Measurement Of Anti-PL2R Antibodies And Detection Of PLA2R Antigens In Idiopathic Membranous Nephropathy:Combination Tells More

Part 1. Serum phospholipase A2 receptor autoantibodies and glomerular antigen deposits in membranous nephropathyObjective To analyze the distribution of serum phospholipase A2 receptor (PLA2R) antibodies and glomerular antigen deposits in idiopathic membranous nephropathy (IMN) patients and the clinical application of these two biomarkers.Methods A total of 572 IMN patients received biopsy and diagnosed IMN were recruited from the National Clinical Research Center of Kidney disease. Serum samples at the time of biopsy and paraffin embedded biopsy tissues were collected. The quantitative serum PLA2R antibody level was measured using the ELISA method while the glomerular antigen deposit were detected by immunofluorescence staining. Glomerular deposit of Thrombospondin Type-1 Domain-containing 7A (THSD7A), another IMN related antigen, were measured by immnuohistochemical staining. Clinical data at baseline and follow up were collected. Kaplan-Meier curves were used to analyze the difference in proteinuria remission and renal function progression. COX multivariate model was used to validate independent predictive factors for proteinuria remission.Results The serum PLA2R antibody (SAb) was present in 68.5% (392/572) patients. Positive glomerular PLA2R antigen (GAg) deposit was observed in 98.9% (387/392) of the SAb positive patients and also in 70.6%(127/180) of the SAb negative patients. The positive rate for glomerular PLA2R antigen deposit was significantly higher than that for serum PLA2R antibody (89.9 vs.68.5%, P<0.001). Patients with serum antibody exhibited higher levels of baseline proteinuria (4.0 vs.2.6 g/24h, P<0.001) and lower level of eGFR (105 vs.109 ml/min-1.73 m2, P=0.008). SAb+/GAg+ patients exhibited higher levels of proteinuria (4.0 vs.2.4 g/24h, P<0.001) and a lower eGFR (105 vs.110 ml/min·1.73 m2, P=0.002) than SAb-/GAg+ patients. SAb+/GAg+ patients also showed lower chance of achieving proteinuria remission (P<0.001) compared with SAb-/GAg+ patients. Among 53 patients that were negative for both PLA2R antibody and antigen, four were positive for glomerular THSD7A antigen deposit.Conclusion Combining the measurement of serum PLA2R antibodies and glomerular antigen deposits can be a more reliable and sensitive method in diagnosing IMN. Compared with glomerular antigen deposits, the serum antibodies were more tightly correlated with disease activity and prognosis. The detection of glomerular THSD7A antigen deposit is necessary in patients without both PLA2R antibody and antigen deposit.Part 2 Dynamic changes of serum phospholipase A2 receptor antibodies and glomerular antigen depositsObjective:To describe the dynamic changes of serum phospholipase A2 receptor (PLA2R) antibody and glomerular antigen deposits, and their relationships with treatment response and prognosis.Methods A total of 52 patients with repeat biopsy and diagnosed idiopathic membranous nephropathy (IMN) were recruited from the National Clinical Research Center of Kidney disease. Serum samples and biopsy tissues at both biopsies were collected. ELISA were used to measure the level of PLA2R antibody while immunofluorescence staining were used to detect antigen deposit. Intensities of antigen deposit were evaluated semi quantitatively. Clinical data at both times of biopsy, interval and follow up were collected. Kaplan-Meier analysis was adopted to analyze difference in chance of remission and relapse.Results Among 52 IMN patients with repeat biopsy, twenty-one patients had not achieved remission at the second biopsy; eleven patients had undergone proteinuria remission but relapsed prior to the second biopsy; while remission was achieved in 20 patients at the second biopsy. In patients failed to achieve remission, serum antibodies tend to maintain positive or increase while the glomerular antigen intensities tend to sustain or increase at the repeat biopsy. In patients achieved remission, the serum antibody tend to decrease or turn negative while the glomerular antigen intensity tend to sustain or decrease. However, in some patients there existed discrepancy between the change of serum antibodies and change of glomerular antigen intensities. Patients who failed to achieve remission showed a much higher serum antibody positive rate at the first biopsy than did patients who achieved remission (90.5% vs.40%, respectively, P<0.001). The glomerular antigen deposit positive rate at the first biopsy in patients who failed to achieved remission was also higher than in patients who achieved remission (100% vs 70%, respectively, P=0.009). Kaplan-Meier analysis indicated a higher risk of relapse in patients with sustained glomerular antigen deposit than in patients without antigen deposit when achieved remission (P=0.027). The Cox regression analysis showed that the intensity of glomerular antigen staining when remission was achieved was an independent risk factor in predicting relapse after adjusting for complete remission/partial remission status (HR=3.1, P=0.009).Conclusion Intensities of glomerular antigen deposit can change as disease progresses or remits. However, the glomerular antigen deposit is more stable and long lasting than serum antibody. Positive glomerular PLA2R antigen deposit, as well as positive serum antibody, correlated with treatment response. Sustained glomerular antigen deposit when remission is achieved is a risk factor for relapse since it may act as trigger to restart immune response. Based on these findings, inducing the disappearance of glomerular antigen deposit may be a new target in the treatment of IMN.