Membrane rafts regulate both apical sorting and endocytosis of aquaporin 2 in renal cells.

AIM: During antidiuresis, vasopressin release causes the translocation of the water channel aquaporin 2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of the collecting duct principal cells, leading to urine concentration. In the inheritable disorder nephrogenic diabetes insipidus (NDI), patients are unable to concentrate their urine in response to vasopressin. Mutations of the vasopressin receptor II (V2R) gene have shown to be causative for 90% of all NDI cases. The comprehension of the molecular mechanisms regulating AQP2 apical sorting/trafficking is functional to the identification of new molecular targets for treatment of NDI. Because patients with V2R mutations express functional AQP2 in their collecting ducts, it would be possible to develop a therapy if an alternative, V2R-independent pathway, leading to AQP2 membrane expression, could be activated. The raft mechanism is currently the most favored apical sorting mechanism thus in this study, we investigate whether AQP2 sorting/trafficking is regulated by association with membrane rafts. METHODS: The study was performed in MCD4 mouse collecting duct principal cells. Immunolocalization of AQP2 was performed by confocal microscopy and its association with membrane rafts was examined on the base of rafts insolubility in non-ionic detergents at low temperature. AQP2 association with membrane rafts along the biosynthetic pathway was also investigated in Brefeldin A-treated cells. Strong or mild cholesterol depletion were performed incubating cells in the presence of the cholesterol-lowering drug Lovastatin in LDL-deficient or complete medium, respectively. RESULTS: In both MCD4 cells and rat kidney, AQP2 was stably associated with Lubrol WX-insoluble membranes regardless of its presence in the storage compartment or at the apical membrane. Block-and-release experiments indicate that, within the secretory pathway, AQP2 associates with detergent-resistant membranes as early as the endoplasmic reticulum. When AQP2 sorting/trafficking was analyzed after strong cholesterol depletion, the apical sorting of AQP2 at the TGN was significantly delayed. Interestingly, in MCD4 cells, mild cholesterol depletion promoted a strong accumulation of AQP2 at the apical plasma membrane in the absence of FK stimulation. Internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. CONCLUSIONS: Taken together, these findings suggest that the association with membrane rafts regulates AQP2 apical sorting, localization at the apical plasma membrane and endocytosis. The lovastatin-induced AQP2 accumulation at the apical membrane is of a particular interest in the view of a possible use of statins in the treatment of those V2R-dependent forms of NDI.