Murine Podocyte Membrane Proteomics
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Glomerular Visceral Epithelial Cell, Kidney
SUBMITTER: 
Stephan Mueller
LAB HEAD: Stefan F. Lichtenthaler
PROVIDER: PXD022213 | Pride | 2021-02-19
REPOSITORIES: Pride
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Project description:Podocyte injury is a key event for progressive renal failure. We previously established a mouse model of inducible podocyte injury (NEP25) and demonstrated relentless progression of glomerular injury toward sclerosis. To further investigate molecular events, we performed polysome analysis of intact and injured podocytes utilizing the NEP25 and RiboTag transgenic mice. We show here the expression profiling of normal podocytes and podocytes injured by immunotoxin.
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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.
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Project description:Background: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins to trigger actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 in podocytes remain to be fully elucidated. Methods: We generated podocytes deficient in Nck1 and Nck2, and used transcriptomic approaches to profile expression differences. We further applied proteomic techniques to identify specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2 along with podocyte injury models to comprehensively verify our findings. Results: Compound loss of Nck1/2 resulted in altered expression of genes involved in actin binding, cell adhesion and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2, and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling and integrin activation at focal adhesions via α-actinin-4. Furthermore, we found that loss of either Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2 deficient mice, consistent with enhanced susceptibility to podocyte injury. Conclusion: These findings highlight distinct yet complementary roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition and sustaining filtration barrier integrity.
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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.
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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.
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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.
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