Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K_ATP channel activation

Purpose: The expression of aquaporin-8 (AQP8), which plays a crucial role in the maintenance of the cellular fluid and electrolyte balance, was shown to be increased in RPE cells under hyperosmotic conditions. The aim of the present study was to investigate the mechanisms of hyperosmotic AQP8 gene expression and the localization of AQP8 in cultured human RPE cells.

Methods: Hyperosmolarity was produced with the addition of 100 mM NaCl or 200 mM sucrose. Hypoxia was induced by cell culture in a 0.2% O₂ atmosphere or the addition of the hypoxia mimetic CoCl₂. Oxidative stress was induced by the addition of H₂O₂. Gene expression was determined with real-time RT-PCR analysis. AQP8 protein localization and secretion of VEGF were evaluated with immunocytochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA).

Results: Immunocytochemical and western blot data suggest that the AQP8 protein is mainly located in the mitochondria. Extracellular hyperosmolarity, hypoxia, and oxidative stress induced increases in AQP8 gene expression. Hyperosmotic AQP8 gene expression was reduced by inhibitors of the p38 MAPK and PI3K signal transduction pathways, and by JAK2 and PLA₂ inhibitors, and was in part mediated by the transcriptional activity of CREB. Hyperosmotic AQP8 gene expression was also reduced by autocrine/paracrine interleukin-1 signaling, the sulfonylureas glibenclamide and glipizide, which are known inhibitors of K_ATP channel activation, and a pannexin-blocking peptide. The K_ATP channel opener pinacidil increased the expression of AQP8 under control conditions. The cells contained Kir6.1 and SUR2B gene transcripts and displayed Kir6.1 immunoreactivity. siRNA-mediated knockdown of AQP8 caused increases in hypoxic VEGF gene expression and secretion and decreased cell viability under control, hyperosmotic, and hypoxic conditions.

Conclusions: The data indicate that hyperosmotic expression of AQP8 in RPE cells is dependent on the activation of K_ATP channels. The data suggest that AQP8 activity decreases the hypoxic VEGF expression and improves the viability of RPE cells which may have impact for ischemic retinal diseases like diabetic retinopathy and age-related macular degeneration.