Figure 8.

MR is required for NF-κB activation by aldosterone. (A) Effect of the GR agonist dexamethasone on NF-κB activity. mpkCCD_cl4 cells transiently were transfected with NF-κB–dependent luciferase-reporter gene, seeded on polycarbonate filters and incubated with 10⁻⁶ M dexamethasone (Dxm) or aldosterone (Aldo) for 6 h before measurement of luciferase activity. Results are expressed as a percentage of control (untreated cells; Ctl). Bars represent mean ± SEM from six independent experiments. *P < 0.05. (B) The effect of aldosterone was prevented by an MR antagonist. mCCD_cl1 cells grown on polycarbonate filters were incubated for 6 h in the presence or absence of 10⁻⁶ M Aldo and/or 10⁻⁵ M of the specific MR antagonist 17OHP. IκBα and PAI-1 mRNA was detected by RT-PCR. Results are expressed as fold of control values set to 1. Bars are mean ± SEM from six independent experiments. *P < 0.05, **P < 0.01. (C and D) Silencing of MR prevented the effects of aldosterone. mCCD_cl1 cells transfected with siRNA targeting MR or scrambled RNA, seeded on plastic support (C) or polycarbonate filters (D), were incubated in the absence (Ctl) or presence of 10⁻⁶ M aldosterone (Aldo) for 6 h, before protein (B) or RNA extraction (C). (C) MR protein abundance, assessed by immunoblot, confirmed the specific knockdown of MR by siRNA. Representative blots from two independent experiments are shown. (D) SGK1, IκBα, PAI-1, or IL-6 mRNA was detected by RT-PCR. Results are expressed as fold of control value set to 1. Bars are means ± SEM from six independent experiments. *P < 0.05 versus aldosterone-stimulated scrambled RNA.