for TNF, Ang II
and AT1R
To investigate the expression of TNF mRNA, Ang II and AT1R real-time PCR (BC living
creature technique company, Shanghai, China) was performed with the Opticon real-time
PCR machine (FX scientific research Inc. Shanghai, China). Briefly, total RNA was extracted
from renal tissues. All of the RNA samples were treated with the RNase-free DNase I
(GIBCO BRC Inc, Shanghai, China) before the RT-PCR. Real-time quantitative one-step RT-
PCR assay was performed to quantify mRNA using real-time PCR machine (FX scientific
research Inc. Shanghai, China). The primers used for real-time RT-PCR were as follows:
TNF-a: forward 5-CTCATTCCCGCTCGTGG-3 reverse 3-CGTTTGGTGGTTCGTCTCC- 5;
Molecular Mechanisms of Nephro-Protective
Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure
179
AT1R: forward 5-CTTGTTCCCTTTCCTTATC -3reverse 3-ACTCCACCTCACTGTCCA -
5. Ang II : forward 5'- ACCTG CATGA GTGTT GATAGG-3' reverse 3'-ACTTCA ATATC
GTCAGT AACTGGAC-5'.
Total RNA of osteoblasts was isolated by using TRIzol reagent (Invitrogen) and reverse
transcription was performed follow manufacturers manual(BioTNT, Shanghai, China).
Quantitative real-time PCR, enabling the quantification of relative gene expression, was
performed using SYBR green DNA binding fluorescent dye. 10 L of QuantiTect TM
SYBR Green PCR Master Mix, 4 L of QuantiTect TM SYBR Green primer assay
(osteocalcin, b-actin; all provided by BioTNT), 5 L of RNase free water and 1 L of cDNA
(1 ng/L) were used for one reaction. Quantitative real-time PCR was performed in
triplicates with the following cycler program: 95C 10 min, denaturation step: 95C 15 s,
annealing step: 60C 15 s, elongation step: 72C 30 s; dissociation: 95C 15 s, 60C 1min,
95C 15 s, 40 cycles were performed in total. B-actin was taken as an endogenous standard
and relative gene expression was determined using the Ct method. Gene expression
was compared by setting control cultures to 1 (reference value) as indicated in the
relevant figures.
Quantitative analyses of TNF, , Ang II and AT1R expression were performed using a
quantitative image analysis system (FR-2000,FR Science and technology Inc, Shanghai
China). Because the pattern of expression of TNF, Ang II and AT1R are diffuse in nature,
the percentage of positive staining in the renal tissue was quantified under a 20 power
field of microscope. Briefly, up to 10 random areas of kidney with the early stage
(media:intima 1) and advanced stage (media:intima <1) were chosen from each tissue
section and examined. The examined area was outlined, the positive staining patterns were
identified, and the percent positive area in the examined area was then measured. Data were
expressed as the percentage of meanSEM.
4. Characterization of monoclonal anti-TGF antibody
The reactivity of the produced monoclonal antibodies with Urine TGF was screened by
enzyme-linked immunosorbent assay (ELISA) using kit produced by Section living creature
technique limited company of Hangzhou, China (NO,13409007) The sample solution (40 l)
was incubated with the monoclonal anti- TGF antibody (40 l) at room temperature for 1
h in an TGFtransferrin attached microplate. After washing with phosphate-buffered
saline (PBS) containing 0.05% Tween 20, 0.1 ml of peroxidase-labelled goat F(ab')2 fragment
to mouse IgG(Fc) was added into the microplate, followed by incubation at room
temperature for 1 h. After washing with PBS containing 0.05% Tween 20, 0.2 ml of o-
phenylenediamine hydrochloride (1 mg/ml) containing 0.0124% H2O2 was added to the
microplate, and then incubated at room temperature for 30 min. The reaction was
terminated with 1.3 M H2SO4. The absorption at 492 nm was measured.
4.1 Statistical analysis
Data obtained from this study are expressed as the means SEM. Statistical analyses were
performed using GraphPad Prism 3.0 (GraphPad Software, Inc., San Diego, CA). Differences
in blood pressure, serum creatinine, blood urea nitrogen, 24h urine protein and Urine TGF
at different time points (weeks 0 to 8) within the groups, and differences of Ang II and
Chronic Kidney Disease
180
AT1R activation, TNF expression and NF-b accumulation in sham, control and HE86-
treated animals were assessed by one-way analysis of variance, followed by t-test. Results
were considered statistically significant when the P value was <0.05.
5. Result
Renal and systemic parameters obtained at 0 (before treatment), 32and 64 days after Nx are
given in Table 1-5, Figure 1-5. Nx groups exhibited limited growth compared with Sham. In
all Nx groups except treatment group, body weights were statistically different from those
observed before treatment. Average food intake was similar among groups.
6. Effects of HE-86 administration on biochemical parameters in uraemic rats
Table2-3 shows the summary of renal function and 24h urine protein level. There was
significant change in body weight between the control uraemic (control) and HE-86 treated
uraemic (treatment) rats, although they were pair-fed. body weight of treatment group was
showed more than control uraemic. Even 4 weeks after 5/6-nephrectomy, the levels of
serum creatinine and BUN were markedly increased as compared to sham rats. Not only at
4 week but also at 8 week, the uraemic rats treated with HE-86 were manifested significantly
decreased levels of serum creatinine, BUN, respectively. Urinary protein excreation was also
suppressed obviously at 8 week as comparing with control uraemic rats.
N BUN(mmol/L) Scr(mol/L)
sham 12 6.790.70 26.251.04
control 12 12.093.37 50.5615.83
treatment 12 9.812.93 38.8312.00
#
Table 2. Serum creatinine and blood urea nitrogen after 4 week treatment.
#
P<0.05,
##
P<0.01,when compared against empty vector-treated controls
N BUN(mmol/L) Scr(mol/L) 24h urine protein(mg)
sham 12 9.311.05 18.881.55 22.344.4
control 12 14.852.83 53.3812.05 41.478.07
treatment 12 13.622.81 41.0010.51
##
29.145.68
##
Table 3. Serum creatinine, blood urea nitrogen and twenty-four-hour urinary protein
excretion after 8 week treatment.
#
P<0.05,
##
P<0.01,when compared against empty vector-
treated controls
7. Effects of HE-86 administration on mean arterial blood pressure in uraemic
rats
After subtotal nephrectomy, hypertension developed in both HE-86 treatment and control
uremic rats. Blood pressure was significantly elevated from second to eighth week after
nephrectomy compared to sham-operated animals (P < 0.05-0.01), and the rise in blood
pressure was equivalent (systolic blood pressure 180 to 200 mmHg) in control group. After
using HE-86 liquid extract, hypertension was obviously suppressed in treatment group,
showing average systolic blood pressure 140 to 160 mmHg (Table 4).
Molecular Mechanisms of Nephro-Protective
Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure
181
Before
treatment
After treatment
Second week Forth week Sixth week Eighth week
sham 137.3114.72 139.1314.06 125.507.15 150.5613.97 129.6329.16
control 140.5023.55* 212.4643.26 199.9223.55 156.3320.72 202.4415.09
treatment 141.7726.45* 148.5038.82
152.4629.54
141.0014.73
176.0030.70
Table 4. Systolic blood pressure. Data represent the means SEM for groups of twelve rats
treated with either HE-86 or empty vector (
#
P<0.05,
##
P<0.01,when compared against empty
vector-treated controls;*P<0.05,**P<0.01, when compared to normal sham-controls).
8. Effects of HE-86 administration on urine TGF1
High excreation of urine TGF1, which express both glomerular and tubulointerstitial
injuries. To demonstrate further the anti-inflammatory effect of HE-86 on rat chronic renal
failure, we determined the TGF1 levels within the urine by ELISA. Results demonstrated
that compared with vehicle, He-86 treatment significantly reduced urinary TGF1 levels,
corrected by decrease level of serum creatinine, throughout the entire disease course (P<0.05),
indicating that HE-86 treatment may primarily suppress the local immune and inflammatory
response within the diseased kidney. In contrast, overexpression of urine TGF1 was found
in control uraemic rats as compared with normal rats (Table 5). The experimental result
showed the administration of HE-86 significantly inversed high expression of urine TGF in
uraemic rats, manifesting HE-86 to attenuate the development of glomerular sclerosis.
N Urine TGF(ug/L)
sham 12 1.830.64
control 12 1.900.56*
treatment 12 1.770.43
#
Table 5. Effect of HE-86 liquid extract on urine TGF excretion in 5/6 nephrectomy in
rats. (
#
P<0.05,
##
P<0.01,when compared against empty vector-treated controls; *P<0.05,
**P<0.01, when compared to normal sham-controls)
9. Effects of HE-86 administration on localization of NFB in renal tissue
Immunohistochemical analysis was performed to determine the localization of NFB in the
renal cortex (Fig.1-2). NF-B, a critical transcriptional factor for controlling inflammatory
response, has been shown to play a central role in inflammatory diseases, including kidney
diseases [33]. In normal rats, only tubular epithelial cells were weakly stained by the
monoclonal anti-NFB antibody, while glomeruli were hardly stained. In control uraemic
rats, however, proximal tubular epithelial cells, especially of dilated tubules, were
intensively stained by the anti-NFB antibody. In contrast, in the HE-86-treated uraemic
rats activation of the NF-B in tubular epithelial cells was less prominent as compared with
that in the control uraemic rats. The staining of NFB as shown in the control uraemic rats
found increased NFB -positive (intensively stained) area in the renal cortex, whereas HE-
86-treated rats showed markedly decreased NFB -positive area as compared to the control
uraemic rats. These data demonstrate that HE-86 markedly reduces the overexpress of NF
B on the remnant tubular cells.
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182
A B
C
D
E
F
Fig. 1. Immunohistochemistry demonstrates that HE-86 inhibits renal NFB accumulation
within the kidney. The accumulation of NFB in the glomerular and tubulointerstitium is
markedly increased in empty vector-treated animals (C, D), compared to normal sham-
controls (A,B), which is substantially inhibited in 5/6 nephrectomized rats treated with HE-
86 (E, F). Original magnifications, x100.
Molecular Mechanisms of Nephro-Protective
Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure
183
Sham group Control group Treatment group
0
2
4
6
8
10
12
A
B
C
*
Fig. 2. Semiquantitative analysis of the therapeutic effect of HE-86 on NFB localization in
the glomerulus and tubulointerstitium using the Quantitative Image System. A: Percentage
of glomerular and tubulointerstitial NFB deposition in sham group. B: Percentage of NF
B localization in glomerular and tubulointerstitial without treatment C: Percentage of
glomerular and tubulointerstitial NFB accumulation in twelve rats treated with HE-86
was decreased significantly. Each bar represents data (mean SEM) #, P < 0.05 and ##,
P < 0.001, when compared to empty vector-treated controls; *, P < 0.05 and **, P < 0.01, when
compared to the normal sham-control.
10. Effects of HE-86 administration on mRNA levels of TNF, Ang II and AT II
R in renal tissue
The effects of HE-86 on the gene expression of Ang II (Figure 3), AT1R (Figure 4) and
TNF (Figure 5) in the renal cortex were examined. We investigated the potential
Chronic Kidney Disease
184
mechanisms whereby HE-86 suppressed rat tubular interstitial fibrosis and glomerular
cirrhosis. TNF-, being key proinflammatory cytokines in anti-GBM glomerulonephritis,
and a group of chemotactic and adhesion molecules including ICAM-1, MCP-1, was
examined. In vehicle-treated chronic renal failure rats, there was a substantial increase in
renal mRNA expression of TNF-, Treatment with HE-86 significantly reduced
upregulation of TNF- inflammatory genes examined (P<0.05). Furthermore, HE-86 was
capable of attenuating renal cortical mRNAs for Ang II and AT1R as compared with the
control uraemic rats when they were administered after the establishment of
nephrectomized. However, the renal mRNA levels of Ang II and AT1R were markedly
increased in control uraemic rats as compared with normal rats. The variation in the
mRNA levels of TNF, Ang II and AT1R in both HE-86-treated and control uraemic rats
are related to variation in the extent of CRF.
Sham group Control group Treatment group
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
A
B
C
*
Fig. 3. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal Ang II
mRNA expression(A). and Semiquantitative analysis of the therapeutic effect of HE-86 on
Ang II mRNA localization in the glomerulus and tubulointerstitium using the FR-2000
Image Analyze System. A: Degree of glomerular and tubulointerstitial Ang II mRNA
expression in sham group. B: Numbers of Ang II mRNA expression in glomerular and
tubulointerstitial without treatment C: Numbers of glomerular and tubulointerstitial cells
with nuclear localization of Ang II mRNA in twelve rats treated with HE-86 was decreased
significantly. Each bar represents data (mean SEM) , P < 0.05 and , P < 0.01, when
compared to empty vector-treated controls; *, P < 0.05 and **, P < 0.01, when compared to
the normal sham-control.
Molecular Mechanisms of Nephro-Protective
Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure
185
Sham group Control group Treatment group
0
0.5
1
1.5
2
2.5
A
B
C
*
Fig. 4. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal
AT1RmRNA expression(B). and Semiquantitative analysis of the therapeutic effect of HE-86
on AT1RmRNA localization in the glomerulus and tubulointerstitium using the FR-2000
Image Analyze System. A: Degree of glomerular and tubulointerstitial AT1RmRNA
expression in sham group. B: Numbers of AT1RmRNA expression in glomerular and
tubulointerstitial without treatment CNumbers of glomerular and tubulointerstitial cells
with nuclear localization of AT1RmRNA in nephrectomized rats treated with HE-86 was
decreased significantly. Each bar represents data (mean SEM) , P < 0.05 and , P <
0.01, when compared to empty vector-treated controls; *, P < 0.05 and **, P < 0.01, when
compared to the normal sham-control
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186
Sham group Control group Treatment group
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
A
B
C
*
Fig. 5. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal
TNFmRNA expression(C). and Semiquantitative analysis of the therapeutic effect of HE-
86 on TNFmRNA within the glomerulus and tubulointerstitium using the FR-2000
Image Analyze System. A: Degree of glomerular and tubulointerstitial TNFmRNA
expression in sham group. B: Numbers of TNFmRNA expression in glomerular and
tubulointerstitial without treatment C: Numbers of glomerular and tubulointerstitial cells
with nuclear localization of TNFmRNA in nephrectomized rats treated with HE-86 was
decreased significantly. Each bar represents data (mean SEM) , P < 0.05 and , P <
0.01, when compared to empty vector-treated controls; *, P < 0.05 and **, P < 0.01, when
compared to the normal sham-control.
11. Discussion
Renal fibrosis is a final common pathway to end-stage renal disease. Recent studies have
shown that hypertensive nephropathy is a major leading cause of end-stage renal disease
and the renin-angiotensin system plays a pivotal role in the development of progressive
renal injury [34-35]. Clinical trials have shown that blocking the effects of angiotensin II
(Ang II) with angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers
Molecular Mechanisms of Nephro-Protective
Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure
187
can prevent or slow the progression of kidney damage in patients with diabetes and
hypertension [34-36].
As expected, 5/6 renal ablation promoted growth retardation, systemic arterial
hypertension, impaired renal function, and severe albuminuria. These functional changes
were accompanied by severe glomerulosclerosis, as well as expansion and intense
macrophage infiltration of the interstitial area. Mounting evidence indicates that these
renal structural abnormalities, which are characteristic of the Nx and other models of
progressive nephropathies, are a consequence of the concerted action of mechanical stress,
caused by glomerular hypertension and hypertrophy [37-38], and inflammatory
phenomena, comprising cell infiltration and/or proliferation and extracellular matrix
accumulation [38-39]. Moreover, a causal relationship appears to exist between these
phenomena, because the distension of the glomerular walls due to intracapillary
hypertension may trigger the local release of cytokines, growth factors, and, particularly,
Ang II and AT-1 receptors [40-41].
The beneficial effect of RAS suppressors was initially attributed to amelioration of the
glomerular hemodynamic dysfunction associated with progressive nephropathies.
However, recent observations suggest that the nonhemodynamic effects of RAS suppressors
may be equally important, given the strong proinflammatory and profibrotic effects of Ang
II [42]. A substantial fraction of this proinflammatory ANG II may originate in the renal
parenchyma, rather than in renal vessels or in the systemic circulation [43]. Increased
intrarenal production of ANG II was described in various models of renal fibrosis [44-46]. A
preliminary report has suggested that, in the 5/6 renal ablation (Nx) model, ANG II is
expressed in renal interstitial cells, paralleling the severity of renal injury [47].
Increasing evidence shows that angiotensin II (Ang II) plays a critical role in cardiovascular
disease and is a key mediator in the process of vascular fibrosis, characterized by reduced
lumen diameter and arterial wall thickening attributable to excessive deposition of
extracellular matrix (ECM). Vascular fibrosis is a major complication of hypertension and
diabetic mellitus. It has been shown that upregulated tissue rennin-angiotensin system is
involved in development of vascular lesions in both human and experimental vascular
diseases [48-49]. This observation is confirmed by the finding that infusion of Ang II is able
to induce vascular fibrosis in rats [50]. The functional importance of Ang II in vascular
fibrosis is further supported by the evidence that blockade of Ang II inhibits vascular
fibrosis in diabetic and subtotal nephrectomy rats and NO-deficient mice [51-53].
Both the hemodynamic and proinflammatory effects of Ang II are mediated by AT-1
receptors (AT1R) [54], extensively expressed in renal tissue. In the normal rat kidney, AT1R
are predominantly expressed in tubular cells and vessels [55]. Recent data obtained with the
Nx model have suggested that AT1R expression is shifted from the glomerular to the
tubulointerstitial compartment 4 wk after ablation [56]. However, the renal distribution of
AT1R in this model and its temporal evolution have not been established.
Beyond its hemodynamic effects, Ang II is recognized as a cytokine with an active role in
cardiovascular remodeling. It is well known that Ang II signals through its Ang II receptor 1
(AT1) receptor to exert most of its biological functions [57]. After binding to the AT1
receptor, Ang II activates multiple downstream intracellular signaling pathways, including
tyrosine kinase, mitogen-activated protein kinase (MAPK), p38, and Janus family kinase
Chronic Kidney Disease
188
[58]. Activation of these pathways leads to numerous heterogeneous downstream events
that play essential roles in the biological activities of Ang II, such as cell growth and
migration, ECM production, and apoptosis [58].
Renal expression of AT1R in rats appeared mostly in tubular cells, and to a lesser extent, at
the interstitial area, whereas weaker expression was seen in vessels and glomeruli. This
pattern was completely disrupted after Nx, when dense AT1R expression could be
demonstrated in interstitial cells, far exceeding in intensity the expression of AT1R in
tubules. The exact meaning of this finding and the cell types involved are uncertain. Several
inflammatory cells known to infiltrate the renal interstitium in the Nx model have the
potential to express AT1R, such as lymphocytes [59] and macrophages [60]. In addition,
AT1R may be expressed by myofibroblasts originating from tubular cell transdifferentiation
[61]. This hypothesis is particularly attractive because it helps to explain the progressive
shift in AT1R expression, from tubules to the interstitial area, observed in Nx rats, and also
because tubular cells already express AT1R under normal conditions. The simultaneous
presence at the interstitial area of large amounts of Ang II and of the AT1R may accelerate
the progression of the nephropathy by a positive-feedback mechanism. Consistent with this
view is the aggravation of the renal structural injury of Nx, which was paralleled by the
intensity of the inflammatory infiltration and of the interstitial expression of Ang II.
It is well accepted that NF-B is a key transcriptional factor to regulate a variety of
inflammatory responses [75]. NF-B is composed of p50 and p65 subunits, among which
p65 is a potent transcriptional activator, strongly promoting inflammatory reaction in
kidney diseases [76]. NFB total protein expression, and inflammation, which may have
resulted from blockade of the oxidative stress pathway [77-78]. This was accompanied by a
substantial attenuation in renal fibrosis, which might have resulted from the modulating
actions of vitamins on lipid peroxidation and profibrotic activity involved in renal tissue
damage [79-82]. In this study, marked activation of NF-B was closely correlated with the
renal inflammation. In our study, using liquid extract isolated from clinical effective Chinese
prescription, we were able to show that overexpression activation of NF-B was
substantially suppressed as compared with control group. These findings are consistent
with the improving renal function and correcting high blood pressure.
Tumour necrosis fator-(TNF-)is a potent pro-inflammatory cytokine which is produced
by many cell types including monocytes/macrophages, and renal mesangial and epithelial
cells. It induces the expression of major histocompatibility complex (MHC) class I and II
molecules, endothelial adhesion molecules and procoagulant activity of endothelium. TNF-
stimulates the release of other pro-inflammatory cytokines, chemokines and growth
factors, including interleukin-1(IL-1), monocyte chemoattractant protein-1 (MCP-1) and
transforming growth factor-(TFG-) [83-84]. The biological effects of TNF- are mediated
by binding to specific receptors which are widely distributed. TNF- binds to two types of
receptor: TNF receptor type 1 and TNF receptor type 2, which have molecular weights of 55
kDa (p55) and 75 kDa (p75), respectively. Both receptors are necessary and act
synergistically for cell proliferation and maturation, cytotoxicity and antiviral activity, but
p55 is responsible for activation of NFB and mediation of apoptosis [85].
TNF- may contribute to renal damage by inciting an inflammatory response within the
kidney via induction of a variety of chemokines and adhesion molecules [86-87]. There is a
Molecular Mechanisms of Nephro-Protective
Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure
189
mounting evidence to implicate TNF- in the pathogenesis of glomeruli of rodents with
experimental nephritis, and is found in renal biopsies, sera and urine of patients with
different types of glomerulonephritis [88-91]; In vitro and in vivo studies document that
TNF- is produced locally within inflamed glomeruli by mesangial and epithelial cells, as
well as by infiltrating monocytes/macrophages [89,91]; Systemic administration of TNF-
results in glomerular damage in rabbits [92] and exacerbates the degree of glomerular injury
in nephrotoxic nephritis in rats [93]; and blocking endogenous TNF- in nephrotoxic
nephritis in rats ameliorates acute glomerular inflammation [94], and down-regulates
glomerular IL-1mRNA and circulating TNF- concentrations [95].
Treatment of Nx rats with the HE-86 promoted a significant regression of hypertension,
high level of creatinine and blood urea nitrogen, albuminuria, and inflammatory signs such
as urine TGF- and renal tissue TNF-, NF-B, Ang II and AT1R expression, whereas the
parameters of renal structural tissue injury were strongly attenuated, compared with
pretreatment levels. The protection achieved with effective unit from clinical prescription
treatment was much greater than that obtained with traditional prescription alone. On the
basis of the present study, we cannot exclude the hypothesis that the success of HE-86 was
due to a particularly effective hemodynamic action, although previous observations from
this laboratory [96] indicated that NOF, a new nonsteroidal anti-inflammatory, had no
significant effect on glomerular hemodynamics. Because treatment with NOF alone had no
effect on blood pressure, it seems unlikely that the hemodynamic effect of NOS was directly
intensified by its association with NOF. Therefore, the efficacy of extract HE-86 was likely
due to the simultaneous blockade of the hemodynamic and proinflammatory actions of Ang
II, AT1R and its derivatives as TNF-, NF-B, TGF- and by abrogation of the complex
interplay between hypertension and inflammation. The present findings support other
scholars observations of the Nx model, which similarly indicated the superiority of the
combination of a RAS suppressor with an anti-inflammatory agent [97-99]. It is noteworthy
that HE-86 afforded partial regression of the nephropathy associated with Nx even though it
was started 4 week after surgery, when renal injury was already established. This
observation suggests that both continued stimulation of Ang II and AT1 receptors and
production of inflammatory factors continue to play an important pathogenic role even
during the late phases of the process, necessitating vigorous and persistent treatment to
prevent further renal deterioration.
Taken together with our previous data and the present results, it is likely that HE-86-
induced reduction of renal rennin-angentensin system is mediated, at least partly, by
reducing the overload of inflammatory factors activity on remnant kidney unit. In summary,
HE-86effective composition coming from clinical validly treating patients with chronic renal
failure especially for early and middle stage, partially reversed the nephropathy and renal
inflammation associated with the Nx model, showing much more effective protection than
with traditional Chinese medicine prescription.
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12
The Effects of Asymmetric Dimethylarginine
(ADMA), Nitric Oxide (NO) and Homocysteine
(Hcy) on Progression of Mild Chronic Kidney
Disease (CKD): Relationship Between Clinical
and Biochemical Parameters
A. Atamer
1
, S. Alisir Ecder
2
, Y. Atamer
3
,
Y. Kocyigit
4
, N. Bozkurt Yigit
5
and T. Ecder
6
1
Haydarpasa Training and Research Hospital,
Department of Internal Medicine, Division of Gastroenterology, Istanbul
2
Goztepe Training and Research Hospital,
Department of Internal Medicine, Division of Nephrology, Istanbul
3
Dicle University Medical Faculty, Department of Clinical Biochemistry, Diyarbakir
4
Dicle University Medical Faculty, Department of Physiology, Diyarbakir
5
Yalova University, Termal Vocational School,
Department of Physical Medicine and Rehabilitation, Yalova
6
Istanbul University, Istanbul Medical Faculty,
Department of Internal Medicine, Division of Nephrology, Istanbul,
Turkey
1. Introduction
Chronic kidney disease (CKD) is a syndrome characterized by the progressive and
irrevocable loss of nephrons due to several diseases. Chronic kidney disease has a varying
spectrum ranging from normal renal function to uremic syndrome. Actually, the stages of
renal failure have interpenetrated each other and it is not possible to draw a clear line
between them. The most important reason of mortality and morbidity of patients with CKD
are cardiovascular diseases and atherosclerotic complications; cardiac insufficiency 15%,
myocardial infarction 10%, pericarditis 3% (1, 2). Development of vascular injury in CKD is
caused by both classic (Framingham) risk factors (hypertension, dyslipidemia, smoking,
diabetes mellitus) and CKD specific factors (anaemia, secondary hyperparathyroidism etc).
Besides, there are papers reporting that recently defined potential risk factors such as
homocysteine (Hcy), C-reactive protein (CRP), interleukin-6 (IL-6), fibrinogen, soluble
intracellular adhesion molecule (sICAM-1), asymmetric dimethyl arginine (ADMA), cardiac
specific troponin-I (cTnI), advanced glycation endproducts have a role in the development
of accelerated atherosclerosis seen in patients with CKD (2-13). Asymmetric
dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO)
synthase and it is a guanidine analogue of L-arginine aminoacid detectable in human urine
Chronic Kidney Disease
198
and plasma synthesized from endothelial cells (Figure 1). It is shown that high ADMA level
increases the cardiovascular incident risk by 34% and mortality risk by 52% (4-8). Increased
ADMA concentration has a high prevalence in hyperhomocysteinemia, coronary artery
diseases, hypercholesterolemia, diabetes mellitus, hypertension, preeclampsia, peripheral
arterial occlusive disease, impaired renal function and other diseases (7,9,10). Reduced nitric
oxide (NO)-dependent vasodilation is regarded as an early indicator of atherosclerotic
diseases (7,14). It is documented that adult patients with renal failure have 2-6 times higher
ADMA than healthy subjects due to reduced renal excretion and reduced enzymatic
degradation (15). NO is synthesized from L-arginine via NO synthase enzyme. NO
inhibition decreases endothelial derived vasodilation and increases vascular resistance.
Reduced NO availability can occur in patients with CKD. Moreover CKD can contribute to
the accelaration of hypertension and cardiovascular complications. It appears that the
increase in endogenic NO inhibitors like ADMA plays a major role in this process (11, 15-
17). It has been shown that Hcy stimulates ADMA formation and plasma ADMA levels
elevate in humans and animals by hyperhomocysteinemia (18-20). Increased serum Hcy
level in adult CKD patients is an independent risk factor for cardiovascular system
mortality. Elevated ADMA and hyperhomocysteinemia may be due to decreased renal
excretion (18-22). It is reported that ADMA formation may be related with Hcy metabolism
(18,19). It was found that there is a significant interaction of serum fibrinogen and CKD with
respect to risk of both fatal/nonfatal coronary events and death (2024).
Fig. 1. Biochemical pathway for generation and degradation of ADMA and homocysteine.
The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy)
on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and...
199
The aim of this study was to investigate the role of uremia-related cardiovascular risk
factors, such as ADMA, NO, Hcy and fibrinogen, in the pathogenesis and progression of
early stage CKD and to evaluate the relation of these parameters with each other.
2. Material and methods
2.1 Subjects
This prospective study was carried out in 65 untreated mild chronic kidney disease (35
men and 30 women; mean age 55.2 9.6 years) and 65 healthy control subjects with
matched age, sex and body mass index (BMI). The creatinine clearance was calculated by
the Cockcroft-Gault Formula (25). Patients having creatinine clearance less than 75 ml/min
were considered to have mild CKD. Body mass index was determined as weight divided
by the square of height (kg/m
2
). The underlying causes of CKD were glomerulonephritis
(n=17), interstitial nephropathy (n=12), autosomal dominant polycystic kidney disease
(n=13), chronic pyelonephritis (n=7) and urological problems (n=5). No cause was
identified in 11 cases. The exclusion criteria were diabetes mellitus, active hepatitis,
malignancy, smoking and infectious disease. Patients using vitamin supplements were also
excluded.
The study protocol was approved by the Ethics Committee of the Dicle University School of
Medicine (Diyarbakir, Turkey) and written informed consent was obtained from each
participant.
2.2 Methods
In all patients, venous blood samples were drawn between 7:00 AM after a 12-h fastened,
and the serum was frozen at -70 C in aliquots until biochemical analysis were performed.
ADMA Measurement: ADMA was measured by HPLC according to the method described by
Chen et al. (26). Mobile phases consisting of 50 mM sodium acetate (pH 6.8), methanol and
tetrahydrofuran (THF) (A, 82:17:1; B, 22:77:1) were used. All separations were performed at
27
0
C and at a flow-rate of 1.0 ml/min. The wavelengths of fluorescence detector were set at
338 nm and 425 nm for excitation and emission, respectively. 20 mg of 5-sulfosalicylic acid
(5-SSA) was added to 1 ml plasma, and the mixture was left in an ice bath for 10 min. The
precipitated protein was removed by centrifugation at 2000 g for 10 min. o-
Phthaldialdehyde (OPA) (10 mg) was dissolved in 0.5 ml of methanol, and 2 ml of 0.4 M
borate buffer (0.4 M boric acid adjusted to pH 10.0 with potassium hydroxide) and 30 l of
mercaptoethanol were added. The derivatization was performed by mixing 10 l of sample
or working standard solution and 100 l of OPA reagent and reacting for 3 min before
autoinjecting onto the column.
NO Measurement: The serum level of NO was measured using a colorimetric method based
on the Griess reaction (27), in which nitrite is reacted with sulphanilamide and N-(1-
naphthyl) ethylenediamine to produce an azo dye that can be detected at 540 nm. This was
carried out after enzymatic reduction of nitrate to nitrite with nitrate reductase.
Hcy Measurement: Serum level of Hcy was measured using HPLC with fluorescence
detection (Shimadzu RF-10A fluorescence detector; Shimadzu Co., Kyoto, Japan).
Chronic Kidney Disease
200
Urea, creatinine, calcium, phosphate, albumin, protein, high sensitive CRP (hsCRP), insulin,
glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density
lipoprotein cholesterol (LDL-C) and triglyceride assays were determined by standard
laboratory methods according to the established methodology. The serum level of
fibrinogen was measured by the Clauss method using a commercial kit. All routine
laboratory measurements were carried out using certified assay methods.
Statistical analysis of the differences between groups of subjects was performed using the
Kolmogorov-Smirnov and unpaired students t-test or by the Mann-Whitney non-
parametric test as appropriate. Pearsons correlation analyses were performed.
3. Results
Serum levels of ADMA, Hcy, creatinine, LDL-C and hsCRP were significantly (p<0.001)
higher in patients with mild CKD than in healthy controls. Also, systolic and diastolic blood
pressures were increased (p<0.001). There were no significant differences in levels of serum
fasting blood glucose, insulin, total cholesterol, HDL-C, triglyceride, calcium and phosphate
between the mild CKD and healthy controls (P>0.05). Serum NO and creatinine clearance
levels were decreased in patients with mild CKD than in healthy controls (p<0.001). Clinical
and laboratory data are reported in Table 1. In multiple linear regression analysis, ADMA
level was negatively correlated with NO (r = -0.861; p<0.001) as shown in Figure 2A, and
positively correlated with Hcy (r = 0.547; p<0.001, Figure 2B) and fibrinogen (r = 0.704;
p<0.01, Figure 2C). ADMA level was positively correlated with creatinine (r=0.510;p<0.001),
LDL-C (r=0.420;p<0.01), hsCRP (r=0.525;p<0.001), systolic (r=0.375; p<0.001) and diastolic
blood pressure (r=0.410;p<0.001) levels. ADMA level was negatively correlated with GFR
(r=-0.720;p<0.001). Also, serum NO level was negatively correlated with homocystein (r = -
0.390; p<0.001, Figure 3). We found no association between ADMA and HDL-C or other
parameters in either subjects with mild CKD.
4. Discussion
The findings of the present study are as follows: (1) Serum ADMA level is increased in
patients with CKD compared with healthy subjects and is associated with decreased NO
and GFR. (2) Elevation of circulating serum ADMA is associated with increased Hcy and
fibrinogen in CKD patients. (3) Serum NO level as dependent variable was also negatively
correlated with Hcy. Our findings suggested that the ADMA levels can reflect a possible
independent role in CKD pathogenesis. Increased ADMA serum levels cause persistent
renal vasoconstriction and sodium retention, and contributes to the development of high
blood pressure (11). In addition, it might influence NO and GFR levels and affect
atherosclerosis formation.
Several studies suggested that ADMA level can be an independent risk factor for
progression of CKD (3-13). Elevated ADMA reduces bioavailability of NO and induces
endothelial dysfunction and may be involved in the pathophysiology of cardiovascular
disease in CKD (8). ADMA fulfils many of the characteristic features of an uremic toxin
(14,15). Elevation of circulated ADMA, an endogenous inhibitor of nitric oxide synthase, is
an independent risk factor for cardiovascular diseases in predialysis patients with CKD
(5,14,15). High ADMA levels lead to NO depletion, impaired endothelium-dependent
The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy)
on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and...
201
Healthy Subjects
(n=65)
Chronic Kidney Disease
(n=65)
Age (years) 54.9 10.1 55.2 9.6
Number of patients (M/F) 35/30 35/30
Body mass index (kg/m2) 24.90 2.1 24.70 2.6
Systolic BP (mmHg) 110.20 10.4 *128.40 22.4
Diastolic BP (mmHg) 72.20 11.6 *84.40 16.3
Creatinine clearance (ml/min) 90.20 15.1 *52.50 15.3
Urea (mg/dl) 31.50 6.2 *61.30 14.6
Creatinine (mg/dl) 1.20 0.42 *1.61 0.73
Calcium (mg/dl) 8.73 1.2 8.91 1.08
Phosphate (mg/dl) 4.10 1.09 4.09 1.2
Albumin (g/dl) 3.82 0.9 3.94 0.5
Protein (g/dl) 6.40 1.1 6.01 0.3
Glucose (mg/dl) 87.90 16.2 90.10 15.4
Insulin (u/ml) 11.60 2.9 12.04 2.83
Triglyceride(mg/dl) 118.30 20.2 120.10 18.5
Total cholesterol (mg/dl) 184.20 22.1 185.60 19.4
HDL-C (mg/dl) 45.80 12.4 42.02 14.3
LDL-C (mg/dl) 113.20 12.8 *142.12 18.6
hsCRP (mg/dl) 1.914 0.667 *7.048 2.249
Fibrinogen (g/L) 2.835 0.646 *4.5740.521
ADMA (mol/L) 0.512 0.116 *0.8370.189
Nitric oxide (mol/L) 75.67 8.626 *44.317.811
Homocystein (mol/L) 6.256 1.629 *18.37 3.192
*P < 0.001; Data are reported as means SD.
BP: Blood Pressure; HDL-C: High Density Lipoprotein Cholesterol; LDL-C: Low Density Lipoprotein
Cholesterol; hsCRP: High sensitive C Reactive Protein; ADMA: Asymmetric dimethylarginine
Table 1. Clinical and laboratory data of patients with CKD and healthy subjects.
Chronic Kidney Disease
202
(a)
(b)
The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy)
on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and...
203
(c)
Fig. 2. Correlation between asymmetric dimethylarginine (ADMA) and (A) nitric oxide
(NO), (B) homocysteine (Hcy), and (C) fibrinogen.
Fig. 3. Correlation between nitric oxide (NO) and homocysteine (Hcy).
Chronic Kidney Disease
204
vasodilation and plaque rupture with thrombus formation (8). In addition, increased ADMA
level in circulation is a combined result of decreased elimination and reduced activity of
ADMA catabolism by dimethylarginine dimethylaminohydrolase (DDAH) (8,9). Elevated
plasma levels of ADMA in patients with end stage renal disease (ESRD) were first reported
by Vallance et al. (15). Several recent studies have already indicated that elevated plasma
ADMA levels could cause cardiovascular morbidity and mortality in progressive chronic
kidney disease (4-13). Mihout et al. (9) demonstrated that high plasma ADMA levels
contribute to the development of hypertension, oxidative stress, and interstitial and
glomerular fibrosis, and peritubular capillary rarefaction. This may be involved in the
decline of renal function. Serum levels of ADMA in CKD are predictive of renal survival
and of cardiovascular damage. High ADMA levels are associated with endothelial
dysfunction and oxidative stres (12). In the study by Young et al. (8) , there was a strong
association of ADMA with prevalent cardiovascular disease and a modest association with
all-cause and cardiovascular disease mortality. ADMA is strongly associated with intima-
media thickness of the carotid artery and left ventricular mass, particularly concentric left
ventricular hypertrophy (11).
Coen et al. (12) suggested that ADMA levels could be influenced by the severity of
hyperparathyroidsm and contribute to cardivascular death linked to parathyroid hormone
(PTH) of hemodialysis patients. Another study conducted by Shi et al (5) has shown that the
circulating level of ADMA is an important risk factor of LVH and predicts CVD in pre-
dialysis CKD patients.
Selcoki et al. (10) reported that ADMA level was to be one of the strongest risk markers for
atherosclerosis in patients with mild and moderate CKD. Ninety percent of ADMA has been
metabolized by DDAH, while the other small portion, 10 %, is excreted by urinary system.
Potential mechanisms of elevated plasma ADMA levels in renal failure are increased protein
methylation, increased proteolysis, impaired renal excretion and impaired metabolism by
DDAH (18). These results are consistent with data from our study. Our results suggest that
high ADMA level can be a significant risk factor for progression of renal dysfunction in the
earlier stages of CKD.
Several recent studies found markedly elevated plasma ADMA levels not only in patients
with ESRD, but also in patients with progressive CKD (2). It is of note that our results are in
line with a recent study by Nakamura
et al. (28), who found that elevation of serum ADMA
levels play a role in the progression of atherosclerosis and CKD in high-risk patients.
Studies in both the general population and the dialysis population showed a strong and
independent link between ADMA, all-cause mortality, and cardiovascular events
(11,12,21,24). As a consequence, elevated serum levels of ADMA may be of relevance not
only in vascular pathology but also in the pathophysiology of hypertension, and in paralel,
in the development of renal damage (13).
When ADMA accumulates in CKD due to defective inactivation and excretion, it is a factor
of impaired NO synthesis. The decrease in the generation of NO lead to endothelial
malfunction and damage (12). Nitric oxide is an important molecule which has many
physiological functions, such as mediating vasodilation, inhibiting atherosclerosis, and
modulating the growth of the myocardium (5). Nitric oxide is produced from its precursor
L-arginine via a reaction catalyzed by endothelial NO synthase (NOS) (8,9). Endothelium-
The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy)
on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and...
205
derived nitric oxide is a potent endothelial vasodilator which balances constrictors to
regulate blood pressure and vascular tone (9). Leone et al. (35) suggested that NO may play
a role in blood pressure regulation. NO is a cardiovascular protective substance because it
causes vasodilation and leucocyte aggregation (10). Nitric oxide also plays a role in
regulating renal sodium excretion and renin release (30). Nitric oxide, synthesised from L-
arginine, contributes to the regulation of blood pressure and to host defence (29). As an
endogenous vasodilator it contributes to renal arteriolar tone and modulates relaxation of
the mesangium, thus contributing to regulation of glomerular microcirculation. It has
antiplatelet and antithrombogenic effects and thus helps prevent thrombosis within the
glomerular capillaries (30).
Clinical and experimental evidence suggest that the elevation of ADMA may cause a low
production of NO (11,14-17,29,30). Synthesis of NO can be blocked by inhibition of nitric
oxide synthase (NOS) activities with guanidino-substituted analogues of L-arginine such as
ADMA (28). Accumulation of endogenous ADMA, leading to impaired NO synthesis, might
contribute to the hypertension and immune dysfunction associated with chronic renal
failure (29). Reduced bioavailability of NO, increased systemic blood pressure, endothelial
cell injury and dysfunction are thought to play an important role in progressive kidney
damage (7). Endothelial dysfunction due to reduced availability of NO is an early step in the
course of atherosclerotic vascular disease (7). Increased ADMA blood levels may contribute
to this process. In addition, NO inhibits key processes of atherosclerosis, such as monocyte
endothelial adhesion, platelet aggregation, and vascular smooth muscle cell poliferation
(31).
In our study, while serum ADMA and Hcy levels were significantly higher in the patients
with CKD than in healthy subject, the NO level was significantly lower. Our findings
were in agreement with previous studies (7,9,10,18). Low NO is a major feature of chronic
kidney diseases. We examined the relationship of ADMA with NO and with Hcy in CKD
patients. In this prospective study, high ADMA level was associated with both decreased
NO and increased Hcy. Similarly, Strong relationships between increased serum Hcy,
fibrinogen, ADMA and decreased NO, GFR and mortality from cardiovascular events
have recently been demonstrated. Several prospective clinical studies have shown that
ADMA, fibrinogen, Hcy, LDL-C and other cardiovascular risk parameters are effected in
patients with CKD, atherosclerosis, hypertension, diabetes and other clinical entities (14-
18,22).
The major factor for high plasma ADMA levels in renal failure seems to be a decrease
DDAH activity, which in turn may be due to increased oxidative stress and/or
hyperhomocysteinemia (18). Recent studies show contradictory data regarding the role of
hyperhomocysteinemia on cardiovascular morbidity and mortality in CKD patients (32).
Rasmussen et al. (22) suggested that elevated homocysteine level is an independent
predictor of cardiovascular events in patients with ESRD. Ninomiya et al. (33) suggested
that baseline Hcy level showed a significantly inverse association with rate of change in
kidney function during the 5 years after being adjusted for confounding factors, including
baseline kidney function.
One study indicates a linkage between hyperhomocysteinemia, oxidative stress and ADMA
metabolism (32). Recently, it was hypothesized that some of the deleterious effects of
Chronic Kidney Disease
206
hyperhomocysteinemia may involve ADMA-related cardiovascular effect in CKD (18-20).
Hyperhomocysteinemia, elevated plasma ADMA concentrations have first been described
in patients with renal failure (18). Plasma levels of homocysteine and ADMA are elevated in
patients with renal failure and both have been associated with cardiovascular events,
possibly due to their negative effects on endothelial function. ADMA in methylation of
homocystein plays an important role. Elevated homocysteine level is strongly related to
renal function and probably due to decreased metabolic clearance (18-20). Homocysteine
and ADMA are aminoacids which are biochemically linked by a common synthetic
pathway. Homocysteine inhibits DDAH, the enzyme responsible for the breakdown of
ADMA. Homocysteine may enhance protein degradation by destabilizing protein structure
or by increasing oxidative stress, resulting in ADMA release (18).
Contraversely, Simic-Ogrizovic et al. (24) suggested that although total serum Hcy level was
not found to be a predictor of overall and cardiovascular mortality, the role of
hyperhomocysteinemia as risk factor for cardiovascular disease cannot be excluded in
hemodialysis patients.
We found a strong association between ADMA levels and hyperfibrinogenemia, and
hyperhomocysteinemia in our study. In addition, as inflammation index, CRP and
fibrinogen were increased. Our results show that increased ADMA, Hcy, hsCRP and
fibrinogen levels contribute to the progression of renal disease. Serum levels of ADMA and
Hcy may interact and modulate the effect of each other, thus contributing to a common
mechanism leading to cardiovascular diseases in CKD. These findings are similar to
observations from previous studies (18-21).
The level of serum fibrinogen (an inflammation marker) is increased in CKD. Increased
serum fibrinogen level independently predicts cardiac events (20). Shishehbor et al. (19)
suggested that Hcy and fibrinogen levels can explain nearly 40% of the attributable
mortality risk from CKD. Bostom et al. (21) suggested that Hcy, lipoprotein(a) (Lp(a)), and
fibrinogen interact to promote atherothrombosis, combined hyperhomocysteinemia,
hyperfibrinogenemia, and, Lp(a) excess may contribute to the high incidence of vascular
disease sequelae experienced by dialysis patients, which is inadequately explained by
traditional cardiovascular disease risk factors. In our present study, the serum level of LDL-
C was significantly higher in the patients with CKD than in the healthy subjects. In addition,
the ADMA level was positively correlated with LDL-C. The association of increased LDL-C
with increased risk of coronary heart disease may be thought as a covariable in the oxidative
activation of ADMA synthesis.
Descamps-Latscha et al. (23) thought that CRP, fibrinogen and advanced oxidation protein
products (AOPP) levels independently predict atherosclerotic cardiovascular events in
patients with CKD in the predialysis phase and might directly contribute to the uremia-
associated accelerated atherogenesis. These findings lend support to the hypothesis that
accumulation of ADMA is an important risk factor for cardiovascular events in CKD (2).
Our findings suggest that high ADMA, fibrinogen and Hcy levels and NO deficiency may
contribute to the process of atherosclerotic cardiovascular disease and other consequeces of
uremia in predialysis patients with CKD. In addition, the ADMA level was associated with
hyperhomocysteineamia and hyperfibrinogenemia.
The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy)
on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and...
207
5. References
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(eds).Little, Brown and Company, Boston 1994: 604-622.
[2] Zoccali C, Mallamaci F, Tripepi G. Traditional and emerging risk factors in end-stage
renal disease. Kidney Int 2003;63(suppl85):S105-S110.
[3] Busch M, Franke S, Miller A, et al. Potential risk factors in chronic kidney disease: EGEs,
total homocysteine and metabolites, and the C-reactive protein. Kidney Int
2004;66:338-347.
[4] Fujimi-Hayashida A, Ueda S, Yamagishi S, et al. Association of asymmetric
dimethylarginine with severity of kidney injury and decline in kidney function in
IgA Nephropathy. Am J Nephrol 2011; 33: 1-6.
[5] Shi B, Ni Z, Zhou W, et al. Circulating levels of asymmetric dimethylarginine are an
independent risk factor for left ventricular hypertrophy and predict cardiovascular
events in pre-dialysis patients with chronic kidney disease. Eur J Intern Med
2010;21(5):444-8.
[6] Abedini S, Meinitzer A, Holme I, et al. Asymmetrical dimethylarginine is associated with
renal and cardiovascular outcomes and all-cause mortality in renal transplant
recipients. Kidney Int 2010;77(1): 44-50.
[7] Fliser D, Kielsten JT, Haller H, and Bode-Bger SM. Asymmetric dimethylarginine: A
cardiovascular risk factor in renal disease? Kidney Int (Supp) 2003;(84):3740.
[8] Young JM, Terin N, Wang X, et al. Asymmetric dimethylarginine and mortality in stages
3 to 4 chronic kidney disease. Clin J Am Soc Nephrol 2009;4(6):11151120.
[9] Mihout F, Shweke N, Bige N, et al. Asymmetric dimethylarginine (ADMA) induces
chronic kidney disease through a mechanism involving collagen and TGF-1
synthesis. J Pathol 2011; 223(1) : 3745.
[10] Selcoki Y, Aydn M, kizek M, Armutcu F, Eryonucu B, Kanbay M. Association between
asymmetric dimethylarginine and the severity of coronary artery disease in
patients with chronic kidney disease. Turk Neph Dial Transpl 2011;20(1):58-64.
[11] Kielstein JT, Simmel S, Bode-Bger SM, et al. Subpressor dose asymmetric
dimethylarginine modulates renal function in humans through nitric oxide
synthase inhibition. Kidney Blood Pres Res 2004;27(3):143-147.
[12] Coen G, Mantella D, Sardella D, et al. Asymmetric dimethylarginine, vascular
calcifications and parathyroid hormone serum levels in hemodialysis patients. J
Nephrol 2009;22(5):616-622.
[13] Kielstein JT, Bger RH, Bode-Bger SM, et al. Low dialysance of asymmetric
dimethylarginine (ADMA)- in vivo and in vitro evidence of significant protein
binding. Clin Nephrol 2004;62(4):295-300.
[14] Vallance P, Leiper J. Blocking NO synthesis: How, where and why? Nature Reviews
Drug Discovery 2002;1(12):939-950
[15] Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous
inhibitor of nitric oxide synthesis in chronic renal failure. Lancet
1992;339(8793):572-575.
[16] Schmidt RJ, Baylis C. Total nitric oxide production is low in patients with chronic renal
disease. Kidney Int 2000;58:1261-1266.
[17] Baylis C. Nitric oxide deficiency in chronic kidney disease. Am J Physiol Renal physiol
2008;294(1):F1-F9.
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[18] van Guldener C, Nanayakkara PW, Stehouwer CD. Homocysteine and asymmetric
dimethylarginine (ADMA): biochemically linked but differently related to vascular
disease in chronic kidney disease.Clin Chem Lab Med. 2007;45(12):1683-7.
[19] Shishehbor MH, Oliveira LP, Lauer MS, et al. Emerging cardiovascular risk factors that
account for a significant portion of attributable mortality risk in chronic kidney
disease. Am J Cardiol. 2008 Jun 15;101(12):1741-6. Epub 2008 Apr 9.
[20] Weiner DE, Tighiouart H, Elsayed EF, et al. The relationship between nontraditional
risk factors and outcomes in individuals with stage 3 to 4 CKD.Am J Kidney Dis.
2008 Feb;51(2):212-23.
[21] Bostom AG, Shemin D, Lapane KL, et al. Hyperhomocysteinemia,
hyperfibrinogenemia, and lipoprotein (a) excess in maintenance dialysis patients: a
matched case-control study. Atherosclerosis. 1996 Aug 23;125(1):91-101
[22] Rasmussen LE, Svensson M, Jrgensen KA, et al. The content of docosahexaenoic acid
in serum phospholipid is inversely correlated with plasma homocysteine levels in
patients with end-stage renal disease. Nutr Res. 2010 Aug;30(8):535-40.
[23] Descamps-Latscha B, Witko-Sarsat V, Nguyen-Khoa T,et al. Advanced oxidation
protein products as risk factors for atherosclerotic cardiovascular events in
nondiabetic predialysis patients. Am J Kidney Dis. 2005 Jan;45(1):39-47
[24] Simic-Ogrizovic S, Stosovic M, Novakovic I, et al. Fuzzy role of hyperhomocysteinemia
in hemodialysis patients' mortality. Biomed Pharmacother. 2006 May;60(4):200-7.
[25] Cockcroft DW and Gault MH: Prediction of creatinine clearance from serum creatinine.
Nephron 1976, 16: 31-41.
[26] Chen BM, Xia LW, Zhao RQ. Determination of NG, NG-dimethylarginine in human
plasma by high performance liquid chromatography. J Chromatogr B Biomed Sci
Appl 1997;692:467-471.
[27] Bories PN, Bories C. Nitrate determination in biological fluids by an enzymatic one-step
assay with nitrate reductase. Clin Chem 1995;41:904-907.
[28] Nakamura T, Sato E, Fujiwara N. Ezetimibe decreases serum levels of asymmetric
dimethylarginine (ADMA) and ameliorates renal injury in non-diabetic cronic
kidney disease patients in a cholesterol-independent manner. Pharm Res
2009;60(6):525-528.
[29] Leone A, Moncada S, Vallance P, Calver A and Collier J. Accumulation of an
endogenous inhibitor of nitric oxide synthesis in chronic renal failure. 1992;
339(8793): 572-575.
[30] Raij L, Jaimes E, del Castillo D, Guerra J and Westberg G. Pathophysiology of the
vascular wall: the role of nitric oxide in renal disease. Prostaglandins, Leukotrienes
and Essential Fatty Acids 1996;54(1):53-58.
[31] Fliser D, Kronenberg F, Kielstein JT. Asymmetric dimethylarginine and progression of
chronic kidney disease: The Mild to Moderate Kidney Disease Study. J Am Soc
Nephrol 2005;16:1-6.
[32] Schmitt B, Wolters M, Kressel G, et al. Effects of combined supplementation with B
vitamins and antioxidants on plasma levels of asymmetric dimethylarginine
(ADMA) in subjects with elevated risk for cardiovascular disease.Atherosclerosis.
2007 Jul;193(1):168-76.
[33] Ninomiya T, Kiyohara Y, Kubo M. Hyperhomocysteinemia and the development of
chronic kidney disease in a general population: The Hisayama study. Am J Kid Dis
2004;44(3):437-445.
13
Neutrophil Activation and Erythrocyte
Membrane Protein Composition in
Stage 5 Chronic Kidney Disease Patients
Elsio Costa
1,2
, Lus Belo
2,3
and Alice Santos-Silva
2,3
1
Instituto de Cincias da Sade da Universidade Catlica Portuguesa
2
Instituto de Biologia Molecular e Celular da Universidade do Porto
3
Faculdade de Farmcia da Universidade do Porto
Portugal
1. Introduction
Anaemia is a frequent complication associated with stage 5 chronic kidney disease (CKD),
and is mainly due to insufficient production of erythropoietin by the kidneys. Accumulation
of uremic toxins, excessive toxic storage of aluminium in the bone marrow (Miyoshi, 2006),
blood loss (either iatrogenic, from the puncture sites of the vascular access and blood
sampling, or from other sources, such as the gastrointestinal tract), and premature
erythrocyte destruction have also been frequently associated with anaemia in stage 5 CKD
patients (Medina, 1994; Pisoni, 2004).
The erythrocyte, presenting a limited biosynthesis capacity, suffers and accumulates
physical and/or chemical changes, which become more pronounced with cell aging, and
whenever an unusual physical or chemical stress develops (Locatelli, 2004a). Erythrocytes
are physically stressed during the haemodialysis process, and metabolically stressed by
the unfavourable plasmatic environment, due to metabolite accumulation, and by the
high rate of haemoglobin autoxidation, due to the increase in haemoglobin turnover, a
physiologic compensation mechanism triggered in case of anaemia (Lucchi, 2000; Stoya,
2002). The erythrocytes are, therefore, continuously challenged to sustain haemoglobin in
its reduced functional form, as well as to maintain the integrity and deformability of the
membrane.
Leukocytosis is essential as the primary host defence, and neutrophils, the major
leukocyte population of blood in adults, play a primordial role. It is well known that
neutrophils have mechanisms that are used to destroy invading microorganisms. These
cells use oxygen-dependent and oxygen-independent microbicidal
artillery to destroy and
remove infectious agents (Witko-Sarsat, 2000). Activated neutrophils also undergo
degranulation, with the release of several components, namely, proteases and cationic
proteins (Witko-Sarsat, 2000).
In this book chapter we review the cross-talk between changes in erythrocyte membrane
protein composition and the release of neutrophil activation products.
Chronic Kidney Disease
210
2. Erythrocyte membrane protein composition
Erythrocyte membrane proteins can be classified into three categories, according to their
functional properties in the membrane struture (An & Mohandas, 2008; Mohandas &
Gallagher, 2008). The first includes cytoskeletal proteins, as spectrin ( and chains),
protein 4.1, and actin; the second includes integral/transmembrane proteins of which the
representative proteins are band 3 and glycophorins; the third includes anchoring/linker
proteins, namely, ankyrin (also known as band 2.1) and protein 4.2. The anchoring/linker
membrane proteins mediate the attachment of cytoskeletal proteins to integral proteins (Fig.
1) (Lucchi, 2000; Gallagher, 2005; Mohandas & Gallagher, 2008).
Fig. 1. Schematic representation of red blood cell membrane, showing the topographical
localization of proteins and their interactions. The membrane is a complex structure in
which a plasma membrane envelope composed of amphiphilic lipid molecules is anchored
to a two dimensional elastic network of skeletal proteins through tethering sites
(transmembrane proteins) embedded in the lipid bilayer. Adapted from An & Mohandas,
2008.
The cytoskeleton is a 3-dimensional network of proteins that covers the cytoplasmatic
surface of the erythrocyte membrane and is responsible for its biconcave shape and the
properties of elasticity and flexibility. It comprises approximately half the membrane
protein mass and is primarily composed of spectrin, protein 4.2 and actin. Spectrin is the
major protein of the cytoskeleton, and, therefore, the primary cause of erythrocyte shape,
integrity and deformability. It is linked to the lipid bilayer, by vertical protein interactions
with the transmembrane proteins, band 3 and glicophorin A (Lucchi, 2000). In the vertical
protein interaction of spectrin with band 3 there are also ankyrin (also known as band 2.1)
and protein 4.2 involved. A normal linkage of spectrin with the other proteins of the
cytoskeleton assures normal horizontal protein interactions. The vertical and horizontal
interactions between membrane constituents account for the integrity, strength, and
deformability of the cell (An & Mohandas, 2008; Mohandas & Gallagher, 2008). Disruption
of vertical interactions because of membrane protein deficiencies favours membrane
vesiculation with loss of surface area and development of spherocytic cells, with increasing
Neutrophil Activation and Erythrocyte Membrane
Protein Composition in Stage 5 Chronic Kidney Disease Patients
211
rigidity of the cell membrane that may lead to premature spleen sequestration and
destruction (An & Mohandas, 2008).
3. Neutrophil activation
Leukocytosis and recruitment of circulating leukocytes into the affected areas are hallmarks
of inflammation. Leukocytes are chimio-attracted to inflammatory regions and their
transmigration from blood to the injured tissue is primarily mediated by the expression of
cell-adhesion molecules in the endothelium, which interact with surface receptors on
leukocytes (Muller, 1999; Sullivan, 2000). This leukocyte-endothelial
interaction is regulated
by a cascade of molecular steps that lead to the morphological changes that accompany
adhesion.
At the inflammatory site, leukocytes release their granular content and may exert
their phagocytic capacities.
In acute inflammation, the leukocyte infiltration is predominantly of neutrophils, whereas in
chronic inflammation an infiltration predominantly of macrophages and lymphocytes is
observed. Leukocyte-endothelial cell interactions are important for leukocyte transmigration
and trafficking in physiological conditions. There is increasing evidences that changes in
those leukocyte-endothelial interactions, due to endothelium damage or dysfunction, might
be implicated in the pathogenesis of diseases, such as inflammatory diseases (Harlan, 1985;
Ley, 2007).
Leukocytosis is essential as the primary host defence, and neutrophils, the major leukocyte
population of blood in adults, play a primordial role. It is well known that neutrophils have
mechanisms that are used to destroy invading microorganisms. These cells use an
extraordinary
array of oxygen-dependent and oxygen-independent microbicidal
weapons to
destroy and remove infectious agents (Witko-Sarsat, 2000). Oxygen-dependent
mechanisms
involve the production of reactive oxygen species
(ROS), which can be microbicidal (Roos,
2003), and lead to the development of oxidative stress. Oxygen-independent
mechanisms
include chemotaxis, phagocytosis and degranulation. The generation of microbicidal
oxidants by neutrophils results
from the activation of a multiprotein enzyme complex,
known as
the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,
which catalyzes the formation of superoxide anion (O
2