Abstract P414: Blockade of Sodium Glucose Cotransporter 2 by Canagliflozin Suppresses High Glucose-induced Angiotensinogen Augmentation in Renal Proximal Tubular Cells

Abstract

Intrarenal angiotensinogen (AGT) is mainly expressed in proximal tubular cells (PTC). AGT is increased by hyperglycemia (HG) in type 1 and 2 diabetes mellitus, which causes elevated intrarenal angiotensin formation contributing to the development of hypertension and kidney injury. Sodium glucose co-transporter 2 (SGLT2) is abundantly expressed in early PTC and may promote increased intrarenal AGT by increasing intracellular glucose levels. This study tested the effects of canagliflozin (CANA), an SGLT2 inhibitor, on HG-induced AGT elevation in cultured PTC. Mouse PTC were treated with 5, 10 or 25 mM glucose. 0-10 μM CANA was applied one hour before glucose treatment. AGT mRNA and protein levels were measured by digital PCR and western blot analysis. Levels of intracellular reactive oxygen species (ROS) were determined with H 2 DCF-DA. Tempol, an antioxidant, was used to test if elevated ROS is involved in HG-induced AGT upregulation. 10 mM glucose increased AGT protein levels at 12 hours (3.06±0.48-fold compared with 5 mM glucose) and treatment with 10 μM CANA attenuated the AGT augmentation (1.68±0.05-fold). AGT protein levels were also increased by 25 mM glucose; but CANA did not suppress the AGT levels caused by this glucose concentration. In PTC treated with 10 mM glucose for 12 hours, the suppressing effect on AGT upregulation was observed with 1 and 10 μM CANA. Lower concentrations of CANA (0.01 and 0.1 μM) did not lower AGT protein levels significantly. Elevated AGT mRNA expression by glucose was also attenuated by CANA. Treatment of PTC with 1 mM pyruvate also increased AGT expression levels, indicating that glycolysis is involved in HG-induced AGT upregulation. After incubation of PTC with 10 mM glucose for 12 hours, intracellular ROS levels were elevated compared to baseline (4.24±0.23-fold) and these were also inhibited by CANA (0.2±0.08-fold). Furthermore, tempol attenuated AGT upregulation in HG-treated PTC. These results indicate that enhanced glucose entry via SGLT2 into PTC elevates intracellular ROS generation by stimulation of glycolysis and consequent AGT augmentation. Thus, SGLT2 inhibition by CANA may limit HG-induced AGT stimulation which suppress intrarenal angiotensin formation and reduce kidney injury in diabetes mellitus.