Abstract P133: NaCl And Insulin Regulate Urate Transporter 1 Via Phosphorylation

Abstract

Objective: Hyperuricemia frequently associates with hypertension and diabetes. Previously, we demonstrated that insulin positively regulates urate transporter 1 (URAT1) in renal proximal tubules; yet the detailed mechanisms remain unclear. We here investigated the role of phosphorylation as a potential mechanism regulating URAT1. Methods: We screened potential phosphorylation sites in HEK cells expressing human URAT1 using phospho-Akt substrate motif antibody. Identified sites were confirmed by in vitro kinase assay and the creation of site-specific phosphorylation antibodies. Functional significance of identified sites was evaluated by cell surface biotinylated assay and Western blotting. As upstream signals, we tested the effects of insulin and NaCl in HEK cells expressing URAT1. Results: Western blot using phospho-Akt substrate motif antibody indicated that URAT1 was phosphorylated in HEK cells. Motif search identified two potential phosphorylation sites, T350 and T408 located at the intracellular regions. In ADP-glo assay, phosphorylation of URAT1 by Akt was significantly reduced when both sites were mutated to non-phosphorylatable Ala (T350A/T408A). T408A but not T350A showed decreased N-glycosylation in Western blotting. Biotinylated Western blotting confirmed that the cell-surface expression was significantly reduced in T408A-URAT1, indicating that T408 phosphorylation regulates URAT1 trafficking. Phosphorylation of T408 was confirmed by creating phospho-specific antibodies against this site. As an upstream signaling, insulin increased URAT1 at the cell surface in HEK cells, an effect that was not observed in T408A-URAT1. In kinase screen, we found that Sgk1, PKA, and PKG also phosphorylate URAT1-T408 besides Akt. NaCl increased Sgk1 and T408 phosphorylation, which resulted in increased cell surface URAT1 levels in HEK cells. In contrast, NaCl did not alter cell surface URAT1 levels in HEK cells expressing T408A-URAT1. Conclusion: These data indicate that insulin and NaCl promote membrane trafficking of URAT1 through T408 phosphorylation via Akt and SGK1, respectively. This mechanism could contribute to the co-occurrence of hyperuricemia in patients with hypertension and diabetes.