Dr Gian Marco Ghiggeri
email: gmarcoghiggeri@gaslini.org

Barbara Seitz-Polski

Joana Justino

Christelle Zaghrini

Christine Payré

Vesna Brglez

Guillaume Dolla

Alberto Sinico

Francesco Scolari

Augusto Vaglio

Marco Prunotto

Giovanni Candiano

Antonella Radice

Nephropathy

Maurizio Bruschi

Dr Gérard Lambeau
email: lambeau@ipmc.cnrs.fr

Gabriella Moroni

Riccardo Magistroni

Domenico Santoro

Antonello Pani

Landino Allegri

Isabella Pisani

Antonio Granata

Pietro Ravani

Monica Bodria

Multi-autoantibody Signature and Clinical Outcome in Membranous Nephropathy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Membranous nephropathy is a common cause of nephrotic syndrome in adults (1)(2)(3). It is characterized by thickening of the glomerular basement membrane with a well-defined pattern of subepithelial immune complex deposits, usually including IgG4 autoantibodies against various autoantigens. The clinical outcome of membranous nephropathy is variable (4). About one third of patients does not require immunosuppressive therapy and reach spontaneous remission while two thirds require immunosuppressive treatment (such as cyclosporine, cytotoxic agents or anti-CD20 antibodies) and respond or not to treatment, with possible progression to end-stage kidney disease over 5-10 years (5)(6)(7)(8)(9)(10).

Antibodies against various podocyte autoantigens have been shown to play a role in the pathogenesis of various forms of membranous nephropathy. In rare familial cases of alloimmune membranous nephropathy, autoantibodies were identified against neutral endopeptidase (11).

These autoantibodies developed in mothers deficient for neutral endopeptidase and crossed the placenta to induce membranous nephropathy in the fetus.

This elegant demonstration of a first causative autoantibody of membranous nephropathy was followed by the identification of phospholipase A2 receptor 1 (PLA2R1) as the major autoantigen in adult membranous nephropathy, with circulating autoantibodies present in about 70% of patients (12). Genetic studies further supported a causal role of PLA2R1 based on association of the disease with 6p21 HLA-DQA1 and 2q24 PLA2R1 loci in cohorts of Caucasian (13) and Asian (14) patients. Multiple epitopes have been identified in the CysR, CTLD1, CTLD7 and CTLD8 domains of PLA2R1 (15-20), which may be reminiscent of a mechanism of epitope spreading reflecting the maturation of the immune response from CysR to other PLA2R1 domains [START_REF] Salant | Does Epitope Spreading Influence Responsiveness to Rituximab in PLA2R-Associated Membranous Nephropathy?[END_REF][START_REF] Beck | Refining Our Understanding of the PLA2R-Antibody Response in Primary Membranous Nephropathy: Looking Forward, Looking Back[END_REF]. Epitope spreading has also been documented in the Heymann rat nephritis model where megalin is the major autoantigen [START_REF] Shah | Intramolecular epitope spreading in Heymann nephritis[END_REF].

Thrombospondin type-1 domain containing 7A (THSD7A) was reported as a second autoantigen in adult membranous nephropathy, with circulating anti-THSD7A autoantibodies present in about 3% of a different group of patients [START_REF] Tomas | Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy[END_REF].

Autoantibodies targeting different intracellular podocyte autoantigens including aldose reductase (AR) [START_REF] Murtas | In vivo characterization of renal auto-antigens involved in human auto-immune diseases: the case of membranous glomerulonephritis[END_REF], superoxide dismutase (SOD2) [START_REF] Prunotto | Autoimmunity in membranous nephropathy targets aldose reductase and SOD2[END_REF] and α-enolase (αENO) [START_REF] Bruschi | Direct characterization of target podocyte antigens and auto-antibodies in human membranous glomerulonephritis: Alfa-enolase and borderline antigens[END_REF] have been identified in a significant number of patients with membranous nephropathy [START_REF] Murtas | Coexistence of different circulating anti-podocyte antibodies in membranous nephropathy[END_REF].

Finally, three new antigenic markers of autoimmunity (exostosin1/exostosin2 and NELL-1) have been very recently identified in immune deposits, with exostosins being more present in secondary cases of membranous nephropathy (i.e. class V lupus nephritis) [START_REF] Sethi | Exostosin 1/Exostosin 2-Associated Membranous Nephropathy[END_REF][START_REF] Sethi | Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy[END_REF]. The exact prevalence and significance of autoantibodies against these new autoantigens in the pathogenesis of membranous nephropathy remain to be determined, with circulating autoantibodies currently found only for NELL-1, and with different prevalence between American and European cohorts of patients [START_REF] Sethi | Exostosin 1/Exostosin 2-Associated Membranous Nephropathy[END_REF][START_REF] Sethi | Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy[END_REF].

In this study, we aimed to 1) detect autoantibodies directed against the well-characterized membrane-bound autoantigens PLA2R1 and THSD7A and the intracellular autoantigens SOD2, AR and αENO in a large retrospective cohort of 285 patients with membranous nephropathy at diagnosis and after 6 and 12 months of follow-up, and 2) evaluate the clinical outcome of patients stratified into different groups based on their combined positivity or not for multiple autoantibodies directed against the different autoantigens.

Materials and Methods

Study design and patient population

This is a cross-sectional study in patients with MN assessing the relationships between detection of different autoantibodies at baseline and eGFR and proteinuria at 12 months. We included 285 patients at the time of a histopathological finding of idiopathic or primary membranous nephropathy (Table S1) and followed them with clinical visits for at least 12 months. They were enrolled in the frame of two studies: 244 patients were enrolled in Italy (Italian Study Group on Membranous Nephropathy-EudraCT 2011-003942-41) and 41 in France (Cohort SOURIS NCT02199145). Criteria for enrollment were (1) biopsy-proven diagnosis of membranous nephropathy associated with proteinuria >0.3 g/day, (2) normal complement profile, (3) negative tests for ANA, anti-dsDNA, ANCA, cryoglobulins and absence of viral markers (hepatitis B surface antigen and HIV), (4) absence of clinical and biochemical signs of cancer and (5) serum samples available at diagnosis and during follow up for determination of antibody levels. Clinical studies were approved by the relevant institutional review boards in the different hospitals and were conducted according to the principles of the Declaration of Helsinki, with written informed consent obtained from all patients. The study was presented at the Ethical Committee of IRCCS Giannina Gaslini. Serum was also obtained from 50 healthy donors recruited at the same institutions that participated to the sample recruitment. Healthy donors had at least one normal urine analysis and normal clinical tests in the prior six months (Table S1).

Detection of circulating autoantibodies

Anti-PLA2R1 autoantibodies (detection of total IgG) against the full-length extracellular domain of PLA2R1 were measured with the standardized ELISA from Euroimmun (Medizinische Labordiagnostika AG, Lübeck, Germany) [START_REF] Dahnrich | Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy[END_REF]. Anti-PLA2R1 autoantibodies (detection of IgG4 subclass) against the specific epitope-containing domains CysR, CTLD1 and CTLD7 were determined as described [START_REF] Seitz-Polski | Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy[END_REF] using recombinant soluble forms of each domain harboring a HA-tag and produced in HEK293 cells. Anti-THSD7A autoantibodies (detection of IgG4 subclass) against the full-length extracellular domain of THSD7A were determined by ELISA as described [START_REF] Zaghrini | Novel ELISA for thrombospondin type 1 domaincontaining 7A autoantibodies in membranous nephropathy[END_REF].

Positivity was confirmed by western-blot [START_REF] Tomas | Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy[END_REF] and indirect immunofluorescence [START_REF] Hoxha | An Indirect Immunofluorescence Method Facilitates Detection of Thrombospondin Type 1 Domain-Containing 7A-Specific Antibodies in Membranous Nephropathy[END_REF] assays.

Autoantibodies against the intracellular antigens AR, SOD2 and αENO (detection of IgG4 subclass) were determined by dot-blot using recombinant proteins as immobilized antigens as described [START_REF] Prunotto | Autoimmunity in membranous nephropathy targets aldose reductase and SOD2[END_REF][START_REF] Bruschi | Direct characterization of target podocyte antigens and auto-antibodies in human membranous glomerulonephritis: Alfa-enolase and borderline antigens[END_REF]. Further details on the detection methods and source of antigens are given in the supplemental material. For anti-PLA2R1 total IgG, we used the normal limit of positivity of 20 RU/mL given by the manufacturer [START_REF] Dahnrich | Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy[END_REF]. For anti-THSD7A and intracellular autoantibodies, normal limits were calculated from ROC curves as described [START_REF] Zaghrini | Novel ELISA for thrombospondin type 1 domaincontaining 7A autoantibodies in membranous nephropathy[END_REF][START_REF] Zweig | Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine[END_REF]. High and low levels of autoantibodies were defined as the titers above median and between median and limit of normality, respectively (Table S2).

Outcome

Clinical outcome was evaluated on the basis of proteinuria levels at diagnosis versus after 12 months of follow-up, and was defined as complete remission (proteinuria <0.3 g/day) or partial remission (proteinuria <3.5 g/day) according to Thompson et al (31). Failure to achieve clinical remission was defined by persistence of proteinuria >3.5 g/day. Kidney function (eGFR) was evaluated based on the CKD-EPI creatinine equation [START_REF] Levey | Estimating GFR using the CKD Epidemiology Collaboration (CKD-EPI) creatinine equation: more accurate GFR estimates, lower CKD prevalence estimates, and better risk predictions[END_REF].

Statistical analysis

Autoantibody levels were expressed as median and interquartile range. The differences in serum concentration of all autoantibodies within patient groups were analyzed using non-parametric U Mann-Whitney test for unpaired samples; Spearman's correlation was used to evaluate correlations among circulating levels of the various types of autoantibodies. The receiveroperating characteristic (ROC) curve analysis was used to discriminate between healthy people and MN patients based on levels of each autoantibody at diagnosis. The best cut-offs were selected as the values that minimized the geometric distance from 100% sensitivity and 100% specificity on the ROC curves. In these analyses, discrimination is assessed as the ability of the test (performance) to distinguish the presence of the outcome beyond chance, defined when the area under the curves (AUC) are significantly >0.5. Odds ratios (OR) and 95% confidence intervals (CI) were tested to determine the associations between autoantibodies positivity or high titer and proteinuria and eGFR using 2x2 contingency tables. Fisher's exact test was used to determine the statistical significance. The P-values were corrected for multiple comparisons with alpha error values of 5%. We used the Kaplan-Meier method to construct survival curves for time to events according to the level of proteinuria. Differences in outcomes were estimated using the log-rank (Mantel-Cox) test and their hazard ratio; p-values ≤0.05 were considered as significant. The performance of single antibody and their synergism for predicting clinical outcome were assessed by two-category net reclassification index (NRI) and area under the curve of receiver operating characteristic analysis (AUC-ROC) [START_REF] Zweig | Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine[END_REF]. We used R for all statistical analyses (http://www.Rproject.org/).

Results

Patients and predictors of clinical outcome

We included 285 patients with biopsy-proven membranous nephropathy whose the clinical characteristics are shown in Tables 1 andS1, according to PLA2R1 and intracellular antibody positivity Most patients were men (n=194, 68%). At diagnosis, 67% of patients had nephrotic syndrome. Seventy-nine percent of patients were treated with immunosuppressive treatments: steroids (33%), cytotoxic drugs (32%), rituximab (12%), and cyclosporine (25%). Antiproteinuric treatment with angiotensin converting enzyme inhibitors (ACEi) was added to the above therapy.

Twenty-one percent of patients received only ACEi. Patients with nephrotic syndrome (n=177, 62%) had persistent high proteinuria after antiproteinuric treatment, with evidence for deterioration of kidney function (eGFR). When the cohort was analyzed as a whole, gender and nephrotic proteinuria at diagnosis were the only two clinical factors associated with the risk of failure to achieve complete remission of proteinuria after 12 months of follow-up (Figure S1), in agreement with previous findings [START_REF] Thompson | Complete and Partial Remission as Surrogate End Points in Membranous Nephropathy[END_REF].

Individual autoantibodies: distribution and levels

All patients were tested at diagnosis for the presence of circulating autoantibodies recognizing PLA2R1 and THSD7A by specific ELISAs [START_REF] Dahnrich | Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy[END_REF][START_REF] Zaghrini | Novel ELISA for thrombospondin type 1 domaincontaining 7A autoantibodies in membranous nephropathy[END_REF]. The presence of circulating autoantibodies specifically recognizing the CysR, CTLD1 and CTLD7 domains of PLA2R1 were also analyzed, defining non-spreaders (patients with detectable reactivity limited to CysR) and spreaders (patients with detectable reactivity to CysR, CTLD1 and/or CTLD7 domains) [START_REF] Seitz-Polski | Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy[END_REF][START_REF] Seitz-Polski | Phospholipase A2 Receptor 1 Epitope Spreading at Baseline Predicts Reduced Likelihood of Remission of Membranous Nephropathy[END_REF]. Patients were also tested for the presence of autoantibodies recognizing the intracellular autoantigens SOD2, AR and αENO [START_REF] Prunotto | Autoimmunity in membranous nephropathy targets aldose reductase and SOD2[END_REF][START_REF] Bruschi | Direct characterization of target podocyte antigens and auto-antibodies in human membranous glomerulonephritis: Alfa-enolase and borderline antigens[END_REF]. Patients with single or multiple positivities to any of these intracellular antigens were referred to as Intracellular + while patients with no detectable reactivity were referred to as Intracellular -. The same analyses were repeated after 6 and 12 months (Figures S2a-S2e). Among all patients, 182 (64%) were positive for anti-PLA2R1 (PLA2R1 + patients, Figures 1a andS2a). The median level of anti-PLA2R1 autoantibodies was 122 RU/mL (IQR 60-267) at baseline and decreased after 6 and 12 months of follow-up (Figure S2a). At baseline, 118 (65%) PLA2R1 + patients were positive for one or more intracellular antigens, defining PLA2R1 + /Intracellular + patients while 64 (35%) were negative, defining PLA2R1 + /Intracellular -patients (Table 1, Figures 1a andS3a). Interestingly, the percentage of Intracellular + patients was higher in patients with high titer of anti-PLA2R1 autoantibodies (Figure 1b). Furthermore, PLA2R1 + patients defined as spreaders had higher anti-PLA2R1 titers than non-spreaders (Figure S3b) and were also more positive for intracellular autoantigens (Figure S3c and Table S3), in particular for αENO (Figure S3d). Among patients, only eight (3%) were positive for anti-THSD7A (THSD7A + , Figures 1a andS2b). One was double-positive for PLA2R1 and THSD7A (Figure S2b). Seven of these patients had additional autoantibodies against at least one intracellular autoantigen (Figures 1a andS3e). Because of their low prevalence, anti-THSD7A + patients were not considered for further analysis.

Thus, among all patients, 95 patients (33%) were identified as double-negative for PLA2R1 and THSD7A. Among these latter patients, 64 (67%) were also positive for one or more intracellular antigens, defining double-negative/Intracellular + patients while 31 (33%) were negative, defining double-negative/Intracellular -patients (Table 1, Figure 1a andS3f).

Overall, autoantibodies against AR, SOD2 and αENO were detected in 85, 85 and 117 patients, respectively (Figures S2c, S2d andS2e). The median levels of each intracellular autoantibody decreased at six and 12 months of follow-up (Figures S2c, S2d andS2e). Positivity and titer levels for anti-PLA2R1, anti-SOD2, anti-AR and anti-αENO autoantibodies at baseline did not correlate to each other (Figure S3g).

Association of the different antibodies with clinical outcome

Considering all patients, positivity for anti-PLA2R1, anti-SOD2 and anti-αENO autoantibodies at diagnosis was variably and independently associated with complete or partial remission of proteinuria and eGFR after 12 months (Table 2). Concomitant positivity of the three antibodies had the highest odds ratio for complete remission of proteinuria after 12 months (OR 5.5 [1.2-24], p=0.01). High titers of anti-PLA2R1 and anti-SOD2 above the median (Table S2, n=91 and n=43, respectively) were similarly associated with lack of partial or complete remission of proteinuria after 12 months compared to those with low titers (Table 2). Kaplan-Meier analysis showed that combined positivity for anti-PLA2R1 and anti-SOD2 or anti-αENO was associated with a higher risk of low eGFR (<60 ml/min/1.73 m 2 ) after 12 months compared to positivity for only anti-PLA2R1 (Figure 2). Additionally, high titers of anti-PLA2R1 and anti-αENO were associated with reduced eGFR after 12 months, as compared to low titers (Table 2). Considering only patients with nephrotic syndrome at diagnosis (67% of the whole cohort), the associations were different (Table 3). Only positivity for anti-SOD2 antibodies and concomitant positivity for anti-PLA2R1 and anti-SOD2 were associated with persistence of proteinuria after 12 months. High titers of anti-PLA2R1 were also associated with persistence of proteinuria. Considering treatment, positivity and high levels of anti-PLA2R1 and anti-SOD2 were the main features associated with lack of partial and complete remission after 12 months in patients who had been treated with cytotoxic drugs, cyclosporine A or rituximab (Table S4).

We further compared the added value of anti-SOD2 and anti-αENO over anti-PLA2R1 to predict clinical outcome using net reclassification improvement (NRI) and area under the receiveroperating-characteristic curve (AUC-ROC) analysis [START_REF] Pencina | Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond[END_REF]. In NRI analysis, the association of two antibodies allowed the reclassification of patients who developed or maintained proteinuria <3.5 g/day after 12 months in 34% and 22% of cases negative for both PLA2R1 and SOD2 or αENO, respectively (Table 4). The same association allowed reclassification of eGFR >60 mL/min/1.73 m 2 in 27% and 24% of patients at 12 months, respectively (Table 4). AUC-ROC analysis discriminated the outcome after 12 months in respect to the titer of a single antibody or its association. In this case, the association of more than one antibody slightly increased the discrimination for patients who developed eGFR <60 mL/min/1.73 m 2 (Table 4).

Stratification of patients based on combined positivity for multiple autoantibodies and association with clinical outcome

Based on combined positivity for autoantibodies against PLA2R1 and intracellular antigens at diagnosis and excluding THSD7A + patients, we stratified the remaining 277 patients into four groups (Table 1). Hundred eighteen patients were anti-PLA2R1 + /Intracellular + (i.e. with additional single or multiple positivity for autoantibodies against intracellular antigens), 64 patients were anti-PLA2R1 + /Intracellular -, 64 patients were anti-PLA2R1 -/Intracellular + , and 31 patients were anti-PLA2R1 -/Intracellular -. At diagnosis, the four groups did not differ for age or sex and had the same percentage of histology classes (Table 1). All patients received comparable therapy (Table 1).

Proteinuria at diagnosis was higher in the two groups anti-PLA2R1 + versus those anti-PLA2R1 - (Table 1 and Figure 3a). Proteinuria after 12 months was lower and eGFR higher in anti-PLA2R1 - /Intracellular -patients versus others (Table 1 and Figures 3a and3b). Differences in outcomes were estimated using Kaplan-Meier curves and the log-rank (Mantel-Cox) test and their hazard ratio. Forty-five percent of anti-PLA2R1 -/Intracellular -patients reached complete remission of proteinuria after 12 months, as compared to 16, 16 and 28% for the other groups (Figure 4a). More anti-PLA2R1 -/Intracellular + patients had a worse clinical outcome after 12 months as compared to anti-PLA2R1 -/Intracellular -patients (Figure 4b). Odds ratios and 95% confidence intervals were utilized to determine the association with reduced eGFR after 12 months. Patients anti-PLA2R1 + /Intracellular + and anti-PLA2R1 -/Intracellular + had about four-fold higher risk of developing eGFR <60 mL/min/1.73 m 2 after 12 months, as compared to patients PLA2R1 -/Intracellular -(Figure 4c). We finally analyzed whether the combined positivity for PLA2R1 epitope spreading and intracellular antigens may be associated with clinical outcome (Table S5).

The concomitance of spreading and anti-αENO positivity was associated with low eGFR at diagnosis and after 12 months (Figure S4) whereas eGFR at diagnosis in non-spreaders was only barely modified by positivity for anti-αENO autoantibodies (not shown).

Discussion

This is the largest retrospective study cohort of membranous nephropathy patients with the combined detection of autoantibodies against the two major membrane-bound antigens PLA2R1 and THSD7A and the three intracellular autoantigens AR, SOD2 and αENO. As expected, PLA2R1 + patients represent the majority of the cohort (64%) while THSD7A+ patients represent only 3%, in line with recent studies [START_REF] Zaghrini | Novel ELISA for thrombospondin type 1 domaincontaining 7A autoantibodies in membranous nephropathy[END_REF][START_REF] Hoxha | An Indirect Immunofluorescence Method Facilitates Detection of Thrombospondin Type 1 Domain-Containing 7A-Specific Antibodies in Membranous Nephropathy[END_REF][START_REF] Sharma | Tissue staining for THSD7A in glomeruli correlates with serum antibodies in primary membranous nephropathy: a clinicopathological study[END_REF]. Our results confirmed that high titers of anti-PLA2R1 autoantibodies are associated with poor outcome in terms of remission of proteinuria and eGFR [START_REF] Hofstra | Anti-phospholipase A2 receptor antibodies correlate with clinical status in idiopathic membranous nephropathy[END_REF][START_REF] Qin | Anti-phospholipase A2 receptor antibody in membranous nephropathy[END_REF][START_REF] Kanigicherla | Anti-PLA2R antibodies measured by ELISA predict long-term outcome in a prevalent population of patients with idiopathic membranous nephropathy[END_REF][START_REF] Hoxha | Phospholipase A2 receptor autoantibodies and clinical outcome in patients with primary membranous nephropathy[END_REF][START_REF] Bech | Association of Anti-PLA2R Antibodies with Outcomes after Immunosuppressive Therapy in Idiopathic Membranous Nephropathy[END_REF][START_REF] Ruggenenti | Anti-Phospholipase A2 Receptor Antibody Titer Predicts washed and patients' sera diluted at 1:100 in PBS/0.1% non-fat dry milk were added (100 μL/well). After 2h incubation at room temperature on a plate shaker, plates were washed and HRP-conjugatedanti-IgG4 secondary antibodies (Southern Biotech) diluted 1:7,500 in SeramunStab ST plus (Seramun Diagnostica) was added (100 μL/well) and incubated for 1h at room temperature on a plate shaker. After four washes, tetramethylbenzidine was added, and the reaction was developed for 15 min and then stop with HCl 1.2N[END_REF]. We also confirmed that epitope spreading was associated with reduced eGFR after 12 months [START_REF] Seitz-Polski | Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy[END_REF][START_REF] Seitz-Polski | Phospholipase A2 Receptor 1 Epitope Spreading at Baseline Predicts Reduced Likelihood of Remission of Membranous Nephropathy[END_REF].

In this study, we add to the above points that the presence of circulating anti-SOD2 and anti-αENO autoantibodies was independently associated with poor outcome in terms of partial and complete remission of proteinuria and eGFR. Furthermore, anti-SOD2, anti-αENO and anti-AR autoantibodies are not correlated to anti-PLA2R1 autoantibodies, and similar percentages of patients were positive for one or more intracellular antigens, irrespective of positivity (n=118, 65%) or not (n=64, 67%) for PLA2R1. Further analysis by Kaplan-Meier curves showed worse clinical outcome for patients positive for anti-PLA2R1 and anti-SOD2 or anti-αENO while NRI and AUC-ROC suggested that intracellular autoantibodies might have an added value over anti-PLA2R1 for refinement of association with clinical outcome. We thus conclude that the positivity for intracellular autoantibodies has an added value over anti-PLA2R1 positivity to predict adverse clinical outcome. It remains to be seen if this might be also true for other membrane-bound antigens such as THSD7A or the new extracellular autoantigens, in particular NELL-1.

Based on the concomitant presence or absence of anti-PLA2R1 and anti-intracellular autoantibodies, we stratified patients into four groups. Extending what was reported above for anti-PLA2R1 positivity, another novel finding is that patients negative for the two categories of autoantibodies have a reduced proteinuria at diagnosis and a better clinical outcome after 12 months of follow-up, in comparison to other patients. Thus, in anti-THSD7A negative patients, the simultaneous measurement of the two categories of autoantibodies (anti-PLA2R1 and anti-SOD2 and anti-αENO) at baseline might be an additive factor to better evaluate clinical outcome.

In this setting, positivity to intracellular antigens may represent per se a negative factor associated with poor clinical outcome. However, we acknowledge limitations in our study including the use of a retrospective cohort with patients with different ages, different clinical characteristics and treatments, and homemade assays to measure autoantibodies to intracellular autoantigens. Our results should thus be confirmed in prospective studies and/or randomized clinical trials.

The second main finding reported here is that positivity for autoantibodies targeting the intracellular autoantigens, especially anti-αENO autoantibodies, is maximal in PLA2R1 + patients with high anti-PLA2R1 titer or those defined as spreaders. The combined positivity associates with a reduction of eGFR, being worse in patients with high anti-PLA2R1 titer (Table 1) and in spreaders (Figure S4) positive for anti-αENO autoantibodies. The lower positivity of anti-αENO autoantibodies or other intracellular autoantigens in patients with low anti-PLA2R1 titer or defined as non-spreaders suggests that the formation of autoantibodies against intracellular antigens represents a secondary phenomenon that occurs after the formation of autoantibodies against PLA2R1 (and possibly THSD7A) as primary triggers of membranous nephropathy. We thus propose that anti-SOD2 and anti-αENO autoantibodies may appear as a second wave of autoantibodies in the pathogenesis of membranous nephropathy. This hypothesis is also supported by the in vitro and in vivo data obtained in the mouse model of THSD7A-associated membranous nephropathy where SOD2 was induced after podocyte injury and in carbonic anhydrase II-associated membranous nephropathy (46, 47).

Anti-SOD2 and anti-αENO autoantibodies are not unique to membranous nephropathy and their occurrence has been described in lupus nephritis [START_REF] Bruschi | Direct characterization of target podocyte antigens and auto-antibodies in human membranous glomerulonephritis: Alfa-enolase and borderline antigens[END_REF]48). However, the IgG subclass is different in membranous nephropathy and lupus nephritis, with IgG4 in membranous nephropathy but IgG2 in lupus nephritis. Therefore, the same autoantigen can trigger the formation of autoantibodies with different IgG isotypes depending on the type of disease. For lupus nephritis, it has been speculated that exposure of methyl-oxidized αENO and/or the DNA-oxidized αENO by neutrophils to TLR9 is a possible mechanism leading to the production of anti-αENO antibodies (48, 49).

SOD2 is a key antioxidant enzyme playing a major role in protecting cells from oxidative injury (50). It is activated as the main antioxidant pathway after formation of autoimmune deposits in glomeruli. Formation of anti-SOD2 autoantibodies may modify the antioxidant protective effect of SOD2 and worsen the clinical outcome.

αENO is a glycolytic enzyme that catalyzes dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and produces pyruvate and ATP in the Embden-Meyerhof-Parnas pathway (51). αENO has also an extracellular localization where it functions as a plasminogen receptor (52). Upon binding to αENO, plasminogen is transformed into plasmin that degrades extracellular matrix components and activates metalloproteases (51, 53). The presence of anti-αENO autoantibodies may reduce its anti-fibrotic role and favor the accumulation of ECM.

Overall, the results of this study show a multi-autoantibody composition in membranous nephropathy, with antibodies that may act in concert, leading to high proteinuria and low eGFR.

Based on the possible functions of SOD2 and αENO after podocyte injury both in vitro and in vivo (46, 47), we propose a multi-hit pathogenic mechanism of membranous nephropathy that first involves the formation of anti-PLA2R1 (or anti-THSD7A or autoantibodies to another similar "primary" autoantigen), that then progresses by a mechanism of both intramolecular (such as within PLA2R1) and intermolecular epitope spreading along which i) the anti-PLA2R1 titer increases and ii) is exacerbated by a second wave of autoantibodies targeting intracellular autoantigens, leading for instance to the production of anti-SOD2 and/or anti-αENO autoantibodies. In patients negative for anti-PLA2R1 and anti-THSD7A autoantibodies, an autoantibody targeting a still unknown membrane-bound autoantigen would take a role similar to anti-PLA2R1 or anti-THSD7A antibodies, possibly represented by patients positive for NELL-1, exostosins or other unknown antigens [START_REF] Sethi | Exostosin 1/Exostosin 2-Associated Membranous Nephropathy[END_REF][START_REF] Sethi | Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy[END_REF].

In conclusion, our study first confirms the relevance of detecting and measuring anti-PLA2R1 (and anti-THSD7A) autoantibodies as fundamental tools to diagnose and identify patients with membranous nephropathy and at risk of severe disease. The study further shows the added value of assessing autoantibodies targeting intracellular podocyte antigens, combined or not with positivity for the above membrane-bound autoantigens. Altogether, our findings pave the way for more personalized medicine in membranous nephropathy.

The Istituto Giannina Gaslini IRCCS (trial sponsor) had provided logistic and financial support to the study through grant from the Ministero della Salute (Ricerca Corrente). People working on the membranous nephropathy project belong to the "Fondazione Malattie Renali del Bambino" of which we acknowledge the financial support. GMG received a grant from Compagnia di San Paolo (ROL 9849). This work was supported by grants from CNRS, the Fondation Maladies Rares (LAM-RD_20170304, GL), the National Research Agency (ANR, grants MNaims (ANR-17-CE17-0012-01, GL) and "Investments for the Future" Laboratory of Excellence SIGNALIFE, a network for innovation on signal transduction pathways in life sciences (ANR-11-LABX-0028-01) with allocated PhD fellowships for CZ and JJ) and the Fondation pour la Recherche Médicale (FRM ING20140129210, SPF20150934219, DEQ20180339193 and FDT201805005509 to GL, VB and JJ).

Disclosures

Some coauthors are co-inventors on the patents "Diagnostics for membranous nephropathy" (GL), "Methods and kits for monitoring membranous nephropathy" (BSP and GL), and "Prognosis and monitoring of membranous nephropathy based on the analysis of PLA2R1 epitope profile and spreading" (BSP, GD and GL). GMG is inventor on a patent on the use of anti-SOD2 and anti-ENO antibodies as biomarkers in membranous nephropathy (EP 3097419).

Supplemental Material

This article contains the following supplemental material: 1. Supplemental Methods 2. Supplemental references 3. Supplemental Tables and Figures Table S1. Epidemiology, histological stage, clinical characteristics and treatment of patients with membranous nephropathy (MN) versus controls (Healthy donors) at diagnosis and after 12 months of follow-up. Table S2. Quantitative data for limits of positivity and distribution of serum levels for each autoantibody. Table S3. Detailed autoantibody positivity for intracellular antigens in non-spreaders and spreaders PLA2R1+ patients. Table S4. Two-way contingency table showing the association of antibodies levels with indexes of kidney outcome and the interaction between autoantibodies against PLA2R1 and intracellular antigens for patients treated with cytotoxic drugs, cyclosporine A or rituximab. Table S5. Clinical characteristics at diagnosis and after 12 months of follow-up for PLA2R1+ patients stratified according to epitope profiles. Figure S1. Prognosis clinical factors associated with complete remission. Figure S2. Circulating levels of the various autoantibodies at diagnosis and during follow-up. Figure S3. Multi-autoantibody composition in the cohort of patients with membranous nephropathy. Figure S4. Kidney function (eGFR) in spreaders positive (n=63) or not (n=84) for anti-αENO autoantibodies.

Table 1. Comparison of clinical characteristics at baseline (T 0 ) and at clinical outcome (T 12 ) of MN patients stratified into four groups according to their antibody profile.

Patients both PLA2R1 and intracellular antibodies positive; patients PLA2R1 positive and intracellular antibodies negative; patients PLA2R1 negative and intracellular antibodies positive; and patients both PLA2R1 negative and intracellular antibodies negative. Data are reported as number and percentage or median with interquartile range. Mann-Whitney or Kruskal-Wallis tests were used for continuous variables, respectively for two or more of two unpaired samples, and chi-squared or Fisher's exact tests were used for categorical variables. Two-tailed p-values ≤0.05 were considered as significant. *p=0. 001

. Two-way contingency table showing the association of antibody levels with indexes of kidney outcome and the interaction between autoantibodies against PLA2R1

and intracellular antigens for the whole cohort. The 2x2 contingency table reports the association with odds ratios (OR) and confidence interval (CI) between antibody titer and clinical outcome. Data are calculated from all patients excluding the eight anti-THSD7A positive patients (n=277). The upper section shows the association between positivity versus negativity of each autoantibody at diagnosis with clinical outcome of proteinuria (complete (≤0.3 g/day) or partial (<3.5 g/day) remission) and eGFR after 12 months. The lower section shows the association of high versus low titers of each antibody with the same parameters of proteinuria and eGFR. The additive effect of positivity for more than one antibody is indicated as P+/S+ and P+/S+/E+ (anti-PLA2R1+/anti-SOD2+/anti-αENO+). *In these cases, P+/S+ were compared to P+/S-and P+/S+/E+ were compared to P+/S-/E-. ** In this case, high P/S (highP titer and S positivity) were compared to high P titer and S negativity.

Proteinuria

Table 3. Two-way contingency table showing the association of antibody levels with indexes of kidney outcome and the interaction between autoantibodies against PLA2R1 and intracellular antigens for nephrotic patients at diagnosis. The 2x2 contingency table reports the association with odds ratios (OR) and confidence interval (CI) between

antibody titer and clinical outcome. Data are calculated from the subset of patients with nephrotic syndrome excluding the eight anti-THSD7A positive patients (n=177). The upper section shows the association between positivity versus negativity of each autoantibody at diagnosis with clinical outcome of proteinuria (complete (≤0.3 g/day) or partial (<3.5 g/day) remission) and eGFR after 12 months. The lower section shows the association of high versus low levels of each antibody with the same parameters of proteinuria and eGFR. The additive effect of positivity for more than one antibody is indicated as P+/S+ and P+/S+/E+. *In these cases, P+/S+ were compared to P+/S-and P+/S+/E+ were compared to P+/S-/E-. ** In this case, high P/S (high P titer and S positivity) were compared to high P titer and S negativity.

Proteinuria

Table 4. Two-category net reclassification improvement (NRI) and area under the receiver-operatingcharacteristic curve (AUC-ROC) analysis for predictors of clinical outcome after 12 months in MN patients by adding intracellular autoantibodies to anti-PLA2R1 positivity.

Data are calculated from all patients excluding the eight anti-THSD7A positive patients (n=277). For NRI, combined negativity of anti-PLA2R1 and anti-SOD2 antibodies allowed the reclassification of 34% of patients with proteinuria <3.5g/day (non-event) and 27% of cases with eGFR>60 mL/min/1.73 m 2 (non-event) after 12 months. Similar results were found for combined negativity of anti-PLA2R1 and anti-αENO. In both cases, the reclassification improvement was in comparison with anti-PLA2R1 positivity alone.

Proteinuria

. Two-way contingency table showing the association of antibody levels with indexes of kidney outcome and the interaction between autoantibodies against PLA2R1

and intracellular antigens for the whole cohort. The 2x2 contingency table reports the association with odds ratios (OR) and confidence interval (CI) between antibody titer and clinical outcome. Data are calculated from all patients excluding the eight anti-THSD7A positive patients (n=277). The upper section shows the association between positivity versus negativity of each autoantibody at diagnosis with clinical outcome of proteinuria (complete (≤0.3 g/day) or partial (<3.5 g/day) remission) and of eGFR after 12 months. The lower section shows the association of high versus low titers of each antibody with the same parameters of proteinuria and eGFR. The additive effect of positivity for more than one antibody is indicated as P+/S+ and P+/S+/E+ (anti-PLA2R1+/anti-SOD2+/anti-αENO+). *In these cases, P+/S+ were compared to P+/S-and P+/S+/E+ were compared to P+/S-/E-. ** In this case, high P/S (highP titer and S positivity) were compared to high P titer and S negativity.

Proteinuria

Table 3. Two-way contingency table showing the association of antibody levels with indexes of kidney outcome and the interaction between autoantibodies against PLA2R1

and intracellular antigens for nephrotic patients at diagnosis. The 2x2 contingency table reports the association with odds ratios (OR) and confidence interval (CI) between antibody titer and clinical outcome. Data are calculated from the subset of patients with nephrotic syndrome excluding the eight anti-THSD7A positive patients (n=177). The upper section shows the association between positivity versus negativity of each autoantibody at diagnosis with clinical outcome of proteinuria (complete (≤0.3 g/day) or partial (<3.5 g/day) remission) and of eGFR after 12 months. The lower section shows the association of high versus low levels of each antibody with the same parameters of proteinuria and eGFR. The additive effect of positivity for more than one antibody is indicated as P+/S+ and P+/S+/E+. *In these cases, P+/S+ were compared to P+/S-and P+/S+/E+ were compared to P+/S-/E-. ** In this case, high P/S (high P titer and S positivity) were compared to high P titer and S negativity. Table 4. Two-category net reclassification improvement (NRI) and area under the receiver-operating-characteristic curve (AUC-ROC) analysis for predictors of clinical outcome after 12 months in MN patients by adding intracellular autoantibodies to anti-PLA2R1 positivity. Data are calculated from all patients excluding the eight anti-THSD7A positive patients (n=277). For NRI, combined negativity of anti-PLA2R1 and anti-SOD2 antibodies allowed the reclassification of 34% of patients with proteinuria <3.5g/day (non-event) and 27% of cases with eGFR>60 mL/min/1.73 m 2 (non-event) after 12 months. Similar results were found for combined negativity of anti-PLA2R1 and anti-αENO. In both cases, the reclassification improvement was in comparison with anti-PLA2R1 positivity alone.

Proteinuria

Proteinuria

Kidney funcMon

Figure 4c

Corporation (Taipei, Taiwan); recSOD2: full-length recombinant protein with GST tag, Abnova Corporation (Taipei, Taiwan); recαENO: full-length recombinant protein with GST tag, Abnova Corporation (Taipei, Taiwan).

Antibodies. Antibodies for specific proteins were utilized for the calibration curve. They were obtained from the following companies: Anti-AR: Abnova Corporation, (Taipei, Taiwan). Anti-SOD2: Abnova Corporation, (Taipei, Taiwan). Anti-αENO: rabbit anti-human Non-Neuronal Enolase (NNE) (alpha-alpha) from AbD Serotec Morpho Sys Ltd, (Endeavour House, Kidlington Oxford, UK). HRP-conjugated secondary antibodies utilized for dot-blot and western-blot were obtained from the following companies: purified mouse monoclonal antibody to human IgG4 (Clone: HP6025) Southern Biotech (Birmingham, AL, USA).

Dot-blot for anti-AR, anti-SOD2 and anti-αENO autoantibodies.

For anti-AR, anti-SOD2 and anti-αENO autoantibody determination, we utilized dot-blot and recombinant proteins as fixed antigen following procedures already described in details 1, 2 . The assays were done with a Bio-Dot apparatus (Bio-Rad, Hercules, CA, USA) following the instruction manual with minor modifications. All samples and the calibrator serum were diluted with Trisbuffered saline (TBS) pH 7.4 (1:100). A calibration curve was prepared by keeping constant the amount of recombinant protein and increasing dilution of specific antibody from 1:500 to 1:32,000. Sera were diluted in the same buffer (1:100) to achieve the desired range of levels, obtained after testing several conditions. Accordingly, the nitrocellulose membrane was pre-wetted in TBS and placed on a sheet of Whatman 3-mm filter paper embedded with the same buffer. Equal amounts of protein (300 ng) were placed in 80 µl of TBS. After removal of air bubbles between the two sheets by gentle pressure, the sample template was placed on the nitrocellulose membrane and a vacuum was applied for a few minutes, to fill up the 96 sample wells with 50 µL using a multi-channel pipette. The vacuum was applied until all the samples were adsorbed. The same operation was repeated five times with 150 µL of buffer each, to wash out the non-adsorbed sample. The nitrocellulose was then gently removed and saturated with 5% w/v bovine serum albumin (BSA) in TBS. Sera were then incubated for six hours at room temperature. At the end of incubation, the membrane was washed six times in 0.15% (v/v) (TBS-T). Incubation with HRP-conjugated anti-human IgG4 (0.5 µg/mL) in 1% w/v BSA in TBS-T was performed for two hours at room temperature. The membrane was then washed four times, 15 min each, with Tween-TBS prior to developing the immunoreaction with SuperSignal West Pico Chemiluminescent substrate (Thermo scientific, Rockford, IL, USA). Chemiluminescence was detected by VersaDoc and computed with QuantityOne software (Bio-Rad) and given as relative optical density [OD unit].

Anti-PLA2R1 epitopes. Anti-PLA2R1 epitope autoantibodies were determined as described 3 using recombinant and soluble forms of CysR, CTLD1 and CTLD7 domains with HA-tag. Plates were coated with anti-HA antibody (Sigma-Aldrich) diluted at 1:5,000 in 20 mMTris pH 8.0 (100 μL/well) at 4°Covernight. Plates were blocked for 2h with SeramunBlock (Seramun Diagnostica). Cell medium from HEK293 cells transfected with soluble forms of PLA2R1 domains (10-100 μL/well) were then added and incubated for 1 h. 4. Two-way contingency table showing the association of antibodies levels with indexes of kidney outcome and the interaction between autoantibodies against PLA2R1 and intracellular antigens for patients treated with cytotoxic drugs, cyclosporine A or rituximab. The 2x2 contingency table reports the association with odds ratios (OR), confidence interval (CI) and p values between antibody titer and clinical outcome. The P values were corrected for multiple comparisons with an alpha error value of 5%. Data are calculated from all patients treated with immunosuppressants after excluding the eight anti-THSD7A positive patients (n=196). The upper section shows the association between positivity versus negativity of each autoantibody at diagnosis with clinical outcome of proteinuria (complete (≤0.3 g/day) and partial (<3.5 g/day) remission) and eGFR after 12 months. The lower section shows the association of high versus low titers of each antibody with the same parameters of proteinuria and eGFR. The additive effect of positivity of more than one antibody is indicated as P+/S+ and P+/S+/E+ (anti-PLA2R1+/anti-SOD2+/anti-αENO+). 1 (0.9-1.3) 1.2 (0.9-1.4) 0.9 (0.8-1.2) 1.2 (1-1.4) ns 1(0.9-1.3) 1.1 (0.9-1.4) ns Month 12 0.9 (0.8-1.4) 1.2 (0.9-1.6) 1 (0.9-1.2) 1.1 (0.9-1.4) ns 0.9(0.8-1.4) 1 (0.9-1.

Proteinuria

FiguresFigure 1 .

 1 Figure 1. Distribution of circulating autoantibodies against the membrane-bound autoantigens (PLA2R1 and THSD7A) further divided according to the combined positivity for intracellular autoantigens (AR, SOD2 and αENO). (a) Patients were first divided according to the presence of circulating autoantibodies targeting the membrane-bound autoantigens PLA2R1 or THSD7A. The three groups of patients (PLA2R1 + , THSD7A + and PLA2R1 -/THSD7A -) were further divided according to positivity for autoantibodies targeting any of the three intracellular podocyte autoantigens (AR, SOD2, αENO). (b) Stratification of PLA2R1 + patients according to anti-PLA2R1 titers below and above the median. *The percentage of patients positive for intracellular autoantibodies was higher in those with high anti-PLA2R1 titers (p=0.02, Fisher exact test).

Figure 2 .

 2 Figure 2. Additive effect of anti-SOD2+ and anti-αENO+ to anti-PLA2R1+ for decreased eGFR after 12 months of follow-up. Kaplan-Meier curve showing the proportion of patients in the different groups who reached the endpoint of reduction of kidney function (eGFR <60 mL/min/1.73 m 2 ) after 12 months of follow-up based on the concomitant positivity or not for anti-PLA2R1 and anti-SOD2 (a) or anti-αENO (b). The Kaplan-Meier curve of the difference was calculated with the Log-rank (Mantel-Cox) test and the hazard ratios with confidence intervals for the different groups.

Figure 3 .

 3 Figure 3. Clinical outcome of proteinuria and eGFR according to circulating autoantibodies defining four groups of patients. (a) Proteinuria and (b) eGFR values (data are presented as median with IQR) at diagnosis and after 12 months of follow-up for patients divided in four groups based on the concomitant positivity or not for anti-PLA2R1 (PLA2R1 + or PLA2R1 -) and antiintracellular antigen antibodies (Intracellular + or Intracellular -). Proteinuria (a) and eGFR (b) values are presented at diagnosis (open bars) and after 12 months (grey bars) for all patients (overall), for patients with nephrotic syndrome (NS) or not (non NS) at diagnosis and for patients who had been treated with an immunosuppressive treatment (IS-treated with cyclophosphamide, cyclosporine A or rituximab) after 12 months of follow-up (black bars).

Figure 4 .

 4 Figure 4. Clinical outcome (complete remission and eGFR) according to circulating autoantibodies defining four groups of patients. (a) Kaplan-Meier curve showing the proportion of patients in the different groups (based on the concomitant positivity or not for anti-PLA2R1

  Figure 4b

Table 2

 2 versus A and B; ** p=0.001 versus A, B and C. ACEi: Angiotensin converting enzyme inhibitors.

	Characteristics	PLA2R1 (n=118) + /Intracellular	+	PLA2R1	+ /Intracellular (n=64)	-	PLA2R1	-/Intracellular (n=64)	+	PLA2R1	-/Intracellular (n=31)	-
	Sex (M/F)		84/34			45/19			47/17			21/10
	Age (years)		61 (50-72)		63 (46-70)		57 (42-67)		55 (43-71)
	MN histological stage
	(%)
	I		18.8			23.6			25.9			44.5
	II		44.2			49.0			50.0			33.3
	III		28.6			23.5			14.8			11.0
	IV		8.4			3.9			9.3			11.1
	Treatment (%)
	Steroids/ACEi	§	80			78			85			78
	Cyclosporine A	18			30			14			16
	Cytotoxic		43			44			33			32
	Rituximab		7			22			16			19
	Clinical characteristics
	Proteinuria (g/day)
	T 0		5.6(3.3-9.7)		6.0 (3.3-9.8)		3.4 (1.8-6.4)*		4.4 (2.6-6.4)*
	T 12		1.7 (0.4-4.4)		1.8(0.6-4.3)		1.3 (0.3-3.5)		0.5 (0.1-2.7)**
	CKD-EPI
	(mL/min/1.73m	2 )
	T 0		69.4(46-99)		65.9(47-100)		72.4 (43-99)		89.2 (63-103)**
	T 12		73.5(48-99)		77.9(59-99)		67.2 (44-101)		95.5 (72-103)**

§ Steroids and ACEi were given alone or in association with other drugs

Table 1 .

 1 Comparison of clinical characteristics at baseline (T 0 ) and at clinical outcome (T 12 ) in patients with iMN stratified into four subgroups according to their antibody profile: patients both PLA2R1 and intracellular antibodies positive; patients PLA2R1 positive and intracellular antibodies negative; patients PLA2R1 negative and intracellular antibodies positive; and patients both PLA2R1 negative and intracellular antibodies negative. Data are reported as number and percentage or median with interquartile range. Mann-Whitney or Kruskal-Wallis tests were used for continuous variables, respectively for two or more of two unpaired samples, and chi-squared or Fisher's exact tests were used for categorical variables. Two-tailed p-values ≤0.05 were considered as significant.

		Proteinuria	eGFR<60
	>0.3 g/day	>3.5 g/day	mL/min/1.73 m2

*p=0.001 versus A and B; ** p=0.001 versus A, B and C. ACEi: Angiotensin converting enzyme inhibitors. § Steroids and ACEi were given alone or in association with other drugs

Table 2

 2

Plates were Supplemental Table 1. Epidemiology, histological stage, clinical characteristics and treatment of patients with membranous nephropathy (MN) versus controls (Healthy donors) at diagnosis and after 12 months

  . N.A., not applicable. ACEi: Angiotensin converting enzyme inhibitors.

	Characteristics	MN (n=285)	Controls (n=50)
	Male gender (n (%))	194 (68)	35 (70)
	Age (years)	61 (11-87)	52 (18-60)
	Histological stage (n (%))
	I	68 (24)	N.A.
	II	131 (46)	N.A.
	III	63 (22)	N.A.
	IV	23 (8)	N.A.
	Clinical characteristics
	eGFR (mL/min/1.73 m 2 )
	Diagnosis	74.2 (47-100)	104 (95-110)
	Month 12	80 (43-104)
	Proteinuria (g/day)
	Diagnosis	5.3 (0.5-25)	0.1 (0.05-0.15)
	Month 12	1.8 (0.1-33)
	Serum albumin (g/dL)
	Diagnosis	2.4 (1.0-4.2)	4.2 (4-4.5)
	Month 12	3.5 (1.5-4.8)
	Treatment (n (%))
	Cytotoxic	90 (32)	N.A.
	Cyclosporine A	72 (25)	N.A.
	Rituximab	34 (12)	N.A.
	Steroids	95 (33)	N.A.
	ACEi §	147 (51)	N.A.

§

ACEi was given alone or in association with other drugs. Twenty-one percent of patients received only ACEi.

Supplemental

Table 2 . Quantitative data for limits of positivity and distribution of serum levels for each autoantibody.

 2 Low levels correspond to values between the limit of positivity and the median; high levels correspond to values over the median. The number of patients in the two groups is shown under parenthesis.

	Autoantibody	Limit of Positivity	Low level limit (n)	High level limit (n)
	Anti-PLA2R1 (RU/mL)	20	20-122 (91)	122-1,751 (91)
	Anti-SOD2 (mg/L)	162	162-286 (42)	286-1,404 (43)
	Anti-AR (mg/L)	84	84-151 (43)	151-2,422 (43)
	Anti-αENO (mg/L)	136	136-205 (58)	205-900 (58)

Table 3 . Detailed autoantibody positivity for intracellular antigens in non- spreaders and spreaders PLA2R1 + patients.

 3 Patients positive for anti-PLA2R1 autoantibodies (n=182) were split according to their epitope profiles as "non-spreaders" (CysR only) or "spreaders" (CysRCTLD1, CysRCTLD7 and CysRCTLD1CTLD7). Spreaders and non-spreaders were further split for the presence of single (anti-AR, anti-SOD2, anti-αENO) or composite anti-intracellular antigen positivity (Intracellular + included all single and multiple positivities for intracellular antigens).

		Anti-PLA2R1 +
		Non-spreaders		Spreaders
		CysR	Overall	CysR	CysR	CysR
				CTLD1	CTLD7	CTLD1
						CTLD7
		(n=35)	(n=147)	(n=34)	(n=46)	(n=67)
	Autoantibody positivity
	Anti-AR + (%)	7	4	1	3	6
	Anti-SOD2 + (%)	0	7	14	3	3
	Anti-αENO + (%)	8	15	17	30	22
	Anti-AR + SOD2 + (%)	10	3	0	3	6
	Anti-AR + αENO + (%)	2	7	6	3	12
	Anti-SOD2 + αENO + (%)	4	13	8	17	16
	Anti-AR + SOD2 + αENO + (%)	10	9	8	13	6
	Anti-Intracellular + (%)	45	58	54	72	71

Table 5 . Clinical characteristics at diagnosis and after 12 months of follow-up for PLA2R1 + patients stratified according to epitope profiles.

 5 Data are reported as median with interquartile range. Mann-Whitney or Kruskal-Wallis tests were used for continuous variables and Chisquared or Fisher's exact tests for categorical variables. Two-tailed p-values ≤0.05 were considered as significant (ns: not significant). ACEi: Angiotensin converting enzyme inhibitors. Note the columns "Anti-CysR" and "Non-spreaders" are identical, but presented twice for clarity.

		>0.3 g/day	Proteinuria ≤0.3 g/day	OR (CI)	P value	Proteinuria >3.5 g/day	Proteinuria ≤3.5 g/day	OR (CI)	P value	eGFR≤60 ml/min/1.73m2	eGFR>60 ml/min/1.73m2	OR (CI)	P value
	Anti-PLA2R1 (P)
	Positive Negative	100 (51%) 46 (20%)	23 (12%) 27 (14%)	2.5 (1.3-4.9)	0.01	34 (17%) 14 (7%)	89 (45%) 59 (30%)	1.6 (0.8-3.3)	0. 2	44 (22%) 23 (12%)	79 (40%) 50 (26%)	1.2 (0.6-2.2)	0.6
	Anti-SOD2 (S)
	Positive Negative	59 (30% ) 87 (44%)	9 (5%) 41 (21%)	3.1 (1.4-6.8)	0.005	22 (11%) 26 (13%)	46 (23%) 102 (52%)	1.9 (1-3.6)	0.08	26 (13%) 41 (21%)	36 (18%) 93 (47%)	1.6 (0.9-3)	0.1
	Anti-αENO (E)
	Positive Negative	64 (33% ) 82 (42%)	15 (8%) 35 (18%)	1.8 (0.9-3.6)	0.1	25 (13%) 24 (12%)	54 (28%) 93 (47%)	1.8 (0.9-3.4)	0.09	29 (15%) 38 (19%)	42 (21%) 87 (44%)	1.6 (0.9-2.9)	0.1
	P+/S+*
	Positive Negative	37 (30%) 63 (51%)	4 (3%) 19 (15%)	2.8 (0.9-9)	0.09	15 (12%) 19 (15%)	26 (21%) 63 (51%)	1.9 (0.8-4.3)	0.1	16 (13%) 28 (23%)	25 (20%) 54 (44%)	1.2 (0.6-2.7)	0.7
	P+/S+/E+*
	Positive Negative	27 (22%) 73 (59%)	2 (2%) 21 (17%)	3.9 (0.8-18)	0.1	10 (8%) 24 (20%)	19 (15%) 70 (57%)	1.5 (0.6-3.7)	0.3	26 (21%) 18 (15%)	42 (34%) 37 (30%)	1.3 (0.6-2.7)	0.6
	Anti-PLA2R1 (P)
	High titer Low titer	50 (41%) 50 (41%)	8 (7%) 15 (12%)	1.9 (0.7-4.8)	0. 2	20 (16%) 14 (11%)	39 (32%) 50 (41%)	1.8 (0.8-4.1)	0.2	25 (20%) 19 (15%)	34 (28%) 45 (37%)	1.7 (0.8-3.7)	0.2
	Anti-SOD2 (S)
	High titer Low titer	32 (47%) 27 (40%)	7 (10%) 2 (3%)	0.3 (0.1-1.8)	0.3	16 (24%) 6 (9%)	20 (29%) 26 (38%)	3.5 (1.1-10)	0.04	16 (26%) 10 (16%)	18 (29%) 18 (29%)	1.6 (0.6-4.5)	0.4
	Anti-αENO (E)
	High titer Low titer	32 (41%) 32 (41%)	8 (10%) 7 (9%)	0.9 (0.3-2.7)	1	19 (24%) 6 (8%)	25 (32%) 29 (37%)	3.7 (1.3-11)	0.02	15 (21%) 14 (20%)	23 (32%) 19 (27%)	0.9 (0.3-2.3)	0.8
	High P+/S+**
	High titer P/ S+ High titer P/ S-	13 (32%) 24 (58%)	2 (5%) 2 (5%)	0.5 (0.1-4.3)	0.6	6 (15%) 9 (22%)	9 (22%) 17 (41%)	1.2 (0.3-4.7)	0.7	6 (15%) 10 (24%)	9 (22%) 16 (39%)	1 (0.3-3.9)	1

*In these cases, P+/S+ were compared to P+/S-and P+/S+/E+ were compared to P+/S-/E-. ** In this case, high P/S (high P titer and S positivity) were compared to high P titer and S negativity.

Supplemental

mL/min/1.73m 2 )

  Wallis test comparing the whole line of data (ie. anti-CysR, CysRCTLD1, CysRCTLD7 and CysRCTLD1CTLD7). § ACEi was given alone or in association with other drugs.

								5)	ns
	Proteinuria (g/day)
	Diagnosis	6 (4.9-7.6)	4.6 (3-8)	6 (3.5-9.9)	7 (4.4-10.9)	ns*	6 (4.9-7.6)	6 (3.6-10)	ns
	Month 12	1.2 (0.2-4)	1 (0.4-4.2)	2.4 (1.1-5.8)	2.8 (0.6-5.4)	ns*	1.2 (0.2-4)	2.2 (0.6-5.7)	ns
	eGFR (Diagnosis	69.8 (48-109)	57.9 (40-75)	77.5 (55-107)	64.9 (44-87)	0.03*	69.8 (48-109)	65.8 (45-93)	ns
	Month 12	89.5 (48-105)	60.2 (44-87)	79.9 (64-102)	62.3 (47-90)	0.04*	89.5 (48-105)	68.6 (48-90)	ns
	*Kruskal-

PLA2R1 + /Intracellular + PLA2R1 + /Intracellular - PLA2R -/Intracellular + PLA2R1 -/Intracellular -

Funding

Author Contributions

G.M.G, B.S.P. and G.L. designed the study; J.J., C.Z., C.P., V.B., G.D., C.M., M.B., G.C and A.S. carried out experiments; G.M.G, B.S.P., M.B., M.P. and G.L. analyzed the data; G.M.G, B.S.P., J.J., M.B. and G.L. made the figures; G.M.G, B.S.P., J.J., M.B. and G.L. drafted and revised the paper; A.S., F.S., A.V., M.P. and B.S.P. had a major role in the inclusion of patients; all authors approved the final version of the manuscript.

Supplemental Methods

Recombinant proteins.

Recombinant proteins utilized for dot-blot were obtained from the companies as follows: recAR: full-length recombinant protein with GST tag, Abnova