The Role Of Complement 5a And Its Receptor C5aR In Podocyte Injury In The Pathogenesis Of Immunoglobulin A Nephropathy

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.