HHS Public Access: Advances in The Pathophysiology of Pre-Eclampsia and Related Podocyte Injury
HHS Public Access: Advances in The Pathophysiology of Pre-Eclampsia and Related Podocyte Injury
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Kidney Int. Author manuscript; available in PMC 2015 February 01.
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Abstract
Pre-eclampsia is a pregnancy-specific hypertensive disorder that may lead to serious maternal and
fetal complications. It is a multisystem disease that is commonly, but not always, accompanied by
proteinuria. Its cause(s) remain unknown, and delivery remains the only definitive treatment. It is
increasingly recognized that many pathophysiological processes contribute to this syndrome, with
different signaling pathways converging at the point of systemic endothelial dysfunction,
hypertension, and proteinuria. Different animal models of pre-eclampsia have proven utility for
specific aspects of pre-eclampsia research, and offer insights into pathophysiology and treatment
possibilities. Therapeutic interventions that specifically target these pathways may optimize pre-
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eclampsia management and may improve fetal and maternal outcomes. In addition, recent findings
regarding placental, endothelial, and podocyte pathophysiology in pre-eclampsia provide unique
and exciting possibilities for improved diagnostic accuracy. Emerging evidence suggests that
testing for urinary podocytes or their markers may facilitate the prediction and diagnosis of pre-
eclampsia. In this review, we explore recent research regarding placental, endothelial, and
podocyte pathophysiology. We further discuss new signaling and genetic pathways that may
contribute to pre-eclampsia pathophysiology, emerging screening and diagnostic strategies, and
potential targeted interventions.
Keywords
endothelial dysfunction; placenta; podocyturia; pre-eclampsia
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Address for Correspondence: Vesna D. Garovic, MD, Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW,
Rochester, MN 55905 USA, Phone: 507-266-1963, Fax: 507-266-7891, [email protected].
Disclosure
Conflict of interest: Dr. Garovic is the inventor of technology referenced in this article. That technology has been patented by Mayo
Clinic, but is currently not licensed.
All other authors report no conflict of interest.
Craici et al. Page 2
INTRODUCTION
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these disorders as a continuum comes from clinical evidence demonstrating that either
chronic or gestational hypertension may progress to pre-eclampsia (commonly evidenced by
new-onset or worsening of proteinuria), while pre-eclampsia may progress to more severe
forms, such as eclampsia or HELLP syndrome. An alternative approach views pre-eclampsia
as a separate disease entity. Either way, it is recognized that pre-eclampsia is a
heterogeneous disease. Different clinical subtypes may reflect distinct underlying
pathological mechanisms.2 For example, it is common in clinical practice to subcategorize
pre-eclampsia as early versus late (before and after 34 weeks of gestation, respectively),3
and mild versus severe,4 based on the absence/ presence of severe hypertension, defined as a
blood pressure ≥160/110 mm Hg, proteinuria ≥ 5 gr/24-hours, neurological/renal/cardiac
impairment, or signs of HELLP. Recent evidence suggests that women with early severe
pre-eclampsia, who are at particularly high risk for adverse pregnancy outcomes, may have a
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more pronounced anti-angiogenic imbalance than those with late pre-eclampsia and more
favorable outcomes.5 Active research in this field may delineate the mechanisms of the
subtypes of pre-eclampsia, commonly referred to as placental versus maternal pre-
eclampsia, based on their etiologies and origins.6, 7
Renal pathology in pre-eclampsia in the form of endotheliosis has long been recognized, and
the kidney manifestations of pre-eclampsia form the basis for a “nephrocentric” view in the
research and clinical arenas.8 In contrast, obstetric literature questions the importance of
kidney injury (as demonstrated by proteinuria) in the diagnosis of pre-eclampsia, suggesting
that a subclass of “non-proteinuric pre-eclampsia” should be added,9 or that detection of
proteinuria should not be mandatory for a pre-eclampsia diagnosis.10 However, similar to
other renal diseases, proteinuria in pre-eclampsia may represent a late marker of renal injury.
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Our recent data suggest that podocyturia i.e., the urinary loss of viable glomerular epithelial
cells (podocytes), may occur prior to the clinical features of pre-eclampsia, potentially
representing an earlier marker of renal injury than proteinuria. These findings set the stage
for future studies of podocyturia in women who meet all of the clinical criteria, for the
diagnosis of pre-eclampsia, except proteinuria.
The main objective of this review is to discuss emerging theories regarding pre-eclampsia
pathophysiology, focusing on the different causal pathways that translate into different
subtypes (clinical phenotypes) of pre-eclampsia; highlight animal models that may advance
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hypoxia is frequently viewed as an early event that may cause placental production of
soluble factors leading to endothelial dysfunction.11 Over the last decade, pre-eclampsia has
been associated with elevated levels of the soluble receptor for vascular endothelial growth
factor (VEGF) of placental origin. This soluble receptor, commonly referred to as soluble
fms-like tyrosine kinase receptor-1 (sFlt-1), may bind and neutralize VEGF, and thus limit
the availability of free VEGF for fetal and placental angiogenesis. Several rodent models
simulate pre-eclampsia by exogenous sFlt-1 administration. In the most direct model,
intraperitoneal sFlt-1 injections produce short-term elevations of sFlt-1.12 In the hours after
sFlt-1 injection, animals develop hypertension, proteinuria, and altered podocyte protein
expression, but do not develop glomerular endotheliosis, the classical renal lesion of pre-
eclampsia. Administration of an adenoviral vector encoding sFlt-1 leads to longer-term
sFlt-1 exposure in rats.13 This model reproduces the findings of hypertension, proteinuria,
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adaptation of the uterine spiral arteries supplying the placenta may lead to placental
ischemia,18 recurrent episodes of ischemia and reperfusion,19 or high-velocity blood flow
that damages placental villous tissue.19 Ultimately, the damaged placenta may release one or
more factors into the maternal circulation that cause systemic endothelial dysfunction.18
Several animal models attempt to emulate the incomplete adaptation of the maternal spiral
arteries that supply the placenta. The reduced uterine perfusion pressure (RUPP) model
surgically reduces uterine blood flow by partial or complete ligation of the aorta or uterine
artery (ies). An excellent review of the RUPP model was recently published.20 The RUPP
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model causes hypertension and proteinuria, but not glomerular endotheliosis; hence, it may
not be a good model for studying renal pathophysiology in pre-eclampsia. However, the
RUPP model has proven valuable for studying the effects, rather than the causes, of fetal-
placental ischemia. Elevated levels of tumor necrosis factor α (TNFα); interleukin (IL)-6;
autoantibodies to the angiotensin II type I receptor (AT1-AA); the potent vasoconstrictor
endothelin-1; sFlt-1; and soluble endoglin reported in pre-eclampsia are also seen in the
RUPP model.21, 22
The baboon utero-placental ischemia model (UPI) is similar to the RUPP rat model, but
results in more specific utero-placental ischemia.23 This technique involves uterine artery
angiography followed by unilateral uterine artery ligation. Compared with sham animals,
pregnant UPI baboons display clinically significant hypertension and proteinuria, with renal
biopsies showing endotheliosis similar to human pre-eclampsia. Of the animal models, the
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baboon UPI model may most closely resemble the human condition. However, the inherent
complexities of using non-human primates in research limit its availability. Similar to the
RUPP model,24 sFlt-1 levels are elevated in this model, indicating that they are a
consequence, rather than the cause, of placental ischemia.
placentation, the pathological substrate for fetal growth restriction, commonly occurs with
early disease,25 which, compared to late pre-eclampsia, is characterized by different
biochemical markers, heritability, and worse pregnancy outcomes.3 In addition, particularly
severe maternal complications have been associated with postpartum pre-eclampsia, which
occurs in 6% of all pre-eclamptic pregnancies15 and in up to 30% of those with HELLP
syndrome.26 Regardless of the origins/etiologies of the initial insults, systemic endothelial
dysfunction appears to be the converging point of different causal pathways, ultimately
leading to the cardinal clinical features of pre-eclampsia.
GENETIC STUDIES
Genetic studies of pre-eclampsia suggest heritability of this condition, but have not
consistently identified the responsible genetic variants using either candidate gene or
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genome scanning approaches.27 The discussion that follows presents recent genetic studies.
Animal studies have shown that abnormal Notch signaling may lead to abnormal
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studies,36, 37 a recent animal study supported the possible involvement of STOX1 in pre-
eclampsia. Transgenic mice overexpressing human STOX1 developed hypertension,
proteinuria, and elevated levels of sFlt-1 and sEng.38 Although the renal histology is similar
to that seen in pre-eclampsia, hypertension begins before placental formation, suggesting
that the pathophysiology might be different than that seen in human disease. These data
indicate that STOX1 may contribute to some cases of pre-eclampsia, but it is unlikely to be a
common cause of the disorder.
Epigenetic studies
Altered DNA methylation contributes to the control of proliferative, invasive, and immune
tolerance in oncogenesis,39 a disease process with many parallels to normal pregnancy.
These conditions share the common goal of providing a nutrient supply and immune
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DYSREGULATION OF ANGIOGENESIS
Over the last decade, the pathway receiving the most attention involves the imbalance
between the pro-angiogenic VEGF and placental growth factor (PlGF), and the anti-
angiogenic sFlt-1 and soluble endoglin. Excessive production of anti-angiogenic sFlt-1 and
soluble endoglin reduces the bioavailability of pro-angiogenic PlGF and VEGF. While
reduced VEGF signaling is central to the sFlt-1 hypothesis, several lines of evidence suggest
that this may be insufficient to cause hypertension and proteinuria when PlGF is present.
sFlt-1, but not sFlk-1 (a type 2 VEGF receptor which only binds VEGF).13 In contrast,
adenoviral expression of both sFlt-1 or sFlk-1 causes hypertension and proteinuria in non-
pregnant rats, which have very low PlGF concentrations.13 On the clinical side, higher blood
pressures early in pregnancy and more preterm deliveries were reported in pre-eclamptic
women with low PlGF from 15 weeks gestation to term, compared to pre-eclamptic women
with normal or high PlGF from 15 weeks gestation to term.2 This suggests that low versus
normal/high PlGF levels may underpin two different clinical subtypes of pre-eclampsia.2
Some researchers have suggested redefining pre-eclampsia by using placenta-derived
biomarkers, which link placental pathology (abnormal placentation) to impaired
angiogenesis (low PlGF levels) and subsequent clinical phenotype (early, severe
preeclampsia).6 While this classification may improve the reliability and reproducibility of
outcomes assessment in pre-eclampsia, wider application is critically dependent on future
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Therapeutic Implications
In humans, sFlt-1 may contribute directly to the pathogenesis of pre-eclampsia. Its removal
by apheresis was associated with reduced hypertension and proteinuria in pre-eclamptic
women.42 However, the dextran columns used for apheresis remove many substances from
the circulation; hence it is not clear whether sFlt-1 was the causative agent.
vasoconstriction and platelet aggregation activities unopposed.45 This latter observation led
to the hypothesis that aspirin might prevent pre-eclampsia.46 While clinical trials reported
conflicting results, a meta-analysis of 59 trials involving 37,560 women found that low
doses of aspirin reduced the absolute risk pre-eclampsia in women who were at high risk and
concluded that future studies are required to assess which women are most likely to benefit,
when treatment is best started, and at what dose.47
eclampsia. These include the presence of agonistic autoantibodies for angiotensin II type 1
(AT1) receptors,48 and heterodimerization between the AT1-receptor and the B2-receptor
for the depressor, bradykinin. The AT1-B2-heterodimer mediates increased responsiveness
to angiotensin II, and also confers resistance to AT1-receptor inactivation by reactive
oxygen species.49
The discussion that follows does not aim to present the complex interactions among a
myriad of vasoactive molecules that may have a role in pre-eclampsia,50 but rather to
discuss more recent discoveries and their potential roles in identifying new therapeutic
targets.
monoxide (CO). Biliverdin reductase then rapidly converts biliverdin to bilirubin. Both
biliverdin and bilirubin are powerful anti-oxidants, and CO may also have beneficial effects.
HO activity appears to be crucial for proper placental vascular development.51, 52 HO-1
knockout mice display abnormal placentation, intrauterine growth restriction (IUGR), and
fetal demise. Heme administration produces a similar phenotype in wild-type mice.
Interestingly, inhaled CO reverses these adverse effects.53 A study of non-smoking women
during late gestation (>31 weeks), demonstrated that women with pre-eclampsia, compared
to those without, have significantly reduced end-tidal CO levels, indicating reduced HO-1
activity.54
Nitric oxide
Nitric oxide (NO), a potent vasodilator that mediates endothelium-dependent relaxation, has
been linked to endothelial dysfunction in pre-eclampsia.58 As NO is regulated by VEGF,59 it
was hypothesized that down-regulation of VEGF in pregnancy may result in a suboptimal
increase in NO and endothelial dysfunction in pre-eclampsia (Figure 2). However, data
concerning NO production in pre-eclampsia have been conflicting. Investigators have
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reported lower,60 unchanged,61 and higher62 production compared to normal controls, even
after controlling for diet, medications and urinary excretion that can affect serum nitrate
levels. It appears that reduced NO bioavailability, rather than changes in absolute NO levels,
may alter endothelial function. Elevated intracellular levels of NO and superoxide react to
produce an excess of peroxynitrate, the presence of which is confirmed by nitrotyrosine
formation. Endothelial dysfunction results from the decreased abilities of specific proteins to
undergo tyrosine phosphorylation once peroxynitrate-induced tyrosine nitration is
Therapeutic Implications
CO, NO and H2S belong to the category of endogenously generated small molecules,
gasotransmitters. These molecules are emerging as potential therapeutics for several disease
entities, including cardiovascular disease and pre-eclampsia. Therapeutic use of CO gas and
CO-releasing molecule was studied in preclinical animal models, which demonstrated anti-
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AT1-AA; hence placental ischemia may be an important stimulus for AT1-AA production.70
AT1-AA infusion in pregnant rats induces hypertension and mitigates impaired vasodilatory
responses to acetylcholine in renal interlobar arteries.71 In addition, recent evidence suggests
that elements of innate immunity, such as TNFα and interleukin 6 (IL-6), may induce a pre-
eclamptic syndrome by generation of AT1-AA,70, 72 and that activation of Toll-like
receptors, also involved in innate immunity, may cause a pre-eclampsia-like syndrome in
rats.73 Additional studies are needed to determine the exact role of AT1-AA in the
pathophysiology of pre-eclampsia and to explore toll-like receptors as potential therapeutic
targets.
Studies of human tissue show that the expressions of podocyte-specific proteins are severely
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affected by pre-eclampsia. A study comparing renal sections from women with pre-
eclampsia, compared to those from women with either normotensive or chronic hypertensive
pregnancies, reported reduced expressions of podocyte-associated proteins, nephrin,
glomerular epithelial protein 1, GLEPP-1, and ezrin in their renal tissue sections.74
Decreased glomerular expressions of nephrin and synaptopodin were seen in renal tissue
sections from women who died from pre-eclampsia compared to those of women with
normal pregnancies who died from other causes.75 Podocin expression, however, was
relatively unchanged. The degree of podocyte dysfunction required for such dramatic
changes in nephrin and synaptopodin expressions might be expected to cause changes in
multiple other proteins important to the integrity of the glomerular filtration barrier and,
possibly, podocyte attachment.
The detection of podocyte products and live podocytes in the urine (podocyturia) suggests
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that podocyte pathology is more severe than might be inferred from renal biopsy studies.
Various methods have been developed to detect urinary podocyte products.76–86 Culturing
of urinary podocytes increases specificity by removing dead and non-specific cells, but is
difficult and time consuming. Cytospin techniques, while quicker and possibly more
amenable to automation, suffer from low sensitivity and specificity due to the large amount
of cellular debris. More sensitive techniques using reverse transcriptase-polymerase chain
reaction (RT-PCR) and mass spectrometry remain in development. Studies utilizing these
methods are outlined below and summarized in Table 1.
Using staining for podocin to detect live podocytes, we have shown 100% sensitivity and
specificity in diagnosing pre-eclampsia at the time of delivery.76 Synaptopodin, nephrin, and
podocalyxin were also useful markers for urinary podocytes, but lacked sensitivity and
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specificity compared to podocin. This is in agreement with autopsy studies showing that
podocin expression is preserved in podocytes from pre-eclamptic patients compared with
nephrin and synaptopodin.75 Podocyturia appears before the onset of proteinuria, and the
number of podocytes positively correlates with the degree of proteinuria, suggesting a cause-
effect relationship between ongoing podocyte loss and the onset and severity of
proteinuria,85 i.e., that these are mechanistically related.
similar to those found in pre-eclampsia and its severe form, HELLP syndrome. Podocyturia
is also seen in patients treated with bevacizumab, although less consistently than with pre-
eclampsia.88
finding only 38% sensitivity and 70% specificity for diagnosing pre-eclampsia.79 Previous
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More novel methods for detecting urinary podocytes and their products may also prove
useful for the early diagnosis of pre-eclampsia. Mass spectrometry and RT-PCR offer
standardized, reproducible techniques for the detection of podocyte products.81, 92 These
methods are operator-independent and highly reproducible, and may facilitate large-scale
studies that will determine the clinical usefulness of podocyturia in larger patient
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populations that are more broadly representative of pregnant women. Critical clinical
questions that remain un-answered include the ability of podocyturia to differentiate
between pre-eclampsia, other complications of pregnancy (such as gestational diabetes
mellitus), and other proteinuric diseases that either predate pregnancy or occur during
pregnancy.
Endothelin 1: a possible final common pathway for endothelial and podocyte dysfunction
Recent data suggest that endothelin-1, one of the most powerful human vasoconstrictors,
may act through the endothelin type A (ETA) receptor to provide a bridge between placental
ischemia and the clinical signs of pre-eclampsia, both hypertension and podocyte damage/
proteinuria. Endothelin-1 may act both in an autocrine or paracrine manner, therefore,
systemic levels do not necessarily reflect local tissue expression or effects. Endothelin-1
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Therapeutic implications
Podocyturia has been shown to decrease with blood pressure control and modulation of the
renin-angiotensin-aldosterone system, by either angiotensin-converting enzyme (ACE)
With respect to future studies of the mechanisms of renal injury later in life, these may be
limited by the fact that humans are the only species known to suffer from spontaneous pre-
eclampsia. Although many animal models have been developed for the study of this
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sFlt-1 and sEng, and spiral artery pathology.113 These changes were absent when sera from
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normal pregnant women were injected, and they were specific to pregnancy, i.e., were
absent after injection of non-pregnant IL 10−/− mice.113 These models closely mimic human
disease, including the renal pathology, and may provide valuable insight into pre-eclampsia
pathophysiology and treatment.
CONCLUSIONS
Over the last decade, our understanding of the pathophysiology of pre-eclampsia and related
disorders has increased dramatically. The heterogeneity of both the causal pathways and
clinical presentations of pre-eclampsia suggests that therapies acting on a particular pathway
will only be effective in patients with aberrations in that particular pathway. Research on
pre-eclampsia prevention and treatment should either focus on pathways common to all
women with pre-eclampsia, or target the subgroup of women who have abnormalities in the
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pathway examined. The potential to identify targeted therapies that address the underlying
causes of disease in distinct pathophysiological subtypes of pre-eclampsia may improve
treatment options for a disease which has seen few therapeutic advances in recent decades.
Acknowledgments
Sources of Support: The project described was supported by the award numbers K08HD051714 from the Eunice
Kennedy Shriver National Institute of Child Health & Human Development, P50AG44170 from the National
Institute on Aging (Vesna D. Garovic), and K12HD065987 from the Office of Women’s Health Research, Building
Interdisciplinary Careers in Women’s Health (Tracey L. Weissgerber).
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Several different signaling pathways may play a role, ultimately converging at the point of
systemic endothelial dysfunction, hypertension, and proteinuria.
Abbreviations: AT1-AA, autoantibodies to the angiotensin II type 1 receptor; AT1-AA-B2
heterodimers, angiotensin II type 1 receptor-bradykinin type 2 receptor heterodimers; carbon
monoxide; CKD, chronic renal disease; CTD, connective tissue disease; DM, diabetes
mellitus; HELLP, hemolysis, elevated liver enzymes, low platelet count; IL-6, interleukin 6;
LFT, liver function tests; PlGF, placental growth factor; PRES, posterior reversible
encephalopathy syndrome; sFlt-1, soluble fms-like tyrosine kinase 1; TNFα, tumor necrosis
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Figure 3. Possible mechanisms of the association between pre-eclampsia and future renal disease
Abbreviations: CKD, chronic kidney disease; ESRD, end-stage renal disease.
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Table 1
and year Study groups Time point(s) Sample preparation Method Results
Garovic et al 15 PE < 24 hours before Podocyte culture IF for podocin, nephrin, Podocin staining present in 15/15 PE and absent in 16/16 NL
(2007)76 16 NL delivery podocalyxin, and Other 3 less sensitive and specific
synaptopodin
Aita et al (2009)77 11 PE 35 weeks Cytospin IF for podocalyxin Podocyturia at 35 weeks and 4 days post in PE
45 NL 4 days post Almost no podocyturia at 1 month post delivery
1 month post 9 of 45 controls showed podocyturia 4 days postdelivery
Correlation between podocyturia and BP, but not proteinuria
Zhao et al (2011)78 16 severe PE 3rd trimester Podocyte culture IF for nephrin Podocyturia present in all cases of severe PE and nephrotic
3 mild PE syndrome
7 NL Podocyturia absent in all NL and all 3 mild PE cases
7 NP with nephrotic
syndrome
Jim et al (2012)79 29 PE < 24 hours before Cytospin IF for synaptopodin Podocyturia in 11 of 29 (38%) of PE, 3 of 9 (33%) with HTN,
9 GHTN and HTN delivery and 0 of 9 NL
9 NL Sensitivity = 38%, Specificity = 70%
Facca et al 25 NL 3rd trimester Cytospin IF for nephrin Mean total number of podocytes 0.9 ± 1.6 for NL versus 9.3 ±
(2012)80 14 PE 16.8 for PE (P=0.212)
Kelder et al 35 PE 31 to 36 weeks gestation Urine centrifugation RT-PCR for nephrin, Elevated mRNA for nephrin, podocin, VEGF in PE compared to
(2012)81 5 GHTN TRIzol RNA isolation podocin, VEGF NL and NP
34 NL Positive correlation between nephrin and VEGF mRNA in PE
12 NP (r=0.82, P<0.0001)
Wang et al 20 PE 3rd trimester ELISA of frozen urine ELISA for nephrin, Urinary nephrin, podocalyxin, and Big-h3 levels are increased in
(2012)82 6 HTN supernatant podocalyxin, Big-h3, and PE
8 NL VEGF Urinary Big-h3 levels correlate with levels of nephrin and
podocalyxin
Chen et al (2013)83 14 PE < 1 week before delivery Cytospin IF for podocalyxin Number of podocytes was higher in PE compared to GHTN
14 GHTN (P<0.05) and NL (P<0.001)
BP, blood pressure; GHTN, gestational hypertension; HTN, hypertension; IF, immunofluorescence; NL, normal pregnancy; NP, not pregnant; PE, pre-eclampsia; VEGF, vascular endothelial growth factor;
HELLP, hemolysis, elevated liver enzymes, low platelet count; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry.
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