Hypertension affects ≈1 billion people worldwide, and suboptimal control of blood pressure (BP) is the number-one attributable risk for death throughout the world. In the United States, 40% of people with hypertension are not being treated, and only 34% of treated hypertensive patients are controlled to BP levels <140/90 mm Hg.1 Hypertension places a strain on the healthcare delivery system because of its prevalence and severity of associated complications.2 Treatment guidelines for hypertension recommend thiazide-type diuretics, angiotensin-converting enzyme (ACE) inhibitors, or calcium channel blockers (CCBs) as acceptable initial drug choices.1 However, most patients require ≥2 antihypertensive agents to achieve adequate BP control, and guidelines recommend combining agents from different classes.1,3 Combination therapy provides different mechanisms of action to enhance BP lowering, improve outcomes, and lessen adverse effects. Diuretics effectively lower BP and reduce cardiovascular morbidity and mortality,1,3,4 but they may cause adverse metabolic effects such as the increased incidence of new-onset diabetes mellitus as seen in the chlorthalidone arm of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT).3 Independent of their BP-lowering effects, ACE inhibitors prevent adverse cardiovascular outcomes and provide protection from end-organ injury through reductions in microalbuminuria and left ventricular hypertrophy (LVH) and improvements in vascular endothelial function.1,5-8 Recent studies suggest that the CCB amlodipine, like ACE inhibitors, improves antioxidant and anti-inflammatory markers and endothelial function independently of reductions in BP.9-11 CCBs effectively lower BP by acting on vascular smooth muscle.12 Higher doses, however, can cause significant side effects such as peripheral edema.12 The incidence of peripheral edema can be decreased by combining the CCB with an ACE inhibitor.13,14 Combination therapy with agents from different classes can, therefore, further reduce BP and ameliorate side effects.1
Clinical studies have demonstrated that ≈50% of hypertensive patients have salt-sensitive hypertension, a well-known independent risk factor for increased cardiovascular morbidity and mortality.15,16 Dahl salt-sensitive (DS) rats given dietary salt develop hypertension accompanied by cardiovascular and renal injury.17 We have demonstrated that DS rats manifest a specific vascular diathesis linked to nitric oxide deficiency, endothelial dysfunction, increased oxidative stress, and functional upregulation of angiotensin II.9,17-19 Given the importance of achieving BP control and the numerous antihypertensive medications available singly and in combination, the purpose of this study was to compare an ACE inhibitor (benazepril), a CCB (amlodipine), a diuretic (hydrochlorothiazide [HCTZ]), or combinations of benazepril plus amlodipine or benazepril plus HCTZ for BP reduction and end-organ protection in a rat model that is a paradigm of salt-sensitive hypertension in humans. Because the typical American diet is relatively high in salt1 and many people do not adhere to a low-salt diet, we mimicked the clinical situation by maintaining the DS rats on a high-salt diet.
MATERIALS AND METHODS
Six-week-old male DS rats purchased from Harlan Sprague-Dawley (Indianapolis, IN) were maintained under controlled conditions of light, temperature, and humidity in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. The study was approved by the Institutional Animal Care and Use Committee at the Miami (Florida) Veterans Affairs Medical Center.
After a 2-week acclimatization to the new environment, the rats were separated into 3 primary groups (control, single therapy, combination therapy) that were further divided into subgroups (Fig. 1). The control groups consisted of rats fed a normal-salt diet (0.5% NaCl, n = 8) or a high-salt diet (4% NaCl, n = 8) for 6 weeks. The single-therapy groups consisted of 8 rats each and were treated with 10 mg/kg amlodipine daily by gavage, 40 mg/kg benazepril daily by gavage, or 75 mg/L HCTZ in drinking water. The combination-therapy groups consisted of 5 rats treated with 40 mg/kg benazepril and 10 mg/kg amlodipine daily by gavage, 9 rats treated with 20 mg/kg benazepril and 5 mg/kg amlodipine daily by gavage, and 9 rats treated with 40 mg/kg benazepril by gavage and 75 mg/kg HCTZ in drinking water daily. All of the rats in the single- and combination-therapy groups were also fed a high-salt diet.
End-organ Damage Analyses
End-organ damage was determined by LVH, aortic hypertrophy, and proteinuria after 6 weeks of treatment. The rats were decapitated, and the heart, kidneys, and aorta were harvested. The left ventricle was dissected from the right ventricle and weighed. The aorta was weighed and measured; the measurement reference points were the arch of thoracic aorta and the origin of mesenteric artery. Twenty-four-hour urinary excretion was collected in individual metabolic cages, and urine protein excretion, expressed in milligrams per 24 h, was determined by BioRad.9,18,19 Systolic BP was measured in conscious rats by the tail-cuff method (Visitech Systems, Apex, NC).
Endothelial Function Analysis
Endothelial function was determined using an organ chamber bath as previously described.9,18,19 Aortic rings were precontracted to 70% of maximal contraction to norepinephrine. Acetylcholine (10−9-10−5 mol/L) was added and aortic endothelium-dependent relaxation (EDR) measured.
Relaxation of aortic rings was expressed as percent inhibition of norepinephrine-induced constriction. The maximal vasorelaxation to acetylcholine (Emax) and the concentrations of an agonist causing a half-maximal response (ED50) were determined from the concentration-response curves using the best fit to a logistic sigmoid function. The results were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed by analysis of variance (ANOVA) with Bonferonni's correction for multiple comparisons, followed by Scheffe's test. Statistical significance was defined as P < 0.05.
Systolic BP was significantly higher with a high-salt diet (187 ± 6 mmHg) than with a normal-salt diet (143 ± 4 mmHg). Figure 2 shows that monotherapy with amlodipine (159 ± 2 mmHg) or HCTZ (161 ± 3 mmHg) but not benazepril (178 ± 4 mmHg) significantly lowered systolic BP compared with the systolic BP of control rats fed a high-salt diet (P < 0.05). All of the combination therapy regimens were more effective in reducing systolic BP than was monotherapy with any drug, and the combinations normalized systolic BP to values comparable to those observed in the normal-salt control group (Fig. 2).
Figure 3A shows that aortic hypertrophy was partially reduced by monotherapy with amlodipine and HCTZ but not benazepril, and Figure 3B shows that LVH was reduced with HCTZ (223 mg/100 g body weight) compared with the high-salt control group (244 mg/100 g body weight; P < 0.05). Compared with monotherapy, all of the combination regimens were more effective in preventing aortic hypertrophy. The combination of benazepril and HCTZ normalized LVH to values comparable to those observed in the normal-salt control group (201 and 207 mg/100 g body weight, respectively).
Proteinuria was significantly reduced by monotherapy with amlodipine, benazepril, and HCTZ compared with high-salt controls, but was not normalized (Fig. 4). Combination therapy with benazepril/amlodipine 40/10, benazepril/amlodipine 20/5, or benazepril/HCTZ normalized urinary protein excretion to levels (31.5, 32.5, and 29 mg/24 h, respectively) similar to those of the normal-salt control group (28 mg/24 h).
Endothelium-dependent relaxation to acetylcholine in the aortic rings of hypertensive high-salt rats was significantly impaired (Table 1). Monotherapy with amlodipine or benazepril but not HCTZ significantly improved endothelium-dependent relaxation to acetylcholine (Table 1), whereas the combination therapy completely normalized the endothelium-dependent relaxation to acetylcholine (Table 1). We and others have demonstrated that endothelium-independent relaxation to nitric oxide donor was not impaired in the aorta of hypertensive DS rats.17,20 Therefore, we surmised that the improvement of vasorelaxation to acetylcholine after antihypertensive therapy was mainly the result of restoration of nitric oxide bioavailability.
The present study shows that in the hypertensive DS rat, a paradigm of salt-sensitive hypertension in humans, combination therapy with benazepril plus amlodipine or benazepril plus HCTZ was more effective in lowering systolic BP and reducing proteinuria, aortic hypertrophy, and LVH than was monotherapy with any of these agents.
Experimental and clinical studies have demonstrated that ACE inhibitors have many beneficial effects on the vasculature beyond their hemodynamic effects, including improvement of endothelial function and reduction of inflammation associated with atherosclerosis.21 It has been proposed that ACE inhibitors may be less effective in older adult and African American patients, populations with a high prevalence of salt-sensitive hypertension.22 In the present study, we showed that the ACE inhibitor benazepril alone did not reduce systolic BP and resulted in minimal reduction in proteinuria. However, despite the absence of a BP-lowering effect, benazepril substantially improved endothelial function in this animal model, suggesting a significant vascular benefit. We have recently demonstrated similar effects with an angiotensin II receptor blocker.18 Furthermore, combination of benazepril with either amlodipine or HCTZ normalized BP, significantly reduced end-organ injury, and was superior to monotherapy.
Amlodipine exhibited similar protective properties in the present salt-sensitive hypertension study as in a previous study of angiotensin II-induced hypertension.9 Amlodipine significantly decreased BP and aortic hypertrophy and preserved endothelial function. Amlodipine stimulates nitric oxide production to an extent similar to ACE inhibitors, and its antioxidant property effectively inhibits oxidative stress-dependent mechanisms involved in angiotensin II-mediated cardiovascular injury.9,10,23
Although monotherapy with amlodipine effectively lowered BP in DS rats on a high-salt diet, it did not prevent end-organ damage or restore endothelial function to that of normal-salt control rats. However, combination therapy with benazepril and amlodipine resulted in significant improvements in LVH and all other markers of target organ damage compared with the high-salt control group. Similar results were obtained with the combination of benazepril plus HCTZ. Although the present study was not specifically designed to investigate different combination therapy dosing regimens, it appears that improvements in systolic BP, aortic hypertrophy, and endothelial function may be dose dependent.
Because of their effects of blunting sodium and water retention, diuretics effectively lower BP in salt-sensitive hypertension.17,22 However, compared with benazepril or amlodipine, HCTZ did not affect endothelial function, whereas the combination of HCTZ and benazepril not only normalized BP and end-organ injury but also improved endothelial function.
Drugs other than antihypertensive agents have been studied for their effect on target organ damage in salt-sensitive hypertension. Atorvastatin improves endothelial function and ameliorates end-organ injury in salt-sensitive hypertension at least in part by inhibiting oxidative stress and upregulating nitric oxide synthesis despite only modest improvements in systolic BP.19
In conclusion, data from the present study support clinical data suggesting that combination antihypertensive drug therapy is more effective than monotherapy for controlling systolic BP and preventing end-organ damage. Complementary mechanisms of action by agents from different antihypertensive drug classes appear to facilitate the increased beneficial effects.
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