Renal Protective Effects of Empagliflozin via Inhibition of the EMT Program Associated with Suppression of Aberrant Glycolysis in Proximal Tubules

Background: The sodium glucose co-transporter 2(SGLT2) inhibitors are beneficial in halting diabetic kidney disease, however complete mechanisms are not elucidated yet. Epithelial to mesenchymal transition (EMT) program has been associated with suppression of sirtuin 3 (Sirt3) and aberrant glycolysis. Here, we hypothesized that SGLT2 inhibitor empagliflozin restored normal kidney histology and function, associated with the inhibition of aberrant glycolysis in diabetic kidney. Methods: In vivo, streptozotocin-induced diabetic CD-1 mice, the model exhibiting prominent kidney fibrosis compared to other strains, were utilized and intervention with empagliflozin or insulin was performed. In vitro analysis was performed in HK-2 tubular cells. Results: CD-1 mice with streptozotocin-induced diabetes displayed kidney fibrosis associated with EMT program at 4 months after diabetes induction. Empagliflozin-intervention for 1 month restored all; blood glucose adjusted by insulin did not. Empagliflozin normalized suppressed Sirt3 levels and aberrant glycolysis characterized by hypoxia-inducible factor-1I± accumulation, hexokinase 2 induction and pyruvate kinase isozymes M2 dimer formation in diabetic kidney. Empagliflozin also suppressed accumulation of by-products of glycolysis in diabetic kidney. High-glucose media induced EMT, associated with Sirt3 suppression and aberrant glycolysis induction, in HK2 proximal tubule cell line; SGLT2-knockdown suppressed EMT with restorations of all. Conclusions: Taken together, SGLT2 inhibitors exhibit reno-protective potential that is partially dependent on the inhibition of glucose-reabsorption, which could be utilized as substrate for aberrant glycolysis in kidney tubule. Funding: Empagliflozin was provided by Boehringer Ingelheim through an MTA. This study was essentially supported by the grant from Japan Diabetes Foundation (2016). This work was partially supported by grants from the Japan Society for the Promotion of Science for KK (23790381 and 23790381) and DK (25282028 and 25670414). This work was partially supported by a Grant for Collaborative Research awarded to DK (C2011-4 and C2012-1) and a Grant for Promoted Research awarded to KK (S2015-3, S2016-3, and S2017-1) from Kanazawa Medical University. Boehringer Ingelheim, Mitsubishi-Tanabe Pharma and Ono Pharmaceutical contributed to establishing the Division of Anticipatory Molecular Food Science and Technology. Declaration of Interest: KK and DK received lecture honoraria from Boehringer Ingelheim and Eli Lilly. KK is under a consultancy agreement with Boehringer Ingelheim. Ethical Approval: Animal experiment was approved by IACUC of Kanazawa Medical University (protocol number 2015-59 & 2017-73).