Figure 1

Tolvaptan induces Nrf2 nuclear translocation and HO-1 expression in mpkCCD cells. (a) Tolvaptan promotes Nrf2 nuclear translocation. (Left panel) Western blotting of Nrf2 in nuclear extract. mpkCCD cells were treated with 200 μM tolvaptan or 5 μM sulforaphane on filter for 12 h, following which nuclear fraction was separated using commercially available reagents for nuclear extraction. Arrow indicates the band of Nrf2. (Right panel) Densitometric analysis of Nrf2. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05. C: control (DMSO), Tol: 200 μM tolvaptan, Sul: 5 μM sulforaphane. P.C.: positive control. (b) Tolvaptan increases HO-1 mRNA expression in a dose-dependent manner. mpkCCD cells were treated with 10–200 μM tolvaptan for 4 h, following which HO-1 mRNA expression was examined using qPCR. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05, **P < 0.01. (c) Tolvaptan induces HO-1 protein expression in a dose-dependent manner. (Top panel) mpkCCD cells were treated with 10–200 μM tolvaptan for 12 h. (Bottom panel) Densitometric analysis of HO-1 is presented. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05, **P < 0.01. (d) An Nrf2 inhibitor, ML385, inhibits the HO-1 induction by tolvaptan. (Top panel) Following the pre-treatment of mpkCCD cells using DMSO (C, T, and S) or 50 μM ML385 (T + M and S + M) for 1 h, mpkCCD cells were treated with 200 μM tolvaptan or 5 μM sulforaphane in the presence or absence of 50 μM ML385 for 12 h. C: control (DMSO), T: 200 μM tolvaptan, M: 50 μM ML385, S: 5 μM sulforaphane. (Bottom panel) Densitometric analysis of HO-1 is presented. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05, **P < 0.01.