Potential therapeutic targets for membranous nephropathy: proteome-wide Mendelian randomization and colocalization analysis.

Background: The currently available medications for treating membranous nephropathy (MN) still have unsatisfactory efficacy in inhibiting disease recurrence, slowing down its progression, and even halting the development of end-stage renal disease. There is still a need to develop novel drugs targeting MN. Methods: We utilized summary statistics of MN from the Kiryluk Lab and obtained plasma protein data from Zheng et al. We performed a Bidirectional Mendelian randomization analysis, HEIDI test, mediation analysis, Bayesian colocalization, phenotype scanning, drug bank analysis, and protein-protein interaction network. Results: The Mendelian randomization analysis uncovered 8 distinct proteins associated with MN after multiple false discovery rate corrections. Proteins related to an increased risk of MN in plasma include ABO [(Histo-Blood Group Abo System Transferase) (WR OR = 1.12, 95%CI:1.05-1.19, FDR=0.09, PPH4 = 0.79)], VWF [(Von Willebrand Factor) (WR OR = 1.41, 95%CI:1.16-1.72, FDR=0.02, PPH4 = 0.81)] and CD209 [(Cd209 Antigen) (WR OR = 1.19, 95%CI:1.07-1.31, FDR=0.09, PPH4 = 0.78)], and proteins that have a protective effect on MN: HRG [(Histidine-Rich Glycoprotein) (WR OR = 0.84, 95%CI:0.76-0.93, FDR=0.02, PPH4 = 0.80)], CD27 [(Cd27 Antigen) (WR OR = 0.78, 95%CI:0.68-0.90, FDR=0.02, PPH4 = 0.80)], LRPPRC [(Leucine-Rich Ppr Motif-Containing Protein, Mitochondrial) (WR OR = 0.79, 95%CI:0.69-0.91, FDR=0.09, PPH4 = 0.80)], TIMP4 [(Metalloproteinase Inhibitor 4) (WR OR = 0.67, 95%CI:0.53-0.84, FDR=0.09, PPH4 = 0.79)] and MAP2K4 [(Dual Specificity Mitogen-Activated Protein Kinase Kinase 4) (WR OR = 0.82, 95%CI:0.72-0.92, FDR=0.09, PPH4 = 0.80)]. ABO, HRG, and TIMP4 successfully passed the HEIDI test. None of these proteins exhibited a reverse causal relationship. Bayesian colocalization analysis provided evidence that all of them share variants with MN. We identified type 1 diabetes, trunk fat, and asthma as having intermediate effects in these pathways. Conclusions: Our comprehensive analysis indicates a causal effect of ABO, CD27, VWF, HRG, CD209, LRPPRC, MAP2K4, and TIMP4 at the genetically determined circulating levels on the risk of MN. These proteins can potentially be a promising therapeutic target for the treatment of MN.

Predictive value of the domain specific PLA2R antibodies for clinical remission in patients with primary membranous nephropathy: A retrospective study.

BACKGROUND: M-type phospholipase A2 receptor (PLA2R) is a major auto-antigen of primary membranous nephropathy(PMN). Anti-PLA2R antibody levels are closely associated with disease severity and therapeutic effectiveness. Analysis of PLA2R antigen epitope reactivity may have a greater predictive value for remission compared with total PLA2R-antibody level. This study aims to elucidate the relationship between domain-specific antibody levels and clinical outcomes of PMN. METHODS: This retrospective analysis included 87 patients with PLA2R-associated PMN. Among them, 40 and 47 were treated with rituximab (RTX) and cyclophosphamide (CTX) regimen, respectively. The quantitative detection of -immunoglobulin G (IgG)/-IgG4 targeting PLA2R and its epitope levels in the serum of patients with PMN were obtained through time-resolved fluorescence immunoassays and served as biomarkers in evaluating the treatment effectiveness. A predictive PMN remission possibility nomogram was developed using multivariate logistic regression analysis. Discrimination in the prediction model was assessed using the area under the receiver operating characteristic curve (AUC-ROC).Bootstrap ROC was used to evaluate the performance of the prediction model. RESULTS: After a 6-month treatment period, the remission rates of proteinuria, including complete remission and partial remission in the RTX and CTX groups, were 70% and 70.21% (P = 0.983), respectively. However, there was a significant difference in immunological remission in the PLA2R-IgG4 between the RTX and CTX groups (21.43% vs. 61.90%, P = 0.019). Furthermore, we found differences in PLA2R-CysR-IgG4(P = 0.030), PLA2R-CTLD1-IgG4(P = 0.005), PLA2R-CTLD678-IgG4(P = 0.003), and epitope spreading (P = 0.023) between responders and non-responders in the CTX group. Multivariate logistic analysis showed that higher levels of urinary protein (odds ratio [OR], 0.49; 95% confidence interval [CI], 0.26-0.95; P = 0.035) and higher levels of PLA2R-CTLD1-IgG4 (OR, 0.79; 95%CI,0.62-0.99; P = 0.041) were independent risk factors for early remission. A multivariate model for estimating the possibility of early remission in patients with PMN is presented as a nomogram. The AUC-ROC of our model was 0.721 (95%CI, 0.601-0.840), in consistency with the results obtained with internal validation, for which the AUC-ROC was 0.711 (95%CI, 0.587-0.824), thus, demonstrating robustness. CONCLUSIONS: Cyclophosphamide can induce immunological remission earlier than rituximab at the span of 6 months. The PLA2R-CTLD1-IgG4 has a better predict value than total PLA2R-IgG for remission of proteinuria at the 6th month.

Clinical implications of serum anti-PLA2R levels and glomerular PLA2R deposits in primary membranous nephropathy.

Introduction. The clinical implications of serum anti-PLA2R with glomerular PLA2R deposits in primary membranous nephropathy (PMN) is scarcely reported. Hence the study was designed to demonstrate the prevalence of serum anti-PLA2R levels and PLA2R staining in glomeruli in PMN and the clinical implications of the two parameters. Objectives. Investigate the prevalence of anti PLA2R positivity in PMN. Ascertain correlation between serum anti-PLA2R levels and glomerular staining for PLA2R with clinical and lab parameters in PMN. Patients and Methods. Fifty PMN patients during the period from October 2017 to December 2018 were included. Labs were done and eGFR was calculated as per MDRD 6. Anti-PLA2R titres were done in all patients. Titres more than 20 RU/ml were considered positive. Glomerular staining for PLA2R was graded on fresh frozen tissue by immunofluorescence technique. Results. Anti-PLA2R antibody positivity and glomerular PLA2R deposition was observed in 42% (21/50) and 86% (43/50) patients respectively. 79.3% (23/29) had positive glomerular PLA2R deposition with negative serum anti PLA2R. Positive correlation were observed between serum PLA2R antibody and serum creatinine (p = 0.0001) and urine protein-creatinine ratio levels with tissue PLA2R staining grades (p = 0.04). Negative association was found between serum albumin (p = 0.026) and tissue PLA2R staining grades. Conclusion. Serum anti-PLA2R wasn't a sensitive marker of primary membranous nephropathy in our study group emphasising the need to consider a compendium of serological markers for diagnosis of primary membranous nephropathy and to rely more on glomerular deposition of PLA2R as a better clinical indicator for PMN.

The diversity of peripheral blood T cells and myeloid-derived suppressor cells in patients with idiopathic membranous nephropathy.

OBJECTIVE: Membranous nephropathy is a leading cause of adult-onset nephrotic syndrome. Peripheral T cells and myeloid-derived suppressor cells (MDSCs) are closely associated with autoimmune diseases, while their exact roles and interaction in these processes are unclear. Here, we studied the roles of T cells, MDSCs, and their subsets in patients with idiopathic membranous nephropathy (IMN). MATERIALS AND METHODS: 35 IMN patients and 30 healthy controls were included in this retrospective study. Flow cytometry was performed to determine the phenotype of human T cells and MDSCs in peripheral blood mononuclear cells (PBMCs). Anti-PLA2R was measured by ELISA. Values ≥ 20 RU/mL were defined as positive and < 14 RU/mL as negative. RESULTS: A higher ratio of CD4/CD8 T cells with a lower proportion of Tregs, a remarkably lower proportion of G-MDSCs (but not M-MDSCs), lower frequency of PD-L2+G-MDSCs, and higher frequency of PD-L1+M-MDSCs were found in IMN patients compared to healthy controls. The ratio of CD4/CD8 T cells was higher, and the frequencies of PD-1+CD4+ T cells, CTLA-4+CD4+ T cells, PD-1+Tregs, and CTLA-4+Tregs were lower in PBMCs of PLA2R-positive IMN patients compared to PLA2R-negative IMN patients. CONCLUSION: Tregs and G-MDSCs were reduced in the circulation of the IMN patients, which may promote understanding of the crucial functions that are mediated by these cells in the pathogenesis of IMN.

Preference for anti-phospholipase A2 receptor antibody assay in patients with suspected membranous nephropathy: a survey study on medical practice after publication of Japanese Guidelines for Nephrotic Syndrome 2020.

BACKGROUND: International practice guidelines advocate for the use of anti-phospholipase A2 receptor (PLA2R) antibody testing to diagnose primary membranous nephropathy (pMN). This study aimed to clarify the current status of anti-PLA2R antibody testing in the diagnosis of pMN in Japan and to scrutinize the factors associated with the implementation of this antibody test. METHODS: Utilizing a web-based questionnaire for nephrologists, responses were collected from 306 facilities and 427 nephrologists between November 2021 and December 2021. Preference for anti-PLA2R antibody testing was also investigated. Factors related to the experience of quantifying anti-PLA2R antibodies were estimated by generalized estimating equations using a robust analysis of variance with clusters of facilities of affiliation. RESULTS: Of the 427 respondents, 140 (32.8%) had previous measurement experience at their current workplace and 165 (38.6%) had previous measurement experience overall. In pMN-suspected cases without contraindications to renal biopsy, 147 (34.4%) of the respondents opted to request anti-PLA2R antibody testing. The respondents' experience with anti-PLA2R antibody quantification at their current place of work was generally higher in university hospitals and increased with the annual number of kidney biopsies and the number of years since graduation. CONCLUSION: The results of this study suggest that a significant proportion of nephrologists in Japan have no experience in performing anti-PLA2R antibody assays, and that the assays may be hampered by the limited capabilities of the current workplace and the financial burden on facilities and patients.

Combined Serologic and Genetic Risk Score and Prognostication of Phospholipase A2 receptor-Associated Membranous Nephropathy.

INTRODUCTION: The aim of this study was to test whether a combined risk score on the basis of genetic risk and serology can improve the prediction of kidney failure in phospholipase A2 receptor (PLA2R)-associated primary membranous nephropathy. METHODS: We performed a retrospective analysis of 519 biopsy-proven PLA2R-associated primary membranous nephropathy patients with baseline eGFR ≥25 ml/min per 1.73 m 2 . The combined risk score was calculated by combining the genetic risk score with PLA2R ELISA antibody titers. The primary end point was kidney disease progression defined as a 50% reduction in eGFR or kidney failure. Cox proportional hazard regression analysis and C-statistics were applied to compare the performance of PLA2R antibody, genetic risk score, and combined risk score, as compared with clinical factors alone, in predicting primary outcomes. RESULTS: The median age was 56 years (range, 15-82 years); the male-to-female ratio was 1:0.6, the median eGFR at biopsy was 99 ml/min per 1.73 m 2 (range: 26-167 ml/min per 1.73 m 2 ), and the median proteinuria was 5.3 g/24 hours (range: 1.5-25.8 g/24 hours). During a median follow-up of 67 (5-200) months, 66 (13%) had kidney disease progression. In Cox proportional hazard regression models, PLA2R antibody titers, genetic risk score, and combined risk score were all individually associated with kidney disease progression with and without adjustments for age, sex, proteinuria, eGFR, and tubulointerstitial lesions. The best-performing clinical model to predict kidney disease progression included age, eGFR, proteinuria, serum albumin, diabetes, and tubulointerstitial lesions (C-statistic 0.76 [0.69-0.82], adjusted R 2 0.51). Although the addition of PLA2R antibody titer improved the performance of this model (C-statistic: 0.78 [0.72-0.84], adjusted R 2 0.61), replacing PLA2R antibody with the combined risk score improved the model further (C-statistic: 0.82 [0.77-0.87], adjusted R 2 0.69, difference of C-statistics with clinical model=0.06 [0.03-0.10], P < 0.001; difference of C-statistics with clinical-serologic model=0.04 [0.01-0.06], P < 0.001). CONCLUSIONS: In patients with PLA2R-associated membranous nephropathy, the combined risk score incorporating inherited risk alleles and PLA2R antibody enhanced the prediction of kidney disease progression compared with PLA2R serology and clinical factors alone.

Rationale and design of the Japanese Biomarkers in Nephrotic Syndrome (J-MARINE) study.

INTRODUCTION: Disease subtyping and monitoring are essential for the management of nephrotic syndrome (NS). Although various biomarkers for NS have been reported, their clinical efficacy has not been comprehensively validated in adult Japanese patients. METHODS: The Japanese Biomarkers in Nephrotic Syndrome (J-MARINE) study is a nationwide, multicenter, and prospective cohort study in Japan, enrolling adult (≥18 years) patients with minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G), and lupus nephritis (LN). Baseline clinical information and plasma and urine samples will be collected at the time of immunosuppressive therapy initiation or biopsy. Follow-up data and plasma and urine samples will be collected longitudinally based on the designated protocols. Candidate biomarkers will be measured: CD80, cytotoxic T-lymphocyte antigen 4, and soluble urokinase plasminogen activator receptor for MCD and FSGS; anti-phospholipase A2 receptor and thrombospondin type-1 domain-containing protein 7A antibodies for MN; fragment Ba, C3a, factor I, and properdin for MPGN/C3G; and CD11b, CD16b, and CD163 for LN. Outcomes include complete and partial remission, relapse of proteinuria, a 30% reduction in estimated glomerular filtration rate (eGFR), eGFR decline, and initiation of renal replacement therapy. The diagnostic accuracy and predictive ability for clinical outcomes will be assessed for each biomarker. RESULTS: From April 2019 to April 2023, 365 patients were enrolled: 145, 21, 138, 10, and 51 cases of MCD, FSGS, MN, MPGN/C3G, and LN, respectively. CONCLUSION: This study will provide valuable insights into biomarkers for NS and serve as a biorepository for future studies.

Serum PLA2R antibody as a predictive biomarker for venous thromboembolism risk in primary membranous nephropathy / El anticuerpo PLA2R sérico como biomarcador predictivo del riesgo de tromboembolia venosa en la nefropatía membranosa primaria

Background Patients with nephrotic syndrome are at high risk of venous thromboembolism (VTE), especially for primary membranous nephropathy (PMN). The phospholipase A2 receptor (PLA2R) is a marker of primary membranous nephropathy activity. This study investigated the predictive value of PLA2R antibodies in PMN for VTE.Methods In this retrospective study, we included 97 PMN patients and evaluated the predictive value of serum PLA2R antibodies for VTE risk. Lower extremity venous ultrasound, renal vein ultrasound, or spiral computed tomography pulmonary arteriography were used to assess VTE events. Serum anti-PLA2R antibodies were detected by enzyme-linked immunosorbent assay (ELISA). The risk of VTE was stratified according to serum albumin levels. Results Twenty PMN patients (21%) had thromboembolic events. Eight (15%) of patients with serum albumin >25g/l developed VTE, 6 of whom were positive for serum PLA2R antibodies. Positive serum PLA2R antibodies were significantly associated with VTE events in patients with serum albumin >25g/l (p=0.01). Age, sex, blood creatinine, serum albumin, and 24-h urine protein levels were not statistically different between the two groups. Kaplan–Meier analysis using the log-rank test revealed anti-PLA2R positive membranous nephropathy patients had more probability of VTE events than anti-PLA2R negative patients. Univariate Cox proportional hazards analysis revealed that lnPLA2R-Ab is an unfavorable predictor for VTE events in patients with serum albumin >25g/l (hazard ratio (HR) 2.1, p=0.01). Conclusions The PLA2R antibody was a risk predictor for thromboembolic events in patients with primary membranous nephropathy with serum albumin >25g/l (AU)

Antecedentes Los pacientes con síndrome nefrótico presentan un alto riesgo de tromboembolia venosa (TEV), especialmente en el caso de la nefropatía membranosa primaria (NMP). El receptor de fosfolipasa A2 (PLA2R) es un marcador de la actividad de la NMP. Este estudio investigó el valor predictivo de los anticuerpos PLA2R en NMP para TEV. Métodos En este estudio retrospectivo, se incluyeron 97 pacientes con NMP y se evaluó el valor predictivo de los anticuerpos PLA2R en suero para el riesgo de TEV. Se utilizaron la ecografía venosa de las extremidades inferiores, la ecografía de la vena renal o la arteriografía pulmonar por tomografía computarizada en espiral para evaluar los episodios de TEV. Los anticuerpos séricos anti-PLA2R se detectaron mediante un ensayo inmunoenzimático (ELISA). El riesgo de TEV se estratificó en función de los niveles de albúmina sérica. Resultados Veinte pacientes con NMP (21%) presentaron episodios tromboembólicos. Ocho (15%) de los pacientes con albúmina sérica>25g/l desarrollaron TEV, 6 de los cuales fueron positivos para anticuerpos PLA2R séricos. La positividad de los anticuerpos PLA2R séricos se asoció significativamente con episodios de TEV en pacientes con albúmina sérica>25g/l (p=0,01). La edad, el sexo, la creatinina en sangre, la albúmina sérica y los niveles de proteínas en orina de 24h no fueron estadísticamente diferentes entre los 2 grupos. El análisis de Kaplan-Meier mediante la prueba de rangos logarítmicos reveló que los pacientes con nefropatía membranosa positivos para anti-PLA2R tenían más probabilidad de sufrir TEV que los pacientes negativos para anti-PLA2R. El análisis univariante de riesgos proporcionales de Cox reveló que ln PLA2R-Ab es un predictor desfavorable de episodios de TEV en pacientes con albúmina sérica>25g/l (hazard ratio 2,1; p=0,01). Conclusiones El anticuerpo PLA2R fue un predictor de riesgo de episodios tromboembólicos en pacientes con NMP con albúmina sérica>25g/l (AU)

Treatment of rituximab in patients with idiopathic membranous nephropathy: a case series and literature review.

BACKGROUND/AIMS: Membranous nephropathy (MN) is a major cause of nephrotic syndrome in adults. This study aimed to evaluate the effect of rituximab (RTX) in patients with idiopathic MN (iMN) who have a high risk of progression. METHODS: We retrospectively analyzed data of 13 patients with iMN, who received RTX treatments from January 2014 to July 2020. RTX was indicated in patients with iMN with severe proteinuria and decreasing estimated glomerular filtration rate (eGFR) in the previous 6 months despite other immunosuppressive therapies. RESULTS: The patients were predominantly males (n = 11) and with a mean age of 55.3 years; median eGFR, 37.0 mL/min/1.73 m2 (interquartile range [IQR], 26.3 to 66.5); serum albumin level, 2.6 g/dL (IQR, 1.9 to 3.1); and spot urine protein-to-creatinine ratio at baseline, 6.6 g/g (IQR, 5.7 to 12.9). In a median follow-up of 22 months, eight patients (61.5%) achieved complete or partial remission. In responder group (n = 8), median eGFR increased from 31.5 to 61.5 mL/min/1.73 m2 (p = 0.049) and serum albumin level increased from 2.3 to 4.2 g/dL (p = 0.017) from RTX initiation to last follow-up. Antiphospholipase A2 receptor antibody (anti-PLA2R-Ab) was positive in six among seven tested patients, which markedly decreased in the responder group. There were no adverse events after RTX. CONCLUSION: This study suggests that RTX is a safe and effective treatment option for patients with iMN who have a high risk of progression. Individualized therapy based on anti-PLA2R-Ab titer would be needed for better outcomes.

Association of serum mannose-binding lectin, anti-phospholipase A2 receptor antibody and renal outcomes in idiopathic membranous nephropathy and atypical membranous nephropathy: a single center retrospective cohort study.

OBJECTIVES: Idiopathic membranous nephropathy (iMN) is a major cause of nephrotic syndrome. Atypical membranous nephropathy (aMN) is a new type of nephropathy in China, characterized by a 'full-house' on immunofluorescent examination, that is IgG, IgA, IgM, C3, C1q positive, but without clinical evidence of a secondary cause. Phospholipase A2 receptor (PLA2R) was the major target antigens in iMN patients. Activation of the mannose-binding lectin (MBL) pathway plays a vital role in the development of MN. Our objective was to investigate the role of PLA2R and MBL in the pathogenesis of iMN and aMN. METHODS: We conducted a retrospective observational study using propensity score matching by age, gender, and eGFR. All clinical, laboratory data, and follow-up data of the patients were collected. Serum levels of anti-PLA2R antibodies and MBL were tested. RESULTS: Finally, 30 iMN patients and 30 aMN patients were included, and 20 healthy controls were retrospectively collected in this study. The 24 h proteinuria level was higher and serum albumin was lower in anti-PLA2R (+) patients than in anti-PLA2R (-) patients in both iMN and aMN groups. In aMN patients, MBL levels were significantly higher in anti-PLA2R (+) patients than in anti-PLA2R (-) patients (p = .045). The serum level of anti-PLA2R positively correlated with no-remission in both iMN and aMN groups. CONCLUSIONS: The complement lectin pathway has an association with the development of MN, especially in patients with positive anti-PLA2R antibodies. Serum MBL cannot differentiate between the two diseases. Serum MBL levels are not associated with clinical manifestations, nor with prognosis.

Negative anti-phospholipase A2 receptor antibody status at three months predicts remission in primary membranous nephropathy.

BACKGROUND: The value of anti-phospholipase A2 receptor antibody (anti-PLA2R ab) monitoring at 3 months after diagnosis in membranous nephropathy (MN) remains uncertain. METHODS: We retrospectively examined the outcome on 1 August 2020 of 59 adult patients (age 54 (44, 68) years, 69% male, SCr 1.0 (0.9, 1.3) mg/dL) diagnosed with MN (kidney biopsy, positive serum anti-PLA2R ab). The outcomes were: kidney survival; partial and/or complete remission. RESULTS: Most of the studied patients (97%) received immunosuppression, cyclophosphamide regimens were the most frequent (87%), followed by cyclosporine (10%). The median time to remission was 12.0 months and the cumulative remission rates were 34% at 6, 54% at 12, and 73% at 24 months. Forty (69%) patients had negative anti-PLA2R ab at 3 months, they had similar age, serum creatinine, albumin, proteinuria, and treatment with the group with positive ab at 3 months. In the Cox proportional hazard model, three months anti-PLA2R ab negativization (HR 0.4 (95%CI 0.1, 0.9)) was an independent predictor for remission, while baseline hypoalbuminemia (HR 3.0 (95%CI 1.5, 5.7)) was associated with absence of remission. Six (10%) patients died, mostly due to cardiovascular disease and infections. A total of five (9%) patients started dialysis. Mean kidney survival time was 50.3 months and there was no survival difference in relation to baseline anti-PLA2R ab titer (p .09) or 3 months negativization (p .8). CONCLUSIONS: Three months anti-PLA2R ab negativization seems to be a late predictor of remission, and lower serum albumin at diagnosis is an early marker for remission absence. Abbreviations: anti-P LA2R ab, anti-phospholipase A2 receptor antibody; eGFR, estimated glomerular filtration rate; ESKD, end stage kidney disease; MN, membranous nephropathy; NELL-1, neural epidermal growth factor-like 1 protein; RAAS: renin-angiotensin-aldosterone system; RBC: red blood cells; RRT, renal replacement therapy; T HSD7A, thrombospondin type-1 domain containing 7A.

Establishment of Galectin-3 Time-resolved Fluoroimmunoassay and its Application in Idiopathic Membranous Nephropathy.

The aim of this study was to establish a time-resolved fluorescent immunoassay (TRFIA) for the detection of serum Galectin-3 (Gal-3) and apply this method to evaluate the clinical significance of serum Gal-3 in predicting Idiopathic Membranous Nephropathy (IMN) progression. The Gal-3-TRFIA was established using the double antibody sandwich method, with the capture antibodies coated on a 96-well microplate and the detection antibodies chelated with Europium (III) (Eu3+). Serum Gal-3 was detected in 81 patients with IMN and 123 healthy controls to further evaluate the value of the Gal-3 in staging of IMN. The sensitivity of the Gal-3-TRFIA assay was 0.85 ng/mL, and the detection range was 0.85-1000 ng/mL. The Gal-3 intra-batch and inter-batch coefficients of variation were 3.45% and 5.12%, respectively. The correlation coefficient (R) between the Gal-3-TRFIA assay and commercially available enzyme-linked immunosorbent assay kits was 0.83. The serum Gal-3 concentration was higher in patients with IMN (65.57 ± 55.90 ng/mL) compared to healthy controls (16.29 ± 9.91 ng/mL, P < 0.0001). In this study, a wide detection range Gal-3-TRFIA assay was developed using lanthanide (Eu3+) chelates for the detection of Gal-3 concentrations in serum. Gal-3 concentration is elevated in patients with IMN.

Clinical diagnostic performance of common laboratory indicators in primary glomerulonephritis.

OBJECTIVE: Immunoglobulin A nephropathy (IgAN) and membranous nephropathy (MN) are common types of primary glomerulonephritis (PGD). A lack of specific clinical features makes diagnosis difficult. Kidney function indicators have been used for their diagnosis. However, the diagnostic performance of these indicators is undetermined. The purpose of this paper is to evaluate their diagnostic potential. PATIENTS AND METHODS: 101 patients with PGD were enrolled, including 50 with MN and 51 with IgAN. The healthy controls included 110 volunteers. The indicators related to kidney function, including TP, ALB, Cre, CysC, eGFR, C1q, Ure, Anti-PLA2R, complement C3, and complement C4 in serum, ACR in urine, and antinuclear antibody profile, IgG staining, IgA staining, IgM staining, C3 staining and C1q staining in tissue samples were evaluated. RESULTS: Statistical differences were found in TP, ALB, Ure, CysC, eGFR, C1q, Anti-PLA2R, complement C3, complement C4 and ACR among the three groups of subjects. ROC analysis showed that Anti-PLA2R and ACR had the highest specificity for identifying IgAN and/or MN from the healthy controls, ACR had the highest sensitivity. The Sp and Se of IgA and IgG in tissue samples for the identification of IgAN and MN were both high. Both IgAN and MN were predicted by anti-PLA2R, especially MN. In tissue samples, MN patients were more likely to be IgG positive and IgAN patients were more likely to be IgA positive. CONCLUSIONS: IgAN and MN may be differentiated using serum Anti-PLA2R, tissue IgG, and tissue IgA. Cre is only useful in middle and late stages of GPDs, ACR is an exclusion marker, and CysC and C1q cannot be used to identify MN.

Peptide GAM immunoadsorption in anti-PLA2 R positive autoimmune membranous nephropathy. The PRISM trial.

Membranous nephropathy associated with anti-PLA2 R autoantibody is a significant cause of nephrotic syndrome worldwide. Treatment remains empiric with a significant side-effect burden despite an increase in our understanding of the disease. We studied the effect of selectively removing this pathogenic autoantibody using immunoadsorption in adult patients with biopsy proven anti-PLA2 R membranous nephropathy. This was a multicenter, single-arm prospective clinical trial carried out in the United Kingdom. Twelve patients underwent five consecutive sessions of peptide GAM immunoadsorption with 12 months follow-up. Primary outcome was anti-PLA2 R titer at week 2. Secondary outcomes were safety and tolerability of therapy, antibody profile, and change in proteinuria, renal excretory function, serum albumin, total immunoglobulin, and quality of life at weeks 12, 24, and 52. Patients were also stratified by the presence or absence of the high-risk allele (heterozygous or homozygous for HLA-DQA1*05). Median pretreatment anti-PLA2 R was 702.50 U/mL, 1045.00 U/mL at week 2 (P-value .023) and 165.00 U/mL at week 52 (P-value .017). The treatment was well tolerated and safe. Two patients required rescue immunosuppression during the follow-up period. There was a significant improvement in serum albumin with a median at baseline of 20.50 g/L rising to 25.00 g/L at week 52 (P-value <.001). There was no statistical difference over the follow-up period in proteinuria or renal function. Patients in possession of a high-risk allele saw improvement in anti-PLA2 R titers, possibly representing a cohort more likely to benefit from immunoadsorption. Immunoadsorption therapy is a safe treatment and well-tolerated treatment in anti-PLA2 R positive autoimmune membranous nephropathy.

Rituximab Immunomonitoring Predicts Remission in Membranous Nephropathy.

Primary membranous nephropathy (pMN) is an autoimmune kidney disease and a common cause of nephrotic syndrome in adults. Rituximab is becoming a first line therapy for patients with persistent nephrotic syndrome with proven safety and efficacy, achieving remission in 60%-80% of cases. For the remaining 20%-40% of patients there is an urgent need to identify early biomarkers of resistance to rituximab to adapt therapeutic management. In nephrotic patients, rituximab is found in the blood more transiently than in other autoimmune diseases without proteinuria, due to rituximab wasting in the urine. However, rituximab immunomonitoring is not routinely performed. We evaluated the predictive value of serum rituximab levels in patients with pMN three months after rituximab injection (month-3) on clinical remission rates six months (month-6) and 12 months (month-12) after injection and investigated predictive factors for serum rituximab levels at month-3. Sixty-eight patients treated with rituximab between July 2015 and January 2020 from two French nephrology centers were included. We identified residual rituximab levels at month-3 as a novel early predictor of remission at month-6 (p <0.0001) and month-12 (p = 0.001). Reduced likelihood of remission in patients with undetectable rituximab at month-3 was associated with lower serum albumin and higher anti-PLA2R1 titers at baseline and with lower serum albumin, higher proteinuria, higher CD19+ counts and higher anti-PLA2R1 titers during follow-up. In multivariate analysis, high baseline proteinuria and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-6 and high baseline weight and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-12. We identified serum albumin at baseline as a predictive factor for serum rituximab levels at month-3. Patients with serum albumin below 22.5 g/L at baseline had an 8.66-fold higher risk of having undetectable rituximab levels at month-3. Therefore, rituximab immunomonitoring in pMN patients treated with rituximab would allow the detection of patients at risk of treatment failure as early as month-3. Studies are needed to assess whether patients with low residual rituximab levels at month-3 may benefit from an early additional course of rituximab.

Characteristics of Anti-Contactin1 Antibody-Associated Autoimmune Nodopathies With Concomitant Membranous Nephropathy.

Background: The concurrence of anti-contactin 1 (CNTN1) antibody-associated chronic inflammatory demyelinating polyneuropathy (CIDP) and membranous nephropathy (MN) has previously been reported in the literature. CIDP with autoantibodies against paranodal proteins are defined as autoimmune nodopathies (AN) in the latest research. In view of the unclear relationship between CIDP and MN, we performed a case study and literature review to investigate the clinical characteristics of anti-CNTN antibody-associated AN with MN. Methods: We detected antibodies against NF155, NF186, CNTN1, CNTN2, CASPR1 and PLA2R in blood samples of a patient with clinically manifested MN and concomitant peripheral neuropathy via double immunofluorescence staining and conducted a quantitative measurement of anti-PLA2R IgG antibodies via enzyme-linked immunosorbent assay (ELISA). Case reports of anti-CNTN1 antibody-associated AN, anti-CNTN1 antibody-associated AN with MN, and CIDP with MN were retrieved through a literature search for a comparative analysis of clinical characteristics. The cases were grouped according to the chronological order of CIDP and MN onset for the comparison of clinical characteristics. Results: A 57-year-old man with anti-PLA2R positive MN was admitted to the hospital due to limb numbness, weakness, and proprioceptive sensory disorder. He was diagnosed with anti-CNTN1 antibody-associated AN and recovered well after immunotherapy. Our literature search returned 22 cases of CIDP with MN that occurred before, after, or concurrently with CIDP. Good responses were achieved with early single-agent or combination immunotherapy, but eight out of the 22 patients with CIDP and concomitant MN ultimately developed different motor sequelae. Five patients had anti-CNTN1 antibody-associated AN with MN. Among these patients, males accounted for the majority of cases (male:female=4:1), the mean age at onset was late (60.2 ± 15.7 years, range 43-78 years), and 40% had acute to subacute onset. Clinical manifestations included sensory-motor neuropathy, sensory ataxia caused by proprioceptive impairment, and elevated cerebrospinal fluid protein levels. Conclusion: The age at onset of CIDP with MN was earlier than that of anti-CNTN1 antibody-associated AN. MN may occur before, after or concurrently with CIDP. The early detection and isotyping of anti-CNTN1 and anti-PLA2R antibodies and the monitoring of isotype switching may be essential for suspected CIDP patients.

Analysis of Glomerular IgG Subclasses Switch in Idiopathic Membranous Nephropathy Classified by Glomerular Phospholipase A2 Receptor Antigen and Serum Antibody.

BACKGROUND: The role of IgG subclass in idiopathic membranous nephropathy (IMN) was unclarified. Recent study found IgG subtype switches from IgG1 to IgG4 in the early pathological stage in IMN. The profile of IgG subclass in phospholipase A2 receptor- (PLA2R-) related and PLA2R-unrelated IMN was unrevealed. Our study is aimed at testifying whether IgG subclass switch existed in PLA2R-related and PLA2R-unrelated IMN, respectively. METHODS: Our study retrospectively enrolled 157 Chinese patients with biopsy-confirmed IMN between September 2017 and November 2019. We measured glomerular PLA2R antigen and serum anti-PLA2R antibody to classify the patients into PLA2R-related (n = 132) and PLA2R-unrelated (n = 25) subgroup. We evaluated glomerular IgG subclass by immunofluorescence (IF) predominance. Our study defined IgG subclass deposition as predominant if the IF score was higher than the other three and ≥1 +, or as codominant if the IF intensity was equal to any other and ≥1 +. We explored the relationship between IF predominance of glomerular IgG subtype and electron microscopic (EM) stages of IMN. RESULTS: We did not find statistical difference of predominant or codominant rate (pre/co-rate) among EM stages in any subclass (P > 0.05). Pre/co-rate of IgG3 linearly associated with EM stage in total and PLA2R-related subgroup (P = 0.044, P = 0.013). PLA2R-related subgroup showed higher IgG4 intensity (2.1 ± 0.6 vs. 1.6 ± 0.7, P = 0.001) and pre/co-rate of IgG4 in stage 1 (97% vs. 57%, P = 0.015) than PLA2R-unrelated group. We found no difference of IgG subclass pre/co-rate in different EM stages or linear association between pre/co-rate of IgG1, IgG2, IgG4, and EM stages (P > 0.05). CONCLUSIONS: Pre/co-rate of IgG3 declined with EM stage in total and PLA2R-related subgroup. We did not find IgG subclass switches from IgG1 to IgG4 in either IMN patients or subgroups.

Utility of non-HDL-C in predicting proteinuria remission of idiopathic membranous nephropathy: a retrospective cohort study.

BACKGROUND: Idiopathic membranous nephropathy (IMN) may have various clinical outcomes. Hyperlipidemia is quite common in IMN. However, the utility of the lipid profile in predicting outcomes remains unknown. This study aimed to explore the correlation between hyperlipidemia and proteinuria remission in IMN. METHODS: 256 patients who diagnosed with IMN confirmed by renal biopsy in Wuhan Tongji Hospital from January 2016 to October 2020 were included in this study. The end point was defined as a combination of partial and complete remission. Cox proportional-hazards regression analysis and Kaplan-Meier curve were applied to assess the prognostic value of the lipid profile for proteinuria remission. RESULTS: A total of 153 (59.8%) patients achieved remission and 103 (40.2%) did not. The levels of total cholesterol, low-density lipoprotein, and non-high-density lipoprotein were significantly lower in the remission group than in the non-remission group. Non-high-density lipoprotein level revealed the strongest correlation with proteinuria (Spearman's rho = 0.42; P < 0.001). The multivariate analysis demonstrated that serum total cholesterol [hazard ratio (HR): 0.883; 95% confidence interval (CI): 0.813-0.958; P = 0.003] and non-high-density lipoprotein cholesterol (HR: 0.892; 95% CI: 0.820-0.970; P = 0.007) levels were independent markers to predict proteinuria remission in IMN. CONCLUSIONS: Among the lipid profile, the non-high-density lipoprotein level exhibited the strongest correlation with proteinuria in IMN. Moreover, elevated serum cholesterol and non-high-density lipoprotein cholesterol concentrations at baseline predicted probability of proteinuria non-remission in IMN.

A Novel Insight into the Role of PLA2R and THSD7A in Membranous Nephropathy.

Membranous nephropathy (MN) is an organ-restricted autoimmune disease mainly caused by circulating autoantibodies against podocyte antigens, including the M-type phospholipase A2 receptor (PLA2R) and thrombospondin domain-containing 7A (THSD7A). Antibodies against PLA2R are present in 70%-80% and against THSD7A in 2% of adult patients, which provides a paradigm shift in molecular diagnosis and management monitoring. Both antigens share some similar characteristics: they are expressed by podocytes and have wide tissue distributions; they are bound by autoantibodies only under nonreducing conditions, and the subtype of most autoantibodies is IgG4. However, the factors triggering autoantibody production as well as the association among air pollution, malignancy, and the pathogenesis of MN remain unclear. In this review, we discuss the similarity between the pathological mechanisms triggered by disparate antigens and their associated diseases. Furthermore, we demonstrated the possibility that PM2.5, malignancy, and gene expression specifically induce exposure of these antigens through conformational changes, molecular mimicry, or increased expression eliciting autoimmune responses. Thus, this review provides novel insights into the pathological mechanism of MN.

Characterization of THSD7A-antibodies not binding to glomerular THSD7A in a patient with diabetes mellitus but no membranous nephropathy.

Membranous nephropathy (MN) is an autoimmune disease caused by autoantibodies against the podocyte antigens phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type 1 domain containing protein 7A (THSD7A) in 80% and 2-3% of patients, respectively. THSD7A antibodies are considered to be pathogenic and highly specific for MN patients. Using an indirect immunofluorescence test (IIFT) we detected THSD7A-antibodies (titre 1:10) in the serum of a patient with high proteinuria who, however, in the kidney biopsy was diagnosed with diabetic nephropathy and MN was excluded as a possible cause of proteinuria. Different immunofluorescence assays and Western blot techniques using recombinant THSD7A (rTHSD7A) or THSD7A from different human tissues revealed that the circulating THSD7A-autoantibodies were only of the IgG3 subclass. The patient serum reacted exclusively with rTHSD7A and only when the antigen was present in reducing Western blot conditions, or on formaldehyde-fixed cells for the IIFT. Our findings show for the first time the existence of circulating THSD7A-antibodies recognizing denatured/reduced rTHSD7A, which do not react with glomerular THSD7A in vivo and are thus presumptively non-pathogenic. As a consequence, kidney biopsy or Western blot analyses of THSD7A under non-reducing conditions should be performed to confirm the diagnosis of THSD7A-associated MN, especially in cases with low THSD7A-antibody levels in the IIFT.