Functional characterization of podocyte-expressed THSD7A in experimental membranous nephropathy
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE300256 | GEO | 2026/02/02
REPOSITORIES: GEO
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Project description:Proteomics experiment 3/3 associated with this work. THSD7A is a key podocyte antigen in membranous nephropathy (MN), an antibody-mediated autoimmune disease. While the pathogenic role of autoantibodies targeting THSD7A in MN is well described, the consequences of autoantibody binding for podocyte homeostasis as well as the functional role of THSD7A in podocytes are incompletely understood. A model of THSD7A-associated MN was induced using rabbit anti-THSD7A antibodies in wild-type (WT) as well as mice lacking THSD7A expression specifically in podocytes (Thsd7a-/- mice). Clinical and histological parameters as well as alterations in the transcriptome and proteome were analyzed. The THSD7A protein interactome was analyzed by pulldown experiments followed by proteomic identification of interaction partners. In the experimental mouse model of THSD7A-associated MN, we observed substantial protein downregulation of key slit diaphragm (SD) proteins such as nephrin and neph1, which was not a consequence of reduced SD protein transcription. Glomeruli showed substantial transcriptomic and proteomic reconfiguration indicative of extensive podocyte injury, including increased interactions between extracellular matrix and receptors, and disruptions in podocyte adhesion, actin-binding, and cytoskeleton-related genes and proteins. These disruptions were at least in part driven by transcription factors such as Egr1, Egr3, Maff, Fosb and Klf4, which are involved in stress responses and inflammation. We also observed upregulation of the ubiquitin-proteasome system, lysosomal pathways, and proteases such as cathepsin S and Q, ADAM10, ADAM15 and ADAM17, which, in combination, may contribute to the loss of SD proteins in MN. While Thsd7a-/- mice only had a mild phenotype under basal conditions, they were completely protected from MN development upon anti-THSD7A antibody transfer. Interactomic analysis of THSD7A revealed interactions with integrin 3 (ITGA3) and other glomerular proteins, implicating THSD7A complexes in pathogenic regulation of cytoskeleton organization, adhesion dynamics, and membrane signaling in MN. Anti-THSD7A antibodies induce transcriptional reprogramming and marked protein reconfiguration, including an upregulation of the proteasome and cleavage, revealing potential new therapeutic targets in MN.
2026-01-30 | PXD065344 | Pride
Project description:Proteomics experiment 2/3 associated with this work. THSD7A is a key podocyte antigen in membranous nephropathy (MN), an antibody-mediated autoimmune disease. While the pathogenic role of autoantibodies targeting THSD7A in MN is well described, the consequences of autoantibody binding for podocyte homeostasis as well as the functional role of THSD7A in podocytes are incompletely understood. A model of THSD7A-associated MN was induced using rabbit anti-THSD7A antibodies in wild-type (WT) as well as mice lacking THSD7A expression specifically in podocytes (Thsd7a-/- mice). Clinical and histological parameters as well as alterations in the transcriptome and proteome were analyzed. The THSD7A protein interactome was analyzed by pulldown experiments followed by proteomic identification of interaction partners. In the experimental mouse model of THSD7A-associated MN, we observed substantial protein downregulation of key slit diaphragm (SD) proteins such as nephrin and neph1, which was not a consequence of reduced SD protein transcription. Glomeruli showed substantial transcriptomic and proteomic reconfiguration indicative of extensive podocyte injury, including increased interactions between extracellular matrix and receptors, and disruptions in podocyte adhesion, actin-binding, and cytoskeleton-related genes and proteins. These disruptions were at least in part driven by transcription factors such as Egr1, Egr3, Maff, Fosb and Klf4, which are involved in stress responses and inflammation. We also observed upregulation of the ubiquitin-proteasome system, lysosomal pathways, and proteases such as cathepsin S and Q, ADAM10, ADAM15 and ADAM17, which, in combination, may contribute to the loss of SD proteins in MN. While Thsd7a-/- mice only had a mild phenotype under basal conditions, they were completely protected from MN development upon anti-THSD7A antibody transfer. Interactomic analysis of THSD7A revealed interactions with integrin 3 (ITGA3) and other glomerular proteins, implicating THSD7A complexes in pathogenic regulation of cytoskeleton organization, adhesion dynamics, and membrane signaling in MN. Anti-THSD7A antibodies induce transcriptional reprogramming and marked protein reconfiguration, including an upregulation of the proteasome and cleavage, revealing potential new therapeutic targets in MN.
2026-01-30 | PXD065323 | Pride
Project description:Proteomics experiment 1/3 associated with this work. THSD7A is a key podocyte antigen in membranous nephropathy (MN), an antibody-mediated autoimmune disease. While the pathogenic role of autoantibodies targeting THSD7A in MN is well described, the consequences of autoantibody binding for podocyte homeostasis as well as the functional role of THSD7A in podocytes are incompletely understood. A model of THSD7A-associated MN was induced using rabbit anti-THSD7A antibodies in wild-type (WT) as well as mice lacking THSD7A expression specifically in podocytes (Thsd7a-/- mice). Clinical and histological parameters as well as alterations in the transcriptome and proteome were analyzed. The THSD7A protein interactome was analyzed by pulldown experiments followed by proteomic identification of interaction partners. In the experimental mouse model of THSD7A-associated MN, we observed substantial protein downregulation of key slit diaphragm (SD) proteins such as nephrin and neph1, which was not a consequence of reduced SD protein transcription. Glomeruli showed substantial transcriptomic and proteomic reconfiguration indicative of extensive podocyte injury, including increased interactions between extracellular matrix and receptors, and disruptions in podocyte adhesion, actin-binding, and cytoskeleton-related genes and proteins. These disruptions were at least in part driven by transcription factors such as Egr1, Egr3, Maff, Fosb and Klf4, which are involved in stress responses and inflammation. We also observed upregulation of the ubiquitin-proteasome system, lysosomal pathways, and proteases such as cathepsin S and Q, ADAM10, ADAM15 and ADAM17, which, in combination, may contribute to the loss of SD proteins in MN. While Thsd7a-/- mice only had a mild phenotype under basal conditions, they were completely protected from MN development upon anti-THSD7A antibody transfer. Interactomic analysis of THSD7A revealed interactions with integrin 3 (ITGA3) and other glomerular proteins, implicating THSD7A complexes in pathogenic regulation of cytoskeleton organization, adhesion dynamics, and membrane signaling in MN. Anti-THSD7A antibodies induce transcriptional reprogramming and marked protein reconfiguration, including an upregulation of the proteasome and cleavage, revealing potential new therapeutic targets in MN.
2026-01-30 | PXD065322 | Pride
Project description:Background: In recent years, an innovative strategy using laser microdissection and mass spectrometry markedly expanded the landscape of antigens associated with membranous nephropathy (MN). Specific associations with phenotypes, diseases and sometimes reversible triggers led to a novel antigen-based classification of MN, paving the way for precision medicine and stressing the need for more routine use of proteomics in MN. Methods: To explore the proteomic landscape of human glomeruli and identify podocyte antigens and disease mechanisms in MN, we expanded the original technique to an integrative approach combining laser capture microdissection, next-generation mass spectrometry and computational analysis. Next to conventional data-dependent acquisition (DDA), we used and assessed the diagnostic yield of the more comprehensive data-independent acquisition (DIA) mass spectrometry, which enables the detection and quantification of every peptide in a sample irrespective of its level of abundance or m/z value. Our proteomic pipeline was applied to residual material from kidney biopsies in 64 individuals, including 31 healthy controls; 5 disease controls; 5 PLA2R-associated MN; and 23 PLA2R-negative MN. Results: Unbiased analyses confirmed the significant enrichment in PLA2R, IgG4 and complement proteins in glomeruli from patients with PLA2R-MN compared with healthy and disease controls, while molecular characterization of complement fragments provided evidence for complement activation in PLA2R-MN. Compared to DDA, DIA mass spectrometry increased the number of glomerular proteins (~3800 vs. ~1200) identified in healthy glomeruli; allowed the detection all known antigens except NELL1 in normal glomeruli; and increased the detection rate of podocyte antigens from 50% to >80% in PLA2R-negative MN. Conclusions: This proof-of-concept study suggests that an integrative approach combining laser microdissection, DIA mass spectrometry and computational biology is a powerful tool, with translational potential, to identify podocyte antigens and unravel disease mechanisms in MN.
2025-03-07 | PXD053837 | Pride
Project description:Background: In recent years, an innovative strategy using laser microdissection and mass spectrometry markedly expanded the landscape of antigens associated with membranous nephropathy (MN). Specific associations with phenotypes, diseases and sometimes reversible triggers led to a novel antigen-based classification of MN, paving the way for precision medicine and stressing the need for more routine use of proteomics in MN. Methods: To explore the proteomic landscape of human glomeruli and identify podocyte antigens and disease mechanisms in MN, we expanded the original technique to an integrative approach combining laser capture microdissection, next-generation mass spectrometry and computational analysis. Next to conventional data-dependent acquisition (DDA), we used and assessed the diagnostic yield of the more comprehensive data-independent acquisition (DIA) mass spectrometry, which enables the detection and quantification of every peptide in a sample irrespective of its level of abundance or m/z value. Our proteomic pipeline was applied to residual material from kidney biopsies in 64 individuals, including 31 healthy controls; 5 disease controls; 5 PLA2R-associated MN; and 23 PLA2R-negative MN. Results: Unbiased analyses confirmed the significant enrichment in PLA2R, IgG4 and complement proteins in glomeruli from patients with PLA2R-MN compared with healthy and disease controls, while molecular characterization of complement fragments provided evidence for complement activation in PLA2R-MN. Compared to DDA, DIA mass spectrometry increased the number of glomerular proteins (~3800 vs. ~1200) identified in healthy glomeruli; allowed the detection all known antigens except NELL1 in normal glomeruli; and increased the detection rate of podocyte antigens from 50% to >80% in PLA2R-negative MN. Conclusions: This proof-of-concept study suggests that an integrative approach combining laser microdissection, DIA mass spectrometry and computational biology is a powerful tool, with translational potential, to identify podocyte antigens and unravel disease mechanisms in MN.
2025-03-07 | PXD054062 | Pride
Project description:we profiled GEC mRNA expression levels in two animal models with podocyte depletion. We used a transgenic mouse model with YFP-labelled GEC for sorting of GEC. Podocyte depletion was induced by injection of mice with a specific anti-podocyte antibody or crossing with a podocyte-specific doxycycline-induced diphtheria toxin A (DTA) expression transgenic mouse model. Both mouse models developed significant proteinuria, glomerulosclerosis, and podocyte depletion. YFP-positive GECs were sorted from these mice for RNA sequencing. Analysis of the differentially expressed genes (DEGs) between the diseased and control mice revealed significant alteration of metabolism and immune system in the antibody-mediated podocyte depletion model while angiogenesis, vascular development, actin cytoskeleton, and focal adhesion pathways were highly enriched in the DTA model. Apoptosis and cell adhesion pathways were enriched in both models. Apoptosis of GEC was confirmed in the kidney of these mice, which leads to the reduction of GEC number. We also identified a list of genes which were altered in both animal models. Among them, we have further validated Cd63, a molecule which is regulated in human DKD and plays a key role in endothelial cell function. In conclusion, this is the first unbiased approach to determine the genomic-wide response of GEC to podocyte depletion in two different animal models. Our study proves that podocyte depletion could induce GEC injury.
2021-06-01 | GSE128736 | GEO
Project description:Membranous nephropathy (MN) is an autoimmune kidney disease with the clinical hallmark of causing high level proteinuria. MN is caused by circulating antibodies binding to targeting antigens on the surface of podocytes – specialized endothelial cells contributing to the formation of the glomerular filtration barrier. Histomorphologically MN is characterized by a deposition of IgG along the glomerular basement membrane (GBM) – predominantly IgG4 in primary MN – as well as an accumulation of the respective target antigen, thickening of the GBM and effacement of podocyte foot processes. The predominant MN target antigen in 70-80% of MN patients is the phospholipase A2 receptor 1. Additional target antigens, which occur with lower frequencies, have recently been described. The aim of the study was the identification of a novel MN target antigen, which fulfills the typical criteria of MN target antigen: being a podocytic membrane protein, which is recognized by IgG4 subclass specific autoantibodies. Performing a mass spectrometry analysis based on a TMT-based relative quantification approach of immunoprecipitated samples resulted in the identification of Netrin G1 (NTNG1) as a novel MN target antigen. The results were validated using immunohistochemistry, Western Blot and ELISA. The molecular characterization of patients with MN will allow a better diagnosis and clinical management of these patients in the future.
2022-08-03 | PXD032713 | Pride
Project description:Background: Minimal change disease (MCD) and focal-segmental glomerulosclerosis (FSGS) are immune-mediated glomerular diseases manifesting as nephrotic syndrome. Autoantibodies against the podocyte slit diaphragm protein nephrin were recently identified in a subset of patients with minimal change disease, but their clinical and pathophysiological significance is largely unknown. Methods: Using immunoprecipitation assays, we performed a blinded screening for anti-nephrin antibodies in diagnostic and follow-up serum samples from adult patients with biopsy-proven MCD, FSGS, IgA nephropathy, and membranous nephropathy in comparison to healthy controls in two independent patient cohorts from Hamburg, Germany, and Bari, Italy. We further established a mouse model of anti-nephrin antibody-induced disease by active immunization using the recombinant murine nephrin ectodomain. Results: Anti-nephrin autoantibodies were detected in 50 of 110 (45%) patients with MCD, 8 of 107 (7%) patients with FSGS, 1 of 50 (2%) patients with membranous nephropathy, 0 of 48 (0%) patients with IgA nephropathy, and 0 of 67 (0%) healthy individuals. During follow-up, presence, and absence of anti-nephrin autoantibodies in patients with MCD and FSGS strongly correlated with active disease and remission, respectively. Immunization of mice induced anti-nephrin autoantibody formation and a highly dynamic phenotype with severe nephrotic syndrome and the histological features of MCD. Mechanistically, anti-nephrin autoantibodies induced nephrin phosphorylation at Tyr1191, cytoskeletal rearrangement, and downregulation of key podocyte proteins. Conclusion: Anti-nephrin antibodies are a valuable biomarker of disease activity in patients with MCD and FSGS, and binding of anti-nephrin antibodies at the podocyte slit diaphragm induces MCD with nephrotic syndrome.
2024-05-27 | PXD047417 | Pride
Project description:Background: Minimal change disease (MCD) and focal-segmental glomerulosclerosis (FSGS) are immune-mediated glomerular diseases manifesting as nephrotic syndrome. Autoantibodies against the podocyte slit diaphragm protein nephrin were recently identified in a subset of patients with minimal change disease, but their clinical and pathophysiological significance is largely unknown. Methods: Using immunoprecipitation assays, we performed a blinded screening for anti-nephrin antibodies in diagnostic and follow-up serum samples from adult patients with biopsy-proven MCD, FSGS, IgA nephropathy, and membranous nephropathy in comparison to healthy controls in two independent patient cohorts from Hamburg, Germany, and Bari, Italy. We further established a mouse model of anti-nephrin antibody-induced disease by active immunization using the recombinant murine nephrin ectodomain. Results: Anti-nephrin autoantibodies were detected in 50 of 110 (45%) patients with MCD, 8 of 107 (7%) patients with FSGS, 1 of 50 (2%) patients with membranous nephropathy, 0 of 48 (0%) patients with IgA nephropathy, and 0 of 67 (0%) healthy individuals. During follow-up, presence, and absence of anti-nephrin autoantibodies in patients with MCD and FSGS strongly correlated with active disease and remission, respectively. Immunization of mice induced anti-nephrin autoantibody formation and a highly dynamic phenotype with severe nephrotic syndrome and the histological features of MCD. Mechanistically, anti-nephrin autoantibodies induced nephrin phosphorylation at Tyr1191, cytoskeletal rearrangement, and downregulation of key podocyte proteins. Conclusion: Anti-nephrin antibodies are a valuable biomarker of disease activity in patients with MCD and FSGS, and binding of anti-nephrin antibodies at the podocyte slit diaphragm induces MCD with nephrotic syndrome.
2024-05-27 | PXD047421 | Pride
Project description:We investigated a glomerulonephritis (GN) model in rats induced by nephrotoxic serum (NTS) which contains antibodies against the glomerular basement membrane (GBM). The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans. Male Wistar Kyoto (WKY) and Sprague Dawley (SD) rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys. The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The ?-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb) and complement component 6 (C6) were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats. Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6. Two different rat strains (WKY and SD) were dosed with different doses of NTS and after 14 days rats were euthanized and kidneys were removed for RNA extraction and hybridization on Affymetrix microarrays. We sought to analyze the deregulation of gene expression during NTS-induced glomerulonephritis and to compare the effect in the two rat strains.
2015-07-06 | E-GEOD-64265 | biostudies-arrayexpress