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Mouse Podocyte Proteome - ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier

ABSTRACT: Label-free expression proteomics analysis of FACS-sorted mouse podocytes versus non-podocytes

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Podocyte (sensu Diptera), Kidney

SUBMITTER: Joern Dengjel

LAB HEAD: Joern Dengjel

PROVIDER: PXD010601 | Pride | 2018-12-14

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20121123_AR_probe3_10.raw	Raw
	20121123_AR_probe3_1_121124135510.raw	Raw
	20121123_AR_probe3_2_121124162257.raw	Raw
	20121123_AR_probe3_3_121124185047.raw	Raw
	20121123_AR_probe3_4_121124211837.raw	Raw

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Publications

ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier.

Schell Christoph C Sabass Benedikt B Helmstaedter Martin M Geist Felix F Abed Ahmed A Yasuda-Yamahara Mako M Sigle August A Maier Jasmin I JI Grahammer Florian F Siegerist Florian F Artelt Nadine N Endlich Nicole N Kerjaschki Dontscho D Arnold Hans-Henning HH Dengjel Jörn J Rogg Manuel M Huber Tobias B TB

Developmental cell 20181129 6

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the devel ...[more]

PMID: 30503751

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Molecular fingerprint of the podocyte reveals novel gene regulatory networks

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 M-bM-^@M-^XomicsM-bM-^@M-^Y 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.

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Deep RNA Sequencing of native mouse podocytes and non-podocytes glomerular cells

Project description:Purpose: Next-generation sequencing (NGS) was used to define the transcriptome of native mouse podocytes and non-podocytes glomerular cells as part of a project aiming to define the molecular fingerprint of mouse podocytes. Method: Glomeruli from 29 Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J x hNPHS2Cre mice at the age of 10 weeks were purified and a single cell solution was prepared to seperate GFP-expressing (podocytes) and GFP-negative (non-podocytes glomerular cells) cells by FACS sorting. RNA was extracted and prepared for further analysis using directional, polyA+ library preparation. An Illumina HiSeq2500 was used for a paired-end sequencing of 100 cycles . Salmon and Sleuth were used for downstream analysis. Results: A total of 100 Million reads each from podocytes and non-podocytes glomerular cells could be used for further analysis.

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Comparison of Glomerular and Podocyte mRNA Profiles in eNOS-/- Diabetic Mice Induced by Streptozotocin

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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Glucocorticoid therapy regulates podocyte motility by inhibition of Rac1

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2017-07-17 | E-MTAB-5235 | biostudies-arrayexpress

Role of calcium/calmodulin-dependent kinase type IV in podocyte function in lupus nephritis

Project description:Kidney podocytes and their slit diaphragms contribute to prevent urinary protein loss. T cell from patients with systemic lupus erythematosus display increased expression of calcium/calmodulin kinase IV (CaMKIV). Here we evaluated the functional role of CaMKIV in lupus nephritis (LN) using kidney biopsy specimens and human podocyte cell line (AB8/13). We found that exposure of podocytes to IgG from LN patients resulted in entry of IgG into the cytoplasm. CaMKIV expression was found to be increased in podocytes of LN kidney biopsy specimens and exposure to IgG from LN patients. IgG entered podocytes using the FcRn receptor because when podocytes where treated with FcRn siRNA less IgG was found in the cytoplasm. The DNA microarray studies of podocytes exposed to LN IgG revealed that genes that are related to the activation of immune cells or podocyte damage were upregulated. These genes included CD86, CaMKIV, PTPN22, PDE5A, CD47 and MALT1. Interestingly, CD86 expression decreased after silencing CaMKIV in podocytes. Also, in situ hybridization experiments showed that the expression of CD86 was reduced in podocytes from MRL/lpr.camkivM-bM-^HM-^R/M-bM-^HM-^R mice. IgG from LN patients may enter podocytes through the FcRn and causes the upregulation of a distinct set of genes which may alter podocyte function. Upregulation of CaMKIV appears to precede that of genes known to be linked to podocyte damage such as CD86. These findings may indicate that inhibition of CaMKIV may prove of clinical use in patients with LN. IgG Purification Kits (Dojindo Molecular Technologies, Inc.) are used for isolation and purification of immunoglobulin G of healthy and normal individual according to the manufacturerM-bM-^@M-^Ys protocol. Flow through the column was used for non IgG binding samples. Cultured human podocytes with IgG purified from sera of normal individuals and LN patient for 24 hr were collected for RNA.

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Expression data for OVE26 diabetic mouse podocyte

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