足细胞损伤在子痫前期蛋白尿发生中的作用及其机制探讨
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摘要
第一部分
肾素-血管紧张素系统(RAS)、血管生成因子及其拮抗因子与足细胞损伤在子痫前期蛋白尿发生中的相关性及其机制探讨
(一)肾素-血管紧张素系统与尿液足细胞丢失在子痫前期蛋白尿发生中的作用
目的：
在各种肾小球疾病中,尿液足细胞丢失可反映足细胞损伤,足细胞作为肾小球滤过屏障的三层结构之一,其损伤与蛋白尿的发生密切相关。子痫前期肾病典型的病理改变是内皮组织增生,而足细胞损伤在其蛋白尿发生中的作用尚不明确。已知肾素-血管紧张素系统(RAS)异常,能够改变全身血流动力学,调节全身水钠代谢,调节血压,尤其是肾脏局部RAS的激活,对于许多慢性肾脏病蛋白尿的产生及肾小球硬化等方面具有重要意义。而子痫前期患者循环RAS、肾脏局部RAS状况及其在肾脏损伤及蛋白尿产生过程中的作用仍不清楚。本研究的目的在于探讨RAS组分与尿液足细胞丢失在子痫前期肾病患者足细胞损伤调节过程中的作用及其相互关系。
方法：
入选符合纳入条件的子痫前期患者14例(n=14),妊娠期高血压患者14例(n=14)和孕周匹配的正常妊娠者13例(n=13)。用抗-podocalyxin单克隆抗体标记尿足细胞作免疫荧光检测。ELISA法检测血清和尿液的血管紧张素-(1-7)[Ang-(1-7)]、血管紧张素II(Ang 1I)和血管紧张素原(AGT)浓度。对尿足细胞数、RAS组分与蛋白尿之间的关系作相关性分析,应用受试者工作特征(receiver operating characteristic, ROC)曲线评价各指标对子痫前期的诊断价值。
结果：
1.分娩前,子痫前期患者尿液中足细胞数[中位数(四分位数间距)：3.10(2.37-5.66)cells/ml]显著高于妊娠期高血压组[1.41(0.71-3.73)cells/ml,P<0.05]和正常妊娠组[0.52(0.01-1.49)cells/ml.P<0.001].分娩后,各组间尿足细胞数无显著差异。子痫前期组分娩前尿足细胞数显著高于分娩后[3.10(2.37-5.66)vs.0.45(0-1.76)cells/ml,P<0.001].
2.子痫前期组Ang-(1-7)浓度(血清：53.88±17.97pg/ml；尿液：69.99±19.09pg/ml)较妊娠期高血压组(血清：70.96±19.83pg/ml,P<0.05；尿液：98.78±23.99 pg／ml,P=0.001)和正常妊娠组(血清72.50±20.59pg/ml,P<0.05；尿液：92.91±18.04pg/ml,P<0.01)显著降低。各组间血清和尿液AngⅡ浓度无显著差异。子痫前期组AGT浓度(血清：69.47±18.68μg/ml；尿液：41.01±64.29 ng/ml)低于妊娠期高血压组(血清：82.93±12.34μg/ml,P<0.05；尿液：86.60±62.75ng/ml,P>0.05)和正常妊娠组(血清：90.94±14.92μg/ml,P=0.001；尿液：116.21±65.59ng/ml,P<0.01).
3.分娩前尿足细胞数与随机尿白蛋白/肌酐比值(ACR)、肾功能和血压呈正相关。血清、尿液Ang-(1-7)和AGT浓度与分娩前尿足细胞数、ACR、肾功能及血压呈负相关。血清AngⅡ、AGT分别与尿液AngⅡ、AGT呈正相关。
4.ROC曲线下面积(AUC)表明,分娩前尿足细胞、血清和尿液Ang-(1-7)是诊断子痫前期有用指标,血清和尿液AGT对其诊断也具有一定价值。
5.选取最高约登指数(Youden index,即：正确诊断指数)的截断值：分娩前尿足细胞数≥1个cell/ml,血清Ang-(1-7)≤55pg/ml,尿液Ang-(1-7)≤80pg/ml,其灵敏度/特异度分别是100%/76.92%,57.14%/84.62%,71.43%/92.31%,不同串联或并联试验可有效提高诊断子痫前期的灵敏度和特异度。
结论：
降低的Ang-(1-7)和下调的肾脏局部RAS组分可能促进了子痫前期肾病患者足细胞损伤、脱落,从而导致蛋白尿的发生。Ang-(1-7)和尿液足细胞检测可能成为诊断子痫前期肾病的有效标志,AGT也具有一定的诊断价值。
(二)血管生成因子及其拮抗因子与足细胞损伤对子痫前期蛋白尿产生的影响
目的：
血管内皮生长因子(vascular endothelial growth factor, VEGF)是体内最具活力的一种血管生长因子,在肾小球内皮细胞、足细胞、肾小管上皮细胞及系膜细胞等均有表达,通过其细胞膜受体(Flk-1, Flt-1, neuropilins等)介导来发挥作用。胎盘生长因子(PIGF)是VEGF家族另一成员,仅能特异地与Flt-1结合。而可溶性fms-样酪氨酸激酶受体1(sFlt-1)是Flt-1的剪接变异体,也是VEGF的天然拮抗剂,可完全阻断VEGF的生物学活性。近年来研究发现,VEGF家族与sFlt-1在子痫前期的发病中起着重要作用,可能与其内皮系统受损有关。然而,鉴于足细胞可直接分泌VEGF,而其受体如neuropilins蛋白也表达于足细胞上,且足细胞损伤在子痫前期蛋白尿发生中的作用逐渐被认识,我们推测血管生成因子VEGF、PIGF及其拮抗剂-sFlt-1之间的平衡失调可能引起肾小球足细胞损伤,而非单一引起内皮受损,继而发生蛋白尿。本研究目的在于检测子痫前期患者血管生成因子及其拮抗剂的表达变化,并将其与尿足细胞排泄进行相关性分析,从而探讨其变化与足细胞损伤对子痫前期蛋白尿的影响。
方法：
入选符合纳入条件的子痫前期患者14例(n=14),妊娠期高血压患者14例(n=14)和孕周匹配的正常妊娠者13例(n=13)作为研究对象。尿足细胞用抗-podocalyxin单克隆抗体标记进行免疫荧光检测并计数。ELISA法定量检测血清和尿液的VEGF、PIGF和sFlt-1浓度。计算血清和尿液P1GF/sFlt-1比值,检测随机尿白蛋白/肌酐的比值(ACR)和肾功能。对足细胞尿、血管生成因子及其拮抗因子和蛋白尿之间的关系进行相关性分析。绘制ROC曲线评估各指标不同截断值诊断子痫前期的灵敏度和特异度。
结果：
1.分娩前尿足细胞检测及计数结果见第一部分(一),结果1。
2.子痫前期组患者血清VEGF浓度(44.05±25.01pg/ml)显著高于正常妊娠组(25.84±14.22 pg/ml, P<0.05),但与妊娠期高血压组(34.75±15.67pg/ml, P>0.05)比较无显著差异。三组间尿液VEGF浓度无显著差异。血清和尿液PIGF浓度在子痫前期组患者中(血清：52.59±7.46pg/ml;尿液：12.39±5.30pg/ml)显著低于妊娠期高血压组(血清：60.39±4.74pg/ml, P=0.001;尿液：26.25±8.25 pg/ml, P< 0.05)和正常妊娠组(血清:65.40±3.76 pg/ml, P<0.001;尿液：31.81±26.65 pg/ml, P<0.01)血清和尿液sFlt-1浓度在子痫前期组(血清：538.33±53.28 pg/ml, P<0.05;尿液：812.09±75.78pg/ml, P<0.01)和妊娠期高血压组患者中(血清：546.11±66.80pg/ml, P<0.05;尿液：813.34±68.03pg/ml,P<0.01)显著高于正常妊娠组(血清：466.82±111.57pg/ml;尿液：679.35±188.88 pg/ml)。血清和尿液的P1GF/sFlt-1比值在子痫前期组(血清：P<0.001；尿液：P<0.01)和妊娠期高血压组中(血清：P<0.001；尿液：P<0.05)都显著低于正常妊娠组。
3.相关性分析表明血清VEGF浓度与分娩前的尿足细胞数、ACR、尿酸(UA)和血压呈显著正相关；血清P1GF浓度、血清P1GF/sFlt-1比值都与分娩前的尿足细胞数、ACR、UA和血压呈负相关。尿液P1GF浓度、尿液P1GF/sFlt-1比值都与ACR呈负相关。血清和尿液的VEGF.P1GF,sFlt-1浓度、P1GF/sFlt-1比值之间也存在着相关关系。
4. P1GF浓度的AUC[血清：0.872(95%CI,0.744-1.000,P<0.001)；尿液：0.811(95%CI,0.681-0.941,P=0.001)]和P1GF/sFlt-1比值的AUC[血清：0.847(95%CI,0.728-0.965,P<0.001)；尿液：0.815(95%CI,0.686-0.943,P=0.001)]优于VEGF[血清：0.675(95%CI,0.483-0.866,P=0.079)；尿液：0.550(95%CI,0.354-0.746,P=0.613)]和sFlt-1[血清：0.590(95%CI,0.414-0.766,P=0.350)；尿液：0.567(95%CI,0.383-0.752,P=0.483)]。
5.选取最高约登指数(正确诊断指数)的截断值：血清P1GF< 60 pg/ml,尿液P1GF< 25 pg/ml,作为诊断子痫前期的预测值,其灵敏度/特异度分别是85.71%/92.31%,100%/46.15%,不同串联或并联试验可有效提高诊断子痫前期的灵敏度和特异度。
结论：
VEGF.P1GF及其拮抗因子sFlt-1的异常调节可能是导致子痫前期足细胞损伤和蛋白尿的发生机制之一。P1GF、PlGF/sFlt-1比值有潜力成为子痫前期的诊断标志物。
第二部分子痫前期患者尿液的定量蛋白质组学研究
目的：
子痫前期患者蛋白尿的程度与母体肾损伤的预后密切相关。作为一种非侵入性的检查,尿液蛋白质组学已成为识别肾脏疾病的有用工具,对于妊娠妇女这一特殊群体尤其适用。本研究通过比较子痫前期患者、妊娠期高血压患者和正常妊娠者的尿液蛋白质组,寻找各组尿液中差异表达的蛋白质,为筛选子痫前期早期诊断的潜在生物标志物、研究子痫前期肾损伤的发病机制提供了线索。尿液蛋白质组学在识别子痫前期肾病方面具有广阔的应用前景。
方法：
冷丙酮／三氯乙酸法提取子痫前期患者(n=10)、妊娠期高血压患者(n=10)和正常妊娠者(n=10)的尿蛋白,去除高丰度蛋白、除盐,Bradford法测蛋白浓度。应用同位素标记的相对和绝对定量(iTRAQ)标签和二维液相色谱与串联质谱联用(2DLC-MS/MS)技术定量和鉴定蛋白质。实验重复两次(记做RUN1和RUN2)。Protein Pilot 3.0软件分析质谱图,IPI (International Protein Index)数据库中搜索肽段信息,根据iTRAQ标记定量的比值(ratio)寻找差异蛋白质；应用Gene Ontology (GO)和Metacore软件进行相关生物信息学分析。ELISA方法验证了差异表达的蛋白质。
结果：
1.在2次2D LC-MS/MS实验中分别鉴定到507和516个蛋白,其中362个蛋白在2次实验中均被鉴定到,5个蛋白无iTRAQ标记的比值信息,两次实验的重复性超过70%,结果可靠。子痫前期组与正常妊娠组比较,有113个蛋白在2次实验中均有明显差异(iTRAQ比值<0.833或>1.2),31个蛋白在三组间比较均有显著差异。
2.这些差异蛋白质与血液凝固、细胞粘附和分化、免疫反应和细胞骨架重塑等生物过程有关。他们在蛋白质的网络中存在相互作用。
3.在差异蛋白质中,角蛋白(Keratin)、α-辅肌动蛋白-4 (Alpha-actinin-4)、Afamin、α-2-巨球蛋白(Alpha-2-macroglobulin)、血清白蛋白(ALB)、α1-抗胰蛋白酶(SERPINA1)、波形蛋白(Vimentin)、β-2C微管蛋白(Tubulin beta-2C chain)在三组间比较均呈高倍差异表达,可能与子痫前期的发病机制密切相关。而α-actinin-4、Vimentin是足细胞骨架结构的重要蛋白,子痫前期患者尿液中α-actinin-4、Vimentin蛋白的高倍差异表达可能与足细胞损伤和蛋白尿的发生密切相关。
4.子痫前期患者尿液的血管紧张素原(angiotensinogen, AGT)表达下调,此发现与ELISA的验证结果一致。
结论：
本研究发现了多种差异表达的蛋白质,为探索子痫前期肾病蛋白尿的发生机制,寻找子痫前期肾病早期诊断及预测的生物标志物提供了线索。降低的尿液血管紧张素原(AGT)有助于识别子痫前期肾病,AGT、SERPINA1和ALB是子痫前期生物标志物的候选蛋白。尿液中足细胞相关骨架蛋白Vimentin和α-actinin-4高倍差异表达,为子痫前期肾病足细胞损伤提供了更充分的证据；部分差异表达蛋白尚需进一步验证。
第三部分
肾素-血管紧张素系统(RAS)组分Ang-(1-7)对子痫前期患者血清体外诱导足细胞损伤的影响及其机制初探
目的：
RAS是一个非常复杂的、多层次的内分泌系统,包括多种组成成分和代谢片段。血管紧张素II(AngII)作为R.AS的核心,主要发挥收缩血管、升高血压和组织损伤等病理和生理学作用。体内外研究证实足细胞能表达血管紧张素受体,是Ang II致肾脏损伤的靶细胞之一。Ang II本身也可造成肾小球足突细胞的形态改变,促进足细胞凋亡,使之数量下降。近年来在RAS中发现的新成员Ang-(1-7)及其特异性受体-原癌基因编码的G蛋白配体受体(Mas)在体内作用广泛,能够对抗AngII的效应而发挥扩张血管的作用。RAS中的对抗调节轴：ACE 2-Ang-(1-7)-Mas轴与ACE-AngⅡ-AT1R轴在子痫前期的发病机制中起着重要作用。在我们第一部分的研究结果中也显示,Ang-(1-7)与子痫前期患者肾小球足细胞损伤和蛋白尿的发生有着密切的相关性；Ang-(1-7)浓度在子痫前期患者的血清和尿液中显著降低,可能是造成肾小球足细胞损伤的原因之—为进一步探讨Ang-(1-7)对子痫前期肾小球足细胞的影响及其可能机制,我们在体外模拟子痫前期环境,用Ang-(1-7)干预培养足细胞,观察足细胞相关蛋白的变化。
方法：
应用体外细胞培养技术,培养条件性永生的小鼠肾小球足细胞,收集临床子痫前期患者血清标本干预足细胞,制备子痫前期足细胞的体外模型,设立正常对照组、正常妊娠者血清组、子痫前期患者血清组、Ang-(1-7)+子痫前期患者血清组进行实验。显微镜下观察足细胞形态变化；免疫荧光法检测足细胞标志蛋白nephrin,CD2AP的表达变化、足细胞骨架蛋白F-actin、足细胞紧密连接分子ZO-1及Mas受体的表达变化；Western blot检测Mas受体的表达及变化。
结果：
1.子痫前期患者血清干预足细胞后,免疫荧光观察其nephrin、F-actin和ZO-1表达均较正常对照组和正常妊娠者血清组显著下降甚至不表达,CD2AP的表达未见显著变化；加入Ang-(1-7)共同培养后,足细胞nephri、F-actin和ZO-1的表达显著增加。
2.免疫荧光及Western blot检测各组足细胞均有Mas受体表达,但子痫前期患者血清组足细胞Mas受体表达显著减少；加入Ang-(1-7)共同培养后,足细胞Mas受体表达增加。
结论：
Ang-(1-7)能够保护子痫前期患者血清体外诱导的足细胞损伤,其作用可能和Ang-(1-7)与Mas受体结合所产生的效应有关,深入机制尚需进一步探索。
PART I
The correlation of renin angiotensin system (RAS) components, angiogenic factors and its antagonist and podocyte injury in development of proteinuria in preeclampsia and its possible mechanisms
1. Effects of RAS and urinary podocyte loss on development of proteinuria in preeclampsia
Objective
Urinary 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.
Methods
Women 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)], angiotensinⅡ(AngⅡ) 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.
Results
1. 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 AngⅡhad no significant difference. AGT concentrations in preeclampsia (serum:69.47Ⅱ18.68μg/ml; urine:41.01±64.29 ng/ml) was lower than gestational hypertension group (serum:82.93±12.34μg/ml, P< 0.05; urine: 86.60±62.75 ng/ml, P> 0.05) and normal control (serum:90.94±14.92μg/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 AngⅡand AGT was positively correlated with urinary AngⅡand 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.
Conclusions
Decreased 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 preeclampsia
Objective
Vascular 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.
Methods
14 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.
Results
1. The results of prepartal urinary podocyte was present as PartⅠsection 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.
Conclusions
Imbalance 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 II
Quantitative proteomic investigation of urine in preeclampsia
Objective
The 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.
Methods
The 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.
Results
1. 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.
Conclusion
These 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.
PARTⅢ
Effects of RAS component Ang-(1-7) on podocyte injury in vitro induced by patients'serum of preeclampsia and its possible mechanisms
Objective
Renin angiotensin system (RAS) is a very complicated and multistrata endocrinium, including multiple components and metabolic fragments. AngⅡ, 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 AngⅡ. AngⅡitself 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 AngⅡand dilate vascular. The antistatic equipment in RAS as ACE 2-Ang-(1-7)-Mas axis and ACE-AngⅡ-AT1R axis play an important role in pathogenesis of preeclampsia. Moreover, The results of PartⅠin 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.
Methods
The 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.
Results
1. 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.
Conclusion
Ang-(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.
肾素-血管紧张素系统(RAS)、血管生成因子及其拮抗因子与足细胞损伤在子痫前期蛋白尿发生中的相关性及其机制探讨
(一)肾素-血管紧张素系统与尿液足细胞丢失在子痫前期蛋白尿发生中的作用
目的：
在各种肾小球疾病中,尿液足细胞丢失可反映足细胞损伤,足细胞作为肾小球滤过屏障的三层结构之一,其损伤与蛋白尿的发生密切相关。子痫前期肾病典型的病理改变是内皮组织增生,而足细胞损伤在其蛋白尿发生中的作用尚不明确。已知肾素-血管紧张素系统(RAS)异常,能够改变全身血流动力学,调节全身水钠代谢,调节血压,尤其是肾脏局部RAS的激活,对于许多慢性肾脏病蛋白尿的产生及肾小球硬化等方面具有重要意义。而子痫前期患者循环RAS、肾脏局部RAS状况及其在肾脏损伤及蛋白尿产生过程中的作用仍不清楚。本研究的目的在于探讨RAS组分与尿液足细胞丢失在子痫前期肾病患者足细胞损伤调节过程中的作用及其相互关系。
方法：
入选符合纳入条件的子痫前期患者14例(n=14),妊娠期高血压患者14例(n=14)和孕周匹配的正常妊娠者13例(n=13)。用抗-podocalyxin单克隆抗体标记尿足细胞作免疫荧光检测。ELISA法检测血清和尿液的血管紧张素-(1-7)[Ang-(1-7)]、血管紧张素II(Ang 1I)和血管紧张素原(AGT)浓度。对尿足细胞数、RAS组分与蛋白尿之间的关系作相关性分析,应用受试者工作特征(receiver operating characteristic, ROC)曲线评价各指标对子痫前期的诊断价值。
结果：
1.分娩前,子痫前期患者尿液中足细胞数[中位数(四分位数间距)：3.10(2.37-5.66)cells/ml]显著高于妊娠期高血压组[1.41(0.71-3.73)cells/ml,P<0.05]和正常妊娠组[0.52(0.01-1.49)cells/ml.P<0.001].分娩后,各组间尿足细胞数无显著差异。子痫前期组分娩前尿足细胞数显著高于分娩后[3.10(2.37-5.66)vs.0.45(0-1.76)cells/ml,P<0.001].
2.子痫前期组Ang-(1-7)浓度(血清：53.88±17.97pg/ml；尿液：69.99±19.09pg/ml)较妊娠期高血压组(血清：70.96±19.83pg/ml,P<0.05；尿液：98.78±23.99 pg／ml,P=0.001)和正常妊娠组(血清72.50±20.59pg/ml,P<0.05；尿液：92.91±18.04pg/ml,P<0.01)显著降低。各组间血清和尿液AngⅡ浓度无显著差异。子痫前期组AGT浓度(血清：69.47±18.68μg/ml；尿液：41.01±64.29 ng/ml)低于妊娠期高血压组(血清：82.93±12.34μg/ml,P<0.05；尿液：86.60±62.75ng/ml,P>0.05)和正常妊娠组(血清：90.94±14.92μg/ml,P=0.001；尿液：116.21±65.59ng/ml,P<0.01).
3.分娩前尿足细胞数与随机尿白蛋白/肌酐比值(ACR)、肾功能和血压呈正相关。血清、尿液Ang-(1-7)和AGT浓度与分娩前尿足细胞数、ACR、肾功能及血压呈负相关。血清AngⅡ、AGT分别与尿液AngⅡ、AGT呈正相关。
4.ROC曲线下面积(AUC)表明,分娩前尿足细胞、血清和尿液Ang-(1-7)是诊断子痫前期有用指标,血清和尿液AGT对其诊断也具有一定价值。
5.选取最高约登指数(Youden index,即：正确诊断指数)的截断值：分娩前尿足细胞数≥1个cell/ml,血清Ang-(1-7)≤55pg/ml,尿液Ang-(1-7)≤80pg/ml,其灵敏度/特异度分别是100%/76.92%,57.14%/84.62%,71.43%/92.31%,不同串联或并联试验可有效提高诊断子痫前期的灵敏度和特异度。
结论：
降低的Ang-(1-7)和下调的肾脏局部RAS组分可能促进了子痫前期肾病患者足细胞损伤、脱落,从而导致蛋白尿的发生。Ang-(1-7)和尿液足细胞检测可能成为诊断子痫前期肾病的有效标志,AGT也具有一定的诊断价值。
(二)血管生成因子及其拮抗因子与足细胞损伤对子痫前期蛋白尿产生的影响
目的：
血管内皮生长因子(vascular endothelial growth factor, VEGF)是体内最具活力的一种血管生长因子,在肾小球内皮细胞、足细胞、肾小管上皮细胞及系膜细胞等均有表达,通过其细胞膜受体(Flk-1, Flt-1, neuropilins等)介导来发挥作用。胎盘生长因子(PIGF)是VEGF家族另一成员,仅能特异地与Flt-1结合。而可溶性fms-样酪氨酸激酶受体1(sFlt-1)是Flt-1的剪接变异体,也是VEGF的天然拮抗剂,可完全阻断VEGF的生物学活性。近年来研究发现,VEGF家族与sFlt-1在子痫前期的发病中起着重要作用,可能与其内皮系统受损有关。然而,鉴于足细胞可直接分泌VEGF,而其受体如neuropilins蛋白也表达于足细胞上,且足细胞损伤在子痫前期蛋白尿发生中的作用逐渐被认识,我们推测血管生成因子VEGF、PIGF及其拮抗剂-sFlt-1之间的平衡失调可能引起肾小球足细胞损伤,而非单一引起内皮受损,继而发生蛋白尿。本研究目的在于检测子痫前期患者血管生成因子及其拮抗剂的表达变化,并将其与尿足细胞排泄进行相关性分析,从而探讨其变化与足细胞损伤对子痫前期蛋白尿的影响。
方法：
入选符合纳入条件的子痫前期患者14例(n=14),妊娠期高血压患者14例(n=14)和孕周匹配的正常妊娠者13例(n=13)作为研究对象。尿足细胞用抗-podocalyxin单克隆抗体标记进行免疫荧光检测并计数。ELISA法定量检测血清和尿液的VEGF、PIGF和sFlt-1浓度。计算血清和尿液P1GF/sFlt-1比值,检测随机尿白蛋白/肌酐的比值(ACR)和肾功能。对足细胞尿、血管生成因子及其拮抗因子和蛋白尿之间的关系进行相关性分析。绘制ROC曲线评估各指标不同截断值诊断子痫前期的灵敏度和特异度。
结果：
1.分娩前尿足细胞检测及计数结果见第一部分(一),结果1。
2.子痫前期组患者血清VEGF浓度(44.05±25.01pg/ml)显著高于正常妊娠组(25.84±14.22 pg/ml, P<0.05),但与妊娠期高血压组(34.75±15.67pg/ml, P>0.05)比较无显著差异。三组间尿液VEGF浓度无显著差异。血清和尿液PIGF浓度在子痫前期组患者中(血清：52.59±7.46pg/ml;尿液：12.39±5.30pg/ml)显著低于妊娠期高血压组(血清：60.39±4.74pg/ml, P=0.001;尿液：26.25±8.25 pg/ml, P< 0.05)和正常妊娠组(血清:65.40±3.76 pg/ml, P<0.001;尿液：31.81±26.65 pg/ml, P<0.01)血清和尿液sFlt-1浓度在子痫前期组(血清：538.33±53.28 pg/ml, P<0.05;尿液：812.09±75.78pg/ml, P<0.01)和妊娠期高血压组患者中(血清：546.11±66.80pg/ml, P<0.05;尿液：813.34±68.03pg/ml,P<0.01)显著高于正常妊娠组(血清：466.82±111.57pg/ml;尿液：679.35±188.88 pg/ml)。血清和尿液的P1GF/sFlt-1比值在子痫前期组(血清：P<0.001；尿液：P<0.01)和妊娠期高血压组中(血清：P<0.001；尿液：P<0.05)都显著低于正常妊娠组。
3.相关性分析表明血清VEGF浓度与分娩前的尿足细胞数、ACR、尿酸(UA)和血压呈显著正相关；血清P1GF浓度、血清P1GF/sFlt-1比值都与分娩前的尿足细胞数、ACR、UA和血压呈负相关。尿液P1GF浓度、尿液P1GF/sFlt-1比值都与ACR呈负相关。血清和尿液的VEGF.P1GF,sFlt-1浓度、P1GF/sFlt-1比值之间也存在着相关关系。
4. P1GF浓度的AUC[血清：0.872(95%CI,0.744-1.000,P<0.001)；尿液：0.811(95%CI,0.681-0.941,P=0.001)]和P1GF/sFlt-1比值的AUC[血清：0.847(95%CI,0.728-0.965,P<0.001)；尿液：0.815(95%CI,0.686-0.943,P=0.001)]优于VEGF[血清：0.675(95%CI,0.483-0.866,P=0.079)；尿液：0.550(95%CI,0.354-0.746,P=0.613)]和sFlt-1[血清：0.590(95%CI,0.414-0.766,P=0.350)；尿液：0.567(95%CI,0.383-0.752,P=0.483)]。
5.选取最高约登指数(正确诊断指数)的截断值：血清P1GF< 60 pg/ml,尿液P1GF< 25 pg/ml,作为诊断子痫前期的预测值,其灵敏度/特异度分别是85.71%/92.31%,100%/46.15%,不同串联或并联试验可有效提高诊断子痫前期的灵敏度和特异度。
结论：
VEGF.P1GF及其拮抗因子sFlt-1的异常调节可能是导致子痫前期足细胞损伤和蛋白尿的发生机制之一。P1GF、PlGF/sFlt-1比值有潜力成为子痫前期的诊断标志物。
第二部分子痫前期患者尿液的定量蛋白质组学研究
目的：
子痫前期患者蛋白尿的程度与母体肾损伤的预后密切相关。作为一种非侵入性的检查,尿液蛋白质组学已成为识别肾脏疾病的有用工具,对于妊娠妇女这一特殊群体尤其适用。本研究通过比较子痫前期患者、妊娠期高血压患者和正常妊娠者的尿液蛋白质组,寻找各组尿液中差异表达的蛋白质,为筛选子痫前期早期诊断的潜在生物标志物、研究子痫前期肾损伤的发病机制提供了线索。尿液蛋白质组学在识别子痫前期肾病方面具有广阔的应用前景。
方法：
冷丙酮／三氯乙酸法提取子痫前期患者(n=10)、妊娠期高血压患者(n=10)和正常妊娠者(n=10)的尿蛋白,去除高丰度蛋白、除盐,Bradford法测蛋白浓度。应用同位素标记的相对和绝对定量(iTRAQ)标签和二维液相色谱与串联质谱联用(2DLC-MS/MS)技术定量和鉴定蛋白质。实验重复两次(记做RUN1和RUN2)。Protein Pilot 3.0软件分析质谱图,IPI (International Protein Index)数据库中搜索肽段信息,根据iTRAQ标记定量的比值(ratio)寻找差异蛋白质；应用Gene Ontology (GO)和Metacore软件进行相关生物信息学分析。ELISA方法验证了差异表达的蛋白质。
结果：
1.在2次2D LC-MS/MS实验中分别鉴定到507和516个蛋白,其中362个蛋白在2次实验中均被鉴定到,5个蛋白无iTRAQ标记的比值信息,两次实验的重复性超过70%,结果可靠。子痫前期组与正常妊娠组比较,有113个蛋白在2次实验中均有明显差异(iTRAQ比值<0.833或>1.2),31个蛋白在三组间比较均有显著差异。
2.这些差异蛋白质与血液凝固、细胞粘附和分化、免疫反应和细胞骨架重塑等生物过程有关。他们在蛋白质的网络中存在相互作用。
3.在差异蛋白质中,角蛋白(Keratin)、α-辅肌动蛋白-4 (Alpha-actinin-4)、Afamin、α-2-巨球蛋白(Alpha-2-macroglobulin)、血清白蛋白(ALB)、α1-抗胰蛋白酶(SERPINA1)、波形蛋白(Vimentin)、β-2C微管蛋白(Tubulin beta-2C chain)在三组间比较均呈高倍差异表达,可能与子痫前期的发病机制密切相关。而α-actinin-4、Vimentin是足细胞骨架结构的重要蛋白,子痫前期患者尿液中α-actinin-4、Vimentin蛋白的高倍差异表达可能与足细胞损伤和蛋白尿的发生密切相关。
4.子痫前期患者尿液的血管紧张素原(angiotensinogen, AGT)表达下调,此发现与ELISA的验证结果一致。
结论：
本研究发现了多种差异表达的蛋白质,为探索子痫前期肾病蛋白尿的发生机制,寻找子痫前期肾病早期诊断及预测的生物标志物提供了线索。降低的尿液血管紧张素原(AGT)有助于识别子痫前期肾病,AGT、SERPINA1和ALB是子痫前期生物标志物的候选蛋白。尿液中足细胞相关骨架蛋白Vimentin和α-actinin-4高倍差异表达,为子痫前期肾病足细胞损伤提供了更充分的证据；部分差异表达蛋白尚需进一步验证。
第三部分
肾素-血管紧张素系统(RAS)组分Ang-(1-7)对子痫前期患者血清体外诱导足细胞损伤的影响及其机制初探
目的：
RAS是一个非常复杂的、多层次的内分泌系统,包括多种组成成分和代谢片段。血管紧张素II(AngII)作为R.AS的核心,主要发挥收缩血管、升高血压和组织损伤等病理和生理学作用。体内外研究证实足细胞能表达血管紧张素受体,是Ang II致肾脏损伤的靶细胞之一。Ang II本身也可造成肾小球足突细胞的形态改变,促进足细胞凋亡,使之数量下降。近年来在RAS中发现的新成员Ang-(1-7)及其特异性受体-原癌基因编码的G蛋白配体受体(Mas)在体内作用广泛,能够对抗AngII的效应而发挥扩张血管的作用。RAS中的对抗调节轴：ACE 2-Ang-(1-7)-Mas轴与ACE-AngⅡ-AT1R轴在子痫前期的发病机制中起着重要作用。在我们第一部分的研究结果中也显示,Ang-(1-7)与子痫前期患者肾小球足细胞损伤和蛋白尿的发生有着密切的相关性；Ang-(1-7)浓度在子痫前期患者的血清和尿液中显著降低,可能是造成肾小球足细胞损伤的原因之—为进一步探讨Ang-(1-7)对子痫前期肾小球足细胞的影响及其可能机制,我们在体外模拟子痫前期环境,用Ang-(1-7)干预培养足细胞,观察足细胞相关蛋白的变化。
方法：
应用体外细胞培养技术,培养条件性永生的小鼠肾小球足细胞,收集临床子痫前期患者血清标本干预足细胞,制备子痫前期足细胞的体外模型,设立正常对照组、正常妊娠者血清组、子痫前期患者血清组、Ang-(1-7)+子痫前期患者血清组进行实验。显微镜下观察足细胞形态变化；免疫荧光法检测足细胞标志蛋白nephrin,CD2AP的表达变化、足细胞骨架蛋白F-actin、足细胞紧密连接分子ZO-1及Mas受体的表达变化；Western blot检测Mas受体的表达及变化。
结果：
1.子痫前期患者血清干预足细胞后,免疫荧光观察其nephrin、F-actin和ZO-1表达均较正常对照组和正常妊娠者血清组显著下降甚至不表达,CD2AP的表达未见显著变化；加入Ang-(1-7)共同培养后,足细胞nephri、F-actin和ZO-1的表达显著增加。
2.免疫荧光及Western blot检测各组足细胞均有Mas受体表达,但子痫前期患者血清组足细胞Mas受体表达显著减少；加入Ang-(1-7)共同培养后,足细胞Mas受体表达增加。
结论：
Ang-(1-7)能够保护子痫前期患者血清体外诱导的足细胞损伤,其作用可能和Ang-(1-7)与Mas受体结合所产生的效应有关,深入机制尚需进一步探索。
PART I
The correlation of renin angiotensin system (RAS) components, angiogenic factors and its antagonist and podocyte injury in development of proteinuria in preeclampsia and its possible mechanisms
1. Effects of RAS and urinary podocyte loss on development of proteinuria in preeclampsia
Objective
Urinary 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.
Methods
Women 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)], angiotensinⅡ(AngⅡ) 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.
Results
1. 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 AngⅡhad no significant difference. AGT concentrations in preeclampsia (serum:69.47Ⅱ18.68μg/ml; urine:41.01±64.29 ng/ml) was lower than gestational hypertension group (serum:82.93±12.34μg/ml, P< 0.05; urine: 86.60±62.75 ng/ml, P> 0.05) and normal control (serum:90.94±14.92μg/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 AngⅡand AGT was positively correlated with urinary AngⅡand 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.
Conclusions
Decreased 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 preeclampsia
Objective
Vascular 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.
Methods
14 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.
Results
1. The results of prepartal urinary podocyte was present as PartⅠsection 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.
Conclusions
Imbalance 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 II
Quantitative proteomic investigation of urine in preeclampsia
Objective
The 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.
Methods
The 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.
Results
1. 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.
Conclusion
These 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.
PARTⅢ
Effects of RAS component Ang-(1-7) on podocyte injury in vitro induced by patients'serum of preeclampsia and its possible mechanisms
Objective
Renin angiotensin system (RAS) is a very complicated and multistrata endocrinium, including multiple components and metabolic fragments. AngⅡ, 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 AngⅡ. AngⅡitself 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 AngⅡand dilate vascular. The antistatic equipment in RAS as ACE 2-Ang-(1-7)-Mas axis and ACE-AngⅡ-AT1R axis play an important role in pathogenesis of preeclampsia. Moreover, The results of PartⅠin 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.
Methods
The 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.
Results
1. 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.
Conclusion
Ang-(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.
引文
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