Identify and characterize the polyploidization process of primary human proximal tubular cells (hPTC) [scRNA-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE242695 | GEO | 2023/09/08
REPOSITORIES: GEO
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Project description:We generated single-cell RNA sequencing (scRNAseq) datasets of primary cultures of human proximal tubular cells at different passages which we have found to contain a polyploid fraction. Within hPTC clusters, we found genes previously associated with different phases of endoreplication-mediated polyploidy and established that YAP1 controls the polyploidization process of hPTC. These observations were verified by treating hPTC with verteporfin (a YAP1 inhibitor), YAP1 silencing and by quantifying the expression of YAP1 downstream targets in sorted polyploid hPTC
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Project description:We found that PTC undergo polyploidization immediately after AKI induced by ischemia. To identify the mechanism controlling polyploidization of PTC, we generated single-cell RNA sequencing (scRNAseq) datasets from healthy mouse kidneys, 2 and 30 days after ischemia reperfusion injury. We found that polyploid PTC become hypertrophic and undergo polyploidization in a YAP1-related manner. In particular, YAP1 controls polyploidization of PTC via E2f7, E2f8 and Akt1. We confirmed these findings by knocking-down those genes in vitro.
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Project description:Microarray analysis reveals up-regulation of retinoic acid and hepatocyte growth factor related signaling pathways by pro-insulin C-peptide in kidney proximal tubular cells: Antagonism of the pro-fibrotic effects of TGF-b1 Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or transforming growth factor beta 1 (TGF-β1) in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor β (RARβ), hepatcoyte growth factor (HGF), cellular retinoic acid binding protein II (CRABPII), vimentin, E-cadherin, Snail and β-catenin was assessed by immunoblotting. The cellular localisation of vimentin and β-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1,458 genes after C-peptide exposure for 18h or 48h respectively. From these, members of the anti-fibrotic retinoic acid (RA) and HGF signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARβ, CRABPII and HGF. We confirmed a role for RA in reversal of TGF-β1-induced changes associated with epithelial-mesenchymal transition (EMT), including expression changes in Snail, E-cadherin, vimetin and redistribution of β-catenin. Importantly, these TGF-β1-induced changes were inhibited by C-peptide. Further, effects of TGF-β1 on Snail and E-cadherin expression were blocked by HGF and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.
2010-02-11 | GSE20247 | GEO
Project description:In order to study the effect of mesenchymal stem cells on miRNAs in renal tubular epithelial cells during renal fibrosis, and to find new treatment methods for renal fibrosis, we used TGF-β1 to stimulate mouse tubular epithelial cells, co-cultured with mesenchymal stem cells for 48 hours, and collected renal tubular epithelial cells .The renal tubular epithelial cells that were only stimulated by TGF-β1 were used as a control group. High-throughput miRNA sequencing was used to detect the increased and decreased miRNAs after co-culture.
2020-04-07 | GSE148144 | GEO
Project description:To identify differentially expressed long noncoding RNAs (lncRNAs) upon TGF-β stimulation in human cultured tubular epithelial cells HK2 and HKC8, we have employed long noncoding RNA microarray expression profiling as a discovery platform to find differentially expressed lncRNAs with TGF-β stimulation in these cells. Cultured human tubular epithelial cells HK2 and HKC8 were stimulated with PBS or TGF-β1. After incubation with TGF-β1 (10ng/ml) for 24 hours, 86 overlapping lncRNAs were upregulated and 47 overlapping lncRNAs were downregulated more than 2 fold vesus cells treated with PBS in these two epithelial cell lines. Expression of ENST00000429588 from this result was quantified in the same RNA samples by real-time PCR, confirming the upregulation upon TGF-β stimulation is repeatable.
2018-08-15 | GSE108554 | GEO
Project description:A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion: Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease. Livers from C57Bl/6 mice were isolated at defined ages: embryonic day 15.5 (n=3; mixed gender), 2 weeks (n=3; male), 3 weeks (n=3, male) and 7 weeks (n=3; male). Differential miRNA expression was assessed using the nCounter Mouse miRNA Expression Assay Kit (nanoString).
2016-03-29 | E-GEOD-68537 | biostudies-arrayexpress
Project description:Tubular epithelial cells (TECs) play a major role in potentiating renal fibrosis. While TGF-β1 is a key inducer of the pro-fibrotic phenotype in TECs, inhibition of TGF-β1 also blocks its protective effects, making this an unsuccessful strategy for treating renal fibrosis. Molecules induced by TGF-β1 to regulate TEC phenotype would serve as more specific and effective targets. To uncover such molecules, we performed high throughput analyses on TGF-β1 treated renal tubular epithelial cells (HKC). RNA sequencing identified 3565 genes and proteomics identified 74 proteins differentially regulated by TGF-β1 in HKC. We then selected 47 genes that were most upregulated in our in vitro datasets and that do not have well-characterized roles in renal fibrosis based on our review of the literature. We searched 8 publicly available datasets containing transcriptomic data from diseased human kidneys for gene expression patterns of the selected genes and found that MARCKS and DOCK2 were amongst the most consistently upregulated in the human datasets. In HKC, MARCKS knockdown decreased TGF-β1 mediated upregulation of pro-fibrotic markers, while DOCK2 knockdown had the opposite effect. Our data suggests novel roles for these proteins in renal TECs as potential promoters the pro-fibrotic phenotype.
2022-07-06 | GSE207291 | GEO
Project description:A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion: Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease.
2016-03-29 | GSE68537 | GEO
Project description:Isolated proximal tubular cells from proximal tubular cell-specific KAT5 knockout mice for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the physiological significance of KAT5 in proximal tubular cells.
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Project description:Smad3 is an important downstream transcriptional factor of TGF-β signaling in the pathogenesis of renal fibrosis. Understanding the target genes is essential to decode the mechanism underlying Smad3-regulated renal fibrosis. To uncover the potential target genes of Smad3 in renal tubular cells, we applied the ChIP-seq technology to analyze the genomic chromatin binding landscape of Smad3 in TCMK-1 cells. The TCMK-1 cells of 90% confluence were starved in low-serum (0.5% FBS) medium for 6 h followed by stimulation with 5 ng/ml TGF-β1 for 30 min. The cells were harvested for ChIP assay. The precipitated chromatin was used for DNA isolation and deep-sequencing.
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