Dapagliflozin Ameliorates High-Fat, Diet-Induced, Megalin-Mediated Autolysosomal Disorders in Mouse Proximal Tubules

Background: Megalin is a multi-ligand endocytic receptor located in the apical membrane of proximal tubules (PTs). Our prior study (JASN 2016) elucidated that megalin-mediated endocytosis of glomerular-filtered lipotoxic substances drives tubulo-glomerular alterations in mice fed a high-fat diet (HFD). In this model, pathological vacuolation appears particularly in segment 2 (S2) of PTs due to megalin-dependent autolysosomal disorders. Receptor-mediated endocytosis (RME) is the primary pathway for substance uptake in S1 (greater than S2), while fluid-phase endocytosis (FPE) prevails in S2, which is vulnerable to metabolic overload due to its less advanced endolysosomal systems. This investigation aims to explore the impact of dapagliflozin, an SGLT2 inhibitor, on autolysosomal disorders in PTs of HFD-fed mice and its underlying mechanism. Methods: Nine-week-old male C57BL/6J mice were fed an HFD with oral administration of dapagliflozin (1mg/kg body weight/day) or vehicle for 28 days (n=10 each). Kidney sections underwent PAS staining, and Image-Pro Plus ver. 7.0 evaluated the vacuolar area/cortical tubular area. Urinary C-megalin, a marker for lysosomal overload in PTs (Diabetes 2017), was also measured. Renal megalin expression was gauged via immunoblotting, qPCR, and immunohistochemistry. Megalin's endocytic function was assessed by measuring the urinary excretion of α1-microglobulin, an endocytic ligand of megalin. RME and FPE were scrutinized on kidney sections post intravenous injection of fluorescent lysozyme and dextran, respectively, in 9-week-old male kidney-specific conditional megalin KO mice and littermate controls, and C57BL/6J mice receiving dapagliflozin or vehicle for 5 days. Results: Dapagliflozin administration significantly reduced the vacuolar area/cortical tubular area and urinary C-megalin excretion in HFD-fed mice. While renal megalin expression remained unaltered, urinary α1-microglobulin excretion increased with dapagliflozin administration. In megalin KO mice, the uptake of fluorescent lysozyme and dextran in PTs decreased. Similarly, the administration of dapagliflozin to mice diminished the uptake of both tracers in PTs. Conclusion: Dapagliflozin ameliorates HFD-induced, megalin-mediated autolysosomal disorders particularly in S2 of PTs by suppressing both RME and FPE dependent on megalin. Funding: Commercial Support - Denka Company Limited, Government Support – Non-U.S.