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Mechanical stress enhances CD9 expression in cultured podocytes

ABSTRACT: Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Warsow et al. (Kidney Int. 84: 104-115, 2013) after application of mechanical stress (Endlich et al., J. Am. Soc. Nephrol. 12: 413-422, 2001) as compared to control conditions. Elevated glomerular pressure represents a high risk for the development of severe kidney diseases and causes an increase of mechanical load to podocytes. In this study we investigated whether mechanical stress alters gene expression in cultured podocytes using gene arrays. We found that tetraspanin CD9 is significantly upregulated in cultured podocytes after mechanical stress. The differential expression of CD9 was confirmed by RT-PCR and Western blot under stretched and unstretched conditions. Furthermore, mechanical stress resulted in a relocalization of CD9. To get an insight into the functional role of CD9, podocytes were transfected with pEGFP-CD9. The expression of CD9 induced the formation of substratum-attached thin arborized protrusions (TAPs). Ca2+ depletion revealed that podocytes over-expressing CD9 possess altered adhesive properties in contrast to the control transfected cells. Finally, elevated CD9 expression increased migration of podocytes in a wound assay. In summary, our results suggest that upregulation of CD9 may play an important role in podocyte morphology, adhesion and migration.

ORGANISM(S): Mus musculus

PROVIDER: GSE66336 | GEO | 2015/02/27

SECONDARY ACCESSION(S): PRJNA276570

REPOSITORIES: GEO

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Mechanical stress enhances CD9 expression in cultured podocytes

Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Warsow et al. (Kidney Int. 84: 104-115, 2013) after application of mechanical stress (Endlich et al., J. Am. Soc. Nephrol. 12: 413-422, 2001) as compared to control conditions. Elevated glomerular pressure represents a high risk for the development of severe kidney diseases and causes an increase of mechanical load to podocytes. In this study we investigated whether mechanical stress alters gene expression in cultured podocytes using gene arrays. We found that tetraspanin CD9 is significantly upregulated in cultured podocytes after mechanical stress. The differential expression of CD9 was confirmed by RT-PCR and Western blot under stretched and unstretched conditions. Furthermore, mechanical stress resulted in a relocalization of CD9. To get an insight into the functional role of CD9, podocytes were transfected with pEGFP-CD9. The expression of CD9 induced the formation of substratum-attached thin arborized protrusions (TAPs). Ca2+ depletion revealed that podocytes over-expressing CD9 possess altered adhesive properties in contrast to the control transfected cells. Finally, elevated CD9 expression increased migration of podocytes in a wound assay. In summary, our results suggest that upregulation of CD9 may play an important role in podocyte morphology, adhesion and migration. Three independent batches were used.

2015-02-27 | E-GEOD-66336 | biostudies-arrayexpress

Differentiated mouse podocytes (SVI)

Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Kabgani et al. (PLoS One 7:e34907, 2012). The transcriptomes of the podocyte cell line were mapped on a protein-protein interaction network of the podocyte (PodNet). Together with other transcriptomes taken from GEO, we analyzed differential gene regulation and differential regulation of protein-protein interactions between cultured podocytes and differentiated in vivo podocytes.

2012-10-16 | GSE41523 | GEO

Differentiated mouse podocytes (SVI)

Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Kabgani et al. (PLoS One 7:e34907, 2012). The transcriptomes of the podocyte cell line were mapped on a protein-protein interaction network of the podocyte (PodNet). Together with other transcriptomes taken from GEO, we analyzed differential gene regulation and differential regulation of protein-protein interactions between cultured podocytes and differentiated in vivo podocytes. Three independent batches were used.

2012-10-16 | E-GEOD-41523 | biostudies-arrayexpress

Mus musculus

Project description:Mechanical stress enhances CD9 expression in cultured podocytes

| PRJNA276570 | ENA

Comparative protein profiling of wild type and zyxin knockout podocytes

Project description:Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. The present study investigated the role of zyxin in mechanically stretched podocytes, a model for glomerular hypertension. Conditionally immortalized differentiated podocytes were seeded on flexible silicon membranes of a six well plate, which was mounted on a manifold connected to a custom-built stretch apparatus (NIPOKA GmbH, Greifswald, Germany), which induced cyclic pressure variations resulting in upward and downward motion of the silicone membranes. We found that zyxin is essential for the adhesion of the postmitotic cell type, the podocytes, in vitro during mechanical stretch as well as in vivo for the maintenance of the kidney filtration.

2024-04-30 | PXD050861 | Pride

Podocyte-specific deletion of tubular sclerosis complex 2 causes focal segmental glomerular sclerosis and progressive renal failure

Project description:Obesity can initiate and accelerate the progression of kidney diseases. However, it remains unclear how obesity affects renal dysfunction. Here, we show that a newly generated podocyte-specific tubular sclerosis complex 2 (Tsc2) knockout mouse model (Tsc2∆podocyte) develops proteinuria and dies due to end-stage renal dysfunction by 10 weeks of age. Tsc2∆podocyte mice exhibit an increased glomerular size and focal segmental glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis and proteinaceous casts. Podocytes isolated from Tsc2∆podocyte mice show nuclear factor, erythroid derived 2, like 2-mediated increased oxidative stress response on microarray analysis and their autophagic activity is lowered through the mammalian target of rapamycin (mTOR)—unc-51-like kinase 1 pathway. Rapamycin attenuated podocyte dysfunction and extends survival in Tsc2∆podocyte mice. Additionally, mTOR complex 1 (mTORC1) activity is increased in podocytes of renal biopsy specimens obtained from obese patients with chronic kidney disease. Our work shows that mTORC1 hyperactivation in podocytes leads to severe renal dysfunction and that inhibition of mTORC1 activity in podocytes could be a key therapeutic target for obesity-related kidney diseases.

2020-03-21 | GSE106828 | GEO

Drosophila nephrocyte proteome

Project description:Podocyte injury is a major cause of chronic kidney disease, but the involved signaling pathways are largely unknown. Here, we used ultrasensitive proteomics to investigate the response of the native renal podocyte to injury and prioritized candidates by aligning proteinuria in individual mice with protein expression data. The analysis revealed large perturbations of signaling pathways known to associate with FSGS, but also showed perturbation in metabolism and proteins responding to mechanical stress. We employed Drosophila nephrocytes for further functional analysis of selected candidates and studied filtration function and endocytosis. Among the candidates identified was the mechanosensor protein Filamin-B which is a central component of a conserved cellular response to mechanical stress in podocytes in vitro. Loss of Cher, the orthologue of Filamin-B, led to an increased endocytosis in nephrocytes. Of note, Filamin-B expression was only elevated in the Doxorubicin-induced proteome of podocytes but not after LPS induced injury despite the regulation of multiple similar pathways on the systems level. The Filamin-B amount in glomeruli of proteinuric PAN rats correlated with the amount of albumin . In conclusion, this study identified conserved mechanisms of podocyte stress response employing state of the art ultrasensitive proteomics in isolated mouse podocytes and Drosophila nephrocytes.

2020-07-29 | PXD012025 | Pride

Expression analysis of control and KLF4-overexpressing podocytes

Project description:To investigate podocyte gene expression and promoter methylation affected by KLF4, we examine about whole genome expression profiles using Agilent microarray and its correlation with promoter methylation profiles using Illumina DNA methylation array (GSE41689) as shown in supplementary data. Genome wide expression profiling of control (vector-transfected n=1) and KLF4-overexpressing (n=1) human podocytes (parental cell line: Saleem et al. JASN 13:630, 2002) and DNA methylation profile of promoter regions were shown.

2014-11-01 | E-GEOD-54395 | biostudies-arrayexpress

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.

2016-03-16 | E-GEOD-79291 | biostudies-arrayexpress

Analysis of podocytes from doxorubicin treated mice

Project description:Podocyte injury is a major cause of chronic kidney disease, but the involved signaling pathways are largely unknown. Here, we used ultrasensitive proteomics to investigate the response of native podocytes from mice with Doxorubicin-induced injury. We observed perturbations of known propagating pathways of glomerular disease such as Jak-Stat signaling and cell adhesion but also in metabolism and proteins responding to mechanical stress. Using a phenotype-guided approach, we prioritized candidates by aligning proteinuria in individual mice with corresponding podocyte protein expression data. The protein Tropomodulin was decreased and correlated negatively with urinary albumin, and Villin and the mechanosensor protein Filamin-B were increased and correlated positively. We employed Drosophila nephrocytes for further functional analysis of prioritized proteins and studied filtration function and endocytosis. Loss of Villin and Tropomodulin led to an impaired function and reduced expression of endocytosis machineries as evidenced by nephrocyte garland proteomes. Loss of Cher, the orthologue of Filamin-B, led to increased endocytosis in nephrocytes, and Filamin-B abundance in single glomeruli correlated with the amount of deposited albumin in the same glomerulus in proteinuric rats. Filamin-B expression could also be increased by applying mechanical stress to podocytes in vitro, but Filamin-B was not increased in podocyte proteomes in LPS-induced injury despite similar regulation of pathways on the global proteome level. In conclusion, this study identified conserved mechanisms of podocyte stress response employing functional proteomics in purified native mouse podocytes and Drosophila nephrocytes.

2020-07-29 | PXD012063 | Pride

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