Physical association of NHE3 and SGLT2 mediated by accessory proteins in the renal proximal tubule

Proximal tubule NHE3 is tightly regulated by factors controlling extracellular volume homeostasis, blood pressure, and acid-base balance. Emerging evidence suggests that glycemic control-related factors also influence NHE3, supporting the concept of integrated regulation of fluid and glucose handling in the proximal tubule. Accordingly, gliflozins, a class of antidiabetic drugs that inhibit the Na + /glucose cotransporter SGLT2, also inhibit NHE3. We previously demonstrated that NHE3 and SGLT2, but not SGLT1, colocalize in the proximal tubule apical membrane. However, whether NHE3 and SGLT2 physically associate within a multiprotein complex has remained unclear. This study investigated whether NHE3 and SGLT2 are indirectly linked through their accessory proteins PDZK1 and MAP17. Using non-denaturing electrophoresis, we found that SGLT2 co-migrates with NHE3, MAP17 and PDZK1 in a ~480-kDa complex in rat renal cortex (~15% of total SGLT2 in the complex population). SGLT1 and Na⁺/K⁺-ATPase, included as negative controls, did not co-migrate with NHE3. The NHE3-PDZK1-MAP17-SGLT2 complex is present in both sexes, although distinct NHE3 and SGLT2 banding patterns may reflect sex-associated differences in complex conformation. SGLT2-NHE3 complex formation occurred irrespective of NHE3 phosphorylation at serine 552. Acute empagliflozin infusion altered the relative migration and colocalization of NHE3 with SGLT2 and MAP17 but did not disrupt overall complex integrity, supporting a conformational mechanism underlying its effects on NHE3. Together, these findings suggest that NHE3 and SGLT2 assemble into a multiprotein complex through PDZK1 and MAP17, providing mechanistic insight into the coordinated regulation of sodium, fluid, and glucose reabsorption in the proximal tubule.