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. 2021 Jan 6;320(4):C619–C634. doi: 10.1152/ajpcell.00177.2019

Figure 3.

Figure 3.

Inhibition of SGK1 reduces high glucose-induced increases in NKCC and NHE1 abundances and activities. Following high glucose and kinase inhibitor exposures, bovine CMEC were analyzed for abundance and activity of NKCC and NHE1. A: Upper panel: representative Western Blot of CMEC NKCC abundance following 24 h exposure to control normoglycemic medium (NG, 5 mM D-Glucose) or high glucose medium (HG, 30 mM D-Glucose) with SGK1 inhibitor GSK-650394 (1 µM) or vehicle. Lower panel: Quantitated abundance of CMEC NKCC following 24 h exposures to control NG medium or HG medium with 1 µM GSK-650394 or vehicle. Data are expressed relative to NG control and are presented as Tukey box and whisker plots. N values are 3,10, and 6, respectively, for NG, HG, and HG with GSK-650394,. B: NKCC activity in CMEC following exposure to the same conditions as in A. NKCC activity was assessed as bumetanide-sensitive K influx using the 86Rb flux assay as described in Methods. Data are expressed relative to NG control and presented as Tukey box and whisker plots. N values are 4, 5, and 6, respectively for NG, HG, and HG with GSK-650394. C: Upper panel: representative Western blot of CMEC NHE1 abundance following 24 h exposure to control normoglycemic medium (NG, 5 mM D-Glucose) or high glucose medium (HG, 30 mM D-Glucose) with SGK1 inhibitor GSK-650394 (1 µM) or vehicle. Lower panel: Quantitated abundance of CMEC NHE1 following 24 h exposures to control NG medium or HG medium with 1 µM GSK-650394 or vehicle. Data are expressed relative to NG control and are presented as Tukey box and whisker plots. N values are 3, 8, and 6, respectively for NG, HG, and HG with GSK-650394. D: NHE activity in CMEC following exposure to the same conditions as in C. NHE activity was assessed as HOE-642-sensitive H flux using the NH4 prepulse method with the pH sensitive dye BCECF as described in Methods. N values are 4, 8, and 6, respectively for NG, HG, and HG with GSK-650394. *Significantly different than NG control vehicle by Kruskal-Wallis test. *P < 0.05, **P < 0.01. #Significantly different than HG vehicle by Mann-Whitney test, **P < 0.01, ***P < 0.001.