Phosphorylation of aquaporin-2 at serine 269 in urinary extracellular vesicles reflects renal vasopressin activity in rats

Through activation of the arginine vasopressin (AVP) receptor 2 in the renal collecting duct, phosphorylation of the C-terminus of aquaporin-2 (AQP2) triggers AQP2 translocation to the apical membrane, thereby enhancing water reabsorption. AQP2 is excreted into urine via urinary extracellular vesicles (uEVs). Recently, phosphorylated S256 and S269 AQP2 (pS256- and pS269-AQP2), both of which are well-characterized phosphorylated AQP2, have been detected in murine uEVs. However, their biological significance in uEVs remains poorly understood. Here, we employed validated antibodies against pS256- and pS269-AQP2 to investigate the effect of AVP on the secretion of these forms into uEVs. In a rat hydration model, uEV pS269-AQP2 showed the most pronounced increase compared with total AQP2 (phosphorylated + non-phosphorylated forms) and pS256-AQP2 after single-dose treatment with a synthetic AVP analogue, dDAVP. A similar response pattern was observed in a long-term dDAVP-treatment model. Unlike total AQP2 and pS256-AQP2, pS269-AQP2 was localized predominantly at the apical membrane of renal collecting duct cells, regardless of dDAVP treatment or hydration status. Furthermore, in a dehydration/hydration model, uEV pS269-AQP2 showed a close association with urinary osmolality and electrolyte concentrations. Although uEV pS256-AQP2 was also associated with these urinary indices, the relationship was more robust for uEV pS269-AQP2. By contrast, uEV total AQP2 did not correlate significantly with urinary osmolality or electrolyte concentrations. These findings suggest that secretion of pS269-AQP2 in uEVs accurately reflects renal AVP activity. Given the technical challenges in measuring blood AVP, pS269-AQP2 in uEVs may hold promise as a non-invasive biomarker of renal AVP activity.