The Association Between Alternative Complement Activation,Abo Blood Group And Progression In Patients With IgA Nephropathy

IgA nephropathy(IgAN)is one of the most common types of primary glomerulonephritis(PGN)in the world,especially in China,since it was first described by Berger and Hinglais in 1968.Although IgAN was initially regarded as a benign disease,20-50% patients developed to end-stage renal disease(ESRD)within 20 years after diagnosis.In spite of its diversities of renal pathology,the diagnostic hallmark of IgAN is the predominant deposition of IgA,with or without IgG,IgM in the glomerular mesangium,it is often accompanied by Complement 3(C3)deposition butrarely with C1 q deposition.In addition,co-deposits of IgA,C3 and properdin is found in 75%–100% of patients otherwise complement factor H(CFH)in 30%–90% of patients.Accordingly,it is widely assumed that the pathogenesis of IgAN is partly mediated by complement system activation,especially by alternative and mannose binding lectin(MBL)pathway other than classic pathway.As a kind of polygenic and multi-factor complex traits disease,the exact etiology and pathogenesis of IgAN remains unclarified yet.Recent studies have demonstrated IgAN as one kind of autoimmune disease which activation of complement involved in its pathogenesis and progression.C3 is the highest component of serum complement and its level reflects the activation ofcomplement system.Previous study of our group has found that the decrease of C3 was an independent risk factor of IgAN progression,nevertheless the underline mechanism remains unknown.Genome Wide Association Studies(GWAS)demonstrated thatgenetic polymorphism of C3(rs3745567)and CFH(rs3753394)was closely related to the circulating level of C3.These findings suggested that complement activation is associated with clinical outcome in patients with IgAN.Complement activation is a very complex process which is regulated by many regulatory factors,for instance that CFH is an important factor for inhibiting excessive activation of alternative pathway.Whether the genetic background of C3 and complement alternative pathway regulatory factors is related to the clinical manifestation and prognosis of IgAN patients is the major problem to be explored in our research,as well as its underlying mechanism and other potential new risk factors of IgAN werestudied and discussed.In the first section of this study,we retrospectively collected 523 primary IgAN patients with follow-up more than 1 year.TagSNPs(C3_rs3745567 and CFH_rs3753394)which were reported to be most relevant to serum C3 level in a recent GWAS study were slected and their genotypes of SNPs in IgAN patients were detected by Sequenom.We found that the serum C3 level was closely related to rs3745567 and rs3753394 in IgAN patients.The effect of rs3753394 on serum C3 level was stronger,it is indicated that serum C3 which analyzed by linear regression was significantly lower in T allele than C allele(B=-0.15,95%CI:-0.03~-0.001,P=0.03).Further analysis displayed that 5% patients who carried both the risk alleles of AT genotype(rs3745567_A and rs3753394_T),their serum C3 was significantly declined than the other three genotypeswhich including AC,GC and GT [91.7(76-116.5)vs 101(87-123),P=0.02].Moreover the proportion of hypocomplementemia C3(hypoC3)incidence was remarkablyhigher in AT genotype(35.2% vs 21.3%,P=0.001).Multiple linear regression showed that AT genotypeas an independent risk factor of C3 decrease after being adjusted by sex,age,eGFR,systolic blood pressure,hemoglobin,urine protein extraction and uric acid(B=-0.03,95% CI:-0.05~-0.01,P=0.004).The above results demonstrated that serum C3 decline of IgAN patientswas associated with C3 and CFH genetic background.In the second part we detected serum CFH and degreeof complement alternative pathway activation(AP)in IgAN patients.Compared to healthy controls,the level of serum CFH was significantly increased in IgAN patients.AP test kits were used to test blood AP degreein low CFH group and high CFH groupaccording to the result of serum CFH in IgAN patients.The AP degree of highCFH group was higher(81% VS 64%,P=0.04)but there was no significant difference between these two groups in clinical baseline data,pathological changes and prognosis.70% IgAN patients were found to have CFH deposition in renal tissues and there was a positive correlation between CFH deposition and IgA,C3 deposition.It is implied that the immune complexes which including IgA could activate complement alternative pathway in kidney and result in the subsequent deposition of C3.Patients with CFH positive deposition had higher urine protein excretion and serum uric acid level than those without CFH deposition,meanwhile,the mesangial proliferation(M),segmental sclerosis(S)and interstitial fibrosis(T)were much more severe.And more importantly,it is showed that patients with CFH deposition had a remarkable higher risk of renal function progression(eGFR decreased 30% or ESRD)(HR=2.91).Even after being adjusted by clinical progression risk parameters(eGFR,Hb,Sbp,Alb),CFH positive deposition was still an independent risk factor for IgAN progression(HR=2.54,95%CI 1.04-6.17),which would increase 1.5 times on the risk.All these data suggested that CFH deposition in kidney would be an important marker of IgAN progression.Subsequently,we sequenced the C terminal of CFH in 102 hypoC3 IgAN patients to further elaborate the genetic background of CFH.According to the results of DNA sequencing and combined with recent studies on single nucleotide polymorphism(SNP)of CFH,we detected candidated SNPs in 617 IgAN patients with long follow-up(more than 1 year)and 1427 healthy controls to explore the correlation between CFH genetic variations and incidence,clinical manifestations,pathological changes and prognosis in IgAN patients.It is found that the risk of IgAN occurrence increased by 35% with every additional T allele of rs1065489(OR=1.35,95%CI 1.18-1.54,P<0.001).Multivariate COX regression analysis showed that rs1065489_GG genotype was an independent risk factor for IgAN progression after fully adjusted by age,sex,eGFR,urinary protein excretion and uric acid(HR=1.68,95% CI 1.005-2.80,P=0.048).These results indicated that CFH_rs1065489 was closely related to the incidence and progression of IgAN.In the third section of this study,we focused on exploring new markers of IgAN progression.We analyzed the distribution of blood type and its association with clinical outcome of IgAN in 919 IgAN patients with more than 1 year follow-up.We found that patients in B antigen group(B/AB)had higher baseline eGFR,lower systolic blood pressure and uric acid than patients in non-B antigen group(A/O).Patients in B antigen group were associated with an independently decreased risk of ESRD(HR= 0.46,95%CI 0.28-0.75,p=0.002)after fully adjusted by age,sex,SBP,eGFR,blood urea nitrogen,hypoalbuminemia,uric acid,serum triglycerides,hemoglobin,serum C3,urine protein and Oxford Classification.We further explored the possible mechanism of the effect of ABO blood group on the clinical manifestation and prognosis of IgAN and then found the whole level of inflammation was elevated in non-B antigen group.LDH and hsCRP were significantly elevated in non-B antigen group.To further investigate the relationship between ABO blood type and pro-inflammatory cytokines level of IgAN patients,IL-6,IL-10 and TNF-? levels were tested in our patients.TNF-? level was elevated in non-B antigen group compared to B antigen group.Based on these data,we assumed that ABO blood type might influence IgAN progression by its potential roles on infection and host immune.In summary,we studied the genetic background of serum complement C3 in IgA patients group which had large sample size and long follow-up data in our hospital.The result indicated that the genotype AT(rs3745567_A and rs3753394_T)had close correlation and impact on serum C3.Genetic variations of CFH,serum alternative pathway activation related factors and CFH deposition were further detectedin renal tissues,which demonstrated that deposition of CFH in the kidney might serve as an important marker for IgAN progression.Meanwhilewe tried to explore new markers of IgAN progression,interestingly,our research found ABO blood type was closely related to clinical manifestations and prognosis of IgAN,of which B antigen group(B/AB)was an independent protective factor.