【作者】 李辉；

【导师】 张颖；

【作者基本信息】 郑州大学 ， 内科学（肾病）， 2021， 硕士

【摘要】 研究背景IgA肾病(IgA nephropathy,IgAN)是我国最常见的原发性肾小球疾病,临床表现多种多样,是导致我国终末期肾脏病(End-stage renal disease,ESRD)的主要原因之一。既往大量数据表明,IgAN患者的不良预后与持续性大量蛋白尿密切相关。已有研究证明,蛋白尿的发生与肾小球滤过屏障受损相关,而足细胞足突间的裂孔隔膜是肾小球滤过屏障的重要组成部分。既往有实验证实了足细胞足突融合、凋亡、去分化及脱落消失是导致蛋白尿、肾小球硬化及滤过衰竭的主要原因。但是IgAN病程中足细胞的损伤机制尚不明确。近年来的研究发现补体系统活化在膜性肾病、局灶节段肾小球硬化症、糖尿病肾病、不典型溶血尿毒综合征等多种原继发肾脏病足细胞损伤中发挥重要作用。IgAN肾组织除了 IgA沉积,常伴C3沉积,证实补体活化参与IgAN的发病及进展。但是补体系统的活化在IgAN足细胞损伤过程中是否发挥作用目前尚不明确。C5a是补体活化的主要效应产物,通过与其受体C5aR结合发挥强大的致炎效应。最近有研究发现膜性肾病足细胞上C5aR表达明显上调。本课题组既往在阿霉素诱导的局灶节段肾小球硬化小鼠模型上证实,C5aR敲除能减轻肾脏足细胞损伤及肾小球硬化,明显减少蛋白尿,保护肾脏功能。本研究从动物实验和细胞实验两个方面探究C5a及其受体C5aR在IgAN足细胞损伤中的作用,从而深入了解IgAN足细胞损伤的机制。研究目的探讨补体C5a及其受体(C5aR)在IgA肾病足细胞损伤过程中的作用。研究方法1.8周龄的雌性Balb/c小鼠共27只,野生型组(Wild Type,WT)组,IgAN组,C5aR敲除(C5aR-/-)组每组各9只,使用有活性的仙台病毒滴鼻联合尾静脉注射诱导IgAN组和C5aR-/-组小鼠建立IgAN模型,检验各组小鼠24小时蛋白尿水平,PAS染色后光学显微镜观察各组小鼠肾组织病理改变,电子显微镜观察小鼠肾组织足细胞超微结构变化及电子致密物的沉积,免疫荧光观察小鼠肾组织nephrin和podocin的表达。2.使用不同浓度Ang-Ⅱ(10-6-10-8M)刺激体外培养的人足细胞24小时,免疫荧光法检测足细胞在病理刺激下C5aR和nephrin的表达,Western blot法及qRT-PCR法观察足细胞在病理刺激下C5aR蛋白和mRNA的表达。3.使用间接法提纯IgAN患者血浆IgA1分子并加热形成聚合体。建立人系膜细胞和人足细胞的体外共培养体系,使用不同浓度IgA1(0 mg/ml,0.1mg/ml,1mg/ml,2mg/ml)刺激共培养的系膜细胞24小时,Western blot法及qRT-PCR检测共培养的足细胞上C5aR,nephrin和podocin蛋白和mRNA的表达。4.共培养的细胞分为三组,空白对照组,IgA1组,PMX-53组,空白对照组不加刺激,IgA1组在共培养的系膜细胞中加入IgA1(1mg/ml),PMX-53组在共培养的系膜细胞中加入IgA1(1mg/ml),同时共培养的足细胞与PMX-53(10-4M)共孵育,每组的细胞均接受不同刺激24小时,Western blot法和qRT-PCR法检测共培养的足细胞上nephrin和podocin蛋白及mRNA的表达。研究结果1.动物实验中,造模14周后,IgAN组及C5aR-/-组小鼠24h蛋白尿水平均高于WT组小鼠(p<0.05),且IgAN组小鼠24h蛋白尿水平明显高于C5aR-/-组(p<0.05)。2.光学显微镜下,IgAN以及C5aR-/-组小鼠肾组织均有不同程度的系膜细胞和系膜基质的增多,而C5aR-/-组小鼠系膜细胞和系膜基质增多的程度较IgAN组减轻,WT组小鼠肾组织未见明显病理改变。3.电子显微镜下,IgAN组以及C5aR-/-组小鼠肾组织均可见足突消失、融合,系膜区有电子致密物沉积,与IgAN组相比,C5aR-/-组小鼠肾组织足突消失及融合的程度减轻,电子致密物沉积减少,WT组小鼠未见足突融合、消失及电子致密物沉积。4.小鼠肾组织免疫荧光结果显示,与WT组小鼠相比,IgAN以及C5aR-/-组小鼠肾组织均可见nephrin和podocin表达减低(p<0.05),C5aR-/-组小鼠肾组织nephrin和podocin的表达又较IgAN组增强(p<0.05)。5.细胞实验中,细胞免疫荧光结果显示:体外培养的人足细胞被不同浓度Ang-Ⅱ刺激后,C5aR的表达升高,且与Ang-Ⅱ浓度呈正相关(p<0.05),nephrin的表达降低,且与Ang-Ⅱ浓度负相关(p<0.05)。Western blot和qRT-PCR结果证实了在受到Ang-Ⅱ刺激后,足细胞上C5aR蛋白及mRNA的表达升高,且与Ang-Ⅱ浓度正相关。6.建立共培养系统后,Western blot和qRT-PCR的结果显示:IgA1刺激共培养的系膜细胞,可使足细胞C5aR蛋白及mRNA的表达随IgA1浓度的升高而升高(p<0.05),nephrin和podocin蛋白及mRNA的表达随IgA1浓度的升高而降低(p<0.05)。7.C5aR拮抗剂(PMX-53)与足细胞共孵育后,Western blot和qRT-PCR的结果显示:与空白对照组相比,IgA1组nephrin和podocin蛋白和mRNA的表达明显降低(p<0.05),PMX-53组nephrin和podocin蛋白及mRNA的表达与空白对照组无统计学差异,与IgA1组相比,PMX-53组nephrin和podocin蛋白及mRNA的表达增加(p<0.05)。研究结论1.IgA1刺激共培养的人系膜细胞,诱导了足细胞上C5aR的表达,促进了足细胞的损伤。2.在体内敲除及在体外拮抗C5a受体均减轻了足细胞的损伤,提示拮抗C5aR可保护IgA肾病足细胞的功能。更多还原

【Abstract】 Immunoglobulin A(IgA)nephropathy(IgAN),the most common primary glomerulonephritis in our country,remains a leading cause of end-stage renal disease(ESRD).Substantial amounts of data demonstrate that poor outcomes are closely correlated with persistent proteinuria in patients with IgAN.A growing body of evidence suggests that the pathogenesis of proteinuria is considered to be associated with the permeability of the glomerular filtration barrier,which involves the function of podocytes.Several studies have recognized that podocyte foot process fusion,dedifferentiation and abscission of podocytes is the major cause of proteinuria,glomerulosclerosis and filtration failure.However,the mechanism mediating podocyte injury in IgAN have not been fully elucidated.Accumulating evidence shows that complement activation plays an important role in podocyte injury,such as membranous nephropathy,focal and segmental glomerulosclerosis,diabetic nephropathy,atypical hemolytic uremic syndrome,et al.One of the most important histologic characteristics broadly observed in the kidney of patients with IgAN is the deposition of IgA as well as complement3(C3)deposition at the glomerular mesangial region.Studies show that complement activation is involved in the pathogenesis and progression of IgAN.Nevertheless,the role of complement activation in the pathogenesis of podocyte injury in IgAN is poorly understood.C5a is a major inflammatory effector of complement activation and exert proinflammatory effects by binding to its receptor.Studies found that C5aR expression is up-regulated on podocyte in membranous nephropathy.We previously found that knockout of C5aR leads to protectiveness of podocyte function in mouse model of adriamycin(ADR)-induced focal and segmental glomerulosclerosis(FSGS).In this study,we demonstrated the effect of C5a and its receptor(C5aR)on podocyte injury in vitro and in vivo,providing insight into the mechanism of podocyte injury in IgAN.ObjectiveTo investigate the mechanism of C5a and its receptor C5aR in podocyte injury in IgAN.Methods(1)Twenty-seven 8-weeks-old female Balb/c mice were randomly divided into three groups:wild-type(WT)group,IgAN group,and C5aR-/-group(9 mice/group).An IgAN mouse model was induced by Sendai virus infection using the mice of IgAN group and C5aR-/-group.24h total urinary protein of every mouse was tested.Light microscopy was used to observe the pathological injury in mouse kidney tissue.Electron microscopy was used to analyze the change of podocyte foot process of every group.Immunofluorescence was used to observe the expression of nephrin and podocin in mouse kidney tissue.(2)Human podocytes were incubated in media containing various concentrations of Ang-Ⅱ(10-6-10-8M)for 24h.The expression of C5aR and nephrin were observed by immunofluorescence.C5aR expression was analyzed by Western blot and quantitative real-time polymerase chain reaction(qRT-PCR).(3)IgAl was purified indirectly from the plasma of patients with IgAN.A Transwell co-culture system of human mesangial cells(HMCs)and human podocytes was developed to mimic the IgAN microenvironment in vivo.HMCs were incubated in media containing various concentrations of IgA1(0 mg/ml,0.1mg/ml,1mg/ml,2mg/ml)for 24h.The expression of C5aR,nephrin and podocin on podocytes in the co-culture system were observed by Western blot and qRT-PCR.(4)The cells of co-culture system were divided into three groups:control group,IgA1 group and PMX-53 group.Control group was stimulated with nothing.Co-cultured HMCs were stimulated with IgA1(1mg/ml)in IgA1 group.Co-cultured HMCs were stimulated with IgA1(lmg/ml)and co-cultured podocytes were stimulated with PMX-53(10-4M)at the same time in PMX-53 group.Western blot and qRT-PCR were used to detect the expression of nephrin and podocin on co-cultured podocytes after 24h.Results(1)In animal experiments,after 14 weeks of immunization no albuminuria was exhibited by the mice in WT group.Proteinuria level in IgAN mice and C5aR-/mice was higher than those in WT group at the end of week 14(p<0.05).Proteinuria level in IgAN mice was significantly greater than C5aR-/-mice after 14 weeks(p<0.05).(2)The IgAN mice showed mesangial hypercellularity with matrix expansion using optical microscope.Mesangial matrix expansion and hypercellularity were reduced in C5aR-/-mice.(3)Podocyte foot process effacement was observed frequently in IgAN mouse tissue using electron microscope.Foot process fusion was less common in C5aR-/-mice.(4)For IgAN mice,the nephrin and podocin staining of the podocytes was markedly attenuated(p<0.05).The weakened nephrin and podocin staining intensity was partly reversed in C5aR-/-mice(p<0.05).(5)In cell experiments,a significant dose-dependent increase was observed in the density of C5aR staining following incubation with angiotensin Ⅱ by immunofluorescence(Ang-Ⅱ,p<0.05).The Ang-Ⅱ dose-dependent up-regulation of C5aR in podocytes was verified using qRT-PCR and Western blot analyses(p<0.05).In contrast,nephrin expression was down-regulated by Ang-Ⅱ stimulation and was negatively associated with Ang-Ⅱ concentrations(p<0.05).(6)A dose-dependent up-regulation of C5aR expression in podocytes co-cultured with HMCs was demonstrated following incubation with IgA1(p<0.05).Likewise,the C5aR mRNA expression level was also up-regulated in a dose-dependent manner in podocytes incubated with IgA1.Conversely,the mRNA levels of nephrin and podocin in podocytes were significantly lower in the group stimulated with IgA1 compared with the control group(p<0.05).The down-regulation of nephrin and podocin protein was confirmed using western blotting(p<0.05).(7)For the group without PMX-53 treatment,the expression level of nephrin and podocin was significantly reduced in podocytes,as shown using western blotting and qRT-PCR(p<0.01).However,the down-regulation of nephrin and podocin was inhibited in the PMX-53-treated group(p<0.01).Conclusion(1)Expression of C5a receptor in podocytes induced by IgA1 stimulation of co-cultured HMCs promotes podocyte injury.(2)Targeting C5aR attenuates podocyte injury in vitro and in vivo.The deficiency of C5a receptor could protect the function of podocytes in IgAN.更多还原