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Extracellular Vesicles From High Glucose-Treated Podocytes Induce Apoptosis of Proximal Tubular Epithelial Cells.

ABSTRACT: Diabetic kidney disease (DKD) is a serious and common complication of diabetes. Extracellular vesicles (EVs) have emerged as crucial vectors in cell-to-cell communication during the development of DKD. EVs may mediate intercellular communication between podocytes and proximal tubules. In this study, EVs were isolated from podocyte culture supernatants under high glucose (HG), normal glucose (NG), and iso-osmolality conditions, and then co-cultured with proximal tubular epithelial cells (PTECs). MicroRNAs (miRNA) sequencing was conducted to identify differentially expressed miRNAs of podocyte EVs and bioinformatics analysis was performed to explore their potential functions. The results showed that EVs secreted from HG-treated podocytes induced apoptosis of PTECs. Moreover, five differentially expressed miRNAs in response to HG condition were identified. Functional enrichment analysis revealed that these five miRNAs are likely involved in biological processes and pathways related to the pathogenesis of DKD. Overall, these findings demonstrate the pro-apoptotic effects of EVs from HG-treated podocytes on PTECs and provide new insights into the pathologic mechanisms underlying DKD.

SUBMITTER: Huang Y

PROVIDER: S-EPMC7667287 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Extracellular Vesicles From High Glucose-Treated Podocytes Induce Apoptosis of Proximal Tubular Epithelial Cells.

Huang Ying Y Li Ruizhao R Zhang Li L Chen Yuanhan Y Dong Wei W Zhao Xingchen X Yang Huan H Zhang Shu S Xie Zhiyong Z Ye Zhiming Z Wang Weidong W Li Chunling C Li Zhilian Z Liu Shuangxin S Dong Zheng Z Yu Xueqing X Liang Xinling X

Frontiers in physiology 20201102

Diabetic kidney disease (DKD) is a serious and common complication of diabetes. Extracellular vesicles (EVs) have emerged as crucial vectors in cell-to-cell communication during the development of DKD. EVs may mediate intercellular communication between podocytes and proximal tubules. In this study, EVs were isolated from podocyte culture supernatants under high glucose (HG), normal glucose (NG), and iso-osmolality conditions, and then co-cultured with proximal tubular epithelial cells (PTECs). ...[more]

PMID: 33224036

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Project description:Hypoxia is the key pathobiological trigger of tubular oxidative stress and cell death that drives the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). The mitochondrial-rich proximal tubular epithelial cells (PTEC) are uniquely sensitive to hypoxia and thus, are pivotal in propagating the sustained tubular loss of AKI-to-CKD transition. Here, we examined the role of PTEC-derived small extracellular vesicles (sEV) in propagating the 'wave of tubular death'. Ex vivo patient-derived PTEC were cultured under normoxia (21 % O2) and hypoxia (1 % O2) on Transwell inserts for isolation and analysis of sEV secreted from apical versus basolateral PTEC surfaces. Increased numbers of sEV were secreted from the apical surface of hypoxic PTEC compared with normoxic PTEC. No differences in basolateral sEV numbers were observed between culture conditions. Biological pathway analysis of hypoxic-apical sEV cargo identified distinct miRNAs linked with cellular injury pathways. In functional assays, hypoxic-apical sEV selectively induced ferroptotic cell death (↓glutathione peroxidase-4, ↑lipid peroxidation) in autologous PTEC compared with normoxic-apical sEV. The addition of ferroptosis inhibitors, ferrostatin-1 and baicalein, attenuated PTEC ferroptosis. RNAse A pretreatment of hypoxic-apical sEV also abrogated PTEC ferroptosis, demonstrating a role for sEV RNA in ferroptotic 'wave of death' signalling. In line with these in vitro findings, in situ immunolabelling of diagnostic kidney biopsies from AKI patients with clinical progression to CKD (AKI-to-CKD transition) showed evidence of ferroptosis propagation (increased numbers of ACSL4+ PTEC), while urine-derived sEV (usEV) from these 'AKI-to-CKD transition' patients triggered PTEC ferroptosis (↑lipid peroxidation) in functional studies. Our data establish PTEC-derived apical sEV and their intravesicular RNA as mediators of tubular lipid peroxidation and ferroptosis in hypoxic kidney injury. This concept of how tubular pathology is propagated from the initiating insult into a 'wave of death' provides novel therapeutic check-points for targeting AKI-to-CKD transition.

Project description:Background Albuminuria is a hallmark of diabetic kidney disease (DKD) that promotes its progression, leading to renal fibrosis. Renal macrophage function is complex and influenced by macrophage metabolic status. However, the metabolic state of diabetic renal macrophages and the impact of albuminuria on the macrophage metabolic state are poorly understood. Methods Extracellular vesicles (EVs) from tubular epithelial cells (HK-2) were evaluated using transmission electron microscopy, nanoparticle tracking analysis and western blotting. Glycolytic enzyme expression in macrophages co-cultured with HSA-treated HK-2 cell-derived EVs was detected using RT-qPCR and western blotting. The potential role of EV-associated HIF-1α in the mediation of glycolysis was explored in HIF-1α siRNA pre-transfected macrophages co-cultured with HSA-treated HK-2 cell-derived EVs, and the extent of HIF-1α hydroxylation was measured using western blotting. Additionally, we injected db/db mice with EVs via the caudal vein twice a week for 4 weeks. Renal macrophages were isolated using CD11b microbeads, and immunohistofluorescence was applied to confirm the levels of glycolytic enzymes and HIF-1α in these macrophages. Results Glycolysis was activated in diabetic renal macrophages after co-culture with HSA-treated HK-2 cells. Moreover, HSA-treated HK-2 cell-derived EVs promoted macrophage glycolysis both in vivo and in vitro. Inhibition of glycolysis activation in macrophages using the glycolysis inhibitor 2-DG decreased the expression of both inflammatory and fibrotic genes. Mechanistically, EVs from HSA-stimulated HK-2 cells were found to accelerate macrophage glycolysis by stabilizing HIF-1α. We also found that several miRNAs and lncRNAs, which have been reported to stabilize HIF-1α expression, were increased in HSA-treated HK-2 cell-derived EVs. Conclusion Our study suggested that albuminuria induced renal macrophage glycolysis through tubular epithelial cell-derived EVs by stabilizing HIF-1α, indicating that regulation of macrophage glycolysis may offer a new treatment strategy for DKD patients, especially those with macroalbuminuria. Supplementary Information The online version contains supplementary material available at 10.1186/s10020-022-00525-1.

Project description:Proximal tubular epithelial cells (PTEC) are central players in inflammatory kidney diseases. However, the complex signalling mechanism/s via which polarized PTEC mediate disease progression are poorly understood. Small extracellular vesicles (sEV), including exosomes, are recognized as fundamental components of cellular communication and signalling courtesy of their molecular cargo (lipids, microRNA, proteins). In this study, we examined the molecular content and function of sEV secreted from the apical versus basolateral surfaces of polarized human primary PTEC under inflammatory diseased conditions. PTEC were cultured under normal and inflammatory conditions on Transwell inserts to enable separate collection and isolation of apical/basolateral sEV. Significantly increased numbers of apical and basolateral sEV were secreted under inflammatory conditions compared with equivalent normal conditions. Multi-omics analysis revealed distinct molecular profiles (lipids, microRNA, proteins) between inflammatory and normal conditions for both apical and basolateral sEV. Biological pathway analyses of significantly differentially expressed molecules associated apical inflammatory sEV with processes of cell survival and immunological disease, while basolateral inflammatory sEV were linked to pathways of immune cell trafficking and cell-to-cell signalling. In line with this mechanistic concept, functional assays demonstrated significantly increased production of chemokines (monocyte chemoattractant protein-1, interleukin-8) and immuno-regulatory cytokine interleukin-10 by peripheral blood mononuclear cells activated with basolateral sEV derived from inflammatory PTEC. We propose that the distinct molecular composition of sEV released from the apical versus basolateral membranes of human inflammatory PTEC may reflect specialized functional roles, with basolateral-derived sEV pivotal in modulating tubulointerstitial inflammatory responses observed in many immune-mediated kidney diseases. These findings provide a rationale to further evaluate these sEV-mediated inflammatory pathways as targets for biomarker and therapeutic development.

Project description:Transfer RNA-derived fragments (tRFs), a novel class of small non-coding RNA produced by the cleavage of pre- and mature tRNAs, are involved in various diseases. Renal tubulointerstitial fibrosis is a common final pathway in diabetic nephropathy (DN) in which hyperglycemia-induced tubular extracellular matrix (ECM) accumulation serves a vital role. The present study aimed to detect and investigate the role of tRFs in the accumulation of tubular ECM. Differentially expressed tRFs were analysed with high-throughput sequencing in primary mouse tubular epithelial cells treated with high glucose (HG). The Gene Ontology (GO) was used to analyze the potential molecular functions of these differentially expressed tRFs, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the associated signaling pathways involved in these differentially expressed tRFs. tRF-1:30-Gln-CTG-4 was overexpressed using tRF-1:30-Gln-CTG-4 mimic, followed by HG treatment. A total of 554 distinct tRFs were detected and 64 differentially expressed tRFs (fold change >2; P<0.05) were identified in tubular epithelial cells following high glucose (HG) treatment, among which 27 were upregulated and 37 were downregulated. Ten selected tRFs with the greatest difference (fold change >2; P<0.05) were verified to be consistent with small RNA-sequencing data, of which tRF-1:30-Gln-CTG-4 showed the most pronounced difference in expression and was significantly decreased in response to HG. GO analysis indicated that the differentially expressed tRFs were associated with ‘cellular process’, ‘biological regulation’ and ‘metabolic process’. An analysis of the KEGG database suggested that these differentially expressed tRFs were involved in ‘autophagy’ and signaling pathways for ‘forkhead box O’, ‘the mammalian target of rapamycin’ and ‘mitogen-activated protein kinase’. Finally, the overexpression of tRF-1:30-Gln-CTG-4 ameliorated HG-induced ECM accumulation in tubular epithelial cells. Therefore, the present study demonstrated that there may be a significant association between tRFs and HG-induced ECM accumulation in tubular epithelial cells; these differentially expressed tRFs warrant further study to explore the pathogenesis of DN.

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Extracellular Vesicles From High Glucose-Treated Podocytes Induce Apoptosis of Proximal Tubular Epithelial Cells.

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Extracellular Vesicles From High Glucose-Treated Podocytes Induce Apoptosis of Proximal Tubular Epithelial Cells.

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