Figure 10. The hyperosmotic induction of AQP5 and VEGF depends on the activity of NFAT5. The mRNA levels (A, B, D-G) were determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control (A, B, D) and NaCl control (E-G), respectively. The level of VEGF-A₁₆₅ protein (C, H) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 429.5±71.4 pg/ml [C] and 324.2±35.1 pg/ml VEGF [H], respectively). A-C. The NFAT5 inhibitor rottlerin (2 and 10 µM, respectively) inhibits the hyperosmotic gene expression of AQP5 (A; n=7) and VEGF (B; n=3), as well as the hyperosmotic secretion of VEGF (C; n=5) from RPE cells. Hyperosmolarity was achieved by the addition of 100 mM NaCl to the culture medium. Vehicle control was made with DMSO (1%). D, E. Transfection of RPE cells with NFAT5 siRNA (siNFAT5; 5 nM) results in a reduction of the NFAT5 mRNA level in RPE cells cultured in isoosmotic (D; n=5) and hyperosmotic (+ 100 mM NaCl) media (E; n=3). Data shown in (D) were obtained 48 h after siRNA transfection. Thereafter, the cells were stimulated with a hyperosmotic medium for 2, 6, and 24 h, respectively (E). As negative controls, nontargeted siRNA (siNon; 5 nM) and a transfection reagent (TR) without siRNA were used. F, G. Knocking down the gene expression of NFAT5 with siRNA (siNFAT5; 5 nM) reduced the levels of AQP5 (F; n=6) and VEGF mRNAs (G; n=6) in cells cultured for 24 (F) and 6 h (G), respectively, in hyperosmotic (+ 100 mM NaCl) medium. As a negative control, nontargeted siRNA (siNon; 5 nM) was used (n=6 each). H. NFAT5 siRNA also reduced the secretion of VEGF induced by hyperosmotic (+ 100 mM NaCl) medium (n=6). Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.