Effect Of Complement C5a/C5aR Pathway On Renal Ischemia/reperfusion-induced Autophagy

BackgroundAcute kidney injury(AKI)is a common clinical syndrome caused by various pathogens.The characteristics of AKI include rapidly decline of renal function,azotemia,accompanied with water and acid-base balance disorders.In recent years,the incidence of AKI increased especially inpatients and severe patients.There is currently no specific treatment for AKI.Although slight damage can be reversed by active intervention,some patients with persistent damage would gradually develop into chronic kidney disease(CKD),and ultimately need kidney replacement therapy.In many AKI causes,ischemia reperfusion injury(IRI)is one of the important reasons.Ischemia-reperfusion(IR)is difficult to avoid,and directly related to the survival of the graft in renal transplant surgery.Therefore,it is important to study the mechanism of IRI to find out new drug targets.Rcent studies have shown that inflammatory responses play a key role in the pathogenesis of AKI.After IR,a large number of inflammatory cell infiltration is observed in kidney and accompanied with the activation of inflammatory factors.Complement system as an important component of innate immunity actives and amplifies the inflammatory responses in IRI,but its specific mechanism is not clear.Complement component C5 has been recognized as a key molecule in complement activation.Studies found that C5aR gene deficiency significantly improved IRI.Simultaneously,autophagy was actived after IR.So,we hypothesize that complement C5a / C5aR mediates IR-induced injury by directly or indirectly regulating the autophagy.On the basis of this hypothesis,we investigated the effect of complement C5a / C5aR pathway on IR-induced autophagy by using C5aR knockout(C5aRKO)mice and wild type(WT)mice.ObjectiveTo investigate the effect of C5a / C5aR pathway on IR-induced autophagy.Methods1.In vivo expreiments1.1 Animal models and study design: Six-to eight-week old male mice(WT and C5aRKO mice)were used at present study.The WT mice and C5aRKO mice were divided into sham and IR group.IR was induced by occluding bilateral renal pedicles with micro-arterial clips fore 35 min.Sham-operated mice had their renal pedicles exposed and manipulated underwent same procedure but not occlude.1.2 The expression of C5aR and autophagy-related proteins LC3 and P62 were detected by immunohistochemistry.1.3 Histopathological changes of the kidneys were observed by HE staining.1.4 The level of BUN was measured by automatic biochemical analyzer after reperfusion at 24 h.1.5 The m RNA levels of C5aR and KIM-1 were measured by RT-PCR.1.6 The expression of autophagy-related protein LC3 was detected by immunofluorescence.1.7 The expression of autophagy-related proteins LC3? and P62 was measured by western-blot.2.In vitro expreiments2.1 The HK2 cells were cultured and stimulated by recombinant C5a with gradient concentration in vitro.The expression of autophagy-related protein LC3? was detected by western blot.2.2 After stimulating with recombinant C5a or C5a combined with C5aR antagonist(C5aRA)for 24 h,and the expression of LC3 was observed by immunofluorescence in HK2 cells.2.3 The HK2 cells were cultured with recombinant C5a or C5a combined with C5aRA for 24 h,and the expression of autophagy-related protein LC3? was measured by western blot.Results1.In vivo expreiments1.1 Compared with sham group,immunohistochemistry and RT-PCR results showed that both protein and m RNA levels of C5aR were significantly increased after IR.1.2 Compared with WT mice,C5aRKO mice significantly reduced the degrees of renal pathological damages,BUN levels,and the expression of KIM-1 m RNA.1.3 The expression of autophagy-related protein LC3 in renal tissue increased with time-dependented after IR.1.4 Compared with WT mice,the expression of LC3 decreased and the level of P62 increased in C5aRKO mice.These results indicated that C5aRKO effectively reduce IR-induced autophagy.2.In vitro expreiments2.1 Western-blot showed that the expression of autophagy-associated protein LC3?increased with C5a stimulation in a dose-depended manner.2.2 Immunocytochemistry suggested that recombinant C5a stimulation increases the expression of LC3.After the add of C5aRA,the expression of LC3 was significantly down-regulated.2.3 Western-blot comfired that recombinant C5a stimulation increases the expression of LC3?,and the phenonomen was reversed by C5aRA.ConclusionComplement C5a / C5aR pathway plays an important facilitated role in IR-induced autophagy.