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

Proximal tubule Na⁺/H⁺ exchanger 3 (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 sodium-glucose cotransporter 2 (SGLT2), also inhibit NHE3. We previously demonstrated that NHE3 and SGLT2, but not sodium-glucose cotransporter 1 (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 PDZ domain containing 1 (PDZK1) and MAP17. Using nondenaturing electrophoresis, we found that SGLT2 comigrates 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 comigrate 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.<b>NEW & NOTEWORTHY</b> This study reveals that NHE3, SGLT2, and their accessory proteins, PDZK1 and MAP17, assemble into a multiprotein complex in the renal proximal tubule. The merged NHE3-SGLT2 signals show distinct intensity in females, suggesting potential sex-specific differences in complex composition or organization. Acute empagliflozin infusion modifies the colocalization patterns of NHE3 with SGLT2 and MAP17 without disrupting complex integrity, supporting a model in which SGLT2 inhibitors regulate NHE3 through conformational rearrangements within this native complex.