Aim: 

Aquaporin 2 (AQP2) is the water channel selectively expressed at the apical plasma membrane of the kidney collecting duct principal cells in response to vasopressin. In this study we investigate whether AQP2 apical sorting is determined by its association with lipid rafts.

Methods: 

The study was performed in MCD4 mouse collecting duct principal cells stably expressing human AQP2. Immunolocalization of AQP2 was performed by confocal microscopy and its association with lipid rafts was examined on the base of the lipid rafts insolubility in different non-ionic detergents at low temperature.

Raft versus non-raft membranes were separated upon centrifugation on a discontinuous sucrose gradient. Detergent lisates from both total cells and purified plasma membrane were used as starting material. AQP2 association with lipid rafts along the biosynthetic pathway from the ER to the plasma membrane was also investigated in Brefeldin A-treated cells.

Results: 

Non-ionic detergents insolubility assay, together with flotation on sucrose gradient, indicate that in MCD4 cells, AQP2 is stably associated with Lubrol WX-insoluble/Triton X-100-soluble lipid rafts. Reversible accumulation of newly synthesized AQP2 in the ER indicates that AQP2 acquires lipid rafts association already in the ER. FK stimulation, while not modifying the overall association of AQP2 with lipid rafts, significantly and specifically increases the expression of AQP2 at the apical plasma membrane in the lipid rafts domains. Interestingly, at the apical plasma membrane, AQP2 is strictly associated with the lipid raft-rich microvilli rather than with the planar region of the membrane and can be extracted by TX-100 but not by Lubrol WX treatment.

Conclusions: 

Taken together, these findings suggest that in renal cell, AQP2 is sorted into the apical route and accumulated into the microvilli upon FK stimulation by association with a novel family of Lubrol WX-insoluble lipid rafts.