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.).

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.

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.

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.

Multi-population genome-wide association study implicates immune and non-immune factors in pediatric steroid-sensitive nephrotic syndrome.

Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is the most common childhood glomerular disease. Previous genome-wide association studies (GWAS) identified a risk locus in the HLA Class II region and three additional independent risk loci. But the genetic architecture of pSSNS, and its genetically driven pathobiology, is largely unknown. Here, we conduct a multi-population GWAS meta-analysis in 38,463 participants (2440 cases). We then conduct conditional analyses and population specific GWAS. We discover twelve significant associations-eight from the multi-population meta-analysis (four novel), two from the multi-population conditional analysis (one novel), and two additional novel loci from the European meta-analysis. Fine-mapping implicates specific amino acid haplotypes in HLA-DQA1 and HLA-DQB1 driving the HLA Class II risk locus. Non-HLA loci colocalize with eQTLs of monocytes and numerous T-cell subsets in independent datasets. Colocalization with kidney eQTLs is lacking but overlap with kidney cell open chromatin suggests an uncharacterized disease mechanism in kidney cells. A polygenic risk score (PRS) associates with earlier disease onset. Altogether, these discoveries expand our knowledge of pSSNS genetic architecture across populations and provide cell-specific insights into its molecular drivers. Evaluating these associations in additional cohorts will refine our understanding of population specificity, heterogeneity, and clinical and molecular associations.

Network-Based Assessment of Minimal Change Disease Identifies Glomerular Response to IL-7 and IL-12 Pathways Activation as Innovative Treatment Target.

Background: Minimal change disease (MCD), a major cause of nephrotic syndrome, is usually treated by corticosteroid administration. MCD unresponsiveness to therapy and recurrences are nonetheless frequently observed, particularly in adults. To explore MCD-related pathogenetic mechanisms and to identify novel drug targets ultimately contributing to novel therapeutic avenues with a certain specificity for MCD, we compared glomerular transcriptomes from MCD with membranous nephropathy (MN) patients and healthy controls. Methods: Renal biopsies from adult patients with MCD (n = 14) or MN (n = 12), and non-diseased controls (n = 8) were selected from the Norwegian Kidney Biopsy Registry. RNA for 75 base-pair paired-end RNASeq were obtained from laser capture micro-dissected (LCM) glomeruli from FFPE sections. Transcriptional landscapes were computed by combining pathway-centered analyses and network science methodologies that integrate multiple bioinformatics resources. Results: Compared to normal glomeruli, cells from MCD displayed an inflammatory signature apparently governed by the IL1 and IL7 systems. While enrichment of IL1 production and secretion was a shared feature of MCD and MN compared to normal tissue, responses involving IL7 pathway activation were unique to MCD. Indeed, IL7R expressed by glomeruli was the most upregulated gene of the interleukin family in MCD versus normal controls. IL7 pathway activation was paralleled by significant enrichment in adaptive immune system processes and transcriptional regulation and depletion in pathways related to energy metabolism and transcription. Downregulation of these organ function-related themes again occurred predominately in MCD and was significantly less pronounced in MN. Immunofluorescence and immunohistochemistry, respectively, confirmed the expression of phosphorylated IL-7 receptor alpha (IL7RA, CD127) and IL12 receptor beta 1 (IL12RB1) proteins. Conclusions: Gene expression profiling of archival FFPE-biopsies identifies MCD-specific signatures with IL7RA and IL12RB1 as novel targets for MCD 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.

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.

Sexual dimorphic response to rituximab treatment: A longitudinal observational study in a large cohort of patients with primary membranous nephropathy and persistent nephrotic syndrome.

Rituximab is one of the first-line therapies for patients with membranous nephropathy (MN) at high risk of progression towards kidney failure. We investigated whether the response to Rituximab was affected by sex and anti-PLA2R antibody levels in 204 consecutive patients (148 males and 56 females) with biopsy-proven MN who were referred to the Nephrology Unit of the Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII from March 2001 to October 2016 and managed conservatively for at least 6 months. The primary outcome was a combined endpoint of complete (proteinuria <0.3 g/24 h) or partial (proteinuria <3.0 g/24 h and >50% reduction vs. baseline) remission. Patients gave written informed consent to Rituximab treatment. The study was internally funded. No pharmaceutical company was involved. Anti-PLA2R antibodies were detectable in 125 patients (61.3%). At multivariable analyses, female gender (p = 0.0198) and lower serum creatinine levels (p = 0.0108) emerged as independent predictors of better outcome (p = 0.0198). The predictive value of proteinuria (p = 0.054) and anti-PLA2R titer (p = 0.0766) was borderline significant. Over a median (IQR) of 24.8 (12.0-36.0) months, 40 females (71.4%) progressed to the combined endpoint compared with 73 males (49.3%). Anti-PLA2R titers at baseline [127.6 (35.7-310.8) vs. 110.1 (39.9-226.7) RU/ml] and after Rituximab treatment were similar between the sexes. However, the event rate was significantly higher in females than in males [HR (95%): 2.12 (1.44-3.12), p = 0.0001]. Forty-five of the 62 patients (72.3%) with anti-PLA2R titer below the median progressed to the combined endpoint versus 35 of the 63 (55.6%) with higher titer [HR (95%): 1.97 (1.26-3.07), p < 0.0029]. The highest probability of progressing to the combined endpoint was observed in females with anti-PLA2R antibody titer below the median (86.7%), followed by females with anti-PLA2R antibody titer above the median (83.3%), males with titer below the median (68.1%), and males with titer above the median (44.4%). This trend was statistically significant (p = 0.0023). Similar findings were observed for complete remission (proteinuria <0.3 g/24 h) and after analysis adjustments for baseline serum creatinine. Thus, despite similar immunological features, females were more resilient to renal injury following Rituximab therapy. These findings will hopefully open new avenues to identify the molecular pathways underlying sex-related nephroprotective effects.

Serum Protein Signatures Using Aptamer-Based Proteomics for Minimal Change Disease and Membranous Nephropathy.

Introduction: Minimal change disease (MCD) and membranous nephropathy (MN) are glomerular diseases (glomerulonephritis [GN]) that present with the nephrotic syndrome. Although circulating PLA2R antibodies have been validated as a biomarker for MN, the diagnosis of MCD and PLA2R-negative MN still relies on the results of kidney biopsy or empirical corticosteroids in children. We aimed to identify serum protein biomarker signatures associated with MCD and MN pathogenesis using aptamer-based proteomics. Methods: Quantitative SOMAscan proteomics was applied to the serum of adult patients with MCD (n = 15) and MN (n = 37) and healthy controls (n = 20). Associations between the 1305 proteins detected with SOMAscan were assessed using multiple statistical tests, expression pattern analysis, and systems biology analysis. Results: A total of 208 and 244 proteins were identified that differentiated MCD and MN, respectively, with high statistical significance from the healthy controls (Benjamin-Hochberg [BH] P < 0.0001). There were 157 proteins that discriminated MN from MCD (BH P < 0.05). In MCD, 65 proteins were differentially expressed as compared with MN and healthy controls. When compared with MCD and healthy controls, 44 discriminatory proteins were specifically linked to MN. Systems biology analysis of these signatures identified cell death and inflammation as key pathways differentiating MN from MCD and healthy controls. Dysregulation of fatty acid metabolism pathways was confirmed in both MN and MCD as compared with the healthy subjects. Conclusion: SOMAscan represents a promising proteomic platform for biomarker development in GN. Validation of a greater number of discovery biomarkers in larger patient cohorts is needed before these data can be translated for clinical care.

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.

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.

Membranous nephropathy.

Membranous nephropathy (MN) is a glomerular disease that can occur at all ages. In adults, it is the most frequent cause of nephrotic syndrome. In ~80% of patients, there is no underlying cause of MN (primary MN) and the remaining cases are associated with medications or other diseases such as systemic lupus erythematosus, hepatitis virus infection or malignancies. MN is an autoimmune disease characterized by a thickening of the glomerular capillary walls due to immune complex deposition. Identification of the phospholipase A2 receptor (PLA2R) as the major antigen in adults in 2009 induced a paradigm shift in disease diagnosis and monitoring and several other antigens have since been characterized. Disease outcome is difficult to predict and around one-third of patients will undergo spontaneous remission. In those at high risk of progression, immunosuppressive therapy with cyclophosphamide plus corticosteroids has substantially reduced the need for kidney replacement therapy. Owing to carcinogenic risk, other treatments (calcineurin inhibitors and CD20-targeted B cell depletion therapy (rituximab)) have been developed. However, disease relapses are frequent when calcineurin inhibitors are stopped and the remission rate with rituximab is lower than with cyclophosphamide, particularly in patients with high PLA2R antibody titres. Other new drugs are already available and antigen-specific immunotherapies are being developed.

Anti-PLA2R1 Antibodies as Prognostic Biomarker in Membranous Nephropathy.

INTRODUCTION: Personalized treatment for patients with membranous nephropathy requires accurate prediction of the disease course at an early stage. In this study, we evaluated the value of baseline anti-phospholipase A2 receptor (PLA2R1) antibody titer as a prognostic biomarker in patients with PLA2R1-associated membranous nephropathy. METHODS: In this cohort study, we included 168 patients (118 men, 50 women) referred to our nephrology center between February 1995 and November 2016. Mean age was 52 ± 13 years. There were 156 patients with new-onset disease and 12 patients with a relapse (n = 10) or recent use of immunosuppressive therapy (n = 2). We measured anti-PLA2R1 titer at baseline and analyzed progression to severe disease (30% increase of serum creatinine or start of immunosuppressive therapy) as a primary study endpoint over 60 months. RESULTS: There was a clear association between anti-PLA2R1 antibody titer and severity of the nephrotic syndrome. In univariate analysis, anti-PLA2R1 antibody titer was also associated with disease progression. However, in Cox proportional hazard models that included proteinuria and serum creatinine, anti-PLA2R1 antibody titer was no longer associated with clinical outcome. Results were similar when limiting the analysis to the patients with new-onset disease. CONCLUSION: Our study questions the relevance of single measurement of anti-PLA2R1 antibodies at baseline as a prognostic biomarker in membranous nephropathy. Future studies are needed to determine the possible role of sequential measurements of anti-PLA2R1 antibodies as a prognostic biomarker of disease progression.