The Role Of Complement Abnormalities In The Development Of Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN)is one of the most important microvascular complications of Diabetes Mellitus(DM)and the main cause of end-stage renal disease(ESRD).In addition to conventional renin-angiotensin-aldosterone system inhibitors,clinical treatment is mainly to strengthen the control of hypertension and blood sugar,but conventional treatment methods cannot reduce the risk of ESRD in DN patients.Therefore,it is urgent to further explore the pathogenesis of DN to provide new ideas for clinical research of new drugs.The pathogenesis of DN is complex.In addition to common hypertension,hyperglycemia,metabolic abnormalities,hemodynamic changes and oxidative stress,more and more evidences show that immune inflammatory response plays an important role in the occurrence and progression of DN.The role of complement as an important part of the innate immune system in the occurrence and development of DN has recently become the focus of attention.There are three pathways of complement activation,namely classical pathway(CP),alternative pathway(AP)and lectin pathway also known as MBL(Mannose-Binding Lectin)pathway.Which complement activation pathway plays a major role in the pathogenesis of DN,whether immune cell infiltration is involved in the disease process,whether complement gene polymorphisms increase the risk of DN in DM patients,and whether complement activation affects the prognosis of DN patients,a series of clinical issues need to be solved urgently.Objective1.To explore the relationship between complement activation and immune cell infiltration by analyzing the glomerular transcriptome data of diabetic nephropathy.2.To detect whether the complement gene polymorphism is related to the occurrence and clinical manifestations of diabetic nephropathy.3.To explore the effect of glomerular complement C1q and C3 deposition on the prognosis of patients with diabetic nephropathy.Methods1.Through the Weighted Gene Co-Expression Network Analysis(WGCNA)and Robust Rank Aggregation(RRA)algorithms in the R language to analyze DNassociated glomerular transcriptome datasets from the Gene Expression Omnibus database(GEO),and then get the intersection of the two methods by using the Venn diagram and finally obtained genes associated with the pathogenesis of DN.The key genes were obtained using the Protein-Protein Interaction(PPI)network and Cytoscape software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on the obtained differential genes using the Clusterprofiler package in R language to identify important pathways.The DN immune infiltration was evaluated by the CIBERSORT package in the R language,and the immune cell differential analysis and correlation analysis of key genes were performed,and finally the Venn diagram method was used to obtain the gene-related immune cells.2.210 patients who were treated in China-Japan Friendship Hospital from September 2019 to January 2022 were selected.Among them,110 patients with pure DN confirmed by renal biopsy were selected as the case group,and 100 patients with DM history≥15 years without combined with DN or other kidney disease were regarded as a control group.DNA was extracted respectively,and the genotypes of 4 loci including rs4589079 of C1QA,rs62125134 and rs163915 of C3,and rs930507 of MBL2 were detected by Snapshot sequencing method and then analyzed the correlation between the complement-related gene polymorphisms and the occurrence and clinical manifestations of DN.3.Patients with simple DN who were pathologically diagnosed by renal biopsy in China-Japan Friendship Hospital from January 2013 to December 2020 and whose eGFR after admission was greater than 15ml/min/1.73m2,the number of glomeruli in renal biopsy was more than 5 and follow-up regularly were selected,finally a total of 151 cases were included.We further explored the relationship between the deposition of complement C1q and C3 in the glomerulus and the clinical,renal pathology and prognosis of DN patients.At the same time,immunohistochemical methods were used to detect the three pathways of complement activation namely C1q,C3,C4d and MBL expression in glomerular tissue of patients and its effect on prognosis.Results1.By taking the intersection of WGCNA and RRA algorithms through Venn diagram,a total of 50 differential genes related to the pathogenesis of DN were obtained,including important genes related to complement such as C3,C1QB,C1QA and C7.GO and KEGG pathway enrichment analysis found that complement activation and humoral immune response are important pathways in the pathogenesis of DN,among which C1QA,C1QB and C3 play important roles in these pathways.Immune infiltration analysis of the correlation between DN complement genes and immune cells confirmed that C3 was associated with B cell memory and T cell gamma delta infiltration,C1QA was associated with NK cells activated infiltration and C1QB was associated with B cell naive and NK cells activated infiltration in DN patients.2.A total of 210 patients were included in this study.Among them,there were 100 cases in the simple DM group,including 63 males and 37 females;110 cases in the DN group,including 88 males and 22 females.The genotypes and gene frequencies of C1QA rs4589079,C3 rs62125134 and MBL2 rs930507 were not significantly different between the DM and DN groups,while the C3 rs163915 genotype and gene frequency was significantly different between the DM and DN groups(P<0.05).At the C3 rs163915 locus,patients with the allele C had a higher risk of DN(P<0.05).There were no significant differences in eGFR and 24h urine protein among the intragroup genotypes of the four loci.3.A total of 151 patients with pure DN were included,of which 90 patients reached the renal end point,and the average follow-up time was 23± 19 months.According to the presence or absence of complement C1q and C3 deposition in the glomerulus,they were divided into three groups:Clq with C3 deposition(n=24),Clq or C3 deposition(n=55),and neither C1q nor C3 deposition(n=72).In terms of clinical indicators,the 24h proteinuria level of the three groups were 6.83±3.46g/24h,5.91 ±3.86g/24h,4.71 ±3.64g/24h,and the differences were statistically significant(P<0.05).In pathological scores according to renal pathological damage,the glomerular pathological class of patients with both C1q and C3 deposition was significantly higher than that of the group without C1q and C3 deposition,and the difference was statistically significant(P<0.05).The results of Kaplan-Meier survival analysis showed that there was a statistically significant difference in the cumulative renal survival time among the three groups(P<0.05).The C1q combined C3 deposition group had the shortest cumulative renal survival time and the worst prognosis.Multivariate Cox proportional hazards regression model results showed that glomerular C1q and C3 co-deposition was independent risk factor for progression to ESRD in patients with DN.Immunohistochemical findings showed that patients with high levels of C1q,C3,or C4d expression in the glomerulus were more likely to develop ESRD,whereas MBL was rarely expressed in the glomeruli.Conclusion1.Complement activation in glomeruli is involved in the occurrence and development of DN disease,and the expression of complement C1QA,C1QB and C3 is related to the infiltration of glomerular T,B lymphocytes and NK cells.2.The polymorphism of complement C3 rs 163915 locus increase the DN susceptibility among DM patients and patients with allele C have higher risk of DN.3.The co-deposition of glomerular complement C1q and C3 is associated with higher proteinuria,higher glomerular pathological stage and poor prognosis in DN patients.The co-deposition of glomerular complement C1q and C3 is an independent risk factor for progression to ESRD in patients with DN.Activation of the glomerular classical complement pathway may accelerate the progression of DN.