Focal Adhesion Kinase inhibition induces membrane accumulation of aquaporin-2 in renal epithelial cells by actin depolymerization and endocytosis inhibition

Vasopressin (VP)-induced cellular trafficking of the water channel aquaporin-2 (AQP2) to maintain systemic water balance is regulated in large part by the actin cytoskeleton in collecting duct principal cells (PC). Actin depolymerization/polymerization is involved in both constitutive AQP2 recycling, and the VP stimulated pathway; in many cells, focal adhesion kinase (FAK) modulates the actin cytoskeleton by inhibiting small GTPases. To understand the contribution of FAK to this process, we examined its involvement in AQP2 trafficking. We first showed that FAK inhibition using the drug VS-4718 caused membrane accumulation of AQP2 in LLC-AQP2 epithelial cells in culture and PC in situ (kidney slices), by immunofluorescence staining and biotinylation. This was associated with significantly reduced endocytosis of AQP2 via the clathrin-mediated pathway, along with a reduction in RhoA activation and F-actin depolymerization. Importantly, AQP2 membrane accumulation induced by VS-4718 also occurred in cells expressing the dephosphorylation mutant of AQP2, S256A. Unlike VP, FAK inhibition did not increase cellular cAMP, nor AQP2 S256 phosphorylation. As expected, treatment with the FAK inhibitor VS-4718 suppressed FAK phosphorylation at Tyr397 in both cultured cells and kidney tissue. However, VP stimulation induced a redistribution of phospho-FAK (Tyr397) from basolateral toward the apical region of collecting duct principal cells in tissue slices, concomitant with AQP2 accumulation, but no overall change in the level of FAK phosphorylation was detectable after VP treatment. These data, therefore, identify FAK signaling as a distinct pathway that could provide a novel therapeutic avenue for regulating AQP2 trafficking in water balance disorders.