Study On Complement C3 And Its Related Pathways In Lupus Nephritis

Background:Complement system is a protein response system with fine regulation mechanism,and it is an important part of the innate and adaptive immunity of the body.Its biological function is diverse,and it is activated mainly through classical pathway(CP),alternative pathway(AP)and mannan-binding lectin(MBL).Dysfunction such as abnormal levels of complement components and excessive activation are closely related to the occurrence and development of autoimmune diseases.Complement component3(C3)is the central component of the complement system in the complement family.C3 is the highest complement component in serum,which is mainly synthesized by the liver.Under the action of C3 invertase,it is decomposed into two fragments of C3 a and C3 b.Complement C3 and its cleavage fragments play an important role in the pathogenesis of lupus nephritis(LN).Although there are many studies on C3,there is still a lack of in-depth study on the role of C3 in LN regulatory network and what new signaling pathway C3 participates in the pathogenesis of diseases.It is certain that C3 plays a key role in autoimmune disease SLE.In-depth study of C3 clinical value and C3 related signaling pathway in lupus nephritis is a profound research topic.Our previous study found that TGFβ1 promoted the formation of platelet-derived growth factor B(PDGF-B).Studies on patients with glomerulonephritis have shown that TGFβ1 and PDGF-B are important mediators for extracellular matrix(ECM)accumulation,glomerular fibrosis and mesangial cell proliferation.Other studies reported that the interaction between TGFβ1 and different complement components aggravated epithelial damage in pulmonary fibrosis.However,the relationship between TGFβ1 and complement components in the pathogenesis of LN remains unclear.Screening of abnormal pathways and complement components in the kidneys of LN patients and NZB / W mice may help to identify complement-related therapeutic targets for LN.Objective:To screen the abnormal pathways and complement levels in the kidneys of LN patients and NZB/W mice,and determine the related therapeutic targets of C3 in LN.Methods:KEGG and GO enrichment assays were used to analyze kidney microarray data of LN patients and NZB/W mice.Microarray analysis of gene expression profiles was based on data from the GEO database.Kidney tissues were from the whole kidneys of NZB/W mice(accession number: GSE32583)or human glomerular and renal tubular tissues(accession number: GSE32591).Samples of human glomeruli and renal tubules were from renal biopsies.The expressions of complement-related proteins such as C1 q,C3,C3 a R,C5,C5 a R1,CR3 and TGFβ1 in kidney were detected by immunohistochemistry and immunofluorescence.The m RNA levels of C1 qa,C3 and C3 a R1 were detected by RT-q PCR.The protein levels of C3 and TGFβ1 in plasma and urine were detected by ELISA.Results:1.Activation of complement pathways and upregulation of complement components in kidneys of patients with LN and NZB/W mice.1)Compared with the healthy control group,the expression of 4754 m RNAs in the glomeruli of LN patients was significantly different,of which 2632 were upregulated and 2122 were down-regulated.There were 3725 differentially expressed genes in renal tubules,of which 2359 were up-regulated and 1366 were down-regulated.Compared with the control group,there were 1,725 differentially expressed genes in the early disease stages of NZB/W mice,of which 1375 were up-regulated and 350 were down-regulated.There were 7966 differentially expressed genes in the late stage of disease,of which 2863 were up-regulated and 5103 were down-regulated.2)Cluster analysis showed close similarities in the expression of C1 qa with C1 qb,of C3 a R1 with C5 a R1,and of C3 with C1 qa and C1 qb in LN patients.The expression of C1 qa,C1qb,C1 qc,C3,C3 a R1,C5 a R1,CR3 and CR4 in NZB / W mice gradually increased during disease progression.3)KEGG enrichment analysis indicated NF-kappa B signaling pathway,B cell receptor signaling pathway,Toll-like receptor signaling pathway,complement and coagulation cascades,T cell receptor signaling pathway,TGFβ signaling pathway,and several other pathways were altered both in LN patients and model mice.4)Microarray analysis showed that the expressions of C1 qa,C1qb,C3,C3 a R1and C5 a R1 in glomeruli of LN patients were increased,and the expressions of other m RNAs in renal tubules except C5 a R1 were significantly higher than those in the healthy control group(P < 0.05).5)Immunohistochemical analysis showed that C1 q,C3,C5,C3 a R,C5 a R1 and CR3 protein expression levels were higher in glomeruli and renal tubules of LN patients.In NZB / W mice,with the increase of age,the deposition of C3 and C3 a R in the kidney gradually increased.2.The level of urine C3 increased and plasma C3 decreased in LN patients1)C3a may be a key pathogenic factor for LN patients.Upregulation of the term of regulation of complement activation(GO:0030449)was significant only in LN patients,and upregulation of the term of complement activation,alternative pathway(GO: 0006957)was significant only in NZB/W mice.2)ELISA showed that urine C3 level in LN group was significantly higher than that in healthy control group,and plasma C3 level in LN group was lower.3)The immunohistochemical results of LN patients showed that the expression of C3 in IV,V and IV + V kidneys was similar,but the expression was greater in those with class III disease than healthy controls.3.TGFβ1 regulates the expression of C3 in LN1)The plasma TGFβ1 level in LN patients was decreased.The protein and m RNA levels of TGFβ1 and C3 in the blood of LN patients were positively correlated.2)The urine TGFβ1 level in LN group was significantly higher than that in SLE without LN group.3)Urinary TGFβ1 was significantly positively correlated with 24 h urinary protein.4)The level of TGFβ1 in the urine of patients was significantly positively correlated with the level of C3.5)Renal immunohistochemical results showed that there was a positive correlation between the staining scores of TGFβ1 and C3.6)The results of NZB/W mice bone marrow cells culture showed that TGFβ1significantly increased C3 m RNA expression,TGFβ1 inhibitor SB431542 significantly decreased the m RNA expression.7)The results of primary renal cell culture of NZB/W mice showed that SB431542 reduced the m RNA expression levels of C3 and C3 a R1,but TGFβ1 had no significant indigenous effect on the m RNA expression levels of C3 and C3 a R1.8)PBMC culture results of LN patients showed that TGFβ1 significantly increased C3 m RNA expression,TGFβ1 inhibitor SB431542 significantly reduced the m RNA expression.9)Immunohistochemical results showed that SB431542 significantly reduced the levels of C1 q,C3,C5,C3 a R and C5 a R1.10)Immunofluorescence staining showed that SB431542 significantly reduced the expression level of C3 in kidney.Conclusion:1.In the pathogenesis of LN,the expression of C3 and complement pathwayrelated factors in the kidney of LN patients and NZB/W mice was significantly increased.2.Complement C3 is involved in the formation of urinary protein,and further participates in the pathogenesis and progression of LN.3.TGFβ1 promotes the synthesis of C3,and TGFβ1 inhibitors block the progression of LN by inhibiting the expression of C3 and other complement components.