Sglt2-independent effects of gliflozins on proximal tubule function

Abstract

Background: Beyond glycemic control, Sglt2 inhibitors (Sglt2i) have protective effects on renal function. Sglt2i-mediated reductions in Na+ reabsorption have been suggested to protect PT cells from damage. The mechanisms responsible for renoprotection are unknown and have been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, and the effects of Sglt2i on endocytic function are unknown. Aim of this study: to dissect the direct and immediate effects of SGLT2i on PT cell functions using opossum kidney (OK) PT cells, which represents a powerful physiologically-relevant system model. Methods: We tested the direct effects of Sglt2i on Na+-dependent fluid transport and endocytic uptake in a highly differentiated cell culture model that recapitulates the morphology, metabolism, and high endocytic and transport capacity of PT cells in vivo. The effect of gliflozins on albumin uptake and on fluid transport was quantified in OK PT cells. The effect of canagliflozin vs the NHE3 inhibitor S3226 on early endosome pH in OK cells was measured using fluorescence ratio imaging. Effects of gliflozins, NHE3 inhibitors, and AMPK pathway perturbants were determined by immunoblotting. 10 week-old male C57BL/6 mice were given canagliflozin or empagliflozin by oral gavage daily following vehicle gavage. Urine was collected via metabolic cages at baseline and after 24 h and 48h. Creatinine and albumin were measured by ELISA. Results: Canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin did not affect endosomal pH, and albumin uptake was further reduced when added together with the selective NHE3 inhibitor S3226. Additionally, canagliflozin did not alter NHE3 phosphorylation when added alone, but prevented the reduction in phosphorylation caused by the selective NHE3 inhibitor S3226. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated animals, suggesting that off-target effects of canagliflozin contribute significantly to reduced Na+-dependent fluid transport in vivo. Conclusions: Canagliflozin reduces fluid transport and albumin uptake in PT cells via effects on mitochondrial function upstream of the AMPK/mTOR axis. Sglt2i-mediated reductions in Na+-dependent fluid transport in the PT are secondary to reduced mitochondrial function and are not NHE3-specific. ASN Pre-doctoral fellowship Award, National Institutes of Health: R01 DK118726, R01 DK125049, S10 OD021627, Dialysis Clinic Inc. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.