Visualize circulating factor by using podocytes as a taget cell in patients with FSGS

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,P0.01),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.