Fig. 6

Intracellular ROS during HG conditions with or without GLUT1 blocker BAY 876, PRO20 and the effect of p-coumaric acid (PCA) on CTGF protein levels. A Significant increases in ROS (***p < 0.0001 vs NG, n = 8) was less evident in cells incubated with the GLUT1 specific inhibitor BAY 876 (*p < 0.05 vs NG, n = 8). PRR pharmacological blockade using PRO20 was not able to prevent the augmentation of ROS during HG (***p < 0.0001, n = 8). Co-treatment with ROS scavenger p-coumaric acid (PCA) at 10^–7 M, was able to prevent ROS formation after 24 h HG treatment. ROS were measured by using L2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) which is intracellularly trapped by esterase activity that removes lipophilic blocking groups. Fluorescencence exhibited by dichlorofluorescien (DCF) resulting from the oxidation of the probe by ROS was normalized by micrograms of protein. B Representative immunoblotting showing that increased CTGF protein levels caused by HG were not observed with PCA treatment. *p < 0.05 vs NG, n = 3. C Intracellular TBARS was assessed in cell lysates after 1, 3 and 6 d of exposure to HG compared to controls. HG increased TBARS at day 3 and 6 while BAY876 slightly prevented this increase at day 6, although was significantly higher than cells treated in NG conditions. *p < 0.05; **p < 0.01. D Cell viability of cultured IMCD cells following exposure to HG during 1, 3 or 6 d demonstrated a reduced cell viability after 6 d, BAY876 (red lined bar) partially prevented this effect. Cell viability was assessed by MTT assay. *p < 0.05; **p < 0.01