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bioRxiv · 10.1101/2024.10.08.617300

Focal Adhesion Kinase (FAK) inhibition induces membrane accumulation of aquaporin2 (AQP2) through endocytosis inhibition and actin depolymerization in renal epithelial cells.

Tchakal Mesbahi, A.·He, J.·Zhu, S.·Huang, M.·Fukushima, K.·Bouley, R.·Brown, D.·Lu, H. A. J.
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Abstract

Cellular trafficking of the water channel aquaporin 2 (AQP2) is regulated by the actin cytoskeleton in collecting duct principal cells (PC) to maintain proper water balance in animals. Critical actin depolymerization/polymerization events are involved in both constitutive AQP2 recycling, and the pathway stimulated by vasopressin receptor signaling. Focal adhesion kinase (FAK) plays an important role in modulating the actin cytoskeleton through inhibiting small GTPases, and multiple studies have shown the involvement of FAK in insulin and cholesterol trafficking through actin regulation. To understand whether FAK contributes to water reabsorption by the kidney, we performed a series of in vitro experiments to examine the involvement of FAK and its signaling in mediating AQP2 trafficking in cultured renal epithelial cells. Our data showed that FAK inhibition by specific inhibitors caused membrane accumulation of AQP2 in AQP2expressing LLCPK1 cells by immunofluorescence staining. AQP2 membrane accumulation induced by FAK inhibition is associated with significantly reduced endocytosis of AQP2 via the clathrin-mediated endocytosis pathway. Moreover, AQP2 membrane accumulation induced by FAK inhibition also occurred in cells expressing the constitutive dephosphorylation mutant of AQP2, S256A. This was confirmed by immunoblotting using a specific antibody against phospho-serine 256 AQP2, supporting a phosphorylation independent mechanism. Finally, we demonstrated that inhibition of FAK caused reduced RhoA signaling and promoted F-actin depolymerization. In conclusion, our study identifies FAK signaling as a pathway that could provide a novel therapeutical avenue for AQP2 trafficking regulation in water balance disorders.

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Tchakal Mesbahi, A., He, J., Zhu, S., Huang, M., Fukushima, K., Bouley, R., Brown, D., Lu, H. A. J.. 2024-10-09. Focal Adhesion Kinase (FAK) inhibition induces membrane accumulation of aquaporin2 (AQP2) through endocytosis inhibition and actin depolymerization in renal epithelial cells.. https://doi.org/10.1101/2024.10.08.617300

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