Background <p>Aquaporin 2 (AQP2), a vasopressin-sensitive water channel in the renal collecting ducts, maintains body water homeostasis. Sepsis reduces AQP2 and vasopressin receptor 2 (V2R) levels, contributing to acute kidney injury. In a heritable nephrogenic diabetes insipidus mouse model with V2R mutation, Wnt-5a, a ligand for frizzled receptors (Fzds), enhances AQP2 expression and translocation through calcium/calmodulin/calcineurin signaling. However, the mechanism by which Wnt-5a reduces sepsis-induced AQP2 inhibition remains unclear. We assessed this mechanism and whether Wnt-5a alleviates sepsis-induced downregulation of renal AQP2.</p> Methods <p>Our study used in vitro and in vivo approaches. To study AQP2 trafficking, lipopolysaccharide (LPS) was applied to mouse inner medullary collecting duct 3 (mIMCD3) cells in vitro<i>;</i> subsequently, phosphorylated AQP2 and apical AQP2 expression as well as calcineurin activity and its upstream regulators (endogenous Wnt-5a, Fzds, and intracellular calcium intensity) were examined. In vivo, cecal ligation and puncture (CLP) mice were used to assess AQP2 levels and urine osmolality as indicators of urinary concentration.</p> Results <p>In mIMCD3 cells, LPS application was associated with reduced V2R expression, a vasopressin-irreversible reduction in AQP2, and increased Fzds turnover and Wnt-5a-stimulated intracellular calcium, enhancing calcineurin signaling. Subsequent application of Wnt-5a to LPS-exposed mIMCD3 cells prevented AQP2 reduction. Moreover, it increased phosphorylated AQP2 at residue Ser269 (Ser269-pAQP2) and decreased at Ser261-pAQP2 without affecting Ser256-pAQP2. These effects were reversed by the calcineurin inhibitor cyclosporin A. In CLP mice, Wnt-5a injection was associated with increased renal AQP2 and urine osmolality.</p> Conclusion <p>Wnt-5a attenuates sepsis-induced AQP2 downregulation through the calcineurin signaling pathway.</p>