Figure 2. Effect of uremic toxins on ROS production in C2C12 myoblast cells.

(A) Effect of uremic toxins on ROS production was determined using CM-H₂DCFDA. C2C12 myoblast cells were starved with serum free medium for 2 hr, and then treated with CM-H₂DCFDA in D-PBS for 30 min. After removal of the D-PBS, the cells were treated with uremic toxins and incubated for 2 hr. Fluorescence intensity was measured at an excitation wavelength of 485 nm and at an emission wavelength of 535 nm. (B) Dose-dependent effect of IS on ROS production was measured in C2C12 myoblast cells (C) Expression of mouse Oat1 and Oat3 in C2C12 cells was detected by Western blotting. To determine the effect of inhibitors of Oats, NADPH oxidase or AHR on the IS-induced ROS production, cells were incubated with CM-H₂DCFDA for 30 min followed by incubation with (D) Oats inhibitors (probenecid (0.5 mM)), (E) NADPH oxidase inhibitor (diphenylene iodonium: DPI (50 μM)) or (F) AHR inhibitor (CH223191 (10 μM)) for 30 min. The cells were then incubated with IS (1 mM) for 1 hr with Oats inhibitors or 2 hr with others. Data are expressed the means ± SEM (n = 6). **p < 0.01 compared with control. ^#p < 0.05, ^##p < 0.01 compared with IS only.