【摘要】 目的:局灶节段性肾小球硬化症(FSGS)患者蛋白尿的发生与循环中存在某类物质相关。但循环因子的本质,其作用的靶细胞等问题都没有研究清楚。足细胞病变在大量蛋白尿发生中的重要性得到了充分的认识。本研究利用体外培养的分化足细胞作为检测循环因子的靶细胞,探讨了该方法的临床应用,并对循环因子的理化性质及其损伤足细胞的作用机制进行了研究。方法:经肾活检明确诊断的FSGS患者80例和足细胞病(podocytopathy)患者74例作为研究对象,同时选取临床表现为肾病综合征,组织形态学改变为膜性肾病(MN)65例、IgM肾病(IgMN)14例,IgA肾病(IgAN)41例,糖尿病肾病(DN)13例,狼疮性肾炎(LN)23例,系膜增殖性肾炎(MPGN)15例作为疾病对照,患者血清以10%浓度作用足细胞24h,免疫荧光法观察足细胞骨架蛋白F-actin和足细胞膜连接分子(ZO-1)分布变化。同时,取上述患者0.1ml血清注射大鼠,0h、4h、8h、12h和24h分别观察大鼠蛋白尿的产生,验证体外实验结果。阳性血清经灭活去补体,硫酸葡聚糖镁去脂,盐析法去IgG,阳性血清与患者或正常人尿液共孵育,及阳性血清与细胞因子IL-13中和抗体共孵育等不同处理后再作用足细胞,观察F-actin和ZO-1的变化,鉴定循环因子性质;Westernblot检测患者血清对足细胞内细胞因子相关信号通路JAK-STAT6,应激和细胞生存相关信号通路MAPK(p38,ERK,JNK)及骨架蛋白调节信号通路Rho-ROCK的影响,进一步深入研究循环因子对足细胞功能影响的分子机制。结果:13例FSGS患者(16.3%)和9例足细胞病患者(12.2%)的血清可明显破坏足细胞的骨架结构和ZO-1的分布,胞质内F-actin表达减弱,细胞骨架断裂,排列紊乱,部分开始向细胞边缘聚集,形成毛刺样结构,足细胞失去原有的细胞张力,胞体回缩;相邻足细胞的紧密连接消失,ZO-1的连续性破坏,出现断裂,皱褶,分层。其它肾病患者(MN,IgMN,IgAN,DN,LN,MPGN)的血清未能引起足细胞上述病变。阳性血清注射大鼠8h即可诱导大鼠产生明显的蛋白尿[(0.5±0.03)mg/mlvs(0.2±0.01)mg/ml阴性对照血清,P<0.01],12h大鼠蛋白尿的产生达到高峰,并持续到16h。阳性血清经去补体、去IgG、去除脂蛋白等处理后,血清对足细胞骨架蛋白损伤作用未受影响。循环因子阳性血清与尿液或正常人血清共孵育足细胞不能中和循环因子的损伤作用。细胞因子IL-13中和抗体未能阻断阳性血清诱导的足细胞损伤。进一步的信号分子研究显示,阳性血清作用30min即可激活足细胞内多条信号通路,STAT6,p-38MAPK和Rho-ROCK底物MYPT1磷酸化水平显著升高,而阴性血清未能激活上述信号通路。结论:循环因子特异性存在于部分FSGS和足细胞病变患者中,这类因子可以直接诱导足细胞损伤和蛋白尿的发生。进一步在临床上观察相应的治疗干预对FSGS患者这类循环因子的影响以及其消长与蛋白尿和预后的关系,有助于正确认识这类循环因子与疾病的关联,从而为最终明确这类循环因子的本质奠定基础。更多还原

【Abstract】 Objective:In order to test for the presence of a circulating factor(CF),an in vitro method for quantifying glomerular permeability in rats was utilized.In this study,we utilized podocytes to determine the presence of CF and explore the mechanism by which it overcomes the glomerular permeability barrier.Methodology:Eighty sera from patients with FSGS and Seventy from patients with podocytopathy were investigated.Sera from nephrotic syndrome patients with MN,IgMN,IgAN,DN,LN or MPGN were also studied as controls.Differentiated podocytes were treated with 1ml of medium containing 10% vol/vol of serum from patients for 24hrs.Changes in both F-actin and ZO-1 patterns in podocytes were observed by confocal microscopy.At the same time,Sprague-Dawley rats were injected with 0.1 ml serum from above patients.The level of urine protein in the rats were determined at 0h,4h,8h,12h and 24h.To characterize the nature of CF,the sera were processed before treated podocytes as decomplement,removed lipoproteins by dextran sulfate and calcium chloride,removed IgG by 70%(NH4)2SO4 saturation,pre-incubated with the homologous nephrotic urine or normal sera,or neutralized with IL-13 antibody.In order to understand the underlying effect of CF on podocytes,the activity of JAK-STAT 6,MAPK and RhoA signal pathways were studied using western blot.Results:CF presented in 16.3% of FSGS and 12.2% of podocytopathy patients by using podocytes as a target cell,while CF was not detected in other nephrotic patients(IgAN,MN,MPGN,LN,DN).Sera from certain patients with FSGS and podocytopathy destroyed the cytoskeleton of podocyte,as well as damaged the tight junction of podocyte.Injection of the sera which damaged podocyte induced proteinuria in rats as fast as 8h(0.5±0.03 mg/ml vs 0.2±0.01 mg/ml control sera,P<0.01),and as peak as 12h.Although decomplement,precipitated at 70% ammonium sulfate and removed lipoproteins,the positive sera still contained the CF activity.Activate the cellular signal transduction pathways(RhoA,STAT and p38 MAPK).Coincubation of serum with homologous urine and normal serum could not neutralize the activation of CF,it did not support the missing factor hypothesis.In addition,IL-13 neutralizing antibody did not affect the bioactivity of CF.Proteins from podocytes injuried by sera from patients with FSGS and podocytopathy showed activation of STAT6,p-38 MAPK and RhoA signal pathways as early as 30 min.Conclusion:Ciculating factor,which was specificity presence in patients with FSGS and podocytopathy,directly acted on podocytes and disrupt cytoskeleton as well as cell-cell interation.Multiple signal pathways could be involved in the effect of CF on podocytes.Our study provides new insights into the pathogenesis of FSGS,thus potentially contributing to the design of individual therapeutic strategies in FSGS.更多还原