Aliskiren Enhances the Protective Effects of Valsartan Against Cardiovascular and Renal Injury in Endothelial Nitric Oxide Synthase–Deficient Mice
The protective effect of aliskiren, a direct renin inhibitor, against hypertensive cardiovascular and renal injury remains to be defined. This study was undertaken to examine the protective effects of the combination of aliskiren and valsartan, an angiotensin receptor blocker, against cardiovascular and renal injury. Endothelial NO synthase–deficient mice, subjected to cuff injury of femoral artery, were divided into 5 groups and were treated with the following: (1) vehicle; (2) aliskiren (25 mg/kg per day); (3) valsartan (8 mg/kg per day); (4) combined aliskiren (12.5 mg/kg per day) and valsartan (4 mg/kg per day); and (5) hydralazine (10 mg/kg per day) for 4 weeks. Aliskiren and valsartan alone markedly and similarly suppressed cardiac hypertrophy, inflammation and fibrosis, and coronary remodeling; prevented cuff injury–induced arterial intimal thickening; and reduced urinary albumin excretion, glomerular inflammation, and glomerulosclerosis in endothelial NO synthase–deficient mice. These beneficial effects of aliskiren and valsartan were associated with the significant attenuation of oxidative stress in these tissues. Hence, aliskiren and valsartan markedly exert the protective effects against cardiovascular and renal injury through the reduction of oxidative stress. Furthermore, compared with monotherapy with aliskiren or valsartan, the combination of a half dose of these drugs more greatly improved the above-mentioned cardiovascular and renal injuries of endothelial NO synthase–deficient mice, which were associated with greater attenuation of tissue oxidative stress by the combination therapy. Thus, the combination of aliskiren and valsartan exerts the synergistic organ-protective effects through synergistic attenuation of oxidative stress. The combination of aliskiren and valsartan seems to be a promising therapeutic strategy for hypertensive organ injury caused by endothelial NO synthase dysfunction.
Accumulating experimental and clinical evidence support the idea the renin-angiotensin system (RAS) plays a key role in the pathophysiology and development of hypertension, cardiac diseases, atherosclerosis, and renal diseases in a broad range of patients.1 Mechanistically, conventional RAS blockers, angiotensin-converting enzyme inhibitor and angiotensin II type 1 (AT1) receptor blocker (ARB), attenuate feedback inhibition of renal renin release by suppressing the production and the action, respectively, of angiotensin II, which results in the increase in plasma renin activity.1 Moreover, plasma renin activity is regarded as a risk factor for myocardial infarction in hypertensive patients.2,3 Therefore, it is still unclear whether treatment with angiotensin-converting enzyme inhibitor or ARB allows optimized RAS suppression in patients with hypertension or cardiovascular and renal diseases.
Aliskiren is the first in a new class of orally effective direct renin inhibitors approved for the treatment of hypertension.4–6 In contrast to conventional RAS blockers, aliskiren blocks the renin system by directly inhibiting plasma renin activity and preventing the formation of both angiotensin I and angiotensin II, as shown by basic and clinical findings.4–6 However, it is unknown whether aliskiren is superior to conventional RAS blockers in terms of the prevention of cardiovascular and renal diseases.
Therefore, in the present study, to examine the protective effect of direct renin inhibition against hypertensive organ injury, we compared the effects of aliskiren, valsartan, and their combination on cardiovascular and renal injury of endothelial NO synthase (eNOS)-deficient mice with hypertension. We obtained evidence that the organ-protective effects of the combination of aliskiren and valsartan were greater than those of either monotherapy alone.
Animals and Drugs
Male eNOS−/− mice7,8 and wild-type mice (C57BL/6J) were used for this study. All of the procedures were in accordance with institutional guidelines for animal research. Valsartan and aliskiren were kindly gifted from Novartis. Hydralazine was purchased from Sigma-Aldrich Co.
Effects of Aliskiren, Valsartan, and Their Combination on eNOS−/− Mice
This study was undertaken to compare the effect of each drug treatment on organ injury of eNOS−/− mice, under similar hypotensive effects. Therefore, in preliminary experiments, we examined the effects of various doses of aliskiren, valsartan, their combination, and hydralazine on blood pressure of eNOS−/− mice to determine the equihypotensive dose of each drug. Mice at the age of 12±2 weeks were used for this experiment. According to our previous method,9 as described in the online Data Supplement (please see http://hyper.ahajournals.org.), we developed a model of vascular remodeling by placing a nonconstrictive polyethylene cuff (2-mm length, PE50, Becton Dickinson) loosely around the left femoral artery of eNOS−/− mice. eNOS−/− mice, subjected to the above-mentioned cuff placement, were randomly assigned to 5 groups, including the following: (1) saline-infused group (control group); (2) hydralazine (10 mg/kg per day)-treated group; (3) valsartan (AT1 receptor blocker, 8 mg/kg per day)-treated group; (4) aliskiren (a direct renin inhibitor, 25 mg/kg per day)-treated group; and (5) combined valsartan (4 mg/kg per day) and aliskiren (12.5 mg/kg per day)-treated group. We chose the dose of 25 mg/kg per day for aliskiren monotherapy, because 25 mg/kg per day of aliskiren is the sufficient dose to sufficiently inhibit renin activity in mice in vivo.10 Drug treatment was performed for 4 weeks. Valsartan, aliskiren, and saline (control) were given to mice via osmotic minipump (ALZA Co). Hydralazine was given to mice as the drinking water. Throughout the experiment, systolic blood pressure of the conscious mice was measured every week with the tail-cuff method (BP98A; Softron Co). After 4 weeks of the drug treatment, a 24-hour urine sample was collected from each mouse with metabolic cages. Then, mice were anesthetized with ether, and the heart, kidney, and cuff-injured femoral arteries were rapidly excised from each mouse to perform histological examination and to evaluate various biochemical and molecular parameters, as described in the online Data Supplement.
Cardiovascular and Renal Phenotypes of eNOS−/− Mice
Blood pressure of eNOS−/− mice was significantly higher than that of wild-type mice (132±3 versus 101±2 mm Hg; P<0.01). Figure S1 (please see the online Data Supplement) shows the detail of phenotypes of eNOS−/− mice. Fifteen-week–old eNOS−/− mice displayed the enhancement of cardiac interstitial fibrosis, coronary arterial thickening, and cardiac macrophage infiltration compared with wild-type mice (Figure S1A). Intimal thickening of the femoral artery caused by cuff injury for 4 weeks was greater in eNOS−/− mice than in wild-type mice (Figure S1B). eNOS−/− mice had the prominent glomerulosclerosis and the increased glomerular macrophage infiltration compared with wild-type mice (Figure S1C). Moreover, superoxide levels in cardiac, injured arterial, and glomerular tissues were greater in eNOS−/− mice than in wild-type mice (Figure S1). Thus, the phenotype of eNOS−/− mice was characterized not only by hypertension but also by cardiovascular and renal injuries and the enhancement of oxidative stress in these tissues.
Expression of AT1 Receptor mRNA and Protein in Cardiac and Renal Tissues of eNOS−/− Mice
As shown in Figure S2, AT1a receptor mRNA levels in both cardiac and renal tissues were greater in eNOS−/− mice than in wild-type mice. Cardiac and renal AT1 receptor protein levels in eNOS−/− mice were also larger than those in wild-type mice.
Effect on Blood Pressure and Plasma Angiotensin II
Aliskiren (25 mg/kg per day) alone, valsartan (8 mg/kg per day) alone, the combination of a half-dose of aliskiren (12.5 mg/kg per day) and valsartan (4 mg/kg per day), and hydralazine significantly reduced blood pressure of eNOS−/− mice throughout the treatment (Figure S3). There were no significant differences in the hypotensive effects among all of these drug treatments throughout the experiment.
As shown in Figure S4, plasma angiotensin II levels in eNOS−/− mice were markedly reduced by aliskiren monotherapy (P<0.01), whereas angiotensin II levels were significantly increased by valsartan monotherapy (P<0.01). Moreover, plasma angiotensin II concentrations in the combination group of valsartan plus aliskiren were lower than those in the valsartan monotherapy group (P<0.01) and the vehicle group (P<0.05). These results confirmed that aliskiren at the doses used in this study sufficiently inhibited plasma renin.
Effect on Cardiac Hypertrophy and Remodeling
As shown in Figure 1, aliskiren or valsartan alone significantly and comparably reduced cardiac weight and cardiac interstitial fibrosis of eNOS−/− mice. On the other hand, hydralazine failed to reduce them. The combination of a half-dose of aliskiren and valsartan reduced cardiac weight and cardiac interstitial fibrosis more than monotherapy with either agent. As shown in Figure 2, aliskiren and valsartan monotherapy, but not hydralazine, significantly and similarly reduced coronary arterial thickening and perivascular fibrosis of eNOS−/− mice. Furthermore, the combination of a half-dose of aliskiren and valsartan reduced these parameters more than either monotherapy. Figure 3 shows cardiac macrophage infiltration and cardiac superoxide levels of each group of eNOS−/− mice. In contrast to no effect of hydralazine on these parameters, valsartan and aliskiren monotherapy similarly and significantly reduced cardiac macrophage infiltration and superoxide levels of eNOS−/− mice. Moreover, their combination reduced cardiac macrophage infiltration and superoxide levels of eNOS−/− mice more than either monotherapy.
Effect on Cuff Injury–Induced Arterial Intimal Hyperplasia
Figure 4 shows the ratio of intima:media area of cuff-injured femoral artery from each group of mice after 4 weeks of the treatment. Hydralazine did not reduce cuff-induced arterial intimal thickening of eNOS−/− mice, whereas aliskiren and valsartan monotherapies significantly reduced arterial intimal thickening of eNOS−/− mice. Furthermore, the reduction of the ratio of intima:media area was greater in eNOS−/− mice receiving the combination of aliskiren and valsartan than in those subjected to either monotherapy. As shown in Figure 5, the combination of aliskiren and valsartan reduced the number of proliferating cell nuclear antigen–positive cells in the intima and the media of the cuff-injured femoral artery more than either monotherapy. As shown in Figure 6, aliskiren and valsartan, but not hydralazine, markedly attenuated superoxide in the intima and the media of the cuff-injured femoral artery of eNOS−/− mice. The combination of alikiren and valsartan reduced superoxide in both the intima and the media to a greater extent than either monotherapy alone.
Effect on Renal Injury of eNOS−/− Mice
As shown in Figure 7, valsartan or aliskiren monotherapy significantly reduced urinary albumin excretion, improved glomerulosclerosis index, reduced glomerular macrophage infiltration, and lessened glomerular superoxide levels of eNOS−/− mice, whereas hydralazine treatment failed to improve these parameters. The combination of aliskiren and valsartan improved all of these parameters more than either monotherapy.
Effect on Cardiac and Renal NADPH Oxidase Activities of eNOS−/− Mice
As shown in Figure 8, cardiac and renal NADPH oxidase activities of eNOS−/− mice were significantly higher than those of wild-type mice. Valsartan and aliskiren monotherapies significantly and similarly reduced NADPH oxidase activity of both cardiac and renal tissues of eNOS−/− mice, whereas hydralazine did not significantly reduce it. Furthermore, the combination of these agents attenuated cardiac and renal NADPH oxidase activities more than either monotherapy alone.
The major finding of our current study was that the combination of aliskiren and valsartan exerted greater organ-protective effects than monotherapy with a higher dose of either agent and was associated with greater attenuation of oxidative stress by the combination. Thus, our present work highlights the combination of aliskiren and valsartan as a promising therapeutic strategy for cardiovascular and renal diseases.
Accumulating clinical and experimental evidence indicates that eNOS, through NO production, plays a protective role not only in hypertension but also in cardiovascular and renal diseases.11–14 Importantly, essential hypertension is characterized by the defect of eNOS and the diminished NO production14,15 and is significantly associated with polymorphism in the eNOS gene.16 Thus, it is a clinically very key issue whether RAS blockers protect against cardiovascular injury caused by eNOS dysfunction. In the current work, aliskiren and valsartan monotherapies both markedly prevented cardiac hypertrophy, inflammation and fibrosis, renal glomerulosclerosis and inflammation, and vascular intimal hyperplasia caused by cuff injury in eNOS-deficient mice, whereas the equihypotensive dose of hydralazine failed to prevent them. All of these findings show that these organ-protective effects of aliskiren and valsartan in eNOS-deficient mice were in part mediated by their direct inhibition of RAS independent of blood pressure, although the blood pressure–lowering effect might still be partially required for the protective effects of these agents.
Recent clinical data show the additive beneficial effects of the combination of aliskiren with conventional RAS blockers on patients with hypertension,17 type 2 diabetic nephropathy,18 and heart failure.19 However, importantly, in all of the previous clinical17–19 and experimental20 studies, the efficacy of combined aliskiren and ARB (or angiotensin-converting enzyme inhibitor) was compared with that of monotherapy with either agent at the same dose but not at a higher dose. Hence, it remains to be determined whether the combination of aliskiren with ARB is more effective than monotherapy with a higher dose of either agent. Therefore, in the present study, we compared the combination of aliskiren and valsartan with monotherapy with a double dose of either agent. The hypotensive effect of the combination of aliskiren and valsartan in eNOS-deficient mice was comparable to that of either monotherapy, providing no evidence for the superiority of the combination over a high dose of either monotherapy in terms of blood pressure–lowering effects. However, of note, the combination of aliskiren and valsartan suppressed cardiac hypertrophy, inflammation and fibrosis, coronary arterial remodeling, cuff injury–induced femoral arterial intimal thickening, renal glomerulosclerosis, and inflammation of eNOS-deficient mice to a greater extent than either monotherapy alone. These findings provide the first evidence that the combination of aliskiren and valsartan exerts greater organ-protective effects than monotherapy with higher dose of either agent alone.
Oxidative stress is involved in the onset and development of cardiovascular and renal diseases.21–24 NO plays antiatherogenic role by counteracting oxidative stress.11,21,25 In the present study, as expected, we found the enhancement of superoxide, the initial product of reactive oxygen species, in the heart, the kidney, and cuff-injured femoral artery of eNOS-deficient mice (Figure S1), supporting the contribution of oxidative stress to organ injuries in eNOS-deficient mice. Notably, the combination of aliskiren and valsartan exerted more attenuation of cardiac, arterial, and renal superoxide in eNOS-deficient mice than either monotherapy alone (Figures 3, 6, and 7⇑⇑), which was attributed to greater suppression of NADPH oxidase activity by the combination therapy (Figure 8). These findings show that greater organ-protective effects of the combination therapy in eNOS-deficient mice than either monotherapy are at least in part attributed to more attenuation of NADPH oxidase–mediated oxidative stress by the combination therapy.
Importantly, hypertensive patients and animals are characterized by partial attenuation of eNOS, differing from complete absence of eNOS in eNOS-deficient mice. Therefore, much caution should be given regarding the interpretation and the implication of our current results obtained from eNOS-deficient mice (a particular hypertensive model). The mechanisms underlying vascular protection of aliskiren and ARBs in hypertensive animals or patients are partially mediated by the improvement of eNOS function, such as the enhancement of eNOS activity and the inhibition of eNOS uncoupling.11,20,26–28 Thus, the use of eNOS-deficient mice in this work did not allow us to examine the potential of the eNOS-mediated benefit of the combination of these agents. Additional study using other hypertensive models is needed to confirm the benefits of their combination therapy.
In conclusion, we obtained evidence that the combination of aliskiren and valsartan exerted greater protective effects against cardiovascular and renal diseases caused by eNOS deficiency than monotherapy with a double dose of either agent through greater attenuation of tissue oxidative stress.
Risk factors, such as hypertension, diabetes mellitus, or dyslipidemia, are significantly associated with eNOS dysfunction. Our current experimental findings highlight the combination therapy of aliskiren with valsartan as the promising therapeutic strategy for cardiovascular and renal diseases.
Sources of Funding
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology and by a grant from Novartis Pharmaceutical Corporation.
- Received April 2, 2009.
- Revision received April 30, 2009.
- Accepted June 19, 2009.
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