RNAseq analysis of glomerular transcriptome of wild-type vs. Podocyte Cosmc knockout
Ontology highlight
ABSTRACT: Cosmc is an obligate chaperone for C1GalT1 T transferase necessary for generating mature mucin-type O-glycans. Animals lacking Cosmc in glomerular podocytes develop proteinuria, glomerulosclerosis and progressive renal failure. This study was designed to examine transcriptome differences in 1 month male animals with and without podocyte Cosmc, a time point when proteinuria is established but glomerular architecture is still relatively intact.
Project description:Obesity can initiate and accelerate the progression of kidney diseases. However, it remains unclear how obesity affects renal dysfunction. Here, we show that a newly generated podocyte-specific tubular sclerosis complex 2 (Tsc2) knockout mouse model (Tsc2∆podocyte) develops proteinuria and dies due to end-stage renal dysfunction by 10 weeks of age. Tsc2∆podocyte mice exhibit an increased glomerular size and focal segmental glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis and proteinaceous casts. Podocytes isolated from Tsc2∆podocyte mice show nuclear factor, erythroid derived 2, like 2-mediated increased oxidative stress response on microarray analysis and their autophagic activity is lowered through the mammalian target of rapamycin (mTOR)—unc-51-like kinase 1 pathway. Rapamycin attenuated podocyte dysfunction and extends survival in Tsc2∆podocyte mice. Additionally, mTOR complex 1 (mTORC1) activity is increased in podocytes of renal biopsy specimens obtained from obese patients with chronic kidney disease. Our work shows that mTORC1 hyperactivation in podocytes leads to severe renal dysfunction and that inhibition of mTORC1 activity in podocytes could be a key therapeutic target for obesity-related kidney diseases.
Project description:The progression of proteinuric kidney disease is associated with podocyte loss but the mechanisms remain unclear. Podocytes reenter the cell cycle to repair damaged ds DNA breaks. However, unsuccessful repair results in podocytes crossing the G1/S checkpoint and undergoes abortive cytokinesis. In this study, we identified Pfn1 as a major contributor in maintaining glomerular integrity and its loss in mice results in severe proteinuria, and kidney failure due to podocyte mitotic catastrophe, characterized by abundant multinucleated cells. Reentry of podocytes were identified by using FUCCI-2aR mice, accompanying the alteration of cell-cycle associated proteins, such as P21, P53, Cyclin B, and Cyclin D. Podocyte-specific translating ribosome affinity purification (TRAP) and RNAseq revealed a reduction of Ribosomal RNA-processing protein 8 (Rrp8) and re-expression of Rrp8 partially rescued the in-vitro phenotype. Clinical analysis of patients with proteinuric kidney disease demonstrated multinucleated podocytes and reduced podocyte profilin1 in kidney tissue. These results suggest that profilin is indispensible in regulating podocyte cell cycle and its disruption contributes to podocyte loss through mitotic catastrophe.
Project description:Podocyte histone deacetylases (HDAC) are essential in maintaining a normal glomerular filtration barrier by modulating podocyte quiescence. Podocyte-specific loss Hdac1 and 2 in mice results in severe proteinuria and sustained DNA damage, likely caused by epigenetic alterations and deficient DNA repair, that result in podocyte senescence. Through glomeruli isolation and RNA-seq profiling from the mutant mice, we demonstrated that senescent podocytes develop a senescence-associated secretory phenotype (SASP) that contribute to the loss of podocytes. The role of HDACs in senescence may provide important clues in our understanding of how podocytes are lost following injury.
Project description:Evidence for reduced expression of cyclin-G associated kinase (GAK) in glomeruli of chronic kidney disease patients was observed in the Nephroseq human database and was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak knockout mice (Gak KO) which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mutant mice to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to this process. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak KO mice. Increased podocyte calpain activity mediated proteolysis of IkB resulted in increased NF-kBp65 induced Gadd45b expression in the Gak KO mice. Our results suggest that loss of podocyte associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.
Project description:Podocytes are highly specialised cells within the glomeruli of the kidney that maintain the filtration barrier by forming interdigitating foot processes and slit-diaphragms. Disruption to these features result in proteinuria and glomerulosclerosis. Studies into podocyte biology and disease have previously relied on conditionally immortalised cell lines due to the non- proliferative nature of this cell type. Here we describe an advanced model to study both podocyte and glomerular biology using isolated glomeruli from kidney organoids derived from human pluripotent stem cells.
Project description:Overexpression of glomerular JAK2 mRNA specifically in glomerular podocytes of 129S6 mice led to significant increases in albuminuria, mesangial expansion, glomerulosclerosis, glomerular fibronectin accumulation, and glomerular basement membrane thickening as well as a significant reduction in podocyte density in diabetic mice. Treatment with a specific JAK1/2 inhibitor partly reversed the major phenotypic changes of DKD
Project description:Available single-cell RNA-seq analyses have revealed that individual cells of the same type differ substantially in gene expression. We wonder whether glomerular podocytes, the cell type that is part of glomerular filtration, also exhibit big difference in gene expression among individual cells; and what biological information could be obtained from the single-podocyte RNA-seq data. Therefore, we isolated mouse glomeruli by Dynabead/magnetic concentration method, and digested them with enzymes to dissociate the cells. We loaded the single cell suspension to a Fluidigm C1 Single-Cell Auto Prep System for single cell cDNA preparation. The cDNA samples were amplified and underwent sequecing using Illumina Highseq 2000 system.
Project description:The specialized glomerular epithelial cell (podocyte) of the kidney is a complex cell that is often damaged in glomerular diseases. Study of this cell type is facilitated by an in vitro system of propagation of conditionally immortalized podocytes. Here, genes that are differentially expressed in this in vitro model of podocyte differentiation are evaluated. Conditionally immortalized undifferentiated mouse podocytes were cultured under permissive conditions at 33*C. Podocytes that were differentiated at the non-permissive conditions at 37*C were used for comparison.
Project description:High fructose diet has been proposed as a major contributor to metabolic syndrome, which is usually accompanied with proteinuria due to glomerular podocyte injury. However, the underlying pathological mechanisms of fructose-induced podocyte injury remain elusive. In this study, we have used an iTRAQ based quantitative proteomic strategy to comprehensively characterize the dynamic proteome changes in glomeruli of high fructose-fed rats, and revealed global fructose-induced glomerular metabolic reprogramming at four different stages during the progression of high fructose modeling.
Project description:Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes excessively permeable leading to massive proteinuria. In childhood NS, dysregulation of the immune system has been implicated and increasing evidence points to the central role of podocytes in the pathogenesis. Children with NS are typically treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids that are activating ligands for the glucocorticoid receptor (GR) transcription factor. Although Gc-therapy has been the cornerstone of NS management for decades, the mechanism of action, and target cell, remain poorly understood. We tested the hypothesis that Gc acts directly on the podocyte to produce clinically useful effects without involvement of the immune system. In human podocytes, we demonstrated that the basic GR-signalling mechanism is intact and that Gc induced an increase in podocyte barrier function. To gain mechanistic insight we performed RNA microarray and ChIP-sequencing and identified Gc regulation of motility genes.