Sodium-glucose co-transporter 2 mediates angiotensinogen augmentation in renal proximal tubular cells under high glucose conditions
Increased activity of the intrarenal renin-angiotensin system (RAS), in which proximal tubular angiotensinogen (AGT) is a key factor, has been implicated in the progression of diabetic nephropathy. AGT expression is upregulated in renal proximal tubular cells (PTC) by high glucose due to elevated reactive oxygen species (ROS) generation. Angiotensin II (Ang II) also promotes ROS generation. Glucose reabsorption in PTC occurs mainly through sodium-glucose co-transporter 2 (SGLT2). This study was performed to demonstrate that SGLT2 mediates AGT augmentation in PTC under hyperglycemic conditions. Furthermore, the enhancing effect of Ang II was investigated. Established mouse PTC were treated with 5 (normal), 15, or 25 mM D-glucose or D-mannitol (osmotic control). Pyruvate was used to investigate the role of glycolysis on AGT regulation. Glycolytic activity was quantified using a Seahorse metabolic analyzer. Tempol, an antioxidant, was used to determine the role of ROS. SGLT2 expression was silenced using shRNA. PTC were treated with high glucose and 10-10-10-7 M Ang II or an Ang II receptor blocker. AGT protein levels were increased by 15 (4.4 ± 0.2-fold over control) and 25 mM (4.6 ± 0.2-fold) glucose. AGT mRNA was also augmented (31.1 ± 3.5-fold) by 25 mM glucose, but not mannitol. AGT expression was stimulated by pyruvate (10.7 ± 1.0-fold over control), and exposure to 10, 15, or 25 mM glucose increased glycolytic activity (3.10 ± 0.28-fold, 2.74 ± 0.20-fold, and 2.75 ± 0.34-fold, respectively), suggesting that enhanced glycolysis stimulates AGT expression. ROS accumulation increased (3.03 ± 0.29-fold) in 25 mM glucose over control. Tempol attenuated glucose-induced AGT augmentation by 77%, suggesting that ROS generation contributes to AGT upregulation. SGLT2 knockdown prevented AGT augmentation in 15 mM glucose, indicating that SGLT2 plays a key role mediating AGT upregulation by high glucose. Ang II receptor blockade did not alter AGT levels, and Ang II did not enhance AGT expression in normal or high glucose. Similarly, SGLT2 expression was unchanged by glucose or Ang II. These results indicate that SGLT2 contributes to AGT upregulation in PTC by high glucose, which helps to explain the mechanisms causing intrarenal RAS activation and consequent diabetic nephropathy.