Protective effects of dapagliflozin against oxidative stress-induced cell injury in human proximal tubular cells

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 H(2)O(2). Intracellular Ca(2+) dynamics was monitored by FlexStation 3 using cell-permeable Ca(2+) 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 H(2)O(2)-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 [Ca(2+)](i) in proximal tubular cells, but did not affect calcium release from endoplasmic reticulum and store-operated Ca(2+) entry. The H(2)O(2)-sensitive TRPM2 channel seemed to be involved in the Ca(2+) 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 Ca(2+) dynamics, and thus exerts its protective effects against cell damage under oxidative stress environment.