Role of phospholipase A2 receptor 1 antibody level at diagnosis for long-term renal outcome in membranous nephropathy

Summary

Background

Membranous nephropathy (MN) is an autoimmune disease induced by circulating antibodies against the podocyte protein phospholipase A₂ receptor 1 (PLA₂R1-ab) in 80% of patients and represents the leading cause of nephrotic syndrome in adults. PLA₂R1-ab levels correlate with disease activity and treatment response. However, their predictive role for long-term renal outcome is not clear.

Methods

The aim of this prospective observational multicenter study was to investigate the predictive role of PLA₂R1-ab levels at the time of diagnosis for long-term outcome in a cohort of 243 patients with newly diagnosed biopsy-proven PLA₂R1-associated MN. Statistical analyses included Cox proportional hazard models. The primary study endpoint was defined prior to data collection as doubling of serum creatinine or development of end-stage renal disease.

Results

During the median follow-up time of 48 months, 36 (15%) patients reached the study endpoint. Independent predictors for reaching the study endpoint were baseline PLA₂R1-ab levels (HR = 1.36, 95%CI 1.11–1.66, p = 0.01), percentage of tubular atrophy and interstitial fibrosis (HR = 1.32, 95%CI 1.03–1.68, p = 0.03), PLA₂R1-ab relapse during follow-up (HR = 3.22, 95%CI 1.36–7.60, p = 0.01), and relapse of proteinuria (HR = 2.60, 95%CI 1.17–5.79, p = 0.02). Fifty-four (22%) patients received no immunosuppressive treatment during the study, in 41 (76%) of them PLA₂R1-ab spontaneously disappeared during follow-up, 29 (54%) patients had a complete remission of proteinuria, and 19 (35%) had a partial remission. Patients not treated with immunosuppression were more often females and had lower PLA₂R1-ab levels, proteinuria, and serum creatinine at baseline compared to patients receiving immunosuppression. However, no conclusion on the efficacy of immunosuppressive therapies can be made, since this was not a randomized controlled study and treatment decisions were not made per-protocol.

Conclusions

PLA₂R1-ab levels are, in addition to pre-existing renal damage, predictive factors for long-term outcome and should therefore be considered when deciding the treatment of patients with MN.

Keywords:

Biology and life sciences – Biochemistry – Physical sciences – Research and analysis methods – Mathematics – Developmental biology – Medicine and health sciences – Pathology and laboratory medicine – Diagnostic medicine – Signs and symptoms – pharmacology – statistics – Mathematical and statistical techniques – Statistical methods – immunology – Pharmaceutics – Drug therapy – Drugs – biomarkers – fibrosis – Creatinine – Proteinuria – Immune suppression – Immunosuppressives – Cyclophosphamide – Regression analysis

Introduction

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. In 80% of the patients the disease is caused by binding of circulating antibodies to the phospholipase A₂ receptor 1 (PLA₂R1), which is expressed on the surface of podocytes and is the major target antigen [1]. Detection of PLA₂R1 antibodies (PLA₂R1-ab) allows the diagnosis of MN [2–5]. Moreover, PLA₂R1-ab levels are associated with treatment response, remission of proteinuria, relapse of proteinuria, and recurrence of disease after renal transplantation [4, 6–8]. A number of studies have shown that a decrease of PLA₂R1-ab precedes clinical remission (i.e. remission of proteinuria) after immunosuppressive treatment [6,9,10]. Furthermore, patients with detectable PLA₂R1-ab have a higher risk for relapse of proteinuria [11, 12]. Therefore, measurement of PLA₂R1-ab is helpful for the management of patients with MN [13] but the significance of PLA₂R1-ab levels for renal endpoints, such as doubling of serum creatinine or development of end-stage renal disease, is not clear because of the retrospective character of the available studies or the short-term follow-up of the patients [14, 15]. While complete remission of proteinuria is considered a surrogate parameter for long-term renal outcome [16], a better understanding of the clinical relevance of PLA₂R1-ab for long-term prognosis would substantially improve treatment decisions and risk stratification of patients with MN. In order to better define the role of PLA₂R1-ab levels at the time of diagnosis on long-term renal outcome we conducted a multicenter open prospective observational study in a large cohort of patients with newly diagnosed biopsy-proven PLA₂R1-associated MN.

Material and methods

Patient cohort and study design

Starting from January 2010 all patients with a biopsy-proven diagnosis of MN who fulfilled the study inclusion criteria and provided informed consent to participate in the study were screened for circulating PLA₂R1-ab. Study enrolment and the first measurement of PLA₂R1-ab had to be performed within six months of renal biopsy. Treatment with immunosuppressive agents prior to study start was not allowed. Follow-up visits were performed every three months. PLA₂R1-ab levels, proteinuria, and serum creatinine were measured at every study visit and data were recorded prospectively after each visit. Treatment decisions in enrolled patients were not made per protocol, but by the treating nephrologists, who decided on the therapeutic strategy based on patient characteristics (i.e. proteinuria, nephrotic syndrome, renal function, etc.) and their clinical experience. The primary study endpoint was defined as doubling of serum creatinine in relation to the time of study inclusion or development of end-stage renal disease, whichever occurred earlier. Depletion of PLA₂R1-ab was defined as PLA₂R1-ab falling below 14 U/ml. Remission of proteinuria was defined as proteinuria < 3.5 g/24h and at least 50% reduction from the time of study inclusion. Complete remission of proteinuria was defined as proteinuria < 0.5 g/24h. Relapse of PLA₂R1-ab was defined as PLA₂R1-ab increasing from < 14 U/ml to a level higher than 20 U/ml. Relapse of proteinuria was defined as proteinuria > 3.5 g/24h and at least doubling of proteinuria compared to the lowest value during the period of remission.

Glomerular disease stages were assessed by electron microscopy according to Ehrenreich and Churg [17]. At study inclusion PLA₂R1-ab measurement was performed by ELISA, indirect immunofluorescence (IFT) and Western blot, as described before [18]. During follow-up the PLA₂R1-ab was measured by ELISA [19]. The study was approved by the local ethics committee of the chamber of physicians in Hamburg and conducted in accordance with the ethical principles stated by the Declaration of Helsinki. Informed consent was obtained from all participating patients.

Statistical analyses

Descriptive analyses of continuous data are presented as median and 1^st and 3^rd quartile unless stated otherwise. For categorical data, absolute counts and percentages are reported. Mann-Whitney U and Kruskal-Wallis tests were employed for comparisons of continuous variables while Fisher’s exact tests were used for group-wise comparisons of categorical variables. Multivariate relationships among variables were explored by nonlinear categorical principal component analysis [20, 21]. A two-dimensional solution was chosen and the component loadings of the variables are graphically presented.

Uni- and multivariate Cox regression analyses were used to assess the effect of independent variables on the time to event for the study endpoint. Time-dependent covariates were computed and used to test the proportional hazards assumption for individual independent variables. Multivariate Cox regression analyses started with an initial model containing only additive terms of independent variables and their corresponding time-dependent terms if either one, the additive, or the time-dependent term, had displayed a significant effect in the univariate analyses. Non-significant terms were stepwise eliminated from the model using a hierarchical backward approach [22]. Results of Cox regression analyses are presented as hazard ratios with corresponding 95% confidence intervals and p-values. Right-skewed covariates were log₂-transformed to reduce the over-proportional impact of extremely high data values on computations of hazard ratios while at the same time trying to ensure linear relationships to outcomes. These relationships were evaluated via visual examinations of scatter plots of martingale residuals of Cox regressions not containing any covariates in the model versus the respective covariate of interest.

Detailed description of the statistical analyses for the primary study endpoint as well as for the secondary endpoints depletion of PLA₂R1-ab, relapse of PLA₂R1-ab, remission of proteinuria, and relapse of proteinuria are included in the supplemental material. Statistical significance was defined as p<0.05. All tests were two-tailed. All statistical analyses were done using SPSS version 25.0 (IBM, Armonk, New York).

Immunohistochemical staining of renal biopsies for PLA₂R1

For PLA₂R1 immunohistochemical analyses slides were deparaffinized, pre-treated in citrate buffer (pH 6.2) for 15 min at 120°C and cooled down in iced water (10 min). After rinsing in 99% ethanol, slides were incubated for 10 min with normal serum (Vector S2000; VectorLaboratories, Burlingame, CA) followed by PLA₂R1-antibodies (polyclonal antibody from rabbit, 1:3000, HPA 012657, Sigma-Aldrich, St. Louis, MO) overnight at 4°C. The slides were then washed in PBS, incubated with polymer 1 (Zytomed Zytochem-Plus AP Polymer-KitPOLAP), rinsed in PBS and incubated with polymer 2 (Zytomed Zytochem-Plus AP Polymer-Kit POLAP). After washing in PBS, slides were stained in new fuchsin naphthol As-Bi phosphate substrate mixture (30 min) followed by 1 min of nuclear staining in hemalaun (Mayer).

Results

Clinical baseline characteristics

A total of 312 consecutive patients with biopsy-proven MN were tested for the presence of PLA₂R1-ab in the serum. At study inclusion PLA₂R1-ab were detectable by ELISA and IFT in 222 patients. In addition, 21 patients were tested positive for PLA₂R1-ab by IFT, but not in the ELISA. These 21 sera were additionally analysed by Western blot and all were positive for PLA₂R1-ab, confirming the results of the IFT. For 152 patients the renal biopsy was stained for PLA₂R1 and confirmed the diagnosis of PLA₂R1-associated MN in all cases. Sixty-nine patients were tested negative for PLA₂R1-ab by ELISA, IFT, and Western blot and were not included in the study. In five of these patients renal biopsy showed an enhanced staining for PLA₂R1. Taken together, 243 consecutive patients with the histologic diagnosis of MN and positive PLA₂R1-ab in the serum were included in this prospective multicenter study. The median follow-up time was 48.0 months (1^st to 3^rd quartile: 27.0 to 63.0 months), resulting in a cumulative follow-up time of 916.7 patient-years. The clinical baseline characteristics of the study cohort are presented in Table 1. After dividing the study cohort in tertiles according to the PLA₂R1-ab level at baseline, patients with the highest antibody levels were older, had higher proteinuria and more tubulointerstitial fibrosis, although the absolute difference in the percentage of tubulointerstitial fibrosis between the groups was small. At baseline 63 (26%) patients had an impaired renal function defined as eGFR < 60 ml/min/1.73m² (S1 Table). In addition to having more severe renal damage (serum creatinine, GFR, tubular atrophy and interstitial fibrosis) these patients were also significantly older and had higher proteinuria compared to patients with preserved renal function (eGFR > 60 ml/min/1.73m²) at baseline. PLA₂R1-ab levels were not significantly different between the two groups.

The component loadings computed by nonlinear categorical principal component analysis to examine relationships among clinical parameters at baseline reveal positive correlations among serum creatinine levels at baseline, extent of tubular atrophy and interstitial fibrosis, and age of patients while these clinical parameters show a strong negative correlation with eGFR. PLA₂R1-ab appears unrelated to any of these clinical baseline parameters (S1 Fig).

Clinical variables associated with the primary study endpoint

Thirty-six (15%) patients reached the study endpoint defined as doubling of serum creatinine or development of end-stage renal disease. These patients reached the study endpoint after a median follow-up time of 18.0 months (1^st to 3^rd quartile: 12.0 to 42.0 months), 14 (5.8%) of these patients developed end-stage renal disease. Results of univariate Cox regression analyses testing all independent variables and their time-dependent terms individually are provided in S2 Table. In the multivariate Cox regression analysis higher PLA₂R1-ab levels at baseline significantly increased the risk for reaching the study endpoint (log₂[PLA₂R1-ab levels]: HR = 1.36, 95%CI 1.11–1.66, p = 0.01, Fig 1). Of all other baseline clinical parameters, only the percentage of tubular atrophy and interstitial fibrosis was a statistically significant risk factor (log₂[tubular atrophy and interstitial fibrosis] HR = 1.32, 95%CI 1.03–1.68, p = 0.03). After analyzing all variables for time-varying effects during follow-up a significant time-dependent change of the variable effect was found for serum creatinine, showing that the variable effect of serum creatinine for the study endpoint significantly increases during the follow-up time. In this multivariate Cox regression analysis we also included parameters associated with disease progression and treatment response during follow-up and found that relapse of PLA₂R1-ab during follow-up and a relapse of proteinuria significantly increased the risk for reaching the study endpoint (HR = 3.22, 95%CI 1.36–7.60, p = 0.01 and HR = 2.60 95%CI 1.17–5.79, p = 0.02, respectively).

Patients who reached the study endpoint had significantly higher PLA₂R1-ab, higher serum creatinine levels, lower eGFR, more extended tubular atrophy and interstitial fibrosis at baseline, while during follow-up they more often failed to deplete PLA₂R1-ab and had significantly less often a complete remission of proteinuria compared to patients who did not reach the study endpoint (Table 2).

Secondary study endpoints

Since relapse of PLA₂R1-ab and proteinuria during follow-up significantly increased the risk for reaching the primary study endpoint, we analysed in a next step which clinical parameters might influence the outcome of PLA₂R1-ab and proteinuria during follow-up (supplemental methods). We first performed a univariate Cox regression analysis to identify clinical variables potentially linked to depletion of PLA₂R1-ab (S3 Table), relapse of PLA₂R1-ab (S4 Table), remission of proteinuria (S5 Table), and relapse of proteinuria (S6 Table). In a second step we performed multivariate Cox regression analyses and identified PLA₂R1-ab levels at baseline to be a significant predictor of depletion of PLA₂R1-ab, remission of proteinuria, and relapse of proteinuria during follow-up (log₂[PLA₂R1-ab levels]: HR = 0.71, 95%CI 0.63–0.79, p<0.001; HR = 0.92, 95%CI 0.86–0.99, p = 0.02; and HR = 1.15, 95%CI 1.03–1.28, p = 0.01, respectively; S2 and S3 Figs). PLA₂R1-ab levels at baseline were also predictive for relapse of PLA₂R1-ab during follow-up in the univariate analysis, however, in the multivariate analysis this association was not statistically significant (log₂[PLA₂R1-ab levels]: HR 1.12, 95%CI 0.99–1.26, p = 0.08). The only other parameter associated with depletion of PLA₂R1-ab in the multivariate analysis was use of immunosuppressive treatment (HR 4.15, 95%CI 2.84–6.06, p<0.001; S2 Fig). We also found a significant time-dependent change of the variable effect for PLA₂R1-ab and age, showing that the effect of these variables for the endpoint significantly changes during the follow-up time.

In addition to the PLA₂R1-ab levels at baseline, depletion of PLA₂R1-ab, and proteinuria at baseline were also significant risk factors for remission of proteinuria (HR = 2.56, 95%CI 1.81–3.61, p<0.001 and log₂[proteinuria]: HR = 0.65, 95%CI 0.50–0.83, p<0.001, respectively; S3A Fig). Moreover, proteinuria and serum creatinine showed a significant time-dependent change of the variable effect during the follow-up time. In addition to PLA₂R1-ab levels, serum creatinine at baseline, relapse of PLA₂R1-ab, and partial remission of proteinuria compared to complete remission significantly increased the risk for a relapse of proteinuria (log₂[serum creatinine]: HR = 1.77, 95%CI 1.24–2.52, p = 0.01; HR = 3.06, 95%CI 1.81–5.16, p<0.001; and HR = 10.00, 95%CI 5.97–16.76, p<0.001, respectively; S3B Fig).

Patients treated with immunosuppression or supportive care only

During the study, 189 (78%) patients were treated with immunosuppressive agents in addition to supportive medication, while 54 (22%) patients received supportive medication only (S7 Table). Patients treated with immunosuppression were significantly more often male (p = 0.03), had higher PLA₂R1-ab levels (p<0.001), higher proteinuria (p<0.001), higher serum creatinine at baseline (p = 0.01), and more often a relapse of PLA₂R1-ab during follow-up (p = 0.04) compared to the 54 patients who were treated with supportive care only. However, the differences in PLA₂R1-ab levels and proteinuria between patients treated with immunosuppression and patients receiving only supportive medication diminished during follow-up, most probably as an effect of the started immunosuppressive therapy, and were no longer significant after six months and nine months, respectively (Fig 2). Patients treated with immunosuppressants had higher serum creatinine levels compared to patients treated with supportive medication only and this difference persisted throughout the study follow-up.

Natural course and outcome of PLA₂R1-associated MN

We further analysed the 54 patients who were treated with supportive care only, in order to better understand the natural course of disease, when no immunosuppressive treatment is given. Forty-one (76%) of these patients spontaneously reduced their PLA₂R1-ab levels during follow-up (S8 Table). The only statistically significant difference between the two patient cohorts at baseline were the PLA₂R1-ab levels. Patients with spontaneous reduction of PLA₂R1-ab levels had significantly more often a remission of proteinuria (98% of patients, 71% complete remission and 27% partial remission) compared to patients with persistent PLA₂R1-ab (62% of patients, 0% complete remission and 62% partial remission). Patients with spontaneous reduction of PLA₂R1-ab reached less often the study endpoint, however, this difference did not reach statistical significance (5% versus 23%, p = 0.08).

Effect of individual immunosuppressive treatments

The initial immunosuppressive agent chosen in most patients was cyclosporine A (81 patients, in 66 of them combined with steroids), followed by oral cyclophosphamide (39 patients, in 38 of them combined with steroids), intravenous (iv) cyclophosphamide (35 patients, in 28 of them combined with steroids), and rituximab (19 patients, in six of them combined with steroids). Immunosuppressive treatment was started at 3.0 months (1^st to 3^rd quartile: 0.0 to 6.0 months) after inclusion in the study. Proteinuria, renal function, and PLA₂R1-ab levels were not significantly different between study inclusion and start of treatment (S9 and S10 Tables).

In some of the patients, data on PLA₂R1-ab, proteinuria, and serum creatinine were collected at a short time after start of immunosuppressive treatment, namely one week and four weeks (Fig 3). We noticed a decrease of PLA₂R1-ab levels already after one week in all treatment groups, which was most pronounced in patients treated with oral cyclophosphamide (86%), followed by rituximab (47%), iv cyclophosphamide (40%), and cyclosporine A (18%) (Fig 3A). After four weeks and three months the PLA₂R1-ab levels had decreased in all groups by 74%– 94% and 85%– 97%, respectively (Fig 3B and 3C). After one week, proteinuria was only reduced in patients treated with cyclosporine A (26%), and rituximab (5%), but not in patients treated with cyclophosphamide (Fig 3D). After four weeks, proteinuria fell by 40% in all groups, except for patients treated with rituximab (Fig 3E). Only after three months a decrease of proteinuria by 25%– 54% was observed in all groups (Fig 3F). Serum creatinine decreased after one week in patients treated with rituximab (7%), but increased by 5%, 14%, and 17% in patients treated with oral cyclophosphamide, cyclosporine A, and iv cyclophosphamide, respectively (Fig 3G). After four weeks, serum creatinine fell by 6%– 12% in all groups, except patients treated with cyclosporine A, in whom serum creatinine increased by 18% (Fig 3H). This pattern was also observed at three months, when a decrease of serum creatinine by 6%– 19% was observed in all groups, except patients treated with cyclosporine A, in whom serum creatinine increased by 18% (Fig 3I). In 43 patients treated with cyclosporine A, we were able to analyse data on serum creatinine at the time when cyclosporine A was stopped and within 3 months after cessation of treatment with cyclosporine A. Within this short time period (median 2.0 months, 1^st to 3^rd quartile: 1.0 to 3.0 months) serum creatinine decreased by 9.1% in these patients.

In every treatment group in almost half of the patients the initial immunosuppressive treatment was changed from one medication to another (42% of patients treated with rituximab, 46% of patients treated with oral cyclophosphamide, 52% of patients treated with cyclosporine A and 57% of patients treated with iv cyclophosphamide). However, no statistically significant differences were observed between the baseline clinical characteristics and outcome parameters of patients who needed a second line immunosuppressive treatment and patients who did not need a second line immunosuppressive treatment (S11 Table).

Discussion

The identification of PLA₂R1 as the major target antigen in MN has led to significant improvements in the diagnosis and treatment of MN [1, 13]. The role of PLA₂R1-ab as a biomarker for disease activity and treatment response has been shown in several studies, but almost all of these studies had a retrospective design and were built on short-term surrogate endpoints like proteinuria [4, 6, 8, 11, 12, 23].The identification of biomarkers for hard renal endpoints in patients with MN is important not only for treatment strategies, but also for the design of future therapeutic studies. Some studies analysing the role of PLA₂R1-ab for the outcome of renal function only had a retrospective design, short term follow-up, and the renal endpoint was defined as a rather small increase in renal retention parameters [14, 15].

This is the first prospective study to investigate the predictive role of PLA₂R1-ab for long-term clinical outcome, i.e. doubling of serum creatinine in a large cohort of patients with newly diagnosed PLA₂R1-associated MN. High PLA₂R1-ab levels at baseline were found to be a significant risk factor for doubling of serum creatinine. The hazard ratio of 1.36 per 2-fold-increase of PLA₂R1-ab levels underscores their relevance for the clinical outcome of patients with MN. At baseline, only parameters indicating renal damage, e.g. tubular atrophy and interstitial fibrosis, were found to be a risk factor for reaching the study endpoint in addition to the PLA₂R1-ab level. The IFT was more sensitive than the ELISA for detection of PLA₂R1-ab, as we have shown earlier [2, 6]. At the same time, PLA₂R1-ab levels were not closely related to any of the baseline clinical characteristics as shown in the nonlinear categorical principal component analysis which indicated that serum creatinine, tubular atrophy and interstitial fibrosis, and age of patients were sharing close relations to a common dimension. Our data also confirmed the relevance of clinical parameters associated with disease progression and treatment response during follow-up. Relapse of PLA₂R1-ab and proteinuria during follow-up were significant factors for loss of renal function. Importantly, high PLA₂R1-ab levels at baseline were identified as a risk factor for almost every parameter of disease activity during follow-up (depletion of PLA₂R1-ab, remission of proteinuria, and relapse of proteinuria), in addition to the study endpoint. Moreover, depletion of PLA₂R1-ab was predictive for remission of proteinuria, while relapse of PLA₂R1-ab was predictive for relapse of proteinuria. As has been shown before [16], patients with a partial remission of proteinuria had a much higher risk for relapse of proteinuria compared to patients with a complete remission of proteinuria (HR = 10.0).

Additionally, we had the chance to study the natural course of PLA₂R1-associated MN, since 22% of the patients were treated with supportive medication only. Since these patients had lower PLA₂R1-ab levels, proteinuria, and serum creatinine at baseline compared to patients treated with immunosuppression, they might represent a cohort of patients with low disease activity. In most of these patients PLA₂R1-ab spontaneously disappeared during follow-up. This is an important observation, since it suggests that in a considerable number of patients unknown mechanisms lead to spontaneous disappearance of PLA₂R1-ab from the circulation. A better pathophysiologic characterisation of the mechanisms responsible for this phenomenon might lead to new treatment options for MN.

In a part of the study cohort we analysed the development of PLA₂R1-ab, proteinuria, and serum creatinine within a very short time after start of immunosuppression. As we had observed in single cases of MN before [24], we found a very rapid decline of PLA₂R1-ab upon immunosuppressive treatment, in some patients within a week. After four weeks, PLA₂R1-ab levels had declined by almost 80%, while proteinuria declined at a slower rate by about 40% at four weeks, which also has been observed by others [9, 10]. An intriguing finding in our cohort was that four weeks and three months after start of immunosuppressive treatment serum creatinine declined in all treatment groups, except in patients treated with cyclosporine A, in whom serum creatinine increased by 18% at both time points. The very short latency of this effect and the fact that within three months after cessation of cyclosporine A serum creatinine decreased by 9.1% suggest that these findings represent a hemodynamic effect of cyclosporine A. Taken together, we found no significant difference in the effect of the different immunosuppressants on PLA₂R1-ab or proteinuria in our study cohort since in every treatment group immunosuppressive treatment had to be changed because of treatment failure or adverse effects in 42% - 57% of the patients. The identification of biomarkers, which may allow a prognosis on the efficacy of a specific immunosuppressive treatment in individual patients would be a significant improvement for the management of patients with MN.

Our study has a number of limitations. This was not a randomized controlled study, therefore no final conclusion can be made on the efficacy of the individual immunosuppressive treatments, especially concerning their long-term efficacy on renal function. Moreover, patients treated only with supportive medication might represent a subclass of patients with low disease activity, rather than the random MN patient in the daily clinical routine. Nonetheless, considering the good outcome of disease in these patients, their identification is an unmet need in clinical routine.

PLA₂R1-ab at baseline are an important risk factor for the long-term renal outcome of patients with MN and should therefore be embedded in the decision making and treatment management of these patients. Spontaneous disappearance of PLA₂R1-ab from the circulation is not uncommon and associated with a very good outcome. A better understanding of the immunologic mechanisms leading to this finding might lead to new treatment options for MN.

Supporting information

S1 Fig [docx]
Component loading vectors of all clinical baseline variables.

S2 Fig [docx]
Multivariate Cox regression analysis for depletion and relapse of PLAR1-ab.

S3 Fig [docx]
Multivariate Cox regression analysis for remission and relapse of proteinuria.

S1 Table [docx]
Clinical baseline characteristics and outcomes of patients with eGFR below or higher than 60 mL/min/1.73 m at baseline.

S2 Table [docx]
Univariate Cox regression analysis to identify clinical parameters predictive for the study endpoint.

S3 Table [docx]
Univariate Cox regression analysis for depletion of PLAR1-ab.

S4 Table [docx]
Univariate Cox regression analysis for relapse of PLAR1-ab.

S5 Table [docx]
Univariate Cox regression analysis for remission of proteinuria.

S6 Table [docx]
Univariate Cox regression analysis for relapse of proteinuria.

S7 Table [docx]
Baseline clinical characteristics of patients who received immunosuppression or supportive treatment only.

S8 Table [docx]
Baseline clinical characteristics of patients who only received supportive treatment.

S9 Table [docx]
Clinical characteristics at baseline and the time of treatment start for patients who received immunosuppressive therapy.

S10 Table [docx]
P-values for all differences of clinical characteristics at baseline and the time of treatment start shown in .

S11 Table [docx]
Clinical baseline characteristics and outcomes of patients who needed a second line immunosuppressive treatment and those who did not.

S1 File [docx]
Supplemental methods.