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Analysis of the effect of CHX on the cultured podocyte proteome

ABSTRACT: We analyzed the effect of Cycloheximide (CHX) on the podocyte proteome in both the differentiated and undifferentiated state.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

SUBMITTER: Markus Rinschen

LAB HEAD: Markus Rinschen

PROVIDER: PXD006886 | Pride | 2018-05-01

REPOSITORIES: Pride

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			Action	DRS
	20161205_AWH_ColID_247_Markusinschen_Benzing_ProjID_282_S01T.raw	Raw
	20161205_AWH_ColID_247_Markusinschen_Benzing_ProjID_282_S02T.raw	Raw
	20161206_AWH_ColID_255_MarkusRinschen_Benzing_ProjID_282_S03T.raw	Raw
	20161206_AWH_ColID_255_MarkusRinschen_Benzing_ProjID_282_S04T.raw	Raw
	20161206_AWH_ColID_255_MarkusRinschen_Benzing_ProjID_282_S05T.raw	Raw

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Publications

Protein half-life determines expression of proteostatic networks in podocyte differentiation.

Schroeter Christina B CB Koehler Sybille S Kann Martin M Schermer Bernhard B Benzing Thomas T Brinkkoetter Paul T PT Rinschen Markus M MM

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20180425 9

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions ...[more]

PMID: 29694247

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Deep mapping of the mouse podocyte proteome

Project description:We analyzed the proteome of mouse FACS-sorted podocytes. We compared podocytes ("green cells") with non-podocyte ("red cells") cells of the glomerulus.

2018-06-21 | PXD003306 | Pride

Analysis of the cultured podocyte phosphoproteome

Project description:The renal filtration barrier is critically maintained by the renal podocyte, a postmitotic, neuron-like epithelial cell type. Cultured podocyte cell lines – both in the differentiated and undifferentiated state - are a widely utilized tool to estimate cytoskeletal rearrangement processes in vitro. Here, we mapped the podocyte proteome at a depth of more than 7000 proteins. The data dissects the dynamic range of the podocyte proteome during differentiation. The data also demonstrates an absolute quantitative analysis of proteins mutated in forms of hereditary disease and demonstrates expression of several proteins not expressed in other, standard cell lines. We validated differentially regulated proteins using immunoblot and functional assays estimating lysosomal and proteasomal function. Comparison with deep mapping RNAseq data from the native podocyte, preliminary fingerprint data from the native proteome and staining intensities from the human protein atlas was performed to estimate similiarities between the cultured and native podocyte proteome, unravelling that at least 26 high-abundant native, podocyte-specific proteins, were not identified in the dataset. Notably, this data detected general perturbations in proteostatic mechanisms as a key alteration during podocyte differentiation, with increased proteasome activity in the undifferentiated state and markedly increased expression of lysosomal proteins in the differentiated state. We also performed a comparative phosphoproteomic analysis with published data from the native glomerul. These data also suggests a preference of phosphorylation of actin associated proteins in the differentiated state. The dataset obtained here, a valuable resource, warrants and provides the means for deep mapping of the native podocyte proteome and phosphoproteome in a similar manner.

2016-07-04 | PXD004013 | Pride

Gene expression in a conditionally immortalized mouse podocyte cell line

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.

2010-08-28 | E-GEOD-23856 | biostudies-arrayexpress

Glucocorticoid therapy regulates podocyte motility by inhibition of Rac1

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.

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.

2014-08-01 | E-GEOD-55768 | biostudies-arrayexpress

Podocyte Focal Adhesion Proteome

Project description:Expression proteomics analysis of focal adhesion complexes isolated from a human immortalized podocyte cell line.

2018-12-14 | PXD010626 | Pride

Podocyte injury in Fabry nephropathy

Project description:Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement (ERT) or chaperone mediated stabilization, thereby alleviating lysosome dysfunction. However, the therapeutic effect in the regression of end-organ damage (ie. kidney damage) is limited. Ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not alter podocyte injury. A novel CRISPR-/CAS9-mediated -Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptomic-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury.

2023-07-20 | PXD029618 | Pride

Deep proteomic mapping of the human podocyte proteome

Project description:We subjected differentiated and undifferentiated podocytes to deep proteomics analysis by tryptic digestion and SCX fractionation.

2018-05-01 | PXD007075 | Pride

Human podocyte cell culture degradomics

Project description:Regulated intracellular proteolysis is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. Altered proteolytic substrate turnover has been associated with various glomerular diseases ranging from diabetic nephropathy to focal and segmental glomerulosclerosis. However, thus far it has not been possible to systematically identify proteolytically cleaved proteins although some of the proteases have been characterized. Here we applied TAILS to map N-termini in the mouse glomeruli proteome and to determine and quantify N termini in podocyte cell cultures challenged with PAN.

2017-07-20 | PXD005140 | Pride

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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