Fig 2: Regulation of Kir4.1 and Kir4.2 gene expression in cultured RPE cells by pathogenic conditions. Real-time RT-PCR was carried out with RNA extracted from cells which were stimulated 2, 6, and 24 h. In (f), a 12-h stimulation was also examined on Kir4.2 gene expression. Relative mRNA levels are shown as folds of unstimulated control (1). (a) Effects of chemical hypoxia, induced by adding 150 µM CoCl2, on the Kir channel gene expression. (b) Effect of cell culture in a 0.2% O2-atmosphere. (c–f) Effects of oxidative stress (20 µM H2O2; (c)), hyperglycemia (25 mM glucose; (d)), extracellular hypoosmolarity (60% osmolarity; (e)), and hyperosmolarity (+100 mM NaCl; (f)). (g) Effects of hyperosmotic media prepared by adding 100 mM NaCl or 200 mM sucrose, or by coaddition of NaCl and sucrose, on the Kir4.2 gene expression. (h) Effect of tetracaine (1 mM) on the Kir4.2 mRNA level. (i) Dose-dependent effect of high NaCl on the Kir4.2 mRNA level. Different concentrations (10–100 mM) of NaCl were added to the culture media. Each bar shows data derived from 3–9 independent experiments with cells of different donors. Significant difference vs. unstimulated control: * p < 0.05.

Fig 3: Kir4.2 knockdown affects the proliferation and viability of cultured RPE cells. The expression levels of the Kir4.2 gene (a) and the VEGFA gene (b), the proliferation rate (c), and the viability (d) were investigated in cells transfected with Kir4.2 siRNA (siKir4.2) and nontargeted siRNA (siNon). The cells were cultured 24 h in the absence and presence of high (+100 mM) NaCl and CoCl2 (150 µM), respectively, as indicated by the panels of the bars. The data are shown as fold (1; (a,b)) and percent of unstimulated, nontransfected control cells (100%; (c,d)), respectively. Each bar represents data obtained in 4–10 independent experiments using cells of different donors. Significant difference vs. unstimulated, nontransfected control cells: * p < 0.05. Significant difference vs. NaCl and CoCl2 control, respectively, and transfection with siNon: ? p < 0.05. Significant difference between siKir4.2 and siNon: ? p < 0.05.

Fig 4: Activities of transcription factors which mediate the hyperosmotic and CoCl2-induced expression of the Kir4.2 gene in cultured RPE cells. mRNA levels were examined with real-time RT-PCR analysis. Relative mRNA levels are shown as fold of unstimulated control (1). (a,b) The cells were maintained 24 h in the absence (control) and presence of high (+100 mM) NaCl (a) and CoCl2 (150 µM; (b)), respectively. The effects of a HIF-1 inhibitor (HIF Inh; 5 µM), the STAT3 inhibitor Stattic (1 µM), the NF-?B inhibitor CAPE (5 µM), the AP-1 inhibitor SR11302 (5 µM), and the CREB inhibitor 666-15 (250 nM) were tested. (c) Transfection of the cells with NFAT5 siRNA (siNFAT5; 5 nM) resulted in a reduction of the NFAT5 mRNA level in RPE cells cultured 24 h in the absence (control) and presence of high (+100 mM) NaCl and CoCl2 (150 µM), respectively. Nontargeted scrambled siRNA (siNon; 5 nM) had no effects. (d) Knockdown of NFAT5 gene expression with siRNA (siNFAT5; 5 nM) did not alter the Kir4.2 mRNA level in cells cultured under the three conditions tested. Each bar represents data obtained in 3–7 independent experiments using cells of different donors. Significant difference vs. unstimulated control: * p < 0.05. Significant difference vs. NaCl and CoCl2 control, respectively: ? p < 0.05. Significant difference between siNFAT5 and siNon: ? p < 0.05.

Fig 5: Kir4.2 gene transcription and mRNA stability. The hyperosmotic and CoCl2-induced expression of the Kir4.2 gene in cultured RPE cells is mediated by stimulation of gene transcription; extracellular hyperosmolarity also induces an increase of Kir4.2 mRNA stability. The relative mRNA level is shown as fold of unstimulated control (1; (a)) and percent of the 0-h control (b). (a) The NaCl- and CoCl2-induced Kir4.2 gene expression was prevented by actinomycin D (ActD; 5 µg/mL) which blocks RNA polymerase II. The cells were cultured 12 h in the absence and presence of high (+100 mM) NaCl and 24 h in the absence and presence of CoCl2 (150 µM), respectively. (b) The stability of the Kir4.2 mRNA differed significantly (p < 0.05; ∆) between cells cultured in the presence of high NaCl compared to cells cultured under control conditions and stimulated by CoCl2, respectively. The cells were first stimulated for 12 h with high (+100 mM) NaCl, and 24 h with CoCl2 (150 µM), respectively. Thereafter, actinomycin D (5 µg/mL) was added, and total RNA was isolated at different times. Each bar represents data obtained in 3–7 independent experiments using cells of different donors. Significant difference vs. unstimulated control: * p < 0.05. Significant difference vs. NaCl and CoCl2 control, respectively: ● p < 0.05. Significant difference vs. 0-h control: ○ p < 0.05.