Autoantibodies Targeting Nephrin in Podocytopathies.

BACKGROUND: Minimal change disease and primary focal segmental glomerulosclerosis in adults, along with idiopathic nephrotic syndrome in children, are immune-mediated podocytopathies that lead to nephrotic syndrome. Autoantibodies targeting nephrin have been found in patients with minimal change disease, but their clinical and pathophysiological roles are unclear. METHODS: We conducted a multicenter study to analyze antinephrin autoantibodies in adults with glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody-associated glomerulonephritis, and lupus nephritis, as well as in children with idiopathic nephrotic syndrome and in controls. We also created an experimental mouse model through active immunization with recombinant murine nephrin. RESULTS: The study included 539 patients (357 adults and 182 children) and 117 controls. Among the adults, antinephrin autoantibodies were found in 46 of the 105 patients (44%) with minimal change disease, 7 of 74 (9%) with primary focal segmental glomerulosclerosis, and only in rare cases among the patients with other conditions. Of the 182 children with idiopathic nephrotic syndrome, 94 (52%) had detectable antinephrin autoantibodies. In the subgroup of patients with active minimal change disease or idiopathic nephrotic syndrome who were not receiving immunosuppressive treatment, the prevalence of antinephrin autoantibodies was as high as 69% and 90%, respectively. At study inclusion and during follow-up, antinephrin autoantibody levels were correlated with disease activity. Experimental immunization induced a nephrotic syndrome, a minimal change disease-like phenotype, IgG localization to the podocyte slit diaphragm, nephrin phosphorylation, and severe cytoskeletal changes in mice. CONCLUSIONS: In this study, circulating antinephrin autoantibodies were common in patients with minimal change disease or idiopathic nephrotic syndrome and appeared to be markers of disease activity. Their binding at the slit diaphragm induced podocyte dysfunction and nephrotic syndrome, which highlights their pathophysiological significance. (Funded by Deutsche Forschungsgemeinschaft and others.).

Mapping the T cell epitopes of the M-type transmembrane phospholipase A2 receptor in primary membranous nephropathy.

The M-type phospholipase A2 receptor (PLA2R) is the major autoantigen of primary membranous nephropathy (MN). Despite many studies on B-cell epitopes recognized by antibodies, little is known about T-cell epitopes. Herein, we synthesized 123 linear peptides, each consisting of 15-22 amino acids with 8-12 amino acid overlaps, across ten domains of PLA2R. Their binding capacity to risk (DRB1∗1501, DRB1∗0301) and protective (DRB1∗0901, DRB1∗0701) HLA molecules was then assessed by flow cytometry. Proliferation of CD4+ T cells from patients with anti-PLA2R positive MN was analyzed after peptide stimulation. Cytokines produced by activated peripheral blood mononuclear cells were measured by cytometric bead arrays. We identified 17 PLA2R peptides that bound to both DRB1∗1501 and DRB1∗0301 molecules with high capacity. Some of these peptides showed decreased binding to heterozygous DRB1∗1501/0901 and DRB1∗0301/0701. Ten of the 17 peptides (CysR1, CysR10, CysR12, FnII-3, CTLD3-9, CTLD3-10, CTLD3-11, CTLD5-2-1, CTLD7-1 and CTLD7-2) induced significant proliferation of CD4+ T cells from patients with MN than cells from healthy individuals. Upon activation by these peptides, peripheral blood mononuclear cells from patients with MN produced higher levels of pro-inflammatory cytokines, predominantly IL-6, TNF-α, IL-10, IL-9 and IL-17. Thus, we mapped and identified ten peptides in the CysR, FnII, CTLD3, CTLD5, and CTLD7 domains of PLA2R as potential T-cell epitopes of MN. These findings are a first step towards developing peptide-specific immunotherapies.

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a likely antigenic target in membranous nephropathy and nonsteroidal anti-inflammatory drug use.

Drugs are an important secondary cause of membranous nephropathy (MN) with the most common drugs associated with MN being nonsteroidal anti-inflammatory drugs (NSAIDs). Since the target antigen in NSAID-associated MN is not known, we performed laser microdissection of glomeruli followed by mass spectrometry (MS/MS) in 250 cases of PLA2R-negative MN to identify novel antigenic targets. This was followed by immunohistochemistry to localize the target antigen along the glomerular basement membrane and western blot analyses of eluates of frozen biopsy tissue to detect binding of IgG to the novel antigenic target. MS/MS studies revealed high total spectral counts of a novel protein Proprotein Convertase Subtilisin/Kexin Type 6 (PCSK 6) in five of the 250 cases in the discovery cohort. A validation cohort using protein G immunoprecipitation, MS/MS, and immunofluorescence detected PCSK6 in eight additional cases. All cases were negative for known antigens. Ten of 13 cases had a history of heavy NSAID use with no history available in one case. The mean serum creatinine and proteinuria at kidney biopsy were 0.93 ± 0.47 mg/dL and 6.5 ± 3.3 gms/day, respectively. Immunohistochemistry/immunofluorescence showed granular staining for PCSK6 along the glomerular basement membrane, and confocal microscopy showed co-localization of IgG and PCSK6. IgG subclass analysis in three cases revealed codominance of IgG1 and IgG4. Western blot analysis using eluates from frozen tissue showed IgG binding to PCSK6 in PCSK6-associated but not in PLA2R-positive MN. Thus, PCSK6 may be a likely novel antigenic target in MN in patients with prolonged NSAID use.

Mayo Clinic consensus report on membranous nephropathy: proposal for a novel classification.

Membranous nephropathy (MN) is a pattern of injury caused by autoantibodies binding to specific target antigens, with accumulation of immune complexes along the subepithelial region of glomerular basement membranes. The past 20 years have brought revolutionary advances in the understanding of MN, particularly via the discovery of novel target antigens and their respective autoantibodies. These discoveries have challenged the traditional classification of MN into primary and secondary forms. At least 14 target antigens have been identified, accounting for 80%-90% of cases of MN. Many of the forms of MN associated with these novel MN target antigens have distinctive clinical and pathologic phenotypes. The Mayo Clinic consensus report on MN proposes a 2-step classification of MN. The first step, when possible, is identification of the target antigen, based on a multistep algorithm and using a combination of serology, staining of the kidney biopsy tissue by immunofluorescence or immunohistochemistry, and/or mass spectrometry methodology. The second step is the search for a potential underlying disease or associated condition, which is particularly relevant when knowledge of the target antigen is available to direct it. The meeting acknowledges that the resources and equipment required to perform the proposed testing may not be generally available. However, the meeting consensus was that the time has come to adopt an antigen-based classification of MN because this approach will allow for accurate and specific MN diagnosis, with significant implications for patient management and targeted treatment.

The role of PLA2R antibody monitoring: what we know and what we do not know.

For a long time, kidney biopsy was the only diagnostic means for membranous nephropathy (MN) and proteinuria and serum creatinine were the only markers of disease activity. The discovery of the phospholipase A2 receptor (PLA2R) antibody in 2009 has induced a paradigm shift in both the diagnosis and monitoring of patients. Two serological tests are routinely used: the enzyme-linked immunosorbent assay (ELISA), which is quantitative, and the immunofluorescence assay (IFA), which is more sensitive. In centres where the two assays are available, the recommendation is to use IFA for screening and diagnosis of immunological remission and ELISA for monitoring the effectiveness of therapy. In patients with positive PLA2R antibody serology, normal kidney function and no evidence of an underlying disease, a kidney biopsy is not mandatory given the almost 100% specificity of the assays. Because MN has different phases, one cannot base a clinical or therapeutic decision on a single measurement of PLA2R antibody at baseline. Risk evaluation of disease progression is a dynamic process that should be performed repeatedly to capture the trajectory of the disease based on both the traditional biomarkers (proteinuria and serum creatinine) and PLA2R antibody levels. The effectiveness of therapy is also evaluated on the PLA2R antibody trajectory, particularly during the first 6 months. Finally, PLA2R antibody monitoring has transformed the management of patients with kidney allografts. Future studies are needed to develop more subtle immunological tests, including monitoring of antigen-specific memory B cells.

Recurrence of Anti-Semaphorin 3B-Mediated Membranous Nephropathy after Kidney Transplantation.

BACKGROUND: Membranous nephropathy (MN) is rare in pediatric patients, although its diagnosis may be underestimated in children who are responsive to corticosteroid therapy prescribed for a suspicion of minimal change disease. It is most often associated with an autoimmune disease, predominantly lupus. We previously reported the occurrence of early-onset MN associated with semaphorin 3B in nine children and two adults. METHODS: Biopsies were performed on native kidney and at 1 and 5 months after transplantation. Semaphorin 3B antigen was detected in immune deposits by immunohistochemistry and confocal microscopy on paraffin-embedded biopsies. Anti-semaphorin antibodies were detected by Western blot and analyzed sequentially. RESULTS: We report the first case of early recurrence after transplantation in a 7-year-old boy who presented with severe nephrotic syndrome and advanced kidney failure. There was no evidence of hereditary or associated autoimmune disease. Abundant, almost coalescent deposits were seen by electron microscopy and bright granular, subepithelial staining was observed for semaphorin 3B antigen. Western blot analysis of serum revealed anti-semaphorin 3B antibodies. Recurrence of MN occurred 25 days after transplantation and manifested as nephrotic range proteinuria despite conventional immunosuppressive therapy. Kidney biopsies confirmed histologic MN recurrence with colocalization of semaphorin 3B antigen and IgG. The patient was treated with rituximab. Anti-semaphorin 3B antibodies, which were detected at transplantation, were not detected 40 days after rituximab. CONCLUSION: This case provides evidence that anti-semaphorin 3B antibodies are pathogenic and should be monitored in patients with MN.

Contactin 1, a Potential New Antigen Target in Membranous Nephropathy: A Case Report.

Several novel antigens have recently been characterized in membranous nephropathy (MN), but those involved in the rare cases of MN associated with inflammatory neuropathies remain elusive. Although several antibodies have been identified in the serum, there is no evidence so far for their deposition in glomeruli. We report the case of a 73-year-old woman who was referred because of subacute onset of proximal asymmetric lower limb weakness together with ataxic gait. She was diagnosed with inflammatory neuropathy. Testing showed an estimated glomerular filtration rate of 73mL/min/1.73m2, hypoalbuminemia (2.89g/dL), and proteinuria (3.6g/d). Autoantibodies (antinuclear antibody, anti-extractable nuclear antigen antibody, anti-double stranded DNA antibody, lupus anticoagulant, anticardiolipin antibody, antineutrophil cytoplasmic antibody) were undetectable. Serum immunoglobulin and complement levels were normal. A kidney biopsy with electron microscopy examination showed a classical picture of MN. Testing for antibodies to phospholipase A2 receptor (PLA2R) gave negative results in the serum, and PLA2R and THSD7A antigens were not detected in kidney tissue. Anti-contactin 1 (CNTN1) antibody was detected by enzyme-linked immunosorbent assay at a 1:100 dilution of serum and shown to be mostly of IgG4 subclass by Western blot. CNTN1 antigen was colocalized with IgG4 within immune deposits by confocal microscopy. This observation suggests a pathophysiological link between inflammatory neuropathies and MN. CNTN1 should be considered as a potential candidate antigen involved in MN and tested in PLA2R-negative forms associated with inflammatory neuropathies.

Protocadherin 7-Associated Membranous Nephropathy.

BACKGROUND: Membranous nephropathy (MN) results from deposition of antigen-antibody complexes along the glomerular basement membrane (GBM). PLA2R, THSD7A, NELL1, and SEMA3B account for 80%-90% of target antigens in MN. METHODS: We performed laser microdissection and mass spectrometry (MS/MS) in kidney biopsies from 135 individuals with PLA2R-negative MN, and used immunohistochemistry/immunofluorescence and confocal microscopy to confirm the MS/MS finding, detect additional cases, and localize the novel protein. We also performed MS/MS and immunohistochemistry on 116 controls and used immunofluorescence microscopy to screen biopsy samples from two validation cohorts. Western blot and elution studies were performed to detect antibodies in serum and biopsy tissue. RESULTS: MS/MS studies detected a unique protein, protocadherin 7 (PCDH7), in glomeruli of ten (5.7%) PLA2R-negative MN cases, which also were negative for PLA2R, THSD7A, EXT1/EXT2, NELL1, and SEMA3B. Spectral counts ranged from six to 24 (average 13.2 [SD 6.6]). MS/MS did not detect PCDH7 in controls (which included 28 PLA2R-positive cases). In all ten PCDH7-positive cases, immunohistochemistry showed bright granular staining along the GBM, which was absent in the remaining cases of PLA2R-negative MN and control cases. Four of 69 (5.8%) cases in the validation cohorts (all of which were negative for PLA2R, THSD7A, EXT1, NELL1, and SEMA3B) were PCDH7-positive MN. Kidney biopsy showed minimal complement deposition in 12 of the 14 PCDH7-associated cases. Confocal microscopy showed colocalization of PCDH7 and IgG along the GBM. Western blot analysis using sera from six patients showed antibodies to nonreduced PCDH7. Elution of IgG from frozen tissue of PCDH7-associated MN showed reactivity against PCDH7. CONCLUSIONS: MN associated with the protocadherin PCDH7 appears to be a distinct, previously unidentified type of MN.

When contactin antibodies hit the podocyte: a new neurorenal syndrome.

Membranous nephropathy can be associated with various etiologies and antigens. In this issue of Kidney International, Le Quintrec et al. described contactin-1 cell adhesion molecule as a novel target antigen shared by the peripheral nerve and podocyte in patients with neurological disease and membranous nephropathy.

The clinicopathologic spectrum of segmental membranous glomerulopathy.

Membranous glomerulopathy (MGN) is characterized by global subepithelial immune deposits that stain most intensely by immunofluorescence for IgG. Here we describe the clinical and pathologic findings in a cohort of patients with MGN in which, by definition, only segmental immune deposits are present. This rare variant, termed segmental MGN (sMGN), is poorly characterized. We retrospectively identified all patients with sMGN diagnosed at Columbia University from January 2010 to October 2018, excluding those with systemic lupus erythematosus. Data on presenting features, pathologic findings, and outcomes were collected. Fifty cases of sMGN were identified, representing 2.5% of MGN. In 21 of 50 biopsies, there was an alternative, predominant disease process. The remaining 29 patients with isolated sMGN had a median creatinine of 0.97 mg/dl, median 24-hour urine protein 3.1 g/day, and 32% had nephrotic syndrome. Staining for NELL-1 (a protein kinase C binding protein) was positive in five of 17 cases. Staining for PLA2R, THSD7A, and exostosin 1 (autoantigens in primary MGN) was negative in all biopsies evaluated. Ultrastructural evaluation revealed predominantly early stage sMGN (stage 1 or 1-2 in 14/29). Follow-up was available for 21 of the 29 patients with isolated sMGN (median 12 months), including seven who received immunosuppression (primarily glucocorticoids). During follow-up, 86% had stable/improved kidney function and 45% achieved complete while 15% achieved partial remission. Among the 15 patients with isolated sMGN without full nephrotic syndrome, only two received immunosuppression; nonetheless, 50% achieved complete while 21% achieved partial remission. Thus, sMGN is a rare PLA2R-negative variant of MGN with 29% NELL-1 positivity and favorable prognosis, even in the absence of immunosuppressive treatment.

HLA-D and PLA2R1 risk alleles associate with recurrent primary membranous nephropathy in kidney transplant recipients.

Recurrence of primary membranous nephropathy after transplantation occurs in up to 44% of patients and is driven by PLA2R antibody. Here, we asked whether genetic determinants could improve risk prediction. First, we sequenced PLA2R1 and HLA-D loci in 248 patients with primary membranous nephropathy and identified two independent single nucleotide polymorphisms (SNPs) at risk for primary membranous nephropathy at each locus. These were rs9271188 (intergenic between HLA-DRB1 and HLA-DQA1,) and rs9275086 (intergenic between HLA-DQB1 and HLA-DQA2) at the HLA-D locus along with rs6726925 and rs13018963 at the PLA2R1 locus. Then we investigated whether primary membranous nephropathy at-risk variants were associated with recurrence in a retrospective cohort of 105 donor-recipient pairs and a replication cohort of 40 pairs. Seven SNPs located between HLA-DRB1 and HLA-DQA1 in linkage disequilibrium with rs9271188, and three SNPs in the PLA2R1 region predicted recurrence when presented by the donor, but not when presented by the recipient. The two SNPs in the HLA-D region most strongly associated with recurrence (rs9271705 and rs9271550) were confirmed in the replication cohort. A genetic risk score based on the two best predictors at each locus (rs9271705, rs9271550, rs17830558, and rs3828323) identified a group of patients with high risk of recurrence. Thus, our results suggest that the graft contributes to recurrence of primary membranous nephropathy through the disease susceptibility HLA-D and PLA2R1 SNPs in an autoimmune milieu. Further studies are needed before implementation of genetic testing for these in donor selection.

Membranous nephropathy: current understanding of various causes in light of new target antigens.

PURPOSE OF REVIEW: Membranous nephropathy is an autoimmune disease caused by antibodies mostly directed to podocyte antigens. PLA2R and THSD7A antigens were described in 2009 and 2014 using classical immunochemical techniques. In the last 2 years, thanks to the combination of laser microdissection of glomeruli and mass spectrometry of solubilized digested proteins, several antigens associated with various causes have been described in patients with membranous nephropathy. The purpose of this review is to report on those "new" antigens and to analyse the clinicopathological correlations that make each of this antigen unique. RECENT FINDINGS: This article covers the literature of the last 2 years devoted to the description of those new antigens and biomarkers including NELL-1 and Semaphorin 3B in primary membranous nephropathy, and exostosins 1 and 2 and NCAM in lupus class V membranous nephropathy, which will be compared with the previously described antigens. These findings will lead to propose a new classification of membranous nephropathy based on serology and tissue antigen identification that could/should substitute for the classical distinction between primary and secondary membranous nephropathy. SUMMARY: The discovery of the latest antigens has major implications for the care of patients with membranous nephropathy as they drive the etiologic investigations and provide invaluable markers for treatment monitoring.

Semaphorin 3B-associated membranous nephropathy is a distinct type of disease predominantly present in pediatric patients.

Membranous nephropathy results from subepithelial antigen-antibody complex deposition along the glomerular basement membrane. Although PLA2R, THSD7A, and NELL-1 account for a majority (about 80%) of the target antigens, the target antigen in the remaining cases is not known. Using laser microdissection of PLA2R-negative glomeruli of patients with membranous nephropathy followed by mass spectrometry we identified a unique protein, Semaphorin 3B, in three cases. Mass spectrometry failed to detect Semaphorin-3B in 23 PLA2R-associated cases of membranous nephropathy and 88 controls. Semaphorin 3B in all three cases was localized to granular deposits along the glomerular basement membrane by immunohistochemistry. Next, an additional eight cases of Semaphorin 3B-associated membranous nephropathy were identified in three validation cohorts by immunofluorescence microscopy. In four of 11 cases, kidney biopsy also showed tubular basement membrane deposits of IgG on frozen sections. Confocal microscopy showed that both IgG and Semaphorin 3B co-localized to the glomerular basement membrane. Western blot analysis of five available sera showed reactivity to reduced Semaphorin 3B in four of four patients with active disease and no reactivity in one patient in clinical remission; there was also no reactivity in control sera. Eight of the 11 cases of Semaphorin 3B-associated membranous nephropathy were pediatric cases. Furthermore, in five cases, the disease started at or below the age of two. Thus, Semaphorin 3B-associated membranous nephropathy appears to be a distinct type of disease; more likely to be present in pediatric patients.

Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.

Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of primary membranous nephropathy cases, respectively. In the remaining cases, the target antigen is unknown. Here, laser microdissection of glomeruli followed by mass spectrometry was used to identify novel antigen(s) in PLA2R-negative membranous nephropathy. An initial pilot mass spectrometry study in 35 cases of PLA2R-negative membranous nephropathy showed high spectral counts for neural tissue encoding protein with EGF-like repeats, NELL-1, in six cases. Mass spectrometry failed to detect NELL-1 in 23 PLA2R-associated membranous nephropathy and 88 controls. NELL-1 was localized by immunohistochemistry, which showed bright granular glomerular basement membrane staining for NELL-1 in all six cases. Next, an additional 23 NELL-1 positive cases of membranous nephropathy were identified by immunohistochemistry in a discovery cohort of 91 PLA2R-negative membranous nephropathy cases, 14 were confirmed by mass spectrometry. Thus, 29 of 126 PLA2R-negative cases were positive for NELL-1. PLA2R-associated membranous nephropathy and controls stained negative for NELL-1. We then identified five NELL-1 positive cases of membranous nephropathy out of 84 PLA2R and THSD7A-negative cases in two validation cohorts from France and Belgium. By confocal microscopy, both IgG and NELL-1 co-localized to the glomerular basement membrane. Western blot analysis showed reactivity to NELL-1 in five available sera, but no reactivity in control sera. Clinical and biopsy findings of NELL-1 positive membranous nephropathy showed features of primary membranous nephropathy. Thus, a subset of membranous nephropathy is associated with accumulation and co-localization of NELL-1 and IgG along the glomerular basement membrane, and with anti-NELL-1 antibodies in the serum. Hence, NELL-1 defines a distinct type of primary membranous nephropathy.

Common risk variants in NPHS1 and TNFSF15 are associated with childhood steroid-sensitive nephrotic syndrome.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (Pmeta=6.71E-28, OR=1.88) and TNFSF15 (Pmeta=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).

Podocyte Antigen Staining to Identify Distinct Phenotypes and Outcomes in Membranous Nephropathy: A Retrospective Multicenter Cohort Study.

RATIONALE & OBJECTIVE: Membranous nephropathy (MN) is characterized by the deposition of immune complexes along glomerular basement membranes. M-Type phospholipase A2 receptor (PLA2R), thrombospondin type 1 domain-containing 7A (THSD7A), exostosin 1 and 2 (EXT1/2), and neural epidermal growth factor-like 1 protein (NELL-1) have been identified as established or potential podocyte antigens in MN. We investigated the association of podocyte antigen staining with MN clinical phenotype and outcomes. STUDY DESIGN: Multicenter retrospective cohort study. SETTING & PARTICIPANTS: 177 consecutive patients with MN unrelated to lupus erythematosus, identified after screening of 3,875 native kidney biopsies performed in the Belgian UCLouvain Kidney Disease Network from 2000 through 2018. PREDICTOR: Positive immunostaining for podocyte antigens on archived kidney biopsy samples. OUTCOMES: Association with different phenotypes (baseline characteristics of patients and pathologic findings on kidney biopsy), time to cancer and to kidney failure. ANALYTICAL APPROACH: Kaplan-Meier estimates and Cox regression analyses to assess time to cancer and kidney failure. RESULTS: 177 patients were followed up for a median of 4.0 (IQR, 1.3-8.0) years. Diagnosis of PLA2R-positive (PLA2R+), THSD7A+, and double-negative (PLA2R-/THSD7A-) MN was made in 117 (66.1%), 6 (3.4%), and 54 (30.5%) patients, respectively. Progression to kidney failure was similar in all groups. Although the number of patients with THSD7A+MN was small, they showed a higher incidence (50%) and increased risk for developing cancer during follow-up (adjusted HR, 5.0 [95% CI, 1.4-17.9]; P=0.01). 8% and 5% of patients with double-negative MN stained positively for EXT1/2 and NELL-1, respectively. Most patients with EXT1/2+MN were women, had features of systemic autoimmunity, and showed glomerular C1q deposits. LIMITATIONS: Retrospective design; small number of patients in the THSD7A group; lack of evaluation of immunoglobulin G subclasses deposition. CONCLUSIONS: Our real-world data describe the relative prevalence of subgroups of MN and support the hypothesis that a novel classification of MN based on podocyte antigen staining may be clinically relevant.

Exostosin 1/Exostosin 2-Associated Membranous Nephropathy.

BACKGROUND: In membranous nephropathy (MN), which is characterized by deposition of immune complexes along the glomerular basement membrane (GBM), phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A are target antigens in approximately 70% and 1%-5% of cases of primary MN, respectively. In other cases of primary MN and in secondary MN, the target antigens are unknown. METHODS: We studied 224 cases of biopsy-proven PLA2R-negative MN and 102 controls (including 47 cases of PLA2R-associated MN) in pilot and discovery cohorts. We also evaluated 48 cases of PLA2R-negative presumed primary MN and lupus MN in a validation cohort. We used laser microdissection and mass spectrometry to identify new antigens, which were localized by immunohistochemistry. RESULTS: Mass spectrometry detected exostosin 1 (EXT1) and exostosin 2 (EXT2) in 21 cases of PLA2R-negative MN, but not in PLA2R-associated MN and control cases. Immunohistochemistry staining revealed bright granular GBM staining for EXT1 and EXT2. Clinical and biopsy findings showed features of autoimmune disease, including lupus, in 80.7% of the 26 EXT1/EXT2-associated MN cases we identified. In the validation cohort, we confirmed that EXT1/EXT2 staining was detected in pure class 5 lupus nephritis (eight of 18 patients) and in presumed primary MN associated with signs of autoimmunity (three of 16 patients); only one of the 14 cases of mixed class 5 and 3/4 lupus nephritis was positive for EXT1/EXT2. Tests in seven patients with EXT1/EXT2-associated MN found no circulating anti-exostosin antibodies. CONCLUSIONS: A subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Autoimmune disease is common in this group of patients.

Novel ELISA for thrombospondin type 1 domain-containing 7A autoantibodies in membranous nephropathy.

Autoantibodies against phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type 1 domain-containing 7A (THSD7A) are emerging as biomarkers to classify membranous nephropathy (MN) and to predict outcome or response to treatment. Anti-THSD7A autoantibodies are detected by Western blot and indirect immunofluorescence test (IIFT). Here, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) optimized for quantitative detection of anti-THSD7A autoantibodies. Among 1012 biopsy-proven MN patients from 6 cohorts, 28 THSD7A-positive patients were identified by ELISA, indicating a prevalence of 2.8%. By screening additional patients, mostly referred because of PLA2R1-unrelated MN, we identified 21 more cases, establishing a cohort of 49 THSD7A-positive patients. Twenty-eight patients (57%) were male, and male patients were older than female patients (67 versus 49 years). Eight patients had a history of malignancy, but only 3 were diagnosed with malignancy within 2 years of MN diagnosis. We compared the results of ELISA, IIFT, Western blot, and biopsy staining, and found a significant correlation between ELISA and IIFT titers. Anti-THSD7A autoantibodies were predominantly IgG4 in all patients. Eight patients were double positive for THSD7A and PLA2R1. Levels of anti-THSD7A autoantibodies correlated with disease activity and with response to treatment. Patients with high titer at baseline had poor clinical outcome. In a subgroup of patients with serial titers, persistently elevated anti-THSD7A autoantibodies were observed in patients who did not respond to treatment or did not achieve remission. We conclude that the novel anti-THSD7A ELISA can be used to identify patients with THSD7A-associated MN and to monitor autoantibody titers during treatment.

Phospholipase A2 Receptor 1 Epitope Spreading at Baseline Predicts Reduced Likelihood of Remission of Membranous Nephropathy.

The phospholipase A2 receptor (PLA2R1) is the major autoantigen in primary membranous nephropathy. Several PLA2R1 epitopes have been characterized, and a retrospective study identified PLA2R1 epitope spreading as a potential indicator of poor prognosis. Here, we analyzed the predictive value of anti-PLA2R1 antibody (PLA2R1-Ab) titers and epitope spreading in a prospective cohort of 58 patients positive for PLA2R1-Ab randomly allocated to rituximab (n=29) or antiproteinuric therapy alone (n=29). At baseline, the epitope profile (CysR, CysRC1, CysRC7, or CysRC1C7) did not correlate with age, sex, time from diagnosis, proteinuria, or serum albumin, but epitope spreading strongly correlated with PLA2R1-Ab titer (P<0.001). Ten (58.8%) of the 17 patients who had epitope spreading at baseline and were treated with rituximab showed reversal of epitope spreading at month 6. In adjusted analysis, epitope spreading at baseline was associated with a decreased remission rate at month 6 (odds ratio, 0.16; 95% confidence interval, 0.04 to 0.72; P=0.02) and last follow-up (median, 23 months; odds ratio, 0.14; 95% confidence interval, 0.03 to 0.64; P=0.01), independently from age, sex, baseline PLA2R1-Ab level, and treatment group. We propose that epitope spreading at baseline be considered in the decision for early therapeutic intervention in patients with primary membranous nephropathy.

Transethnic, Genome-Wide Analysis Reveals Immune-Related Risk Alleles and Phenotypic Correlates in Pediatric Steroid-Sensitive Nephrotic Syndrome.

Background Steroid-sensitive nephrotic syndrome (SSNS) is a childhood disease with unclear pathophysiology and genetic architecture. We investigated the genomic basis of SSNS in children recruited in Europe and the biopsy-based North American NEPTUNE cohort.Methods We performed three ancestry-matched, genome-wide association studies (GWAS) in 273 children with NS (Children Cohort Nephrosis and Virus [NEPHROVIR] cohort: 132 European, 56 African, and 85 Maghrebian) followed by independent replication in 112 European children, transethnic meta-analysis, and conditional analysis. GWAS alleles were used to perform glomerular cis-expression quantitative trait loci studies in 39 children in the NEPTUNE cohort and epidemiologic studies in GWAS and NEPTUNE (97 children) cohorts.Results Transethnic meta-analysis identified one SSNS-associated single-nucleotide polymorphism (SNP) rs1063348 in the 3' untranslated region of HLA-DQB1 (P=9.3×10-23). Conditional analysis identified two additional independent risk alleles upstream of HLA-DRB1 (rs28366266, P=3.7×10-11) and in the 3' untranslated region of BTNL2 (rs9348883, P=9.4×10-7) within introns of HCG23 and LOC101929163 These three risk alleles were independent of the risk haplotype DRB1*07:01-DQA1*02:01-DQB1*02:02 identified in European patients. Increased burden of risk alleles across independent loci was associated with higher odds of SSNS. Increased burden of risk alleles across independent loci was associated with higher odds of SSNS, with younger age of onset across all cohorts, and with increased odds of complete remission across histologies in NEPTUNE children. rs1063348 associated with decreased glomerular expression of HLA-DRB1, HLA-DRB5, and HLA-DQB1.Conclusions Transethnic GWAS empowered discovery of three independent risk SNPs for pediatric SSNS. Characterization of these SNPs provide an entry for understanding immune dysregulation in NS and introducing a genomically defined classification.