The Role Of Podocyte Injury In Preeclampsia And Its Possible Mechanisms

PART IThe correlation of renin angiotensin system (RAS) components, angiogenic factors and its antagonist and podocyte injury in development of proteinuria in preeclampsia and its possible mechanisms1. Effects of RAS and urinary podocyte loss on development of proteinuria in preeclampsiaObjectiveUrinary podocyte loss reflects podocyte injury in various glomerular diseases. Podocyte, as a structure of glomerular filtration barrier, its injury was associated with proteinuria development. The typical pathological change in preeclampsia is glomerular endotheliosis, but the effect of podocyte injury on proteinuria development in preeclampsia is unclear. It has been known that the dysfunction of RAS could change the hemodynamics and regulate the blood pressure. Activation of renal local RAS has important meaning for proteinuria development and glomerulosclerosis in chronic kidney disease. However, the change of circulating, intrarenal RAS and their effect on renal injury and proteinuria development is still obscure. This study aims to investigate that the effect of RAS components and urinary podocyte loss on the regulation of podocyte injury in preeclampsia as well as their correlation. MethodsWomen with preeclampsia (n=14), gestational hypertension (n=14) and normal pregnancy (n=13) were enrolled in the study. Urinary podocytes were detected by immunofluorescence using anti-podocalyxin monoclonal antibody. Serum and urinary angiotensin-(1-7) [Ang-(1-7)], angiotensin?(Ang?) and angiotensinogen (AGT) concentrations were quantified by enzyme-linked immunosorbent assays. The relationship among podocyturia, RAS components, and proteinuria was investigated and their receiver operating characteristic curves were analyzed.Results1. Before labour, the level of urinary podocytes was significantly higher in patients with preeclampsia than gestational hypertension [median (IQR):3.10(2.37,5.66) vs.1.41(0.71,3.73) cells/ml of urine, P< 0.05] and normal pregnancy [0.52 (0.01, 1.49) cells/ml of urine, P< 0.001]. After labour, there was no significant difference in podocyturia among groups. In preeclamsia group, level of prepartal urinary podocytes was significantly higher than postpartum [3.10(2.37,5.66) vs. 0.45 (0,1.76) cells/ml of urine, P< 0.001].2. Ang(1-7) concentrations in preeclampsia (serum:53.88±17.97 pg/ml; urine: 69.99±19.09 pg/ml) (mean±SD) was significantly decreased compared to gestational hypertension group (serum:70.96±19.83 pg/ml, P<0.05; urine: 98.78±23.99 pg/ml, P=0.001) and normal pregnant women (serum:72.50±20.59 pg/ml, P<0.05; urine:92.91±18.04 pg/ml, P<0.01). Serum and urinary Ang?had no significant difference. AGT concentrations in preeclampsia (serum:69.47?18.68?g/ml; urine:41.01±64.29 ng/ml) was lower than gestational hypertension group (serum:82.93±12.34?g/ml, P< 0.05; urine: 86.60±62.75 ng/ml, P> 0.05) and normal control (serum:90.94±14.92?g/ml, P =0.001; urine:116.21±65.59 ng/ml, P<0.01).3. The correlation analysis indicated that prepartal urinary podocyte number was positively correlated to ACR, renal function and blood pressure. Serum and urinary Ang-(1-7), AGT concentrations were inversely associated with prepartal urinary podocyte number, ACR, renal function and blood pressure. Serum Ang?and AGT was positively correlated with urinary Ang?and AGT, respectively.4. The ROC curves analysis of serum and urinary Ang-(1-7) as well as antepartum podocyturia confirmed that they were useful for identifying preeclampsia from normal controls. AGT was also valuable for its diagnosis.5. The cutoff value with the highest Youden's index (correct diagnosis index) was defined as the optimal predictors of preeclampsia:antepartum podocyturia?1 cell/ml, serum Ang-(1-7)?55pg/ml, and urinary Ang-(1-7)?80pg/ml, then their sensitivity/specificity were 100%/76.92%,57.14%/84.62%,71.43%/92.31%, respectively. Different combination of serum, urinary Ang-(1-7) or podocyturia detection in parallel or in series boosted identification of preeclampsia.ConclusionsDecreased Ang-(1-7) and downregulated intrarenal RAS may contributed to podocyte loss and injury, which lead to proteinuria in preeclampsia. Ang-(1-7) and podocyturia will be powerful makers for predicting preeclampsia and AGT is valuable for its diagnosis. 2. Effects of Angiogenic Factors and its antagonist and podocyte injury on development of proteinuria in preeclampsiaObjectiveVascular endothelial growth factor (VEGF) is a most vigorous vascular growth factor which was expressed on glomerular endothelial cell, podocyte, renal tubular epithelial cell and mesangial cell and mediated by the membrane receptor (Flk-1,Flt-1, neuropilins etc.) to produce a marked effect. Placenta growth factor (PIGF) is another member of VEGF family, which could only connect with Flt-1 specially. Soluble fms-like tyrosine kinase receptor 1 (sFlt-1) is the splicing variant of Flt-1 and a natural antagonist of VEGF, which could fully block the biologic activity of VEGF. Recent studies suggested that VEGF family and sFlt-1 play an important role in the mechanism of preeclampsia, it may be associated with the injury of endothelial system. However, in view of podocyte could secrete VEGF directly and its receptor such as neuropilins also expressed on the podocyte and the effects of podocyte injury on development of proteinuria in preeclampsia have been paid attention gradually, we suppose that the imbalance of angiogenic factors VEGF, PIGF and its antagonist sFlt-1 may contribute to podocyte injury but not single to endothelium injury and then develop proteinuria. The purpose of the present study is to test the change of angiogenic factors and its antagonist, analyze their correlation with urinary podocyte excretion and investigate the effects of these changes and podocyte injury on proteinuria in preeclampsia.Methods14 women with preeclampsia,14 with gestational hypertension and 13 gestational age-matched normotensive pregnant women were included in this study. Urinary podocytes were stained by immunofluorescence using anti-podocalyxin (PCX) monoclonal antibody. Maternal serum and urinary VEGF, PIGF, sFlt-1 concentration were detected using a sandwich enzyme immunoassay. The ratio of serum and urinary P1GF/sFlt-1 was calculated. Random urine albumin/creatinine ratio (ACR) and renal function were examined. The correlation among podocyturia, angiogenic factors, and proteinuria were investigated. Receiver operating characteristic (ROC) curves were established to estimated reciprocal impact on sensitivity and specificity. Results1. The results of prepartal urinary podocyte was present as Part?section 1.2. Serum VEGF concentration in women with preeclampsia (44.05±25.01 pg/ml) was significantly higher than normal control (25.84±14.22 pg/ml, P< 0.05), but no significant difference versus gestational hypertension (34.75±15.67 pg/ml, P> 0.05); Urinary VEGF concentration had no significant difference among three groups. Serum and urinary P1GF concentrations in women with preeclampsia (serum:52.59±7.46 pg/ml; urine:12.39±5.30 pg/ml) were significantly lower than gestational hypertension (serum:60.39±4.74 pg/ml, P=0.001; urine:26.25±8.25 pg/ml, P<0.05) and normal pregnancy (serum:65.40±3.76 pg/ml, P< 0.001; urine:31.81±26.65 pg/ml, P<0.01). Serum and urinary sFlt-1 concentrations in women with preeclampsia (serum:538.33±53.28 pg/ml, P<0.05; urine:812.09±75.78 pg/ml, P<0.01) and gestational hypertension (serum:546.11±66.80 pg/ml, P<0.05; urine:813.34±68.03 pg/ml, P<0.01) were significantly higher than normal control (serum:466.82±111.57 pg/ml; urine:679.35±188.88 pg/ml). Serum and urinary PIGF/sFlt-1 ratio in women with preeclampsia (serum:P< 0.001; urine:P<0.01) and gestational hypertension (serum:P<0.001; urine:P< 0.05) was significantly lower than normal pregnancy.3. Correlation analysis suggested that serum VEGF concentration was positively correlated to prepartal urinary podocyte number, ACR, uric acid (UA) and blood pressure. Serum P1GF, P1GF/sFlt-1 ratio was inversely correlated to prepartal urinary podocyte number, ACR, UA and blood pressure. Urinary P1GF, P1GF/sFlt-1 ratio was inversely correlated to ACR. There also had correlation among serum and urinary VEGF, P1GF, sFlt-1 and P1GF/sFlt-1 ratio.4. The AUC (Area Under Curve) of P1GF [serum:0.872(95% CI,0.744-1.000, P< 0.001); urine:0.811(95% CI,0.681-0.941, P=0.001)], P1GF/sFlt-1 ratio [serum: 0.847(95% CI,0.728-0.965, P<0.001); urine:0.815(95% CI,0.686-0.943, P= 0.001)] was greater than that of VEGF [serum:0.675 (95% CI,0.483-0.866, P= 0.079); urine:0.550 (95%CI,0.354-0.746, P=0.613)] and sFlt-1 [serum:0.590 (95%CI,0.414-0.766, P=0.350); urine:0.567 (95% CI,0.383-0.752, P=0.483)].5. The cutoff value with the highest Youden's index (correct diagnosis index) was defined as the optimal predictors of preeclampsia:serum P1GF< 60 pg/ml, urinary P1GF< 25pg/ml, then their sensitivity/specificity was 100%/76.92%, 85.71%/92.31%,100%/46.15%, respectively. Different combination of serum, urinary P1GF or podocyturia test in parallel or in series boosted identification of preeclampsia.ConclusionsImbalance of VEGF, P1GF and their antagonist sFlt-1 may be one of mechanisms of podocyte injury and proteinuria in preeclampsia. P1GF and PlGF/sFlt-1 ratio may serve as diagnostic markers for preeclampsia. PART IIQuantitative proteomic investigation of urine in preeclampsiaObjectiveThe extent of proteinuria in preeclampsia is closely associated with the prognosis of maternal renal injury. As a noninvasive examination, urinary proteomics has become a very useful tool for identifying renal disease, especially applicable for gravida, the special population. This study compared the urinary proteomics among women with preeclampsia, gestational hypertension and normal pregnancy to search differentially expressed proteins in urine, which would provide clues for screening early diagnostic biomarker and study the mechanism of renal injury in preeclampsia. Urinary proteomics may have extensive prospects in identifying preeclampsia.MethodsThe urinary proteins of women with preeclampsia (n=10), gestational hypertension (n=10) and normal pregnancy (n=10) were extracted with cold acetone/ trichloroacetic acid (TCA) and the high-abundance proteins and salt were removed. The concentrations of protein samples were quantified by the Bradford method and identified by isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2D LC-MS/MS). The experiment was repeated twice (RUN1 and RUN2). The Protein Pilot 3.0 software was used for mass spectrum analysis and International Protein Index (IPI) database for searching peptide information. The differential expressed proteins were defined as fold change of iTRAQ ratio. The bioinformatics were analyzed by IPI and Gene Ontology Database with Metacore software. One of differential proteins angiotensinogen (AGT) was verified by ELISA.Results1. We identified 507 and 516 proteins in RUN 1 and RUN 2 respectively, and 362 common identified proteins were found both in two experiments.5 proteins were not labeled iTRAQ ratio information. The repeatability of two RUNs exceeded 70% which suggested that the results were reliable.113/362 proteins were expressed differentially (the fold change cutoff ratio< 0.83 or> 1.20) between preeclampsia and normal pregnant group and 31/113 differential expressed proteins were found compared among three groups.2. These differential expressed proteins were associated with biological processes of blood coagulation, cell adhesion and differentiation, immune response and cytoskeleton remodeling etc. They were interacted with each other in the network.3. In these differential expressed proteins, Keratin, Alpha-actinin-4, Afamin, Alpha-2-macroglobulin, Serum albumin (ALB), Isoform 1 of Alpha-1-antitrypsin precursor (SERPINA1), Vimentin and Tubulin beta-2C chain were all differentially expressed with high fold changes compared among three groups. These proteins may be related closely to the mechanisms of preeclampsia. Moreover, Alpha-actinin-4 and Vimentin are important proteins of podocyte cytoskeleton reconstruction. The high fold differential expression of urinary Alpha-actinin-4 and Vimentin may be associated with podocyte injury and development of proteinuria in preeclampsia.4. The urinary angiotensinogen (AGT) was down-regulated in preeclampsia, which was consistent with the ELISA validation results.ConclusionThese studies found a multitude of differential expressed proteins which might provide clues for investigating the mechanisms of proteinuria development and the discovery of early diagnostic and prognostic biomarkers in preeclampsia. Low urinary angiotensinogen levels were useful for identifying preeclampsia. AGT, SERPINA1 and ALB are candidate biomarker for preeclampsia. The high fold differential expression of urinary Alpha-actinin-4 and Vimentin provided more sufficient evidences for podocyte injury and other differentially expressed proteins needed further validation. PART?Effects of RAS component Ang-(1-7) on podocyte injury in vitro induced by patients'serum of preeclampsia and its possible mechanismsObjectiveRenin angiotensin system (RAS) is a very complicated and multistrata endocrinium, including multiple components and metabolic fragments. Ang?, as the core of RAS, primarily play the pathological and physiological action of contracting blood vessels, raising blood pressure and tissue damage etc. Studies had confirmed that podocyte could express angiotensin receptors so that it was one of target cell of renal injury induced by Ang?. Ang?itself could lead to the morphous change of podocyte and promote podocyte apoptosis. Recently the new member of RAS Ang-(1-7) and its specific receptor Mas were found that they have extensive effects, which could counteract the effects of Ang?and dilate vascular. The antistatic equipment in RAS as ACE 2-Ang-(1-7)-Mas axis and ACE-Ang?-AT1R axis play an important role in pathogenesis of preeclampsia. Moreover, The results of Part?in our study also suggested that Ang-(1-7) was correlated with podocyte injury and proteinuria in preeclampsia; Decreased serum and urinary Ang-(1-7) may be a cause of podocyte injury in preeclampsia. In order to further investigate the effect of Ang-(1-7) on podocyte in preeclampsia and its possible mechanisms, we use patients' serum of preeclampsia to preincubate podocyte and then add Ang-(1-7), to observe the changes of podocyte specific proteins and cytoskeletal proteins.MethodsThe podocyte was cultured in vitro and the patients'serum of preeclampsia was collected sterile for preincubating podocyte in order to prepare the podocyte's model of preeclampsia in vitro. They were divided into 4 groups:normal control; normal pregnant serum group; preeclampsia model group and Ang-(1-7)+preeclampsia group. The morphologic change of podocyte was observed by microscope; the changes of podocyte specific proteins such as nephrin and CD2AP, the cytoskeletal protein of podocyte F-actin, the tight junction protein ZO-1 and Mas receptor was examined by immunofluorescence. Western blot was used to examine the expression of Mas receptor.Results1. In preeclampsia model group, it was observed by immunofluorescence that the expression of nephrin, F-actin and ZO-1 on podocytes was significantly decreased than normal control and normal pregnant serum group; the expression of CD2AP had no significantly difference. In Ang-(1-7)+preeclampsia group, the expression of nephrin, F-actin and ZO-1 was significantly increased than in preeclampsia model group.2. There exist Mas receptor on podocytes in all groups which was examined by immunofluorescence and Western blot. The expression of Mas receptor on podocytes in preeclampsia model group was significantly decreased than normal control and normal pregnant serum group; after adding Ang-(1-7), it was increased significantly.ConclusionAng-(1-7) could protect podocyte from injury in vitro induced by patients' serum of preeclampsia. The effect may be associated with integration of Ang-(1-7) and its specific Mas receptor.