The Diagnosis Value Of PLA2R In Urine And Serum Antibodies For Idiopathic Membranous Nephropathy

Objective: Idiopathic membranous nephropathy（IMN）is an autoimmune renal disease,and it is a common cause of nephrotic syndrome in adults.IMN is considered to be caused by autoantibodies in serum targeting autoantigens in the glomerular podocytes,forming immune complex deposited in the glomerular basement membrane leading to the injury of podocytes and glomerular filtration barrier.Phospholipase A2 receptor（PLA2R）identified in 2009 is the primary autoantigen in IMN,which accounts for 70%-90% in IMN.The gold standard for the diagnosis of IMN is renal biopsy,but it is a traumatic method,and the development of serum anti-PLA2R antibody（sPLA2Rab）detection has greatly promoted the non-invasive diagnosis and treatment of IMN,which plays an important role in the diagnosis and monitoring of IMN.The specificity of sPLA2Rab for the diagnosis of IMN is extremely high,but its sensitivity is limited,the positive rate of sPLA2Rab for IMN diagnosis is merely 60%.Therefore,it is of great significance to explore more biomarkers to assist the diagnosis and monitoring of IMN.Urine examination is a more non-invasive method,which has great advantages in the diagnosis and treatment monitoring of renal diseases,and the molecules in urine can reflect the pathological changes of the kidney.Podocytes injury was often observed in IMN patients under electron microscope.The foot process was disordered,effaced,and even detached from the glomerular basement membrane,which mentioned that injured podocytes in IMN patients may shed into urine.PLA2R is a transmembrane protein expressed in podocytes and it is also the primary autoantigen of IMN.Its expression level in urine has not been reported previously.We aim to the explore the difference of PLA2R expression in urinary sediment between IMN patients and other nephropathy patients,and to evaluate the association of its expression level with the clinical manifestations and disease progress in IMN.Then,we investigate the diagnostic value of PLA2R in urinary sediment and sPLA2Rab for IMN to provide a new idea for the noninvasive diagnosis of IMN.Methods: We collected serum and urine samples of 159 IMN patients（96 sPLA2Rabpositive IMN and 63 sPLA2Rab-negative IMN patients）and 153 patients with other nephropathy including 76 IgA,40 FSGS,20 DN,12 LN and 5 MCD patients diagnosed by renal biopsy.Urine sediment and supernatant were obtained by centrifugation.Firstly,to clarify clinical characteristics in patients with different renal diseases,we compared clinical characteristics between patients with IMN and other nephropathy,and those were also compared between sPLA2Rab-positive IMN patients and sPLA2Rab-negative IMN patients.Then relationship between sPLA2Rab level and IMN clinical and pathological manifestations was completed by correlation analysis.To evaluate the diagnostic value of PLA2R in urine for IMN,we firstly performed immunofluorescence staining of Synaptopodin which is the biomarker of podocytes and PLA2R to observe the distribution and expression of PLA2R in podocytes of IMN patients and other nephropathy patients.Next,semi-quantitative analysis of PLA2R expression level in urinary sediment was performed by western blotting（WB）to evaluate its expression level in sPLA2Rab-positive IMN group,sPLA2Rab-negative IMN group and other nephropathy group.Meanwhile,we detected the expression level of PLA2R in urinary supernatant which was another soluble secreted form protein by enzyme linked immunosorbent assay（ELISA）to compare its expression trend with the PLA2R in urinary sediment.Furthermore,correlation analysis was performed to assess the association of PLA2R expression in urinary sediment with laboratory and pathological results to identify its association with IMN clinical manifestations and disease progress.Finally,we analyzed the diagnostic efficacy of PLA2R and other podocytes related index in urinary sediment and sPLA2Rab alone or combined for IMN using receiver operating characteristic curve（ROC）.Results: 1.sPLA2Rab,24h-UTP,TG,TC and LDL-C in IMN patients were higher than those in other nephropathy patients,serum TP,Alb,Urea,and IgG level in IMN patients were lower than those in other nephropathy patients（P < 0.05）.The positive rate of sPLA2Rab（sPLA2Rab > 20 RU/m L）in IMN patients was 60.38%.24h-UTP,TC,LDLC,C4,Cr,and Cys C in sPLA2Rab-positive IMN patients were higher than those in sPLA2Rab-negative IMN patients,and Ca²⁺,TP,Alb,and eGFR were lower than those in sPLA2Rab-negative IMN patients（P < 0.05）.2.sPLA2Rab level in IMN patients were positively correlated with 24h-UTP（r = 0.331,P < 0.0001）and TC（r = 0.412,P < 0.0001）,and negatively correlated with TP（r =-0.307,P < 0.0001）and Alb（r =-0.345,P < 0.0001）.sPLA2Rab levels were higher in IMN patients with stronger glomerular PLA2R antigen deposition intensity（P < 0.01）.3.Podocytes and PLA2R in urinary sediment were obviously observed through immunofluorescence staining in IMN patients whether sPLA2Rab was positive or negative.PLA2R mainly expressed in the membrane and cytoplasm of podocytes in IMN patients,while its expression was weak in patients with other nephropathy.4.Western blotting results showed that the levels of Synaptopodin and PLA2R in urinary sediment in sPLA2Rab-positive and sPLA2Rab-negative IMN groups were higher than those in other nephropathy groups（P < 0.01）,and the expression level of PLA2R in urinary sediment increased more than that of Synaptopodin.Comparing to other nephropathy patients,the PLA2R level in urinary sediment in sPLA2Rab-positive IMN group increased more than 20-fold and it increased 10-fold in sPLA2Rab-negative IMN group.5.There was no significant difference of soluble PLA2R concentration in urinary supernatant between IMN groups with other nephropathy group.The soluble PLA2R concentration in urinary supernatant of sPLA2Rab-negative IMN group was slightly lower than sPLA2Rab-positive IMN group（P < 0.05）.6.PLA2R expression level in urinary sediment of IMN patients was positively correlated with sPLA2Rab（r = 0.347,P < 0.0001）、24h-UTP（r = 0.400,P < 0.0001）、MA（r = 0.306,P < 0.0001）、Cr（r = 0.300,P < 0.001）、TC（r = 0.333,P < 0.0001）,and negatively correlated with TP（r =-0.303,P < 0.001）and Alb（r =-0.415,P < 0.0001）.There was no significant difference of PLA2R level in urinary sediment among IMN patients with different glomerular PLA2R antigen deposition intensity and pathological stage.7.The sensitivity and specificity of PLA2R/β-actin in urinary sediment for the diagnosis of IMN were 87.4% and 69.9%,AUC was 0.853,and the sensitivity and specificity of PLA2R/Synaptopodin in urinary sediment for the diagnosis of IMN were 76.7% and 92.8%,AUC was 0.900.The sensitivity and specificity of sPLA2Rab for the diagnosis of IMN were 85.5% and 97.4%,AUC was 0.940（cut-off value was 2.44 RU/m L）.The sensitivity and specificity of PLA2R/Synaptopodin in urinary sediment combined with sPLA2Rab for the diagnosis of IMN were 93.1% and 96.1%,AUC was 0.973,which had the best diagnosis efficacy for IMN.Conclusion: Both sPLA2Rab level and PLA2R level in urinary sediment were correlated with clinical manifestations of IMN patients.The expression level of PLA2R in urinary sediment was significantly increased in IMN patients compared to other nephropathy patients,and its elevated expression was also observed in sPLA2Rab-negative IMN patients,but PLA2R in urine supernatant was not significantly increased in IMN patients.PLA2R in urine sediment showed high sensitivity in diagnosis for IMN,and the combination of PLA2R/Synaptopodin and sPLA2Rab showed the best diagnostic efficacy for IMN.