Mechanism Study Of Alternative Complement Pathway In Patients With C3 Glomerulopathy

C3 glomerulopathy(C3G)is a set of diseases with distinct renal biopsy findings:dominant glomerular capillary loop C3 fragment deposition on immunofluorescence with absence or near absence immunoglobulins.Two major subgroups of C3G are dense deposit disease(DDD)and C3 glomerulonephritis(C3GN).DDD is characterized with the presence of strong osmiophilic electron dense deposits within glomerular basement membrane.The concept of C3 nephropathy highlights the pathogenesis of alternative complement pathway.It is of great meaning for further study of mechanism and future treatments to study the clinical and pathological characteristics of C3 glomerulopathy,especially the specific characteristics of its subtypes resulting from abnormal complement alternative pathway.In this study,we explored the genetic characteristics of alternative complement pathway in Chinese patients with C3 glomerulopathy and the further analyse the complement deposition within glomeruli in these patients.Part IGenetic Characterization of Alternative Complement Pathway in Chinese Patients with C3 GlomerulopathyBackground and ObjectiveThe alternative pathway is continuously and spontaneously activated under physiological conditions by the hydrolysis of the tioester bond in the central component C3.This process is controlled by more than 10 regulatory proteins.Misdirected complement activation due to genetic alterations and/or autoantibodies to complement components may cause various kidney diseases.Cohort studies mainly including Caucasian population and a few case reports confirmed the genetic defect of the alternative pathway of complement in DDD and C3GN patients.There is a lack of studies of gene mutations concerning about Asia patients.We performed complement gene mutation analysis in patients with C3 nephropathy and observed the alternative complemnent genetic features of patients with C3G.Then we explored how the genetic disfunction effects clinical manifestations,histopathological features and prognosis in patients with C3G.All these problems being solved will lead us to a better understanding of pathogenesis of C3G with respect of complement alternative pathway.Materials and Methods1.42 patients with C3 glomerulopathy(including 10 with DDD and 32 with C3GN)were retrospectively reviewed,as well as a control group of 24 patients with IC induced-MPGN type I.2.Gene analysis and mutation sorting:We perform genetic screening for exons of 11 complement alternative pathway related genes(C3,CFB,CFH,CFI,MCP,THBD,CFHR1,CFHR2,CFHR3,CFHR4,CFHR5)in all patients.Nonsynonymous mutations and frameshift mutations with minor allele frequency(MAF)<3%in the 1000 Genomes Project were recruited.Pyrosequencing was applied for verification.We predict pathogenic mutations with five mutation functional prediction software(SIFT、Align-GVGD、Pmut、SNAP、PolyPhen2.0).Then all recruited variants were aligned to the database and previous papers.Variants were then grouping into 3 types:disease related mutation,rare polymorphism which has not been reported in a certain disease,novel mutation newly found in this study.3.Compared the clinical,pathologic feature and prognosis in C3G patients with gene mutation to those without mutation.Student’s t or Mann-Whitney test or chi square test were used accordingly in comparison between groups.Renal survival rate was determined by Kaplan-Meier analysis.Results1.Patients with C3 nephropathy are more young on onset(P=0.010),with higher incidence of hematuria(P=0.042),lower incidence of hypertension(P=0.043)and lower circulation C3(0.48 vs 0.81g/L,P<0.001)when compared with those with MPGN.Patients C3 nephropathy presented with severer global sclerosis(P=0.042),and a higher proportion of electron dense material deposition in tubular basement membrane(P=0.042).Compared with DDD patients,patients with C3GN presented with lower glomerular crescents(P=0.043),with less complement C3 or electron dense deposition within tubular basement membrane(P=0.005),C3GN patients were characterized by more neutrophil and mononuclear cell infiltration inside glomerular capillary loop(P=0.020).A total of 32 mutations were identified in 11 complement alternative pathway related genes in 42 cases of C3 nephropathy patients and 24 patients with MPGN,of which 14 mutations predicted to be pathological.2.Of the 32 mutations,8 mutations were disease associated that have been reported previously,11 were rare polymorphisms and another 13 mutation were first discovered in this research.The overall incidence was 47%(31/66)in 66 cases of patients with different diseases.C3 nephropathy patients carried mutations in a proportion of 52.4%(22/42).Patients with MPGN Type I was carrying the gene mutation lower in rate without statistical difference.Mutation rate of C3GN patients and DDD patients was about 50%,and there was also no statistical difference.3.MCP,CFH and CFB mutation were common among patients with C3 nephropathy.Incidence rate of MCP mutations in patients with C3 nephropathy was 16.7%,but it did not reach statistical differences between diseases.The CFH mutation incidence in C3 glomerulopathy was 14.3%,which showed no statistical difference compared with patients with MPGN I.while CFH mutation prevalence in patients with DDD rate was up to 40%,much higher than that of patients with C3GN(p<0.05).Of all detected mutations,10 of them can be found in C3 glomerulopathy as well as in patients with MPGN I,accounting for 35.7%and 71.4%,respectively.Five mutations can be found in both DDD and C3GN.4.A considerable proportion of the patients carried mutations affected two or more genes.six patients with C3 glomerulopathy and 2 patients with MPGN carried combined mutations,accounted for 27%(6/22)and 22%(2/9),respectively.The incidence of combined mutations in patients with C3 glomerulopathy and MPGN mutation were similar.Of 6 patients with C3 glomerulopathy who carried combined mutantions,four were C3GN(4/17)and 2 were DDD(2/5).5.Patients with C3 glomerulopathy who carried gene mutation tended to be early onset(20.5 vs 26.7 yrs)compared to those without mutations.They had similar clinical and pathological manifestations.Renal survival rate was slightly better in patients with gene mutation compared with those without mutation,but did not reach statistical difference(P=0.618).Patients with C3GN(n=32)who carried gene mutation(n=17)tend to be early onset,gross hematuria,less accompanied by acute kidney injury,when compared to those without mutations,but did not reach statistical difference.Renal survival rate was slightly better in patients with C3GN carried gene mutation compared with those without mutation,but did not reach statistical difference(P=0.307).Patients with DDD(n=10)who carried gene mutation(n=5)tend to be early onset(12.7 vs 17.4 yrs,P=0.076)compared to those without mutations.Patients with DDD who carried gene mutation showed lower level of Proteinuria(3.64 vs 6.03g/24h),lower serum creatinin and higher eGFR,but did not have statistical difference.Two patients with DDD who carried gene mutation were crescentic glomerulonephritis(40%).Those with mutation tend to show electron dense deposits location in tubular basement(80%vs 20%,P=0.206).Renal survival rate showed no statistical difference(p=0.682).Conclusions1.We investigated gene mutation profile of complement alternative pathway in patients with C3 glomerulopathy from Asia for the first time,screening 11 genes relatived to complement alternative pathway.We found 8 disease associated mutations,11 rare polymorphisms and another 13 novel mutations first discovered in this research.2.We found that about one half of patients with C3 glomerulopathy carried gene mutations of complement alternative pathway.MCP,CFH,CFB mutation prevelance were the highest among patients with C3 nephropathy.Some mutations can be detected in both C3 glomerulopathy and MPGN I.Some patients with C3 glomerulopathy and MPGN carried combined mutations.3.Patients with C3 glomerulopathy who carried gene mutation tended to be early onset compared to those without mutations.Renal survival rate seemed better in patients with gene mutation compared with those without mutation but did not have statistical difference in small samples.Part ⅡGlomerular protein profile of Alternative Complement Pathway in Patients with C3 GlomerulopathyBackground and ObjectiveC3 glomerulopathy(C3G)is characterized by glomerular deposition of C3 on immunofluorescence with absence or near absence immunoglobulins.Previous study of mass spectrometry analysis have confirmed the dominant deposition of complement alternative pathway components within the glomeruli in patients with C3G.The deposition of C3,as well as other complement component resulting from complement degradation,are considered to play an important role in the pathogenesis of C3G.It is unknown whether there is a specific complement protein profile between diseases(C3 nephropathy and immunocomplex induced MPGN)or between C3G subtypes(C3GN and DDD)and what the relationship is between glomerular complement protein profile,genoype and phenotype.It is also to be solved the differences of overall distribution of complement cleavages deposition between diseases.So we performed microdissection and mass spectrometry to explore the characteristics of complement component deposition within glomeruli in patients with C3G.All these problems being solved will lead us a better understanding of pathogenesis of C3G with respect of complement alternative pathway.Materials and Methods1.Four patients with C3 glomerulopathy(including two patients with DDD and two patients with C3GN)and two patients with IC-induced MPGN were selected.2.Ten mm-thick sections of formalin-fixed tissues were deparaffinized.Glomeruli were photographed and then microdissected(50～128 glomeruli each sample).samples were reduced and alkylated,then digested with trypsin.3.The trypsin-generated digests were used for protein identification by nano-flow liquid chromatography electrospray tandem mass spectrometry.The results are combined and assigned peptide and protein probability scores in proteinpilot database.4.Protein profile within glomerular was analysed and the relative amount of complement proteins within glomerular was assessed with label-free quantitation.We also analysed the complement cleavage products with peptide imformations extracting from LC-MS raw data.Results1.Six patients,two patients with C3GN,two with DDD and two with MPGN were selected to glomerular micro-dissection combined with mass spectrometry analysis.A mean of 245±57 proteins,of which 15 were complement components or complement regulators,were identified in gloneruli.C3,C4 and C9 were indentied in all six patients.The most abundant complement proteins were C3,followed by C9 and C4.Immunoglobulins(IgA,IgM,IgG)were identified in all the patients within glomeruli in different degrees.2.The dominant C3 cleavage products deposited within glomeruli were C3d and C3g but not C3c.Anaphylatoxin C3a did not deposit within glomeruli.The dominant C3 cleavage product deposited was C4 alpha chain,followed by C4 beta chain with a far more less spetra count.C9a and C9b deposited within glomeruli with similar levels.The cleavage product distributions of certain complement protein were similar between diseases.3.Patients with C3 glomerulopathy and patients with MPGN showed similar spectrum in complement components or complement regulators.Deposition of C9 in glomerular of patients with C3 glomerulopathy was about two times in amount of those in patients with MPGN.Patients with C3 glomerulopathy and patients with MPGN showed similar C3 and C4 deposition in quantity.Patients with DDD showed much higher relative quantity in C3,C4,C9 within glomeruli compared with those patients with C3GN(1.5,1.3 and 5.9 times as the latter).No obvious difference in glomerular deposition of complement components was showed in relative quantity between patients with C3 glomerulopathy carried mutations and those without mutations.ConclusionsWe first studied the glomerular protein profile in patients with C3 glomerulopathy from Asia.We confirmed the deposition of immunoglobulins and components of classical complement pathway in patients with C3 glomerulopathy.Patients with C3 glomerulopathy and patients with MPGN showed similar spectrum in complement components or complement regulators.The dominant C3 cleavage products were C3d and C3g.Deposition of C9 in patients with C3 glomerulopathy was more than patients with MPGN.Patients with DDD showed much higher relative quantity in C3,C4,C9 within glomeruli compared with those patients with C3GN.