The Effect And Regulatory Mechanisms Of Mesenchymal Stem Cells On Activated Complement C5 In Lupus Nephritis

Background:Systemic lupus erythematosus(SLE)is a chronic systemic autoimmune disease with lupus nephritis(LN)as one of the most common and severe complications.Corticosteroids and immunosuppressive agents are conventional therapies for lupus nephritis,but are associated with substantial side effects and suboptimal efficacy.A considerable number of refractory LN patients eventually lead to end-stage renal failure due to uncontrolled active disease.Excessive activation of the complement system is involved in the development and progression of lupus nephritis.Recently,allogeneic umbilical cord mesenchymal stem cells(MSCs)transplantation has achieved good clinical efficacy for refractory SLE,however,the exact mechanism remains unclear.Objeetive:The present study was to investigate the aetiological role of activated C5 in the pathogenesis of LN,the impact of MSCs transplantation on C5 activation in SLE model mice(B6.MRL/1pr,B6.1pr),and explore the mechanisms of MSCs regulating the activation of C5.Methods:Sixty-six patients with SLE were recruited,among which 33 cases were classified as active LN,17 cases were in remission,and the remaining were non-LN group.Forty blood donors served as healthy control group.Renal tissues were obtained from 40 patients with LN and 9 patients with nephrectomy due to urological tumor.7 LN paitents with MSCs transplantation were also included.26-week-old female B6.1pr mice were randomly allocated in four groups(8 per group),which were given the following treatments:Cyclophosphamide(CTX),MSCs,C5a receptor 1 antagonist(C5aRA),and an equal volume of 0.5%DMSO for blank control group.Six age-matched female C57BL/6 mice were served as normal control group.Levels of proteinuria,albumin,and creatinine were measured by biochemical methods.Levels of C3,C5a,soluble C5b-9(sC5b-9)5 anti-dsDNA antibody,mannose binding lectin(MBL),factor H(FH),clusterin(CLU),and interferon-?(IFN-?)were determined by ELISA.SLEDAI scores of SLE patients were recorded at the enrollment.Histopathological evaluation of renal lesions was undertaken by HE,PAS,PASM and Masson stains under light microscopy.Podocyte foot processes were assayed by the transmission electron microscopy when necessary.Deposits of C5a,membrane attack complex(MAC),C5aR1(CD88),MBL,IgG,IgM,IgA,C3,Clq,and Factor P(FP or Properdin)in the glomeruli were detected by imnunohistochemistry or immunofluorescence assay.MSCs(passage 4-10)were cultured in serum free medirm and different concentrations of IFN-?1b were added into each well.The supernatants of MSCs were collected at 0 h,12 h,24 h,48 h,and 72 h.Total RNA was extracted from the adherent MSCs,the relative expression of C3,C3a receptor(C3aR),CD88,Factor B(FB),FH,CD46,CD55,CD59,and CLU were detected by real-time PCR.The functional characteristics of FH and CLU derived from MSCs were identified by complement cofactor assay and polymeration test of C9 induced by Zn2+ in vitro.Results:Significantly elevated plasma C5a and sC5b-9 were found in SLE,LN,and non-renal involvement active SLE patients.Urine levels of C5a and sC5b-9 were also significantly higher in LN patients than those in healthy subjects.Compared to LN patients in remission,plasma level of C5a was significantly elevated in active LN(43.93±5.04 vs.28.63 ±3.20 ng/ml,P<0.05).There are significant potisitive correlations between plasma C5a and SLEDAI or 24 hours proteinuria in SLE or LN patients respectively,and similar association was observed between plasma and urine levels of sC5b-9 in LN patients.The deposition of C5a and MAC and percentage of CD88 positive cells in glomeruli were significantly enhanced in LN patients when compared to control group.In addition,the deposition of C5a and MAC in glomeruli of types ?,?,and ? LN were significantly greater than those in types ? and ? LN.Further analysis found that both C5a and CD88 in glomeruli were positively correlated with the active index of LN,while,the deposition of MAC in glomeruli was positively correlated with the chronic index and tubular interstitial lesion.Compared to blank control group,proteinuria of mice in CTX,MSCs,and C5aRA treated groups were significantly reduced,and renal function was also improved in both MSCs and CTX treated groups.Compared with those in blank control group,plasma level of C3 was significantly elevated in mice of MSCs C5aRA groups,and mice in MSCs group also appeared a remarkably decreased C5a in the circulation(50.86±17.07 vs.170.60±47.15 ng/ml,P<0.05).Pathological analysis showed that the proliferation glomerulonephritis was significantly inhibited in CTX,MSCs and C5aRA treated mice compared to mice in blank control group,and the decreased accumulation of perivascular inflammatory cells was also found in MSCs and C5aRA treated mice.Deposits of C5a,MAC,and MBL were significantly decreased in the MSCs transplantation group,and deposition of MAC was also decreased in and CTX treated mice compared with the blank control group.The expression of IgG,IgA,C3,Clq,and FP were significantly reduced in MSCs treated mice,meanwhile,the expression of IgG,C3,and FP were also significantly decreased in CTX and C5aRA treated mice when compared to mice in blank control group.All of the above 9 mRNA of complement molecules and regulatory proteins were expressed by MSCs.The concentrations of FH and CLU in MSCs medium significantly increased after IFN-alb stimulation in vitro.MSCs derived FH could assist factor I(FI)in cleaving C3b in vitro,and MSCs derived CLU could inhibit the polymerization of C9 induced by Zn2+ in vitro.Plasma level of IFN-? in SLE and LN patients was significantly higher than that in healthy controls.Levels of FH and CLU were significantly decreased in active LN patients when compared to healthy subjects(369.30±22.92 vs.524.90± 36.58 ?g/ml,p<0.001:22.23±1.50 vs.26.61±1.17 ?ml,p<0.05).Plasma levels of FH and CLU were significantly raised in MSCs treated mice compared to control lupus mice,and plasma level of FH was also significantly increased in LN patients at 24h after MSCs transplantation(630.90±140.20 vs.481.30±158.70 ?g/ml,p<0.05).Conclusion:Excessive activation of complement C5 was involved in the development and progression of lupus nephritis.Allogeneic MSCs transplantation effectively improved the glomerulonephritis of lupus mice,and inhibited the activation of complement C5 in the circulation and kidneys.Treating mechanisms of MSCs included up-regulating of FH and CLU,and interrupting the activation of classical,alternative,and lectin pathways.