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Proximal tubular epithelia-specific transcriptomics of diabetic mice treated with dapagliflozin.

ABSTRACT: Based on recent clinical trials using sodium-glucose co-transporter 2 inhibitor (SGLT2i) demonstrating the significant improvement of outcomes of diabetic kidney disease (DKD), the paradigm shift from "glomerulocentric" to "tubule centric" pathophysiology in DKD progression has been highlighted. Several responsible mechanisms for renoprotective effects by SGLT2i have been proposed recently, but the changes in proximal tubule-specific gene expression by SGLT2i in diabetic mice have not been elucidated. We report the analysis of the proximal tubular-specific pathway, demonstrating the downregulation of oxidative phosphorylation in dapagliflozin-treated db/db mice, a type 2 diabetic model. After 8-week treatment of dapagliflozin for db/db mice having a proximal tubule-specific tdTomato reporter, tdTomato-positive cells were isolated by FACS. Pathway analysis of RNA sequencing of isolated tubular epithelia revealed that oxidative phosphorylation was downregulated in dapagliflozin-treated mice. However, depletion of renal tissue ATP content in db/db mice was ameliorated by dapagliflozin administration. Pimonidazole staining demonstrated renal cortical tissue hypoxia in db/db mice, which was improved by dapagliflozin administration. This study suggests that dapagliflozin can ameliorate the excessive oxygen and ATP consumption, and subsequent tissue hypoxia in the diabetic kidney, which may explain, in part, the responsible mechanisms of the renoprotective effects of dapagliflozin.

SUBMITTER: Uehara-Watanabe N

PROVIDER: S-EPMC9485043 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Proximal tubular epithelia-specific transcriptomics of diabetic mice treated with dapagliflozin.

Uehara-Watanabe Noriko N Okuno-Ozeki Natsuko N Nakamura Itaru I Nakata Tomohiro T Nakai Kunihiro K Yagi-Tomita Aya A Ida Tomoharu T Yamashita Noriyuki N Kamezaki Michitsugu M Kirita Yuhei Y Matoba Satoaki S Tamagaki Keiichi K Kusaba Tetsuro T

Heliyon 20220913 9

Based on recent clinical trials using sodium-glucose co-transporter 2 inhibitor (SGLT2i) demonstrating the significant improvement of outcomes of diabetic kidney disease (DKD), the paradigm shift from "glomerulocentric" to "tubule centric" pathophysiology in DKD progression has been highlighted. Several responsible mechanisms for renoprotective effects by SGLT2i have been proposed recently, but the changes in proximal tubule-specific gene expression by SGLT2i in diabetic mice have not been eluci ...[more]

PMID: 36148274

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Project description:Background and objectiveSodium glucose cotransporter 2 (SGLT2) is the main luminal glucose transporter in the kidney. SGLT2 inhibition results in glycosuria and improved glycaemic control. Drugs inhibiting this transporter have recently been approved for clinical use and have been suggested to have potential renoprotective benefits by limiting glycotoxicity in the proximal tubule. We aimed to determine the renoprotective benefits of empagliflozin, an SGLT2 inhibitor, independent of its glucose lowering effect.Research design and methodsWe induced diabetes using a low dose streptozotocin protocol in 7-8 week old endothelial nitric oxide (eNOS) synthase knockout mice. We measured fasting blood glucose on a monthly basis, terminal urinary albumin/creatinine ratio. Renal histology was assessed for inflammatory and fibrotic changes. Renal cortical mRNA transcription of inflammatory and profibrotic cytokines, glucose transporters and protein expression of SGLT2 and GLUT1 were determined. Outcomes were compared to diabetic animals receiving the angiotensin receptor blocker telmisartan (current best practice).ResultsDiabetic mice had high matched blood glucose levels. Empagliflozin did not attenuate diabetes-induced albuminuria, unlike telmisartan. Empagliflozin did not improve glomerulosclerosis, tubular atrophy, tubulointerstitial inflammation or fibrosis, while telmisartan attenuated these. Empagliflozin did not modify tubular toll-like receptor-2 expression in diabetic mice. Empagliflozin did not reduce the upregulation of macrophage chemoattractant protein-1 (MCP-1), transforming growth factor β1 and fibronectin mRNA observed in the diabetic animals, while telmisartan decreased transcription of MCP-1 and fibronectin. Empagliflozin increased GLUT1 mRNA expression and telmisartan increased SGLT2 mRNA expression in comparison to untreated diabetic mice. However no significant difference was found in protein expression of GLUT1 or SGLT2 among the different groups.ConclusionHence SGLT2 inhibition does not have renoprotective benefits independent of glucose lowering.

Project description:Elevated reactive oxygen species (ROS) in type 2 diabetes cause cellular damage in many organs. Recently, the new class of glucose-lowering agents, SGLT-2 inhibitors, have been shown to reduce the risk of developing diabetic complications; however, the mechanisms of such beneficial effect are largely unknown. Here we aimed to investigate the effects of dapagliflozin on cell proliferation and cell death under oxidative stress conditions and explore its underlying mechanisms. Human proximal tubular cells (HK-2) were used. Cell growth and death were monitored by cell counting, water-soluble tetrazolium-1 (WST-1) and lactate dehydrogenase (LDH) assays, and flow cytometry. The cytosolic and mitochondrial (ROS) production was measured using fluorescent probes (H2DCFDA and MitoSOX) under normal and oxidative stress conditions mimicked by addition of H2O2. Intracellular Ca2+ dynamics was monitored by FlexStation 3 using cell-permeable Ca2+ dye Fura-PE3/AM. Dapagliflozin (0.1-10 μM) had no effect on HK-2 cell proliferation under normal conditions, but an inhibitory effect was seen at an extreme high concentration (100 μM). However, dapagliflozin at 0.1 to 5 μM showed remarkable protective effects against H2O2-induced cell injury via increasing the viable cell number at phase G0/G1. The elevated cytosolic and mitochondrial ROS under oxidative stress was significantly decreased by dapagliflozin. Dapagliflozin increased the basal intracellular [Ca2+]i in proximal tubular cells, but did not affect calcium release from endoplasmic reticulum and store-operated Ca2+ entry. The H2O2-sensitive TRPM2 channel seemed to be involved in the Ca2+ dynamics regulated by dapagliflozin. However, dapagliflozin had no direct effects on ORAI1, ORAI3, TRPC4 and TRPC5 channels. Our results suggest that dapagliflozin shows anti-oxidative properties by reducing cytosolic and mitochondrial ROS production and altering Ca2+ dynamics, and thus exerts its protective effects against cell damage under oxidative stress environment.

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Proximal tubular epithelia-specific transcriptomics of diabetic mice treated with dapagliflozin.

Publications

Proximal tubular epithelia-specific transcriptomics of diabetic mice treated with dapagliflozin.

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