Abstract
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.
Highlights
Dapagliflozin, named Farxiga1, is a glucose-lowering therapeutic drug in the gliflozins family [1,2]
Two primer sets across introns were used for sodium glucose co-transporter 2 (SGLT-2) and β-actin was set as a positive control, the bands for SGLT-2 were much stronger than that for sodium glucose co-transporter-1 (SGLT-1) and β-actin (Fig 1), suggesting that SGLT-2 is highly expressed in the proximal tubular cell line, which confirms the specific cell marker for the proximal tubular cells used in the study
Cell proliferation was monitored by water-soluble tetrazolium-1 (WST-1) assay, which is proportional to the number of viable cells
Summary
Dapagliflozin, named Farxiga, is a glucose-lowering therapeutic drug in the gliflozins family [1,2]. Clinical trials have demonstrated that the inhibition of glucose uptake by proximal tubules leads to lower glucose and glycated haemoglobin (HbA1c) levels in the blood and reduces the risk of developing later life diabetic complications, such as cardiovascular events and diabetic kidney disease [2,6,7] Such beneficial effects and underlying mechanisms of SGLT-2 inhibitors are largely unknown. The reduction of ROS production was observed in rat heart after a 10-week treatment with the dapagliflozin analogue empagliflozin [11] These studies suggest that SGLT-2 inhibitors may have anti-oxidative properties. Dapagliflozin decreases the amplitude of intracellular Ca2+ transient and L-type Ca2+ current in cardiac myocytes isolated from normal and diabetic rats [12], suggesting it may affect intracellular calcium dynamics
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