Detection Of M-type Phospholipase A2 Receptor 1(PLA2R1) In Patients With Idiopathic Membranous Nephropathy And Serum IgA/C3 Ratio In IgA Nephropathy

BackgroundWith the changes of environment, the use of drugs, the increase in the incidence of cancer, the increase renal biopsy in elderly patients, the spectrum of nephritic diseases has changed a great. The ratio of membranous nephropathy also increased year by year. According to the data from Ruijin hospital, the ratio of membranous nephropathy (MN) in renal biopsy of glomerulonephritis increased from 7.1% in 2000 to 22.7% in 2009. A research from Professor Wang Haiyan shows that membranous nephropathy had become the second pathological type following IgA nephropathy. And in over 60-years old elder population, membranous nephropathy is the most common pathological type. In the past half century, scientists did much work in the pathogenesis of membranous nephropathy, including successful manufacturing Heymann’s membranous nephropathy animal model and find neutral endopeptidase (NEP) in the children with membranous nephropathy. In 2009, Beck et al identified M-type A2 receptor 1 by mass spectrometry analysis and found anti-PLA2R1 antibodies in 70% patients with idiopathic membranous nephropathy (IMN). And it considered that the antigen was the target antigen of IMN, and then PLA2R1 has received more and more attention. Chinese have different genes, diet and environment compared with foreign people. The study of PLA2R1 in the renal tissue of patients with idiopathic membranous nephropathy in Chinese is rare.IgA nephropathy (IgAN) is the most common primary glomerular disease in the world, and also in China, accounting for 45%～50% primary glomerular nephritis. The serum IgA/C3 ratio is a simple and feasible item, but its value in distinguishing IgA nephropathy from other nephropathies is still controversial. In addition, there is rare research on the relationship between the serum IgA/C3 ratio and the Oxford classification or Lee’s classification of IgA nephropathy.Objective1. To investigate the relationship between PLA2R1 antigen in renal tissue of IMN, and the difference expression of PLA2R1 antigen in renal tissue and anti-PLA2R1 antibody in serum.2. To evaluate the value of serum IgA/C3 ratio in the diagnosis of IgA nephropathy and its relationship with clinical and pathological changes of IgAN patients.Methods1. Detection of PLA2R1 antigen in renal tissue and anti-PLA2R1 antibody in the serum of patients with IMN.1.1 ObjectsA total of 114 adult patients with biopsy-proved glomerular diseases were enrolled in this study (41 with IMN,8 with hepatitis B virus associated membranous nephropathy,8 with type Ⅴ lupus nephritis,27 with IgAN,19 with minimal change disease,5 with mild mesangial proliferative glomerular nephritis,6 with focal segmental glomerulosclerosis). They were accepted renal biopsy in Third Affiliated Hospital of Southern Medical University from May 2010 to May 2014. IMN was diagnosised based on renal biopsy, and excluded type Ⅴ lupus nephritis, hepatitis B virus associated membranous nephropathy, tumors and other factors caused membranous nephropathy. None of all these participants were long-term treated with corticosteroids and/or immunosuppressants at the time of renal biopsy. All of the patients were informed.1.2 Sample collect and methodsUltrasound guided renal biopsy was performed. All biopsy specimens were processed for light microscopy, immunofluorescence studies and electron microscopy (EM). IgG, IgA, IgM, C3, C1q and C4 staining were performed by direct immunofluorescence method. HE, PAS, PASM, MASSON were performed under light microscope. Paraffin embedded tissue were applied using indirect immunofluorescence to detect PLA2R1 antigen in IMN, secondary membranous nephropathy and other glomerular disease.Cllection of serum samples:cllect fasting venous blood from the day perfroming renal biopsy, all specimens were 5 ml, placed in promoting agent, immediately with the centrifuge for 8 minutes, the speed of 3000R/min, then separation of serum, frozen in-80℃.1.21 Detection of PLA2R1 antigen in renal tissuesMaterials:Rabbit anti human PLA2R1 polyclonal antibody (Sigma, HPA012657), FITC labeled anti rabbit IgG (CW0114), BSA solid (Sigma company).For paraffin section:slice; toast; dewaxing; 1:20 proteinase K digestion for 30min in 37℃; closed by 5% of BSA at room temperature; add rabbit anti-human PLA2R1 polyclonal antibody (sigma) diluted to 1:500 by PBS; about 1.5 hours incubation; PBS washed three times; after drying; adding mouse anti-rabbit IgG (CWIBO); incubation I hour; PBS washing 3 times; application buffer glycerol mounting. Using the OLYMPUS laser scanning confocal microscope observation.Fluorescence intensity was divided into negative, ±,+,++,+++,++++.Negative controls were set for each disease with PBS instead of primary antibodies. Positive was judged as PBS negative and primary antibodies positive.1.22 Detection of anti-PLA2R1 antibody in serum by indirect immunofluorescenceAccording to the instructions of phospholipase A2 receptor 1(PLA2R1) IgG (FA 1254-1005-50 IgG) diluted samples, incubation, flushing, adding secondary antibody labeled by fluorescence, incubation, flushing, mounting, observation under the fluorescence microscopy (excitation filter:488 nm).1.3 Clinical characteristicsPatient age, sex, serum creatinine,24 hours of urinary protein, serum albumin were collected. According to the pathological changes of light and electron microscope, the 41 cases with idiopathic membranous nephropathy were graded respectively to I-V.2. The role of serum IgA/C3 ratio in the diagnosis of IgA nephropathy and its relationship with clinical and pathological features of IgA nephropathyMETHODS2.1 Subjects66 patients with biopsy-proven IgA nephropathy,111 patients with other glomerular diseases (40 MN,33 MCD,27 proliferative glomerulonephritis without mesangial IgA deposition,11 FSGS) and 40 healthy individuals were involved in the study in the Third Affiliated Hospital of Southern Medical University from December 2009 to February 2015. Patients with the following features were excluded:secondary cause of IgAN such as systemic lupus erythematosus, Henoch-Schonlein purpura, small-cell carcinoma, lymphoma and other systemic diseases; estimated glomerular filtration rate (eGFR) was<10 ml/min perl.73 m2at the time of renal biopsy; clinical data was incomplete. IgA nephrology diagnosis was based on histology and immunofluorescence examination. IgA deposition as the predominant or co-dominant immunoglobulin in mesangial, accompanied with deposition of the C3 component of complement and variable codeposition of IgG and IgM. None of all these participants was long-term treated with corticosteroids and/or immunosuppressants at the time of renal biopsy. All participants gave their informed consent.2.2 Clinical characteristicsClinical data collected include age, sex, serum creatinine (Scr),24-h urinary protein, serum albumin (Alb), hemoglobin (Hb), blood urea nitrogen (BUN), uric acid, serum high-density lipoprotein (HDL), cholesterol and triglyceride (TG) levels, low density lipoprotein cholesterol (LDL-C). Estimated glomerular filtration rate (eGFR) was estimated by using the simplified Modification of Diet in Renal Disease (MDRD) study equation that was defined as eGFR= [175×(Scr)-1.234 x (Age)-0.179× (if female, 0.79)]. IgA, IgM, C3, IgG were evaluated by using CRM470 adjusted standardized immune turbidimetric method.2.3 Histological classificationAll biopsy specimens were processed for evaluation of light microscopy, immunofluorescence assay and electron microscopy (EM). IgA nephrology diagnosis was based on histology and immunofluorescence examination. For IgA nephrology, Lee’s histological grades were measured by two blinded pathologists. In addition to Lee’s histological grades of IgA nephropathy, we also used Oxford Classification to measure the patients. Renal biopsy specimens were evaluated by two independent pathologists. In cases of disagreement, the final decision was made by consensus among the authors.Results1. Detection of PLA2R1 antigen in renal tissue and anti-PLA2R1 antibody in the serum of patients with IMN.1.1 Clinical characteristics of the patients with different pathological patternsThere were significant differences in age, sex, serum creatinine, serum albumin, and 24h urinary protein between different pathological types (P<0.05). The renal injury, age, gender were not consistent in different pathological.1.2 Clinical characteristics of IMN patients with PLA2R1 positive and negative in renal biopsyThere was no significant difference in the age, sex, serum creatinine, serum albumin and 24 hours urinary protein between PLA2R1 antigen positive patients and PLA2R1 antigen negative patients in IMN (P>0.05).1.3 The positive rate of PLA2R1 in IMN and secondary membranous nephropathyIn 41 cases of IMN,35 have PLA2R1 antigen deposition. And PLA2R1 was deposits in fine granular along the glomerular capillary loops. PLA2R1 antigen deposition was not found in renal tissue of 6 patients with idiopathic membranous nephropathy.PLA2R1 antigen was not found in renal tissue of secondary membranous nephropathy (hepatitis B virus associated membranous nephropathy, lupus nephritis) and other glomerular diseases.1.4 Comparison of the positive rate of PLA2R1 in renal tissue and serum anti-PLA2R1 antibody in IMNAnti-PLA2R1 antibody was positive in serum of 30 patients with idiopathic membranous nephropathy (positive rate was 73.17%).27 cases were found PLA2R1 in renal tissue and serum,8 cases of renal tissue PLA2R1 antigen positive and serum anti-PLA2R1 antibody negative,3 cases of renal tissue PLA2R1 antigen negative and serum anti-PLA2R1 antibody positive,3 cases were negative for both. There was no significant difference between the two methods (P=0.276).2. The role of serum IgA/C3 ratio in the diagnosis of IgA nephropathy and its relationship with clinical and pathological features of IgA nephropathy2.1 Baseline characteristicsThe mean age of subjects with IgAN, other nephropathy, healthy adults was 37.42±12.07 years,43.49±16.16 years,46.33±15.01 years respectively. As compared with individuals with other nephropathy, individuals with IgAN were more likely to be lower Hb, higher serum creatinine, higher albumin, lower proteinuria, lower eGFR, lower cholesterol, lower triglyceride, lower LDL, lower HDL, higher serum IgG, lower serum C3 and lower serum IgM. As compared with individuals with healthy controls, individuals with IgAN were more likely to have higher BUN, higher Scr, higher proteinuria, lower eGFR, higher uric acid. As compared with individuals with healthy controls, individuals with other nephropathy were more likely to have higher BUN, higher serum creatinine, lower albumin, higher proteinuria, higher cholesterol, higher triglyceride, higher LDL, higher HDL, lower serum IgG and higher serum IgM. As compared with individuals with other nephropathy and healthy controls, individuals with IgAN were more likely to be higher serum IgA (IgAN Vs Other primary, P<0.001; IgAN Vs healthy controls, P<0.001) and serum IgA/C3 (IgAN Vs Other primary, P<0.001; IgAN Vs healthy controls, P<0.001).Receiver-operating characteristic (ROC) plots showed that the sensitivity and specificity discriminant of serum IgA/C3 was 78.79% and 65.6% respectively. For serum IgA/C3 ratio the area under curve was 0.776 (higher than serum IgA). And the cut-off of serum IgA/C3 ratio was 2.23.2.2 Comparison of high and low serum IgA/C3 ratios in patients with IgA nephropathyAccording to the cut-off value of 2.23, we divided IgAN into serum IgA/C3 ratio >2.23 group and serum IgA/C3 ratio<2.23 group. As compared with individuals with serum IgA/C3 ratio<2.23 group, individuals with serum IgA/C3 ratio≥2.23 group were more likely to be young, lower cholesterol, lower triglyceride. The proteinuria (g/24 h) level in the IgA/C3 ratio<2.23 group was significantly higher than the IgA/C3 ratio>2.23 group (P=0.016). And higher proteinuria (g/24 h)≥1 rate also can be seen in IgA/C3 ratio<2.23 group (P=0.031). The mean serum IgA level and serum IgA/C3 ratios of the IgA/C3 ratio>2.23 group was significantly higher than the IgA/C3 ratio< 2.23 group (P<0.001). The mean serum C3 level of the patients in the IgA/C3 ratio>2.23 group was significantly lower than the IgA/C3 ratio< 2.23 group (P< 0.001). There were no differences in gender, Hb, BUN, Scr, Alb, eGFR, uric acid, LDL, HDL, serum IgG, serum IgM, SBP, DBP, the rate of Lee’s grade IV-V(%), mesangial proliferation, endothelial hypercellularity, glomerular sclerosis, T/I fibrosis >25% and MEST score>3.2.3 MEST and clinical data in patients with IgA nephropathyWe divided IgAN into MEST score>3 group and score<3 group. Patients with MEST score>3 are more likely to be higher BUN, higher Scr, higher proteinuria (g/24 h), proteinuria (g/24 h)>1 rate and lower eGFR. And there were no differences in age, gender, Hb, alb, serum IgG, serum IgA, IgA/C3 ratio, Lee’s grade FV-V (%) rate.Conclusion:1. In adult patients with idiopathic membranous nephropathy, the positive rate of PLA2R1 antigen was 85.37%, the rate of anti-PLA2Rl antibody was 73.17%. There was no significant difference in the positive rates of PLA2R1 antigen and serum anti -PLA2R1 antibody in IMN. And there was no significant difference in clinical manifestations between PLA2R1 antigen positive and negative patients.2. Patients with IgA nephropathy, have a higher serum IgA/C3 ratio than other glomerular disease group and health controls. The area under ROC curve for serum IgA/C3 ratio in prediction of IgA nephropathy is 0.776. Elevated serum IgA/C3 ratio with poor IgA nephropathy pathological prognosis has yet to see a clear correlation. In the hospital without renal biopsy and patients who didn’t accept renal biopsy, serum IgA/C3 ratio can play a supplementary role in the diagnosis of IgA nephropathy.