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


Authors: Maida Mahmud aff001;  Hans O. Pinnschmidt aff002;  Linda Reinhard aff001;  Sigrid Harendza aff001;  Thorsten Wiech aff003;  Rolf A. K. Stahl aff001;  Elion Hoxha aff001
Authors place of work: III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany aff001;  Institute of Medical Biometry and Epidemiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany aff002;  Division of Nephropathology, Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany aff003
Published in the journal: PLoS ONE 14(9)
Category: Research Article
doi: 10.1371/journal.pone.0221293

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:

Medicine and health sciences – Diagnostic medicine – Signs and symptoms – Proteinuria – Immune suppression – Pathology and laboratory medicine – Signs and symptoms – Proteinuria – Immune suppression – Pharmacology – Drugs – Immunosuppressives – Cyclophosphamide – Immunology – Immune suppression – Pharmaceutics – Drug therapy – Biology and life sciences – Biochemistry – Biomarkers – Creatinine – Immunology – Immune suppression – Developmental biology – Fibrosis – Research and analysis methods – Mathematical and statistical techniques – Statistical methods – Regression analysis – Physical sciences – Mathematics – Statistics – Statistical methods – 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 [25]. Moreover, PLA2R1-ab levels are associated with treatment response, remission of proteinuria, relapse of proteinuria, and recurrence of disease after renal transplantation [4, 68]. 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 1st and 3rd 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 (1st to 3rd 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.

Tab. 1.

Clinical baseline characteristics of patients with low (tertile 1), medium (tertile 2), or high (tertile 3) PLA2R1-ab level.

<h2>Clinical baseline characteristics of patients with low (tertile 1), medium (tertile 2), or high (tertile 3) PLA<sub>2</sub>R1-ab level.</h2>

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 (1st to 3rd 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).

<h2>Multivariate Cox regression analysis.</h2>
Fig. 1.

Multivariate Cox regression analysis.


PLA2R1-ab levels and the extent of tubular atrophy and interstitial fibrosis were the only variables identified as risk factors at baseline for reaching the study endpoint. Both variables were transformed to their binary logarithm prior to using them in these analyses. Relapse of PLA2R1-ab and proteinuria were the follow-up parameters found to be risk factors for reaching the study endpoint. We adjusted the analysis of all variables for time-varying effects during follow-up and found a significant time-dependent change of the variable effect for serum creatinine. However, the effect of serum creatinine for the study endpoint was not significant. 95%CI: 95% Confidence Interval; HR: hazard ratio; PLA2R1-ab: PLA2R1-antibody; DSC: doubling of serum creatinine.

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).

Tab. 2.

Clinical characteristics of patients who reached the primary study endpoint compared to patients who did not.

<h2>Clinical characteristics of patients who reached the primary study endpoint compared to patients who did not.</h2>

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.

<h2>PLA<sub>2</sub>R1-ab levels, proteinuria, and serum creatinine in patients treated with immunosuppression, or supportive medication only.</h2>
Fig. 2.

PLA2R1-ab levels, proteinuria, and serum creatinine in patients treated with immunosuppression, or supportive medication only.


PLA2R1-ab levels (A) and proteinuria (B) significantly decreased in patients treated with immunosuppression after three months (*) and between three and six months (ǁ). Serum creatinine increased in patients treated with immunosuppression after three months, but remained stable thereafter (C). At baseline patients treated with immunosuppressants had significantly higher PLA2R1-ab levels, proteinuria, and serum creatinine compared to patients who received no immunosuppression (§). The statistical significance was lost at six months for PLA2R1-ab, at nine months for proteinuria, but persisted for serum creatinine. In patients who received supportive treatment only the decrease of PLA2R1-ab levels and proteinuria reached statistical significance after nine months and six months, respectively. Time “0” shows data at the time of the first PLA2R1-ab measurement. ISupp.: Patients treated with immunosuppression. No ISupp.: patients treated with supportive medication only. “*” shows a statistically significant difference between the single time point and the start of the study (p<0.05). “ǁ” shows a statistically significant difference between the single time point and the time 3 months after study start (p<0.05). “§” shows a statistically significant difference between patients treated with immunosuppressive treatment and those who received supportive treatment only (p<0.05). PLA2R1-ab: PLA2R1 antibody.

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 (1st to 3rd 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, 1st to 3rd quartile: 1.0 to 3.0 months) serum creatinine decreased by 9.1% in these patients.

<h2>Changes in PLA<sub>2</sub>R1-ab levels, proteinuria, and serum creatinine following immunosuppressive therapy.</h2>
Fig. 3.

Changes in PLA2R1-ab levels, proteinuria, and serum creatinine following immunosuppressive therapy.


Data are presented as the relation of PLA2R1-ab (Fig 3A, 3B and 3C), proteinuria (Fig 3D, 3E and 3F) and serum creatinine (Fig 3G, 3H and 3I) at the given time point (one week: Fig 3A, 3D, 3G; four weeks: Fig 3B, 3E, 3H; three months: Fig 3C, 3F, 3I) to the time of start of immunosuppression (Time 0). Data are presented for patients treated with oral cyclophosphamide, iv cyclophosphamide, cyclosporine A, or rituximab. Time “0” shows data at the time of the start of immunosuppression. CYC: cyclophosphamide; CsA: cyclosporine A; RTX: rituximab; iv: intravenous; PLA2R1-ab: PLA2R1 antibody.

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.


Zdroje

1. Beck LH Jr, Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med. 2009;361(1):11–21. doi: 10.1056/NEJMoa0810457 19571279

2. Hoxha E, Kneißler U, Stege G, Zahner G, Thiele I, Panzer U, et al. Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy. Kidney Int. 2012;82(7):797–804. doi: 10.1038/ki.2012.209 22673885

3. Svobodova B, Honsova E, Ronco P, Tesar V, Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy. Nephrol Dial Transplant. 2013;28(7):1839–1844. doi: 10.1093/ndt/gfs439 23223223

4. Hofstra JM, Debiec H, Short CD, Pellé T, Kleta R, Mathieson PW, et al. Antiphospholipase A2 receptor antibody titer and subclass in idiopathic membranous nephropathy. J Am Soc Nephrol. 2012;23(10):1735–1743. doi: 10.1681/ASN.2012030242 22956816

5. Timmermans SA, Ayalon R, van Paassen P, Beck LH Jr, van Rie H, Wirtz JJ, et al. Anti-phospholipase A2 receptor antibodies and malignancy in membranous nephropathy. Am J Kidney Dis. 2013;62(6): 1223–1225. doi: 10.1053/j.ajkd.2013.07.019 24021909

6. Hoxha E, Thiele I, Zahner G, Panzer U, Harendza S, Stahl RA. Phospholipase A2 receptor autoantibodies and clinical outcome in patients with primary membranous nephropathy. J Am Soc Nephrol. 2014;25(6):1357–1366. doi: 10.1681/ASN.2013040430 24610926

7. Stahl R, Hoxha E, Fechner K. PLA2R autoantibodies and recurrent membranous nephropathy after transplantation. N Engl J Med. 2010;363(5):496–498. doi: 10.1056/NEJMc1003066 20818871

8. Ruggenenti P, Debiec H, Ruggiero B, Chianca A, Pellé T, Gaspari F, et al. Anti-phospholipase A2 receptor antibody titer predicts post-rituximab outcome of membranous nephropathy. J Am Soc Nephrol. 2015;26(10):2545–2558. doi: 10.1681/ASN.2014070640 25804280

9. Beck LH Jr, Fervenza FC, Beck DM, Bonegio RG, Malik FA, Erickson SB, et al. Rituximab-induced depletion of anti-PLA2R autoantibodies predicts response in membranous nephropathy. J Am Soc Nephrol. 2011;22(8):1543–1550. doi: 10.1681/ASN.2010111125 21784898

10. Dahan K, Debiec H, Plaisier E, Cachanado M, Rousseau A, Wakselman L, et al. Rituximab for Severe Membranous Nephropathy: A 6-Month Trial with Extended Follow-Up. J Am Soc Nephrol. 2017;28(1):348–358. doi: 10.1681/ASN.2016040449 27352623

11. Hoxha E, Harendza S, Pinnschmidt H, Panzer U, Stahl RA. PLA2R antibody levels and clinical outcome in patients with membranous nephropathy and non-nephrotic range proteinuria under treatment with inhibitors of the renin-angiotensin system. PLoS One. 2014;9(10):e110681. doi: 10.1371/journal.pone.0110681 25313791

12. Bech AP, Hofstra JM, Brenchley PE, Wetzels JF. Association of anti-PLA2R antibodies with outcomes after immunosuppressive therapy in idiopathic membranous nephropathy. Clin J Am Soc Nephrol. 2014;9(8):1386–1392. doi: 10.2215/CJN.10471013 25035272

13. De Vriese AS, Glassock RJ, Nath KA, Sethi S, Fervenza FC. A Proposal for a Serology-Based Approach to Membranous Nephropathy. J Am Soc Nephrol. 2017;28(2):421–430. doi: 10.1681/ASN.2016070776 27777266

14. Kanigicherla D, Gummadova J, McKenzi EA, Roberts SA, Harris S, Nikam M, et al. Anti-PLA2R antibodies measured by ELISA predict long-term outcome in a prevalent population of patients with idiopathic membranous nephropathy. Kidney Int. 2013;83(5):940–948. doi: 10.1038/ki.2012.486 23364522

15. Hoxha E, Harendza S, Pinnschmidt H, Panzer U, Stahl RA. M-type phospholipase A2 receptor autoantibodies and renal function in patients with primary membranous nephropathy. Clin J Am Soc Nephrol. 2014;9(11):1883–1890. doi: 10.2215/CJN.03850414 25267554

16. Thompson A, Cattran DC, Blank M, Nachman PH. Complete and Partial Remission as Surrogate End Points in Membranous Nephropathy. J Am Soc Nephrol. 2015;26(12):2930–2937. doi: 10.1681/ASN.2015010091 26078365

17. Ehrenreich T, Churg J. Pathology of membranous nephropathy. Path Ann. 1968;3:145–186.

18. Hoxha E, Beck LH Jr, Wiech T, Tomas NM, Probst C, Mindorf S, et al. An Indirect Immunofluorescence Method Facilitates Detection of Thrombospondin Type 1 Domain-Containing 7A-Specific Antibodies in Membranous Nephropathy. J Am Soc Nephrol. 2017;28(2):520–531. doi: 10.1681/ASN.2016010050 27436855

19. Dähnrich C, Komorowski L, Probst C, Seitz-Polski B, Esnault V, Wetzels JF, et al. Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy. Clin Chim Acta. 2013;421:213–218. doi: 10.1016/j.cca.2013.03.015 23541686

20. Linting M, Meulman JJ, Groenen PJF, van der Koojj AJ. Nonlinear principal components analysis: introduction and application. Psychol Methods. 2007;12(3):336–358. doi: 10.1037/1082-989X.12.3.336 17784798

21. Linting M, van der Kooij AJ. Nonlinear principal components analysis with CATPCA: a tutorial. J Pers Assess. 2012;94(1):12–25. doi: 10.1080/00223891.2011.627965 22176263

22. Kleinbaum DG, Klein M. Logistic regression. A Self-learning Text. 2nd ed. New York: Springer; 2002. pp. 513.

23. Wei SY, Wang YX, Li JS, Zhao SL, Diao TT, Wang Y, et al. Serum anti-PLA2R antibody predicts treatment outcome in idiopathic membranous nephropathy. Am J Nephrol. 2016;43(2):129–140. doi: 10.1159/000445361 27058841

24. Hoxha E, Harendza S, Zahner G, Panzer U, Steinmetz O, Fechner K, et al. An immunofluorescence test for phospholipase-A₂-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis. Nephrol Dial Transplant. 2011;26(8):2526–2532. doi: 10.1093/ndt/gfr247 21633097


Článek vyšel v časopise

PLOS One


2019 Číslo 9

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

Léčba bolesti v ordinaci praktického lékaře
nový kurz
Autoři: MUDr. PhDr. Zdeňka Nováková, Ph.D.

Revmatoidní artritida: včas a k cíli
Autoři: MUDr. Heřman Mann

Jistoty a nástrahy antikoagulační léčby aneb kardiolog - neurolog - farmakolog - nefrolog - právník diskutují
Autoři: doc. MUDr. Štěpán Havránek, Ph.D., prof. MUDr. Roman Herzig, Ph.D., doc. MUDr. Karel Urbánek, Ph.D., prim. MUDr. Jan Vachek, MUDr. et Mgr. Jolana Těšínová, Ph.D.

Léčba akutní pooperační bolesti
Autoři: doc. MUDr. Jiří Málek, CSc.

Nové antipsychotikum kariprazin v léčbě schizofrenie
Autoři: prof. MUDr. Cyril Höschl, DrSc., FRCPsych.

Všechny kurzy
Kurzy