👤 Karl T Debiec

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. 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. 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. MN associated with the protocadherin PCDH7 appears to be a distinct, previously unidentified type of MN. Show less

Membranous nephropathy (MN) is a rare auto-immune disease where the glomerulus is targeted by circulating auto-antibodies mostly against podocyte antigens, which results in the formation of electron-dense immune complexes, activation of complement and massive proteinuria. MN is the most common cause of nephrotic syndrome in adults leading to severe thrombotic complications and kidney failure. This review is focused on the recent therapeutic and pathophysiological advances that occurred in the last two years. For a long time, we were lacking a head-to-head comparison between cyclophosphamide considered as the gold standard therapy and other medications, notably rituximab. Substantial progress has been achieved owing to three randomized controlled trials. MENTOR (Membranous Nephropathy Trial of Rituximab) and STARMEN (Sequential Therapy with Tacrolimus and Rituximab in Primary Membranous Nephropathy) conclusively established that calcineurin inhibitor-based regimens are slower to result in an immunologic response than rituximab or cyclophosphamide, achieve fewer complete clinical remissions, and are less likely to maintainremission. Rituximab Versus Steroids and Cyclophosphamide in the Treatment of Idiopathic Membranous Nephropathy (RI-CYCLO) suggested that competition between cyclophosphamide and rituximab remains open. Given the technological leap combining laser microdissection of glomeruli and mass spectrometry of solubilized digested proteins, four "new antigens" were discovered including NELL-1 and Semaphorin 3B in so-called primary MN, and exostosins 1 and 2 and NCAM 1 in lupus MN. NELL-1 is associated with about 8% of primary MN and is characterized by segmental immune deposits and frequent association with cancer (30%). Semaphorin 3B-associated MN usually occurs in children, often below the age of two years, where it is the main antigen, representing about 16% of non-lupus MN in childhood. Exostosins 1/2 and NCAM 1 are associated with 30% and 6% of lupus MN, respectively. Exostosins 1/2 (EXT1/2) staining is associated with a low rate of end-stage kidney disease (ESKD) even in mixed classes III/IV+V. These findings already lead to revisiting the diagnostic and therapeutic algorithms toward more personalized medicine. Show less

Membranous nephropathy (MN) is characterized by the deposition of immune complexes along glomerular basement membranes. M-Type phospholipase A Multicenter retrospective cohort study. 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. Positive immunostaining for podocyte antigens on archived kidney biopsy samples. Association with different phenotypes (baseline characteristics of patients and pathologic findings on kidney biopsy), time to cancer and to kidney failure. Kaplan-Meier estimates and Cox regression analyses to assess time to cancer and kidney failure. 177 patients were followed up for a median of 4.0 (IQR, 1.3-8.0) years. Diagnosis of PLA Retrospective design; small number of patients in the THSD7A group; lack of evaluation of immunoglobulin G subclasses deposition. 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. Show less

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in Caucasian adults. Phospholipase A2 receptor (PLA2R)- and exostosin 1 (EXT1)/exostosin 2 (EXT2)-associated MN represent the most common primary and secondary forms of MN. The complement profile using a proteomics approach has not been studied in these 2 common forms of MN. We used laser microdissection and mass spectrometry (MS/MS) to dissect glomeruli and identify glomerular complement proteins in PLA2R-associated ( MS/MS identified high total spectral counts for PLA2R and EXT1/EXT2 in corresponding cases of PLA2R- and EXT1/EXT2-positive MN. Both PLA2R- and EXT1/EXT2-associated MN had high spectral counts of complement proteins C3, C4, C5, C6, C7, C8, and C9. Complement protein C1 was present in low spectral counts in EXT1/EXT2-associated MN. Regulators of complement activation that were detected in MN included higher spectral counts of FH, FHR-1, FHR-5, clusterin, vitronectin and lower spectral counts of FHR-3, FHR-4, and CD59. Low spectral counts of FB and properdin, key components of the alternative pathway, also were detected. IgG4 and IgG1 were the most abundant IgG subclasses in PLA2R- and EXT1/EXT2-associated MN. Lower spectral counts for C3, C4, and C5 were detected in control cases when compared with MN. Significant complement activation is present in MN as evidenced by large spectral counts of complement proteins from C3- and C4-based pathways, including regulatory proteins of complement pathways. These data suggest that anticomplement drugs may be effective in treatment for MN. Show less

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. 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. 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. A subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Autoimmune disease is common in this group of patients. Show less

Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of fibroblast growth factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. However, in vivo whole-organism screens of zebrafish identified compound 1 (BCI) as an allosteric inhibitor of DUSP6. Here we designed and synthesized a panel of analogues to define the structure-activity relationship (SAR) of DUSP6 inhibition. In vivo high-content analysis in transgenic zebrafish, coupled with cell-based chemical complementation assays, identified structural features of the pharmacophore of 1 that were essential for biological activity. In vitro assays of DUSP hyperactivation corroborated the results from in vivo and cellular SAR. The results reinforce the notion that DUSPs are druggable through allosteric mechanisms and illustrate the utility of zebrafish as a model organism for in vivo SAR analyses. Show less