Eplerenone suppresses aldosterone/ salt-induced expression of NOX-4

Abstract

Introduction: Salt-induced hypertension in the Dahl rat is associated with increases in angiotensin II, aldosterone, free radical generation and endothelial dysfunction. However, little is known about the specific mechanism(s) associated with the end-organ damage effects of aldosterone. We hypothesised that eplerenone reduces kidney damage by blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.

Methods: Dahl salt-sensitive rats fed either a low-salt (LS) or high-salt (HS) diet were treated with aldosterone in the presence of eplerenone or apocynin. Indirect blood pressure was measured prior to start of diet and weekly thereafter. Levels of plasma nitric oxide (NO) and urinary 8-isoprostane were measured following treatment. Protein levels of selected subunits of NADPH were assessed by western blot.

Results: Eplerenone and apocynin inhibited the rise in blood pressure induced by HS and/or aldosterone. This observation was accompanied with a parallel change in kidney protein levels of NADPH oxidase 4 (NOX-4) and p22phox. Aldosterone and high salt were associated with lower NO levels and greater renal oxidative stress.

Conclusions: NADPH oxidase is associated with the vascular and renal remodelling observed in high dietary salt intake. Aldosterone-induced expression of NOX-4 plays a pivotal role in the end-organ damage effect of aldosterone, as eplerenone tended to reduce kidney damage and inhibit NOX expression.

Figure 1

Effect of eplerenone and apocynin on systolic blood pressure and urinary 8-isoprostane levels. Figure 1A represents the systolic blood pressure after 4 weeks on either a low-salt (LS) or a high-salt (HS) diet, with or without (control) treatment with aldosterone in the presence of eplerenone or apocynin. Figure 1B shows urinary 8-isoprostane levels following 4 weeks on the treatment protocols. Data are represented as mean ± SEM for six animals per group. Significant difference (p < 0.05) from the LS group’s systolic blood pressure is denoted by #; * denotes significant difference (p < 0.05) from the control group (no drug treatment: low and high salt); ** denotes significant difference (p < 0.05) from aldosterone treatment only.

Figure 2

Effect of eplerenone and apocynin on plasma nitric oxide levels in Dahl salt-sensitive rats on an aldosterone/salt diet. Data are represented as mean ± SEM for six animals per group. # Denotes a significant difference (p < 0.05) from control (on diet with no drug treatment) value. * Denotes significant difference (p < 0.05) from the low-salt group.

Figure 3

Representative western blots of aldosterone/salt-induced protein levels of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX-4) subunit of NADPH following treatment with eplerenone. Figure 3B is a histogram reflecting the data in figure 3A. LS: low salt, HS: high salt.

Figure 4

Representative western blots (A) of aldosterone/salt-induced protein levels of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX-4), p22phox and p47phox subunits of NADPH following treatment with apocynin. Figure 4B–C is histogram reflecting the data in figure 4A for p22phox and NOX-4 respectively. LS: low salt, HS: high salt.