RPTEC/TERT1
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB29471 | ENA |
REPOSITORIES: ENA
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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:Microphysiological systems are a promising new experimental tool for drug and chemical safety evaluation. These models are rapidly evolving, and concerns exist about the reproducibility, robustness and relative benefits when compared to traditional in vitro cultures. This study used OrganoPlate® 3-lane 40, a microfluidics-based model of the renal proximal tubule and compared it to multi-well static cultures using a number of human renal proximal tubule epithelial cell (RPTEC) types. We followed previously published protocols but adapted them to the varying cell proliferation rates of different RPTECs; primary cells required up to 7 days in culture before experiments, while some immortalized cell lines could be used in 3 days. Function of various tubule-specific transporters, permeability of small molecules (cisplatin, tenofovir and perfluorooctanoic acid) and fluorescent dextrans (60-150 kDa), basal and chemical-effected gene expression, and cell viability were tested. Different RPTEC types and culture conditions (OrganoPlate® 3-lane 40 vs multi-well plates) were compared. We found that there are advantages offered by OrganoPlate® 3-lane 40 as compared to multi-well cultures – presence of media flow, albeit intermittent, and fairly high throughput. However, this model appears to offer only limited (e.g., MRP-mediated transport) advantages in terms of either gene expression or transporter function when compared to the multi-well plate culture conditions. It can also be used to study cellular uptake and direct toxic effects of small molecules; still, it may have limited utility for studies of pharmaco-/toxico-kinetics because drug transport between blood and tubule compartments is considerably affected by the gel barrier.
2023-11-08 | GSE233406 | GEO
Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed sequencing bias and reproducibility using an equimolar pool of 1,152 small RNA sequences ranging from 15-90 nt, and primarily comprised of annotated human microRNAs. We observed extensive protocol-specific and sequence-specific bias that was largely mitigated in protocols employing sequencing adapters with randomized end-nucleotides. We find that sequencing bias is highly reproducible across labs using the same library preparation technologies, and use the data to calculate inter-protocol bias correction factors. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.
2018-07-09 | GSE94584 | GEO
Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols. This SuperSeries is composed of the SubSeries listed below.
2018-07-09 | GSE94586 | GEO
Project description:Sample preparation protocols using different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents were comprehensively evaluated in the aspect of inducing over-alkylation in this work.
2022-08-15 | PXD036053 | iProX
Project description:Chronic kidney disease (CKD) is a progressive condition characterized by sustained alterations in kidney structure and function. Long-term kidney fibrosis is marked by glomerulosclerosis, vascular sclerosis, and tubulointerstitial fibrosis. Therefore we investigated the therapeutic potential of BMSC-CM on RPTEC/TERT1 in a fibrotic environment. Using PAA gel platforms, we mimicked the stiffness typical of chronic kidney disease patients' kidneys.
2023-12-20 | GSE250271 | GEO
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.
2023-10-20 | GSE216153 | GEO
Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed reproducibility and accuracy of relative expression measurements using two pools of synthetic small RNA sequences, where subsets of the sRNAs vary in relative amount between pools A and B. The pools each contain 334 small RNAs, varying by 15 different ratios between pools A and B (10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 1:5, 1:8, 1:10). We find that although the sequencing bias varies extensively between protocols, the relative abundance measured between samples A and B is largely reproducible and accurate across labs and protocols. These results suggest that measurements of differential expression should be comparable across institutions and library preparation technologies.
2018-07-09 | GSE108138 | GEO
Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed reproducibility and accuracy of relative expression measurements using two pools of synthetic small RNA sequences, where subsets of the sRNAs vary in relative amount between pools A and B. The pools each contain 334 small RNAs, varying by 15 different ratios between pools A and B (10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 1:5, 1:8, 1:10). We find that although the sequencing bias varies extensively between protocols, the relative abundance measured between samples A and B is largely reproducible and accurate across labs and protocols. These results suggest that measurements of differential expression should be comparable across institutions and library preparation technologies.
2018-07-09 | GSE94585 | GEO