Project description:Recent studies have shown the crucial role of podocyte injury in the development of diabetic nephropathy. Ubiquitination/deubiquitination is a post-translational modification of proteins, which is widely involved in the occurrence and development of diseases. The purpose of this study was to investigate the role of OTUD5, a deubiquitination enzyme, in podocyte injury and reveal its regulatory mechanism in the pathogenesis of diabetic nephropathy. RNA-seq analysis indicated a significantly increased expression of OTUD5 in HG/PA-stimulated podocytes.
Project description:In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of “immediate early response” genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.
Project description:In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of “immediate early response” genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.
Project description:In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of “immediate early response” genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.
Project description:Extracellular vesicles were isolated from the cell culture supernatants of podocytes under high glucose(HG), normal glucose(NG) and iso-osmolality stimulation (three replicates/group). miRNA sequencing was performed to identify differentially expressed miRNAs in podocyte-derived extracellular vesicles. After sequencing, A total of 1915 miRNAs were annotated from all samples . A comparison of the HG and NG groups showed that 11 miRNAs were differentially expressed (4 for up-regulated and 7 for down-regulated,|log2(fold change)| > 1, p-value < 0.05), while a comparison of the HG and iso-osmolality groups showed that 18 miRNAs were differentially expressed (1 for up-regulated and 17 for down-regulated, |log2(fold change)| > 1, p-value < 0.05). This study provides the results of miRNA alteration in podocytes extracellular vesicles under HG stimulation.
Project description:The effect of miRNA delivery from glomerular endothelial cells (GEnCs) to podocytes in vitro was examined by miRNA epression in podocytes untreated and treated with extracellular vesicles from GEnCs under varied activation conditions.
Project description:The effect of miRNA delivery from glomerular endothelial cells (GEnCs) to podocytes in vitro was examined by mRNA epression in podocytes untreated and treated with extracellular vesicles from GEnCs under varied activation conditions.
Project description:Purpose: Next-generation sequencing (NGS) was used to define the transcriptome of native mouse podocytes and non-podocytes glomerular cells as part of a project aiming to define the molecular fingerprint of mouse podocytes. Method: Glomeruli from 29 Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J x hNPHS2Cre mice at the age of 10 weeks were purified and a single cell solution was prepared to seperate GFP-expressing (podocytes) and GFP-negative (non-podocytes glomerular cells) cells by FACS sorting. RNA was extracted and prepared for further analysis using directional, polyA+ library preparation. An Illumina HiSeq2500 was used for a paired-end sequencing of 100 cycles . Salmon and Sleuth were used for downstream analysis. Results: A total of 100 Million reads each from podocytes and non-podocytes glomerular cells could be used for further analysis.
Project description:The growth hormone plays a significant role in normal renal function and overactive growth hormone signaling has been implicated in proteinuria in diabetes. Earlier studies from our group have shown that the glomerular podocytes, which play an essential role in renal filtration, express the growth hormone receptor, suggesting the direct action of growth hormone on these cells. Nevertheless, the precise mechanism and the downstream pathways that are induced by the excess growth hormone in these podocytes leading to diabetic nephropathy are not clearly established. To compressively understand the growth hormone’s effect on podocytes at transcript level we performed RNA-Sequencing. Conditionally immortalized human podocytes were employed in this study.