AMPK Phosphorylates Urea Transporter A1 and Aquaporin 2 in Rat Inner Medullary Collecting Ducts

Abstract

Individuals with nephrogenic diabetes insipidus (NDI) cannot concentrate urine due to a defective vasopressin response. When this results from congenital defects in the vasopressin receptor, or from drug toxicity blocking cAMP production, the transporter system responsible for urine concentration remains intact. We explored whether adenosine monophosphate activated kinase (AMPK) might replace vasopressin and improve urine concentration by phosphorylation of urea transporter A1 (UT-A1) and aquaporin 2 (AQP2). Kidney inner medullas (IM) dissected from rats were enzymatically digested to yield suspensions of IMCDs. AMPK was stimulated with metformin (800µM). Phosphorylation of transporters was assessed by metabolic radiolabeling of IM tissue followed by immunoprecipitation of transporters and autoradiography. AMPK stimulation increased phosphorylation of both UT-A1 (by 70 ± 19%) and AQP2 (by 284 ± 96%). Membrane accumulation was determined by ex vivo biotinylation followed by Western analysis of membrane associated transporters. The membrane amount of AQP2 was increased by 76 ± 26% with AMPK stimulation. To determine cellular localization, rats were injected with metformin, kidneys were perfusion fixed and analyzed by immunohistochemistry. Metformin injection of the rats resulted in increased membrane association of AQP2. Summary: UT-A1 and AQP2, two transporters that are crucial for kidney concentrating ability, are both phosphorylated by AMPK. AQP2 membrane association is increased by metformin stimulation of AMPK. This is the first demonstration that AMPK phosphorylates UT-A1 and AQP2 in the kidney inner medulla. These results suggest that metformin may be a potential therapeutic agent for treatment of patients with NDI.