Canagliflozin impairs citric acid cycle in renal proximal tubule cells

Abstract

Introduction: Canagliflozin is a pharmaceutical indicated for treatment of type 2 diabetes mellitus. Currently approved gliflozins specifically inhibit sodium/glucose transporter 2 (SGLT2). The protein is mainly expressed in renal proximal tubule epithelial cells and its inhibition increases urinary glucose excretion. Pre-clinical toxicological findings differ strikingly among the three approved and marketed gliflozins suggesting distinct off-target profiles. We therefore investigated and compared gliflozins for their off-target toxicities in human proximal tubule epithelial cells in vitro.Objectives: Human proximal tubule epithelial cells (RPTEC/TERT1) were exposed to the SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin and the impact on the cellular energy metabolism was studied.Material and Methods: Lactate and glucose were quantified in cell culture supernatant. Seahorse XF24 was used to assess mitochondrial respiration in intact and permeabilized cells. Cellular amino acids were quantified using HPLC. Cytotoxicity was assessed by MTT reduction and LDH leakage.Results: Canagliflozin (50 μM) but not the other two gliflozins markedly increased cellular glucose consumption and lactate secretion rates, indicative of impaired mitochondrial function. Measurements of cellular oxygen consumption revealed that canagliflozin acutely reduced mitochondrial respiration. This finding suggests a direct interference with components of the electron transport chain (ETC). Analysis of respiration in permeabilized cells ruled out inhibition of complexes II to V, but was consistent with complex I as potential target. A quantification of cellular amino acids revealed that canagliflozin but not other SGLT2 or ETC inhibitors caused a pronounced accumulation of glutamine, glutamate and alanine. These data suggest that canagliflozin disrupts the entry of glutamine into the citric acid cycle (anaplerosis), possibly by inhibiting the 2-oxoglutarate dehydrogenase complex (OGDC). Consistent with the latter, cells relying on glutaminolysis for energy generation were much more sensitive to canagliflozin-induced toxicity. Consequently, canagliflozin appears to affect mitochondrial metabolism, whereas this was not observed for the other two gliflozins tested.