The Role And Mechanism Of C3a-C3aR In Autosomal Dominant Polycystic Kidney Disease

Objective:The pathogenesis of ADPKD is complex,the role of abnormal activation of complement alternative pathway and inflammation in the pathogenesis of ADPKD are being paid more attention.The purpose of this study was to determine the expression,role and mechanism of C3a-C3 a R in the progression of ADPKD,and to observe the therapeutic effect of C3 a R inhibitor SB290157 on Pkd1 knockout mice.Method:1.The kidney tissue samples and serum of wild-type mice and Pkd1 knockout mice were collected,as well as normal people and ADPKD patients.The contents of C3 a in kidney homogenate and serum were detected by Elisa,and the expression of C3 a R was detected by immunohistochemistry,Western blot and RT-PCR.2.The Pkd1 mice were divided into:(1)control group;(2)treatment group: daily intraperitoneal injection of SB290157(1mg/kg).In addition,the wild group was set.Each group continued giving drugs for 16 d and then sacrificed.The renal tissue samples and serum were collected to compare the renal function and cyst related indexes between each group;the content of C3 a in the renal tissue homogenate and serum were detected by Elisa;HE staining was used to compare the growth of cysts;the Ki67 staining were used to compare the proliferation of cyst lining epithelial cells.TUNEL staining was used to observe the apoptosis of cyst epithelial cells.Western blot was used to detect the expression of C3 a R and ADPKD related signal pathway proteins in renal tissue.3.Western blot and RT-PCR were used to compare the expression of C3 a R in RCTE and WT9-12 cells.MTT was used to observe the inhibition of SB290157 on WT9-12 cells proliferation.Flow cytometry was used to analysis cell cycle.Western blot was used to detect the regulation of SB290157 on ADPKD related signal pathways in vitro.4.F4/80 and C3 a R in polycystic kidney were co-located by immunofluorescence,and compare the infiltration of macrophages in renal tissue of each group.RAW 264.7 cells were induced to differentiate into M1 and M2 macrophages by LPS and IL-4 respectively,and the untreated cells were M0 macrophages.The expression of C3 a R in M0,M1 and M2 macrophages was compared by Western blot,RT-PCR and immunofluorescence.LPS was used as the stimulator and C3 a was used as the intervention factor to treat RAW264.7cells,the concentration of TNF-? in the supernatant of cell culture between each group was detected by Elisa.RT-PCR was used to detect the expression of i NOS,TNF-?,IL-6 m RNA.Western blot was used to detect the expression of p-Akt,p-ERK,p-P65,p-STAT1 and TNF-? in each group.Results:1.Compared with the control group,the expression of C3a-C3 a R was significantly up-regulated in kidney tissues of ADPKD patients and Pkd1 conditional knockout mice,and was positively correlated with the progress of the disease.2.SB290157,a C3 a R inhibitor,can significantly improve the renal function of polycystic kidney mice.The expression of C3a-C3 a R in renal tissues are down regulated,the growth of cysts is limited and the apoptosis of cyst lining epithelial cells is increased,and the expression of ADPKD related signal pathway proteins such as p-Akt,p-ERK,p-p65 are down regulated.3.Compared with RCTE cells,the up-regulation of C3 a R expression in WT9-12 cells was not significant,which indicated that the main differential expression position of C3 a R was not cyst lining epithelial cells.In vitro,SB290157 had no obvious direct inhibition on the proliferation of WT9-12 cells,and the changes of ADPKD related signal pathway proteins such as p-ERK,p-P65 were not significant.All of these indicate that although C3a-C3 a R promotes the progress of ADPKD,it does not directly affect cyst lining epithelial cells.4.There was co-localization between C3 a R and F4 / 80 in polycystic kidney and C3 a R was expressed on macrophage membrane.C3 a R inhibitor can reduce macrophage infiltration in polycystic kidney,indicating that C3a-C3 a R may have chemotaxis on ADPKD renal tissues.C3 a R was mainly expressed in M1 macrophages and C3a-C3 a R induces M1 macrophages to secrete TNF – ? by promoting Akt,ERK,STAT1 and NF-? B activation.Conclusions:In ADPKD,C3 a is increased by abnormal activation of complement alternative pathway,which leads to macrophages infiltration and activation,promotes TNF-? secretion by M1 macrophages,and then promotes the progress of ADPKD.C3 a R inhibitor can reduce inflammatory cell infiltration,inhibit cyst growth and improve renal function and may be a new therapeutic target of ADPKD.