Abnormal Activation Of Complement Alternative Pathway In Kidney Disease—clinicopathologic Features And Mechanism

The complement system is an important part of innate immunity of human and this system has three activation pathways, including complement alternative pathway. This pathway is also the complement cascade enzymatic reaction, controlled by more than 10 regulatory proteins and involved in the early defense against the infection. Under physiological conditions, the alternative pathway is continuously and spontaneously activated by the hydrolysis of the tioester bond in the central component C3. Misdirected or exaggerated complement activation due to genetic alterations and/or autoantibodies to complement components may cause various kidney diseases. By now, atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) are directly associated with the abnormal activation of the complement alternative pathway. The activation of this pathway has also aggravated the process of IgA nephropathy, lupus neohritis, ischemia-reperfusion injury and other kidney diseases. The study of the pathophysiological process is important to guide the diagnosis and treatment of these diseases.European and American cohort studies were mainly including Caucasian population and small samples of cases with aHUS, DDD and C3GN from Asia were reported. The studies of gene mutations were only reported in aHUS patients fromAsia. This study provides a detail description of the histopathologic features and prognosis observed in the largest cohort of patients with C3G in Asia. Although detailed serologic and genetic data are lacking, this study also identifies important clinicopathologic distinctions between patients with DDD and C3GN. This is helpful to recognize the feature of this rare disease and promote to diagnose and treat this disease correctly. Because more reports of gene mutations of complement regulatory proteins in aHUS, we investigated these gene mutations in a meta-analysis in order to recognize the feature and pathogenicity of these mutations. We analyzed genetic screening of 24 patients with aHUS dignosed in our hospital and the relation between genotype and phenotype.Part I A retrospective analysis of clinicopathology and prognosis in C3 glomerulopathyObjective:To observe the clinicopathologic features and the prognosis of the patients with C3 glomerulopathy (C3G) including two subtypes:dense deposit disease (DDD) and C3 glomerulonephritis (C3GN).Methods:54 patients with C3 glomerulopathy diagnosed by renal biopsy between 1985 and 2014 were retrospectively reviewed including 15 with DDD and 39 with C3GN. We recorded clinical, histologic data and compared with clinicopathologic feature and prognosis of patients in two groups.Results:There were 32 (59%) males and 22 (41%) females with average age of 31.7 (8～71) years at the time of renal biopsy.44% of the patients presented nephrotic syndrome at the onset of C3G.37% and 35% of the C3G patients were complicated by hypertension and anemia, respectively. At the time of the first diagnostic renal biopsy, the mean proteinuria was 4.12±3.61g/24h and the mean serum creatinine level was 99.01±81.33umol/1.82% of the C3G patients presented the low level of serum complement C3 and the mean level of complement C3 was 0.52±0.33 g/L (normal 0.8～1.8g/L).23 of the 38 (61%) patients with antistreptolysin-O titer (ASOT) measured had a titer higher than the normal range. Patients with C3G presented multiple types of renal glomerular pathology. Renal biopsy revealed membrane proliferative glomerulonephritis in 33 patients (61%) and mesangial proliferative in 15 patients (28%) with C3G. Immunohistology showed that bright staining for C3 was evident within capillary wall in 98% of patients, in the mesangium in 63% of patients.22 (41%) cases presented only C3 deposition in kidney by immunofluorescence and 30 (56%) were accompanied by the deposition of IgM.Compared with patients with C3GN, those with DDD were younger (P=0.011), the similar level of proteinuria and surem complement C3 and lower level of serum albumin. More patients (46%) with C3GN were accompanied by hypertension than those with DDD (P=0.031). In DDD group, more patients showed membrane proliferative and crescentic glomerulonephritis, but this difference was not statistically significant. More patients in DDD group presented C3 deposition within renal tubular basement membrance and interstitial vascular wall (P<0.001) besides mesangium and capillary wall. Electron microscopy identified hyperosmiophilic dense deposits mainly occupying the lamina densa of the glomerular basement membrane, tubular basement membrane and Bowman’s capsule in DDD group, while intramembranous, mesangial, subepithelial and subendothelial deposits were found in C3GN biopsies. There was no difference in treatment between two groups and cumulative incidence of poor renal outcome using Kaplan-Meier survival probabilities.Conclusion:This study provides a detail description of the histopathologic features and prognosis observed in the largest cohort of patients with C3G in Asian. Although detailed serologic and genetic data are lacking, this study also identifies important clinicopathologic distinctions between patients with DDD and C3GN. This is helpful to recognize the feature of this rare disease and promote to diagnose and treat this disease correctly. But the number of patients with C3G was small and more validations were needed.Part II Preliminary research on the genetic mechanisms of aHUSObjective:Genetic defection of complement regulatory proteins plays an inprotant role in the pathogenesis of aHUS. There were a lot of reports about gene mutations in patients with aHUS, but a small number of these mutations were validated the changes of the functionality. We investigated these gene mutations in a meta-analysis and predicted the functionality by bioinformatics methods in order to found a research system and analyzed the relation between genotype and phenotype. This is helpful to recognize the genetic mechanisms of aHUS.Methods:We meta-analyzed all reported mutations related to complement regulatory proteins of alternative pathway before May 1,2014. We evaluated five kinds of the mutation functional prediction software according to mutations reported function and used the software with the most accurate rate to predict pathogenic mutations. We observed the relation between the distribution of gene mutations and the function of related proteins. We analyzed genetic screening of 24 patients with aHUS dignosed in our hospital and the relation between genotype and phenotype.Results:Meta-analysis identified 375 mutations in 12 genes associated with about 6513 patients with aHUS, including 262 missense mutations, of which accounted for 99.2 percent Caucasians, only 65(0.5%) cases from Asia. There were large variation in the number of patients involved in these studies and the incidence of mutatios.112 missense mutations of CFH and the higher mutation incidence of CFH (66%), C3 (54%) and MCP (31%) were reported. Accoding to 71 functional mutations among 262 missense mutations, we assessed accuracy and stability of PolyPhen2、SIFT、 SNAP、Align GVGD、Pmut and joint prediction and the sensitivity, negative and positive predictive value, accuracy and MCC value of SNAP were the highest. So SNAP was used to predict the functions of mutations associated with aHUS and secondary structure and distribution of 11 missense mutations were drawn.42.8% mutations of CFH were on the C terminal CCPs 19-20 and 48.3% of the total mumber of pathogenic mutations.21(47.7%) mutations of CFI were in the serine zone and 48% of pathogenic mutations. Mutations of membrane proteins including MCP and THBD were maily in transmembrane region.15 new and 2 reported missense mutations were found by exon capture sequencing in 24 patients with aHUS.12 patients carried 17 gene mutations and the ratio of gene mutations in our patients was 50%. Three new genetic mutations were found in CD46、C3、CFB and CFHR5, respectively and one mutation in CFH、CFHR3 and THBD, respectively. According to the prediction of SNAP,8 mutations were harmful. Based on gene mutations, we divided patients with aHUS into two groups and there was no significant difference in clinical manifestations and prognosis between two groups, except the level of serum complement C3 (P=0.003).Conclusions:We investigated gene mutations of complement alternative pathway in a meta-analysis in order to recognize the feature and pathogenicity of these mutations in patient with aHUS. We analyzed genetic screening of 24 patients with aHUS dignosed in our hospital and the relation between genotype and phenotype. This is helpful to recognize the genetic mechanisms of complement alternative pathway and aHUS.