Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells

1 Department of General and Environmental Physiology, University of Bari, Bari, Italy; and ; 2 Department of Cell and Molecular Physiology, Yale Medical School, New Haven, Connecticut Submitted June 25, 2009 ; accepted in final form November 16, 2009 Vasopressin causes the redistribution of the water channel aquaporin-2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of collecting duct principal cells, leading to urine concentration. The molecular mechanisms regulating the selective apical sorting of AQP2 are only partially uncovered. In this work, we investigate whether AQP2 sorting/trafficking is regulated by its association with membrane rafts. In both MCD4 cells and rat kidney, AQP2 preferentially 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 1 ) AQP2 associates with detergent-resistant membranes early in the biosynthetic pathway; 2 ) strong cholesterol depletion delays the exit of AQP2 from the trans -Golgi network. Interestingly, mild cholesterol depletion promoted a dramatic accumulation of AQP2 at the apical plasma membrane in MCD4 cells in the absence of forskolin stimulation. An internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. Taken together, these data suggest that association with membrane rafts may regulate both AQP2 apical sorting and endocytosis. membrane rafts Address for reprint requests and other correspondence: G. Procino, Dept. of General and Environmental Physiology, Univ. of Bari, Via Amendola 165/A, 70126 Bari, Italy (e-mail: g.procino{at}biologia.uniba.it ).