Three-dimensional spherical cellular aggregates of RPTEC as an in vitro tool for evaluation of human renal proximal tubular toxicity and drug disposition
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PROVIDER: PRJNA892454 | ENA |
REPOSITORIES: ENA
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Project description:The proximal tubule plays an important role in the secretion and reabsorption of drugs in the kidney and is a major site of drug interaction and toxicity. Kidney toxicity analysis via in vitro assays is challenging as few provide appropriate proximal tubular cell function. In this study, we aimed to develop a simple and reproducible method to culture human proximal tubular epithelial cells (RPTECs), used in pharmacokinetic and toxicological evaluation by monitoring organic anion transporter (OAT)1 as a selection marker. As a result, by culturing RPTECs in spherical cellular aggregates, OAT1 protein expression, which does not increase in conventional 2D culture, increased over time and reached a similar level to that in human renal cortices from 5 different donors. The expression of proximal tubule markers, AQP1 and CDH6, was maintained, indicating that 3D RPTEC spheroids had proximal tubule characteristics. For SLC transporters, the 3D spheroid culture improved the protein expression of about 7% of the 139 transporter proteins detected and 2.3% of 4,800 proteins detected to about 5-fold that in human renal cortices. Furthermore, the protein expression levels of about 4800 proteins in 3D RPTEC spheroids (cultured for 12 days) were maintained over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroid is a simple and robust in vitro experimental system for pharmacokinetic and toxicological evaluation in drug development.
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Project description:Expression data from Stx2-treated human renal proximal tubular epithelial cell (RPTEC)
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Project description:Renal Tubular Epithelial Cells (RPTEC) from two donors were cultivated in vitro until irreversible growth arrest was observed. Total RNA from young (replicating) as well as growth arrested cells was harvested and hybridized to LNA based microRNA microarrays using a looped design. After data processing, differential expression of miRNAs in senescent vs. young RPTEC cells was calculated.
2010-04-19 | E-MEXP-2393 | biostudies-arrayexpress
Project description:Plasmacytoid dendritic cells (pDCs) play a key role in the activation of the autoimmune response in LN. Recently we demonstrated that these cells infiltrate the kidney of patients with LN at tubulointerstitial level. The pDCs are the main producers of IFN-alpha, whose effects on the renal tubule are poorly understood. The aim of the study was to investigate the effects of INF-alpha in renal epithelial cells (RPTEC). We investigated the effect of IFN-alpha on the whole-genome gene expression profile in RPTEC cells,. Genomic analysis showed that after stimulation, 108 genes are up-regulated and only 7 are down-regulated, with a fold-change> 2. The Gene Set Enrichment analysis confirmed that IFN-alpha induced the pathway of antigen presentation and the inflammatory signaling in RPTEC. Among the up-regulated genes involved in these pathways, there were HLA-I, the ubiquitins (FBXO6 and DTX3L) and the immunoproteasome subunits LMP7. We performed the validation of these genes by real time PCR and citometry experiments on RPTEC stimulated with IFN-alpha. Immunohistochemical analysis of LMP7 and MXA protein, specific marker of IFN-alpha, showed a significant upregulatation of both proteins in renal biopsies of patients with LN class IV compared to class I. Confocal microscopy showed the colocalization of LMP7-MXA in tubular epithelium of patients with LN class IV and activation of inflammatory signaling via NF-kB in the MXA+ tubules. Our data suggested that inhibition of INF-alpha pathways could represent a novel therapeutic strategy to reduce renal tubular damage in patients with LN. To identify genes specifically modulated by INF-alpha in RPTEC, we stimulated 3 different cell clones with 100U/ml INF-alpha for 48 hours and compared their whole-genome gene expression profiles with that from 3 RPTEC clones cultured without stimuli.
2015-12-15 | E-GEOD-48551 | biostudies-arrayexpress
Project description:Confluent cultures of a human renal fibroblast cell line (TK-173) and a human renal proximal tubule epithelial cell line (RPTEC/TERT1) were treated with 10 micromolar tacrolimus (FK-506) for one and three days.
2012-01-01 | E-MTAB-778 | biostudies-arrayexpress
Project description:Plasmacytoid dendritic cells (pDCs) play a key role in the activation of the autoimmune response in LN. Recently we demonstrated that these cells infiltrate the kidney of patients with LN at tubulointerstitial level. The pDCs are the main producers of IFN-alpha, whose effects on the renal tubule are poorly understood. The aim of the study was to investigate the effects of INF-alpha in renal epithelial cells (RPTEC). We investigated the effect of IFN-alpha on the whole-genome gene expression profile in RPTEC cells,. Genomic analysis showed that after stimulation, 108 genes are up-regulated and only 7 are down-regulated, with a fold-change> 2. The Gene Set Enrichment analysis confirmed that IFN-alpha induced the pathway of antigen presentation and the inflammatory signaling in RPTEC. Among the up-regulated genes involved in these pathways, there were HLA-I, the ubiquitins (FBXO6 and DTX3L) and the immunoproteasome subunits LMP7. We performed the validation of these genes by real time PCR and citometry experiments on RPTEC stimulated with IFN-alpha. Immunohistochemical analysis of LMP7 and MXA protein, specific marker of IFN-alpha, showed a significant upregulatation of both proteins in renal biopsies of patients with LN class IV compared to class I. Confocal microscopy showed the colocalization of LMP7-MXA in tubular epithelium of patients with LN class IV and activation of inflammatory signaling via NF-kB in the MXA+ tubules. Our data suggested that inhibition of INF-alpha pathways could represent a novel therapeutic strategy to reduce renal tubular damage in patients with LN.
2015-12-15 | GSE48551 | GEO
Project description:Epigenetic mechanisms as DNA methylation can affect allograft outcome after kidney transplantation, accelerating renal aging. Complement system is the major player of ischemia reperfusion (I/R) injury and Renal Proximal Tubular Cells (RPTEC) are known to express C5a receptor (C5aR). However, little is known about the downstream effect of C5a-C5aR interaction. We performed a whole-genome DNA methylation analysis on C5a stimulated RPTEC and found several regions regulating genes involved in cell cycle control, DNA damage checkpoints and WNT signaling. These most represented genes were BCL9, CYP1B1 and CDK6. We showed that C5a induced-aberrant gene methylation significantly influences the expression of these genes. Moreover, the stimulation of RTEC by C5a led to senescence by up-regulating SA-β Gal positivity and increasing p53 gene and p21 protein level. IL-6, MCP-1, CTGF gene levels increase indicated the tubular SASP (Senescence associated Secretory Phenotype) acquirement. In accordance, in a swine model of I/R injury, we found that the increase expression of WNT4/βcatenin expression correlated with the augment in SA-β Gal, p21, p16 and IL-6 positivity after 24h from reperfusion. The treatment with C1-INH, a complement inhibitor, efficiently antagonized SASP restoring SA-β Gal, p21, p16, IL-6 expression at basal level and abrogating the WNT4/βcatenin activation. Thus, C5a affects the DNA methylation of genes involved in tubular senescence leading to a persistent, low-grade, inflammatory state called inflammaging. Targeting epigenetic programs may offer novels strategies to protect tubular cells from aging and promote kidney repair and recovery.
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