ABSTRACT: Podocytes form filtration barrier through foot process around glomerualar basement membrane and selectively permit permeability of molecular smaller than albumin. Diabetes can cause podocyte pathological changes leading to high urine albumin level. Diabetic mouse model OVE26 has extremly high urine albumin and previously studies indicated its podocyte damaged. Here we try to find the key genes change in OVE26 diabetic mouse model podocyte by microarray assay while normal FVB mouse podocyte set as control. Overall design: Podocyte eGFP transgenic mice were made on FVB background and crossbred to OVE26 diabetic model. Glomeruli isolated from OVE-GFP mice were digested by trypsin into signal cell. Podocytes with GFP were sorting out by FACS.
Project description:Podocytes form filtration barrier through foot process around glomerualar basement membrane and selectively permit permeability of molecular smaller than albumin. Diabetes can cause podocyte pathological changes leading to high urine albumin level. Diabetic mouse model OVE26 has extremly high urine albumin and previously studies indicated its podocyte damaged. Here we try to find the key genes change in OVE26 diabetic mouse model podocyte by microarray assay while normal FVB mouse podocyte set as control. Podocyte eGFP transgenic mice were made on FVB background and crossbred to OVE26 diabetic model. Glomeruli isolated from OVE-GFP mice were digested by trypsin into signal cell. Podocytes with GFP were sorting out by FACS.
Project description:We compared mRNA profiles of isolated glomeruli versus sorted podocytes between diabetic and control mice. IRG mice crossed with eNOS-/- mice were further bred with podocin-rTTA and TetON-Cre mice to permanently label podocytes before the diabetic injury. Diabetes was induced by injection of streptozotocin. mRNA profiles of isolated glomeruli and sorted podocytes from diabetic and control mice at 10 weeks after induction of diabetes were examined. Consistent with the previous reports, expression of podocyte-specific markers in the glomeruli were down-regulated in the diabetic mice compared to controls. However, these differences disappeared when mRNA levels were corrected for podocyte number per glomerulus. Interestingly, the expression of these markers was not altered in sorted podocytes from diabetic mice, suggesting that the reduced expression of podocyte markers in isolated glomeruli is likely a secondary effect of reduced podocyte number, rather than the loss of differentiation markers. Analysis of the differentially expressed genes in diabetic mice also revealed distinct up-regulated pathways in the glomeruli (mitochondrial function and oxidative stress) and podocytes (actin organization). In conclusion, our data suggest that podocyte-specific gene expression in transcriptome obtained from the whole glomeruli may not represent those of podocytes in the diabetic kidney. We compared mRNA profiles of isolated glomeruli versus sorted podocytes between diabetic and control mice.
Project description:A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+) and microvillus-rich apical membranes (podocalyxin+), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Overall design: 6 biological sample were isolated from wild type and PODXL mutant kidney organoids (12 samples in total). RNA-seq was performed on each sample.
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:Podocytes are cells of the visceral epithelium in the kidneys and form a crucial component of the glomerular filtration barrier, contributing to size selectivity and maintaining a massive filtration surface. We are interested in pursuing a microarray analysis to identify the glycosylation-related genes that are modulated in podocytes during insulin and glucose stimulation. As such, we propose to isolate RNA from cultured AB 8/13 human podocyte cell line in their normal state and treated with different concentrations of insulin and glucose. Initially, we would like to analyze 12 samples (Normal control, 2 Starved controls, Insulin experiments 1-4, and glucose experiments 1-5). These experiments would give us information on the gene regulation changes in different healthy and diabetic conditions. In addition to providing possible identification of the unknown proteins involved with diabetic nephropathy, such analysis would provide novel information about early responses by podocytes in response to insulin and glucose level changes in vitro. The results from this analysis will be utilized to focus our research with regard to glycosylation of the proteins in the podocyte slit diaphragm.
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. Overall design: 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:Podocyte injury is a major determinant in proteinuric kidney disease and identification of potential therapeutic targets for preventing podocyte injury has clinical importance. Here, we show that histone deacetylase Sirt6 protects against podocyte injury through epigenetic regulation of Notch signaling. Sirt6 is downregulated in renal biopsies from patients with podocytopathies and its expression negatively correlates withglomerular filtration rate. Podocyte-specific deletion of Sirt6 exacerbates podocyte injury and proteinuria in two independent mouse models including diabetic nephropathy and adriamycin-induced nephropathy. Sirt6 has pleiotropic protective actions in podocytes including anti-inflammatory and anti-apoptotic effects, is involved in actin cytoskeleton maintenance, and promotes autophagy. Sirt6 also reduces urokinase plasminogen activator receptor expression, which is a key factor for podocyte foot process effacement and proteinuria. Mechanistically, Sirt6 inhibits Notch1 and Notch4 transcription by deacetylating the histone H3K9. We suggest Sirt6 as a potential therapeutic target in proteinuric kidney disease. Overall design: Overexpression of Sirt6 by a Sirt6-adenovirus transfection was used in this study. Cells were stimulated with high glucose for 24h (a final concentration of 40 mmol/l in culture medium).
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 Overall design: Glomerular RNA was extracted using the RNeasy Mini Kit and processed for hybridization on Affymetrix Mouse Gene 2.1 ST chip microarrays
Project description:The thorough characterization of the transcriptome of endogenous podocytes has been hampered by low yields of cell isolation procedures. Here we introduce a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation yielding more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA transcriptional profiling revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. In summary, the presented ‘omics’ approach will facilitate the discovery and integration of novel gene, protein and organelle regulatory networks that deepen our systematic understanding of podocyte biology. To compare gene expression of glomerulus podocytes versus non-podocytes 5 replicates of 4 pooled mice were used. Data were normalized by robust multi-chip analysis. Exon expression values were summarised to the core meta probesets, as defined by Affymetrix, using the oligo library from R.
Project description:Glomerular podocytes are highly differentiated kidney cells that constitute one of the key components responsible for removing toxins and metabolic waste while preserving nutrients in the bloodstream in the renal glomerulus. In addition, podocytes contribute significantly to the formation of the glomerular basement membrane and the integrity of the vascular endothelium. Thus, podocyte injury and/or loss of podocytes results in impaired blood filtration and causes many common renal diseases characterized by severe proteinuria (protein in the urine) and hypoalbuminemia (low levels of blood albumin). Small lipopholic molecules such as steroids, fatty acids, prostaglandins and vitamin metabolites control many aspects of animal development, cell proliferation and differentiation, and homeostasis through binding to their intracellular receptors. Upon binding to their cognate ligands, these nuclear receptors (NRs) are capable of turning on or off of an array of gene networks. By doing so, they regulate a whole spectrum of cellular activities. The ability of small molecule hormones to regulate NR activity makes them excellent pharmaceutical targets. Clinical evidence and animal studies have implicated several NRs in contributing to podocyte development and disease. Recent studies from animals and cultured human or mouse podocytes indicate that synthetic hormones including estradiol, glucocorticoid, retinoid, pioglitazone, vitamin D3 and WY-14643 protect or rescue podocytes from experimental injury. Post treatment of injured podocytes with ligands for the above-mentioned NRs restores cytoskeletal architecture of the podocytes. Nonetheless, the mechanisms underlying the ability of these hormones in protecting podocytes remain largely unexplored. This is partly due to our limited knowledge of the target genes controlled by these hormones. In order to elucidate the mechanisms by which Dex, VD3 and ATRA elicit their renoprotective activity, we initiate a gene expression profiling study to identify the target genes of these hormones in cultured human podocytes. The temperature-sensitive cultured human podocytes (HPCs) were maintained in culture media of RPMI supplemented with 10% FBS, 1% antibiotics and the Insulin-Transferrin-Selenium at the permissive (undifferentiated) temperature of 33 Celsius degree. The 70%~80% confluent HPCs were induced to in vitro differentiate by culturing at 37 Celsius degree for 2 days then with incubation of vehicle (equal volume of DMSO), 100 nM all-trans retinoic acid (ATRA), 100 nM dexamethasone (Dex), or 100 nM vitamin D3 (VD3) treatment for 3 more days. The undifferentiated HPCs with vehicle (equal volume of DMSO) treatment was included as a control. The total RNAs of these cells were isolated using USB prepEase RNA spin kit following the manufacturer's instructions. Total 1 microgram RNA at 100 nanogram per microliter for each sample were sent for a single-channel gene expression microarray studies using HumanRef-8 chip (V3_0_R3_11282963) on Illumina BeadStation platform. The RNA were converted to aRNA and appropriately labelled using MessageAmp Biotin aRNA Amplification Kit (Ambion) following the manufacturer's instructions. The raw data were collected by scanning the chip on Illumina BeadStation. Background subtracted raw data were exported and the following analysis was done in R/Bioconductor environment. Thus, we were able to compare the gene expression profiles between differentiated and undifferentiated HPCs with or without hormone treatment.