Improvement of idiopathic membranous nephropathy diagnosis with ultrasensitive quantitative detection of anti-phospholipase A2 receptor

To the Editor: The diagnosis of glomerulonephritis is generally based on kidney biopsy, which few diagnostic serologic markers have been described. M-type phospholipase A2 receptor (PLA2R) is a type I transmembrane protein abundantly expressed on glomerular podocytes. Antibodies against PLA2R were described by Beck et al1Beck Jr., L.H. Bonegio R.G. Lambeau G. Beck D.M. Powell D.W. Cummins T.D. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.New Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1504) Google Scholar as serum markers for idiopathic membranous nephritis (IMN). Recently, anti-PLA2R antibodies were found to correlate with disease activity and proteinuria in IMN.2Hanko J.B. Mullan R.N. O'Rourke D.M. McNamee P.T. Maxwell A.P. Courtney A.E. The changing pattern of adult primary glomerular disease.Nephrol Dial Transplant. 2009; 24: 3050-3054Crossref PubMed Scopus (91) Google Scholar Anti-PLA2R antibody levels decreased or disappeared on treatment induction or spontaneous remission in patients with IMN. In serial investigations, anti-PLA2R antibodies were found in 70% of IMN and a small percentage (5% to 25%) were positive in sera from patients with secondary MN by qualitative analysis, such as Western immnunoblot, indirect immunofluorescence cell based assay (IIF-CBA), and ELISA,3Qin W. Beck Jr., L.H. Zeng C. Chen Z. Li S. Zuo K. et al.Anti-phospholipase A2 receptor antibody in membranous nephropathy.J Am Soc Nephrol. 2011; 22: 1137-1143Crossref PubMed Scopus (346) Google Scholar, 4Svobodova B. Honsova E. Ronco P. Tesar V. Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy.Nephrol Dial Transplant. 2013; 28: 1839-1844Crossref PubMed Scopus (154) Google Scholar, 5Hofstra J.M. Wetzels J.F. Anti-PLA2R antibodies in membranous nephropathy: ready for routine clinical practice?.Neth J Med. 2012; 70: 109-113PubMed Google Scholar which negatively affected the differential diagnosis of IMN. A quantitative assay is needed to distinguish IMN from secondary MN. Taking advantage of the highly sensitive time-resolved fluoroimmunoassay (TRFIA) format, we developed a quantitative method for detection of anti-PLA2R antibody in serum and used it in the evaluation of renal patients. In this study, recombinant PLA2R was generated by cloning into 293T cells. Goat antihuman IgG antibodies (Jackson Immuno Research, West Grove, Pa) were labeled with Eu3+. The optimal coating concentration of PLA2R antigen was approximately 5 μg/mL. The addition of a fluorescence intensifier enhanced the original fluorescence by 1 million times and improved the sensitivity and range in the anti-PLA2R-IgG-TRFIA. AutoDELFIA1235 (PerkinElmer, Waltham, Mass) was used to read Eu3+ fluorescence in microtiter wells. Serum samples from 286 healthy volunteers without evidence of nephropathy, gastroduodenal disorder, or liver diseases were collected from Jiangyuan Hospital (China). Serum samples collected from 161 kidney disease patients were from the Affiliated Wuxi People's Hospital of Nanjing Medical University (China). Each patient had renal biopsy and pathological examination at the time of blood sampling. Of these cases, 77 were IMN; 53 were IgA nephropathy; 14 were lupus erythematosus nephropathy; 2 were Henoch-Schonlein purpura nephritis; 5 were diabetic nephropathy; 2 were malignant hypertension arteriolar nephrosclerosis; and 8 were hepatitis B virus-associated glomerulonephritis. The institutional review boards of Jiangyuan Hospital and the Affiliated Wuxi People's Hospital of Nanjing Medical University approved the study, respectively. Written informed consent was obtained from all subjects. IMN was diagnosed by renal biopsy in patients who lacked features suggestive of secondary membranous nephropathy. Moreover, pathologic sine qua non of MN is the presence of subepithelial immune-complex deposit, which was best demonstrated by electron microscopy. Serum anti-PLA2R-IgG concentrations were detected by anti-PLA2R-IgG-TRFIA. Statistical analyses were performed with Prism 5.0 software (GraphPad Software, San Diego, Calif). The detection range of anti-PLA2R-IgG-TRFIA was 0.02 to 340 mg/L. The intra- and interassay coefficients of variation were 3.2% and 5.6%, respectively. Based on the disease pathology in kidney tissue samples, the 77 cases of IMN were divided into IMN I, which was 44 cases characterized by the presence of scattered or more regularly distributed small immune-complex-type electron-dense deposits in the subepithelial zone, and IMN II, which was 33 cases characterized by projections of basement membrane material around the subepithelial deposits. Serum from healthy volunteers and patients with IgA nephropathy, lupus nephropathy, and other kidney disease were analyzed (see Fig 1 and Table I).Table IThe serum anti-PLA2R-IgG levels in different test groupsHealthy volunteer (negative control)(n = 286)IgA nephropathy (n = 53)Lupus nephropathy (n = 14)Other kidney disease (n = 17)IMN(n = 77)Range of concentrations (mg/L)0.07-0.880.14-1.780.22-1.990.15-0.840.34-90.89Mean ± SD (mg/L)0.50 ± 0.160.76 ± 0.361.17 ± 0.610.47 ± 0.289.28 ± 15.25Positive rates (anti-PLA2R-IgG > 0.91 mg/L)035.8%50%5.88%88.3%Positive rates (anti-PLA2R-IgG > 1.99 mg/L)000074.0% Open table in a new tab Using a cutoff of <0.9 mg/L (based on the mean of the healthy volunteers +2.58 SD), the positive rate for the IMN was 88.3%. However, 35.8% IgA nephropathy cases and 50.0% lupus nephropathy cases would also be positive. To improve the clinical utility, we adjusted the cutoff value to 1.99 mg/L based on the ROC curves (see Fig E1 in this article's Online Repository at www.jacionline.org), so that all cases of IgA nephropathy, lupus erythematosus, and other kidney diseases were below the cutoff, whereas 57 of 77 of IMN (74.0%) were positive. Thus, the positive rate (sensitivity) was 74.0% for INM group and the specificity was 100%. These suggested that IMN could be distinguished from IgA nephropathy, lupus nephropathy, and other renal disease using appropriate thresholds of serum anti-PLA2R-IgG levels for different clinical purposes. To clarify the association of the quantity of anti-PLA2R in serum with the fluorescence intensity of PLA2R antigen in tissue biopsy, a histological examination was performed with the biopsy sample from each patient. Staining with unrelated primary antibody plus secondary antibody was negative in all biopsy samples. Staining with anti-PLA2R antibody resulted in a range of intensities. Subsequently, the agreement of these 2 methods was analyzed (see Fig E2 in this article's Online Repository at www.jacionline.org). The serum anti-PLA2R level was not directly comparable to the indirect immunofluorescence results. The discovery and determination of anti-PLA2R-IgG in serum provides an ideal marker to aid in diagnosis, assess activity and treatment timing, select drugs, and evaluate curative effects in IMN. To our best knowledge, this is the first time that a sensitive, quantitative analysis of serum anti-PLA2R-IgG-TRFIA has been established. The observation that some patients with IMN do not have PLA2R autoantibodies could be explained by limitations of current immunoassays. The analytical sensitivity of our method is 0.02 mg/L and 88.3% IMN could be detected, which was better than other reports. Why were anti-PLA2R antibodies not detected in all patients with IMN irrespective of the test used? There are some possible explanations for this discrepancy. First, there are 2 alleles that account for the genetic risk to develop IMN, major histocompatibility complex, class II, DQ alpha 1 (HLA-DQA1) and PLA2R1.6Stanescu H.C. Arcos-Burgos M. Medlar A. Bockenhauer D. Kottgen A. Dragomirescu L. et al.Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy.New Engl J Med. 2011; 364: 616-626Crossref PubMed Scopus (386) Google Scholar Interestingly, the association with HLA-DQA1 was stronger than with PLA2R1, which suggests that HLA-DQA1 allele might facilitate autoantibody development targeting not only PLA2R but also other antigens. Thus, existence of anti-PLA2R antibodies could provide further classification for IMN and enable prognostic analysis. Second, there are previous reports that a small percentage (5% to 25%) of samples were positive in sera from patients with secondary MN detected by qualitative methods, such as Western immnunoblot, IIF-CBA, and ELISA,3Qin W. Beck Jr., L.H. Zeng C. Chen Z. Li S. Zuo K. et al.Anti-phospholipase A2 receptor antibody in membranous nephropathy.J Am Soc Nephrol. 2011; 22: 1137-1143Crossref PubMed Scopus (346) Google Scholar, 4Svobodova B. Honsova E. Ronco P. Tesar V. Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy.Nephrol Dial Transplant. 2013; 28: 1839-1844Crossref PubMed Scopus (154) Google Scholar, 5Hofstra J.M. Wetzels J.F. Anti-PLA2R antibodies in membranous nephropathy: ready for routine clinical practice?.Neth J Med. 2012; 70: 109-113PubMed Google Scholar which could affect the differential diagnosis of IMN. In our study, it is possible to distinguish weakly positive IMN from normal control or secondary MN patients through setting suitable thresholds with sensitivity over 74% and specificity up to 100%. In conclusion, quantitative analysis with high sensitivity could distinguish idiopathic and secondary membranous nephropathy, and anti-PLA2R-IgG-TRFIA offers the opportunity to measure a marker to help diagnose, classify, and eventually monitor the course of patients with IMN. Fig E2Comparison of serum anti-PLA2R-IgG to PLA2R antigen in situ for IMN. PLA2R was detected by indirect immunofluorescence in tissue biopsy samples. Anti-PLA2R-IgG was used to detect PLA2R levels in biopsy samples from each patient to compare with TRFIA results. A-D, Fluorescence intensity increased in these samples. E, Comparison of detection by immunofluorescence and TRFIA. Bars = 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)