Gene expression profile at single cell level of podocyte-specific I-PpoI expressing mice kidney cortex
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ABSTRACT: To investigate the alterations in cell populations in the kidney after podocyte DNA DSBs, we performed single-cell RNA sequencing (scRNA-seq) on the renal cortex of 12-week-old podocyte-specific I-PpoI expressing mice that had not yet developed glomerulosclerosis or fibrosis.
Project description:To investigate the alterations in cell populations in the kidney after proximal tubular epithelial cell DNA DSBs, we performed single-cell RNA sequencing (scRNA-seq) on the renal cortex of 16-week-old proximal tubular epithelial cell specific I-PpoI expressing mice.
Project description:Using reduced representation bisulfite sequencing (RRBS) analysis, we examined how I-PpoI-induced DSBs affected DNA methylation patterns in peripheral blood cells of 16-week-old I-PpoI mice.
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:DNA double-strand breaks (DSBs) and their repair can cause extensive epigenetic changes. As a result, DSBs have been proposed to promote transcriptional and, ultimately, physiological dysfunction via both cell-intrinsic and cell-non-autonomous pathways. Studying the consequences of DSBs in higher organisms has, however, been hindered by a scarcity of tools for controlled DSB induction. Using a mouse model for both tissue-specific and temporally controlled DSB formation, we investigated the transcriptional response to break repair. Transcriptional profiling of lymphocytes in spleen and thymus by RNA-Seq, with and without I-PpoI knock-in.
Project description:Podocyte injury is the hallmark of proteinuric kidney diseases, such as Focal Segmental Glomerulosclerosis and Minimal Change Disease. Therapeutic strategies in the management of proteinuric diseases are limited. Although agents such as glucocorticoids, cyclosporine, and rituximab have direct effects on the podocyte by stabilizing its actin cytoskeleton, these drugs are riddled with systemic toxicity and off-target effects that hinder their chronic use. We previously demonstrated that the loss of the kidney-enriched zinc finger transcription factor, Krüppel-like factor 15 (KLF15), increases susceptibility to proteinuric kidney disease as well as attenuates the salutary effects of retinoic acid and glucocorticoids in the podocyte. Here, we show that podocyte-specific induction of KLF15, using the tetracycline-inducible system, attenuated podocyte injury, glomerulosclerosis, tubulointerstitial fibrosis and inflammation, while improving renal function and overall survival in HIV-1 transgenic mice. Enrichment analysis of mRNA sequencing of isolated glomerular extracts from this model shows that podocyte-specific induction of KLF15 activates pathways involved in stabilization of actin cytoskeleton, focal adhesion, and podocyte differentiation. Transcription factor enrichment analysis, with further experimental validation, suggests that KLF15 activity is in part mediated by Wilms Tumor 1 (WT1), a transcription factor known to be critical for podocyte differentiation. Further, we confirmed the benefits of podocyte-specific induction of KLF15 in the adriamycin-induced proteinuric murine model. Collectively, these observations suggest that induction of KLF15 might be a potential therapeutic target in the treatment of proteinuric kidney disease.
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:We found that podocyte-specific bPIX deficient (KO) mice developed progressive proteinuria starting at ~8 weeks of age, and glomerulosclerosis and podocyte loss by 13 weeks of age. To investigate the mechanism of podocyte loss induced by bPIX deficiency, we analyzed mRNA expression by RNA-sequence using isolated glomeruli from control (CTRL) and KO mice.
Project description:The goal of this study was to identify transcriptomic changes of mouse kidney cortex in mice with podocyte-specific deletion of Kruppel-like factor 4, a zinc-finger transcription factor.