Proteomic profiling of kidney biopsies in nephrotic syndrome
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ABSTRACT: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are patterns of kidney injury observed in the filtering units of the kidney known as glomeruli. From the NURTuRE cohort of individuals with nephrotic syndrome (NS), we performed laser microdissection and mass spectrometry analysis of kidney biopsy samples to identify proteomic patterns of disease. 56 individuals with idiopathic NS segregated by histological pattern (37 MCD and 19 FSGS) across three age groups: early childhood (0-6 years), late childhood (6-18 years) and adult (>18 years). We identified global differences in glomerular cell and extracellular matrix composition related to both histological pattern and age.
Project description:To search for biomarkers to differentiate Adult-Onset Steroid Sensitive focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD). Compared the profiles of glomerular transcriptomes between patients with FSGS and patients with MCD using microarray analysis. This dataset is part of the TransQST collection.
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.
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.
Project description:Podocyte injury is the hallmark of both focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) and is ultimately reflected in foot process effacement proteinuria. Triggers and pathogenic pathways leading to podocyte cytoskeleton rearrangements are however incompletely explained. Here, we aimed to contribute to the understanding of these pathways using tissue bottom-up proteomic profiling of laser capture micro dissected glomeruli from MCD and FSGS.
Project description:Focal segmental glomerulosclerosis (FSGS) is a leading cause of end stage renal disease and remains without specific treatment. To identify novel actors involved in the progression of FSGS, we used an experimental model of nephroangiosclerosis with FSGS in rats and performed a differential transcriptomic analysis. Among the genes that were several-fold upregulated in glomeruli, was Isthmin-1 (ISM), an ‘unknown’ to the kidney gene. The objective of the present study was to find if and what role plays ISM to renal pathophysiology.
Project description:In order to provide a better understanding of molecular interactions/responses of snail host hemocyte and early-developing schistosome larvae (sporocyst stage) we perfomed comparative proteomic analyses on hemocytes during active encapsulation of sporocysts under in vitro conditions
Project description:Kinase deletion strains treated with or without nitrogen starvation Background: Protein kinase-mediated signaling pathways play a pivotal role in regulation of stress responses. Nitrogen starvation (NS) induces sexual development when mating partner available or entry into a quiescent state (G0) in heterothallic background. However, it is unclear whether transcriptional profiles in response to NS allows distinguishing between kinase deletion strains defective in sexual development and/or G0-arrest. Results: In this study, we used DNA microarray to analyze transcriptional profiles in 68 protein kinase deletion strains prior to and after NS. Microarray analyses indicated that NS-induced transcriptional profiles of all kinase strains plus wild type are highly correlated with one another (i.e., Pearson¡¯s correlation coefficient r > 0.7) except for that of pka1¦¤, which showed no high correlation with other 11 kinase strains. Significantly, 9 of 11 strains were defective in sexual development, suggesting that the NS-induced profile of pka1¦¤ serves as a signature with no high correlation with profiles of kinase strains defective in sexual development. Furthermore, we show that expression of the NS-induced genes is elevated in pka1¦¤ and cyr1¦¤ strains prior to NS. On the other hand, levels of the NS-induced genes expression in pka1¦¤, cyr1¦¤, sty1¦¤, and byr1¦¤ are significantly lower than that of wild type. Conclusions: Taken together, our analyses indicate that the cAMP-dependent Pka1-mediated signaling pathway play a major role in regulation of the NS-induced genes prior to and after NS. In addition, the ROS-activated kinase Sty1 and the pheromone-induced kinase Byr1 are also involved in up-regulation of NS-induced genes upon NS stress. 68 kinase deletion strains and other 5 deletion strains were treated with (60m) or without (0min) nitrogen starvation
Project description:Focal segmental glomerulosclerosis (FSGS) is characterized by damage to podocytes, a crucial pathological feature. While several mechanisms of podocyte injury have been suggested, many questions remain unanswered. Rho-associated, coiled-coil-containing protein kinase 2 (ROCK2), a serine/threonine kinase with diverse cellular functions, appears to play a significant role. We observed activation of ROCK2 in podocytes of mice with FSGS induced by adriamycin (ADR), as well as in cultured podocytes exposed to ADR. To delve deeper, we used conditional knockout mice where the ROCK2 gene was specifically disrupted in podocytes (PR2KO). These mice showed resistance to ADR-induced albuminuria, glomerular sclerosis, and podocyte damage. Additionally, pharmacological inhibition of ROCK2 significantly improved podocyte loss and kidney sclerosis in a mouse model of FSGS by counteracting profibrotic factors. RNA sequencing of podocytes treated with a ROCK2 inhibitor confirmed ROCK2's role as a regulator of the cyclic nucleotide signaling pathway. Our findings underscore the potential of ROCK2 inhibition as a therapeutic avenue for FSGS.