Our results demonstrate that Dahl SS rats exhibit a slow progression of hypertension when maintained on a low-salt diet, and consumption of a high-salt diet markedly accelerated the development of hypertension. Eplerenone completely blocked and significantly attenuated the progressive rise in SBP in Dahl SS rats under low-salt and high-salt conditions, respectively. This observation concurs with several recent reports that treatment with eplerenone substantially prevented the development of high salt-induced hypertension in Dahl SS rats (4
). However, in contrast to these findings, other investigators reported that eplerenone has no effects on BP in salt-loaded Dahl SS rats (14
). It is possible that differences in experimental protocol and technique may contribute to these different findings. For example, differences in the age of animals used and in different salt diets employed could influence the time course and magnitude of the hypertension and renal injury. As demonstrated here, the BP lowering efficacy of eplerenone is quite different in animals maintained on low- and high-salt diets. It is well documented that long-term exposure to a high-salt diet from a young age provokes severe hypertension and organ damage (14
), which may be refractory to mineralocorticoid antagonism. A second possible explanation for the different effects of eplerenone on BP in this and other studies could be related to the techniques used to measure BP. We employed radiotelemetry, generally viewed as the “gold standard” for BP measurement (33
), in conscious freely moving animals. However, studies that used the indirect tail cuff plethysmographpy (14
) carry limitations in accuracy and do not monitor BP during an entire 24-h period such that minor differences in BP could have been missed. Finally, the doses of eplerenone used by Ohtani et al. (14
) (12.5 or 40 mg·kg−1
) and Nagata et al. (15
) (30 or 100 mg·kg−1
) could be below those required for BP lowering and plasma exposure data for eplerenone were not provided in their studies. It is important to note that in our study, eplerenone was administered con-comitantly with salt-loading at adult age, and the salt content is lower than that employed in most of the reports (4
With respect to mechanism of action, it is suggested that MR antagonists lower BP via enhanced natriuresis/diuresis or nonrenal mechanisms, including antagonizing MR in the central nervous system or cardiovascular tissues (34
). In the present study, no natriuretic/diuretic effect of eplerenone was detected under either low-salt or high-salt conditions by weekly monitoring of renal excretory function. We suspect that an early effect on natriuresis/diuresis could have been missed by assessing renal excretory function after eplerenone had been administered for 1 week. Accordingly, we performed an acute study with eplerenone (100 mg·kg−1
for 3 days) in age-matched Dahl SS rats (without telemetry device implantation), and determined a significant natriuretic effect on the first day of treatment in the low-salt diet condition but not in the high-salt diet condition. The initial natriuretic/diuretic effect of eplerenone in animals on a low-salt diet could lead to a leftward shift of the steady state pressure-natruresis relationship curve, whereby sodium balance is maintained at a lower BP level. It is notable that the BP lowering effect of eplerenone in animals on a high-salt diet is quite different from the effect seen in animals on a low-salt diet, and the anti-hypertensive effect of eplerenone in high-salt fed animals cannot be explained by acute changes in natriuresis/diuresis. However, the contributions of nonrenal mechanisms such as antagonizing MR in the brain, heart, and vasculature to its BP efficacy remain to be determined.
The second purpose of the present study was to evaluate the effects of eplerenone on organ damage. It is interesting that Dahl SS rats, even on a low-salt diet, developed proteinuria. This effect may be attributed to the glomerular phenotype (loss of foot processes in podocytes) of this rat strain (38
). Although glomerular hypertension could accelerate proteinuria, the increase in glomerular pressure may not be a dominant contributor to the development of proteinuria in Dahl SS rats on low salt diet. Accordingly, a modest decrease in BP by eplerenone in Dahl SS rats on low salt diet, may not decrease urinary protein excretion. Animals on a high-salt diet developed more extensive renal injury as demonstrated by increased kidney injury biomarkers and histopathologic alterations in glomeruli, vasculature, and interstitium. The kidneys were enlarged, and proteinuria was dramatically accelerated. All the above-mentioned changes induced by a high-salt challenge were attenuated by eplerenone treatment. These findings further support the concept that eplerenone has renoprotective effects in salt-sensitive hypertensive renal injuries (7
). With respect to cardiac structure and function, it is apparent that a high-salt diet caused cardiac hypertrophy and increased heart rate; the latter could potentially reflect an early stage of cardiac dysfunction. Chronic treatment with eplerenone significantly reduced the cardiac changes observed in animals on a high-salt diet. Consistent with our findings, Ohtani et al. (14
) and Nagata et al. (15
) observed that salt-loading could induce heart failure when 8% salt was introduced to 7 weeks old Dahl SS rats for 5-6 weeks, subsequent treatment with eplerenone for 7-8 weeks results in attenuation in left ventricular hypertrophy and heart failure. Thus, MR antagonism is beneficial to the heart under high-salt conditions. It is not the scope of the present study to investigate the mechanism of organ protection afforded by eplerenone. However, it is reasonable to speculate that hemodynamic and/or nonhemodynamic, as well as other direct actions, might contribute to beneficial effects of eplerenone on tissues. An extensive body of evidence suggests salt-sensitive hypertension is associated with endothelial dysfunction, oxidative stress, inflammation, and fibrosis (28
), and eplerenone has been shown to suppress the induction of oxidative stress (4
), fibrosis/apoptosis, and inflammation (7
) in Dahl SS rats fed high-salt diet.
It is generally accepted that high-salt intake suppresses the renin-angiotensin system and aldosterone levels and low-salt intake has the opposite effect (39
). Previous reports showed that salt-loading in Dahl SS rats reduced plasma aldosterone levels (8
); however, tissue (heart and kidney) levels of aldosterone (40
) or MR mRNA or protein levels of MR (8
) were upregulated. These findings suggested that high dietary salt evokes inappropriate activation of the local aldosterone/MR pathway, which may play a causal role in the development of salt-sensitive hypertension. In the present study, high dietary salt did not change serum aldosterone levels, contrary to reports from other groups (8
). The underlying reason for this divergent finding is unclear at this stage. However, we speculate that the blood sampling method and time of blood collection may affect the measurement of serum aldosterone. In the present study, blood samples were collected around 10AM by jugular venepuncture in conscious animals. It is known that aldosterone secretion exhibits circadian rhythm in rats (41
) and humans (43
). Preliminary data (unpublished) from our laboratory indicates that aldosterone levels measured in conscious animals are at their lowest in the early morning. In addition, the stress caused by the procedure of jugular venepuncture in a conscious state might have an impact on aldosterone secretion. However, it is difficult to make an appropriate comparison with other published studies in which blood collection was conducted under anesthesia and the timing of the sample collection was not reported (8
). We did not determine tissue levels of aldosterone in the present study. Interestingly, Bayorh et al. (40
) reported that tissue levels of aldosterone are increased under conditions of high-salt administration to Dahl SS rats. Thus, it is conceivable that the beneficial effects of eplerenone (hemodynamic or organ protection) may be a consequence of inhibiting the effects of elevated tissue levels of aldosterone.
A concern associated with the clinical use of MR antagonists is hyperkalemia (45
). In the present study, urinary potassium excretion was slightly less in those animals that received eplerenone but the change did not reach statistical significance. Furthermore, plasma potassium levels were not statistically different among groups, although there was a trend for an increase in plasma potassium for those animals on a high-salt diet with eplerenone treatment. The somewhat modest effect on urinary potassium excretion and the absence of elevated plasma aldosterone in these studies may both be a consequence of submaximal MR blockade. Although we do not have a direct in-vivo
measure of the extent of receptor blockade achieved, we do know that we achieved plasma levels of eplerenone of less than 100 nM (77.3 nM in animals on a low-salt diet and 90.6 nM in animals on a high-salt diet). Published data (37
) indicated that in rats, the IC50
of eplerenone for the aldosterone receptor was 360 nM. Thus, plasma levels of eplerenone of less than 100 nM would not be expected to produce complete receptor blockade, which may account for the minimal changes in potassium excretion and little change in the plasma concentration of aldosterone. Furthermore, it is also possible that higher doses of eplerenone, and hence higher plasma exposure, could produce a greater extent of BP lowering and organ protection.
In conclusion, the findings of the present study demonstrate that MR antagonism attenuates the development and progression of hypertension and provides target organ protection in a model of salt-sensitive hypertension. Importantly, these data further support the promise of MR antagonists for the treatment of hypertension.