Annexin A2 mediates apical trafficking of renal Na‐K‐2Cl cotransporter (892.22)

Abstract

Transport activity of the thick ascending limb (TAL) Na‐K‐2Cl‐cotransporter (NKCC2) critically depends on its association with lipid raft membrane microdomains (LR). The mechanisms involved in the recruitment of NKCC2 into LR have not been elucidated so far. In the present study we hypothesized that the lipid raft scaffolding protein annexin A2 serves as an essential mediator in this process. Detergent‐resistant LR were isolated from rat renal outer medullary tissue samples using discontinuous sucrose density gradient and ultracentrifugation (floating assay). Interaction between NKCC2 and Anxa2 was analyzed by immunoprecipitation, GST pull down, and peptide spot assays. Distribution of the products in the apical TAL membrane was studied by immunofluorescence and immunogold electron microscopy. Trafficking was studied in AVP‐deficient rats and cultured TAL cells using the AVP analogue desmopressin (dDAVP) or low chloride hypotonic stress. Functional relevance of Anxa2 for NKCC2 trafficking was studied using RNAi‐mediated knockdown strategies. Floating assay, immunoprecipitation and colocalization studies confirmed the parallel presence of NKCC2 and Anxa2 in apical membrane LR of the TAL. Binding assays approved physical interaction of Anxa2 and the non‐phosphorylated N‐terminus of NKCC2. Moreover, dDAVP and low chloride hypotonic stress caused parallel trafficking of NKCC2 and Anxa2 in rats and in cultured TAL cells and this response was inhibited by RNAi‐mediated knockdown of Anxa2. In conclusion our data demonstrate that Anxa2 is an essential component of the NKCC2 trafficking protein complex and mediates its recruitment into LR microdomains.