The Clinical Value Of PLA2R And THSD7A In Membranous Nephropathy And The Pathogenesis In PLA2R-Related Membranous Nephropathy

Objective Idiopathic membranous nephropathy(IMN)is one of the main causes of nephrotic syndrome(NS)in adults.In the Chinese population,the prevalence of membranous nephropathy(MN)has increased rapidly in recent years.Although some of the patients with IMN may undergo a spontaneous remission of disease,one-third of cases progress to end-stage renal disease(ESRD).IMN is regarded as an organ-specific autoimmune disease.In 2009,M-type phospholipase A2 receptor(PLA2R)was considered as the major target antigen in IMN and Thrombospondin type-1 domain-containing 7A(THSD7A)is the most recently recognized new target antigen in patients with MN.The purpose of our study is to estimate the value of PLA2 R and THSD7 A in the diagnosis of IMN and to analyze the correlation between the expression of serum anti-PLA2 R antibody and renal PLA2 R.We also assess the relationship among glomerular PLA2 R expression with anti PLA2 R autoantibodies,the severity of the disease and the outcomes as well.Up to date,it lacks specific biomarkers for differentiating the malignancy associated MN from IMN coincident with cancer,we investigate clinicalpathological characteristics and the expression of antigens and autoantibodies of MN patients with cancer to evaluate the relationship between MN and malignancy.Since the different pathogenesis between PLA2 R related MN and PLA2 R unrelated MN remains unknown,we aim to understand the involvement of complements pathway and role of gene polymorphisms between them.Methods Adults and children patients with biopsy-proven MN and other glomerular diseases at Xin Hua Hospital and Shanghai Children’s Hospital were included.Clinicalpathological data were obtained from all of the patients at the time of renal biopsy.Serum specimens and urine samples were also collected at baseline and during the follow-up.IHC staining of PLA2 R,THSD7A,Ig G4,C4 d,MBL,S-100 and neurofibromin were performed and circulating anti-PLA2 R antibodies and THSD7 A antibodies were tested using enzyme linked immunosorbent assay(ELISA)and indirect immunofluorescence test(IFT)respectively.Genotype analysis was done by Taq Man assays for seven single-nucleotide polymorphisms in PLA2R1 and HLA-DQA1.Results A total of 85.4% of patients(164/192)with IMN had positive glomerular PLA2 R deposits,while 15% and 75% of cases showed positive of PLA2 R expression in non IMN and UAMN(undetermined atypical membranous nephropathy)group respectively.4 MN patients secondary to HBV infection and 5 MN with cancer showed positive of glomerular PLA2 R deposits.Among IMN patients,the proportion of hypertension were significantly higher whereas serum albumin was significantly lower in the PLA2R-associated patient group,compared with those in the non-PLA2R-associated group(p<0.05).There were no significant differences between two groups with respect to 24-h urinary protein level,serum albumin,serum creatinine levels,e GFR and remission rates during the follow-up.THSD7 A deposits were found in 3(1.6%)IMN patients and all of them presented PLA2 R deposits negative.In the child IMN patients only 16(42.1%)were positive for PLA2 R expression and a significantly higher rate of PLA2 R staining was also observed in the adolescent group when compared to the young child group(81.8% versus 25.9%;p<0.05).Hematuria,proteinuria,serum creatinine levels and e GFR were all significantly different between the two groups(p <0.05).65.8% of patients(50/76)with IMN had positive PLA2 R antibodies,two of five MN patients secondary to HBV infection and three of seven MN with cancer showed positive of anti-PLA2 R antibodies.Of 76 patients,positive rates of glomerular PLA2 R deposition were 92.1%(70/76).30%(21/70)of IMN patients with positive glomerular PLA2 R deposition were negative for anti-PLA2 R antibodies.Patients with PLA2 R antibodies had a higher level of proteinuria and low level of serum albumin than those who lacked PLA2 R antibodies(p <0.05).Patient antibody levels were weakly correlated with baseline proteinuria(p=0.047,r=0.372)and serum albumin(p=0.026,r=-0.358).There were no significant differences with respect to remission rates between patients with PLA2 R antibodies positive and negative during the follow-up.One of three patients with glomerular THSD7 A deposits positive was found to be positive for anti-THSD7 A antibodies.IFT was repeated at the point of partial remission after immunosuppressive therapy,and serum anti-THSD7 A antibody levels turned negative.MN Patients with cancer were significantly older than patients with IMN(p<0.05).MN patients with cancer had significantly higher serum creatinine and lower e GFR than IMN patients(p<0.05).Glomerular PLA2 R and Ig G4 deposits showed positive in 4 of 13 cases with cancer.Meanwhile,2 of 4 patients had serum PLA2 R antibodies positive.All of patients showed negative for glomerular THSD7A deposits and anti THSD7A antibodies.One case with neurofibromatosis 1 was identified with duel PLA2 R and THSD7 A antigen.In cases with PLA2 R related IMN and PLA2 R unrelated IMN,glomerular C4 d deposits were detected in 83.3%(25/30)of patients and 80%(16/20)of patients,respectively.In 41 patients with C4 d positive,activation of the lectin pathway was observed in 12(29.3%)patients.All of 9 C4 d negative patients showed negative for MBL.Allelic frequency distributions of 4 SNPs within PLA2R1 rs3828323,rs4664308,rs3749117 and rs2715928)were significantly different between IMN subjects and healthy control.There were significant differences in genotypic frequency distributions of rs3828323 between patients with PLA2 R related IMN and PLA2 R unrelated IMN.Conclusion The expression of glomerular PLA2 R is significantly higher in IMN patients than in non IMN patients,hence it could be considered as an available method for diagnosis of IMN.IMN patients with positive glomerular PLA2 R expression are associated with more severe proteinuria and higher proportion of hypertension,indicating that glomerular PLA2 R expression is correlated with the severity of disease.Although the incidence of THSD7 A expression is relatively low in IMN cases,it is still valuable for the diagnosis of IMN.The clinical features and prevalence of PLA2 R staining in adolescents with IMN were similar to what was seen in the adult patients,indicating that they probably have a closer etiology and pathogenesis of the disease.Most of the young children with IMN were negative for PLA2 R expression,with a more varied and underlying etiology than in the adults.Most of cases with serum PLA2 R autoantibody positive showed enhanced expression of glomerular PLA2 R,whereas serum PLA2 R antibody could be negative in patients with glomerular PLA2 R positive,suggesting that serum PLA2 R antibody has a good specificity but a little lower sensitivity for diagnosis of IMN.Serum PLA2 R antibody as glomerular PLA2 R expression may reflect the disease activity.One patient with anti-THSD7 A antibodies positive showed negative for anti-THSD7 A antibody at the point of partial remission after immunosuppressive therapy,indicating that anti-THSD7 A antibody may also reflect the severity of disease.Absence of glomerular PLA2 R deposition,together with Ig G4 could be useful clues to differentiate malignancy associated MN from IMN.The positivity of glomerular C4 d and MBL expression suggested that the lectin and/or classical pathway of complement were activated in IMN cases,which showed no difference between PLA2 R related IMN and PLA2 R unrelated IMN.Allelic frequency distributions of 4 SNPs within PLA2R1 rs3828323,rs4664308,rs3749117 and rs2715928)were significantly different between the IMN and healthy control.There were significant differences in genotypic frequency distributions of rs3828323 between patients with PLA2 R related IMN and PLA2 R unrelated IMN,suggesting that PLA2 R related IMN may have specific genetic factor to cause the disease.