Our recent studies have demonstrated that salt excess in the spontaneously hypertensive rat (SHR) produces a modestly increased arterial pressure while promoting marked myocardial fibrosis and structural damage associated with altered coronary hemodynamics and ventricular function. The present study was designed to determine the efficacy of an angiotensin II type 1 (AT1) receptor blocker (ARB) in the prevention of pressure increase and development of target organ damage from high dietary salt intake. Eight-week-old SHRs were given an 8% salt diet for 8 wk; their age- and gender-matched controls received standard chow. Some of the salt-loaded rats were treated concomitantly with ARB (candesartan; 10 mg·kg−1·day−1). The ARB failed to reduce the salt-induced rise in pressure, whereas it significantly attenuated left ventricular (LV) remodeling (mass and wall thicknesses), myocardial fibrosis (hydroxyproline concentration and collagen volume fraction), and the development of LV diastolic dysfunction, as shown by longer isovolumic relaxation time, decreased ratio of peak velocity of early to late diastolic waves, and slower LV relaxation (minimum first derivative of pressure over time/maximal LV pressure). Without affecting the increased pulse pressure by high salt intake, the ARB prevented the salt-induced deterioration of coronary and renal hemodynamics but not the arterial stiffening or hypertrophy (pulse wave velocity and aortic mass index). Additionally, candesartan prevented the salt-induced increase in kidney mass index and proteinuria. In conclusion, the ARB given concomitantly with dietary salt excess ameliorated salt-related structural and functional cardiac and renal abnormalities in SHRs without reducing arterial pressure. These data clearly demonstrated that angiotensin II (via AT1 receptors), at least in part, participated importantly in the pressure-independent effects of salt excess on target organ damage of hypertension.
- left ventricular function
- coronary circulation
- angiotensi II type 1 receptor blocker
left ventricular (LV) hypertrophy (LVH), frequently observed in essential hypertension, is one of the major risk factors underlying cardiovascular morbidity and mortality (9, 10, 41). All components of the heart may be affected, including cardiac and vascular smooth muscle cells as well as endothelial cells and fibroblasts. Therefore, in addition to increased cardiomyocyte size, interstitial and perivascular fibrosis, hypertrophy of vascular smooth muscle, and endothelial dysfunction importantly contribute to coronary hemodynamic impairment and ventricular dysfunction.
Recent studies from our and other laboratories have proposed that salt excess is an important determinant of cardiovascular and renal derangements in hypertension (12, 13, 23, 26, 43). Moreover, in addition to its hemodynamic effects, dietary salt excess exerts additional nonpressure-related cardiac effects in different forms of experimental and human essential hypertension (5–7, 12, 13, 24, 26, 43). Various mechanisms have been suggested to explain these adverse cardiovascular effects of dietary salt excess, and some evidence supports the role of the renin-angiotensin system (RAS) in their development (18, 21, 33, 36, 45).
Our recent studies have demonstrated that salt excess in the spontaneously hypertensive rat (SHR), a well-characterized experimental model of naturally occurring hypertension, promotes a modestly increased arterial pressure while inducing marked myocardial fibrosis and structural damage associated with altered coronary hemodynamics and ventricular function (1, 38). The present study was designed to determine the efficacy of an angiotensin II type 1 (AT1) receptor antagonist in the prevention of arterial pressure increase and development of severe target organ damage due to high dietary salt intake.
Male SHRs, purchased from Harlan Laboratories (Indianapolis, IN), were maintained in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle. All rats were handled in accordance with National Institutes of Health guidelines, and our Institutional Animal Care and Use Committee approved the study protocol in advance. Before the study was initiated, all rats were studied echocardiographically to exclude comorbid congenital cardiac abnormalities (30). Eight-week-old SHRs were then given either a control diet (1%; group 1; n = 10) or a high-salt diet (8%; n = 20) for the ensuing 8 wk. Salt-loaded SHRs were randomized to one of two groups: group 2 (n = 10) was untreated and group 3 (n = 10) was treated with candesartan (10 mg·kg−1·day−1). All rats were permitted free access to chow and tapwater. An additional group of rats (n = 7) was kept under identical experimental conditions as group 3 and was used exclusively for pathological examination. Comparisons were made with our previously published data (38).
Twenty-four-hour urinary measurements.
At the end of the study, rats were placed in individual metabolic cages for 3 consecutive days. On the third day, urinary output was measured, and 24-h urinary protein (Lowry method) excretions were determined.
LV structure and function by transthoracic echocardiography.
At the end of the salt loading period, rats were anesthetized with pentobarbital (50 mg/kg ip), and transthoracic echocardiography (TTE) analysis was performed using an echocardiographic system (Agilent Technologies Sonos 2000 with an 7.5-MHz transducer) to evaluate LV geometry (1, 38). The pulsed-wave Doppler of mitral inflow was recorded, and peak flow velocities of early and late filling waves were measured after xylazine was added. Peak velocities of early and late diastolic wall movements at the level of lateral mitral annulus were also recorded using pulsed Doppler tissue imaging (1, 38).
Systemic and ventricular hemodynamics and aortic stiffness.
After echocardiographic examination, the right carotid artery was cannulated with a transducer-tipped catheter (Micro-Tip 3F, Millar Instruments), which was advanced into the ascending aorta for recording arterial pressure. A second catheter was placed into the abdominal aorta through the femoral artery. Both catheters were connected to a multichannel recorder (Grass Instruments) interfaced to an IBM computer with a digital data-acquisition system (EMKA Technologies) (38). For the determination of pulse wave velocity (PWV), pulse contours from the two catheters were recorded simultaneously on the same channel, and PWV was calculated from the aortic length between the two (measured postmortem) points and the time difference between their diastolic notches. After aortic functional measurements had been made, the catheter already placed in the ascending aorta was advanced further into the LV. LV maximal and end-diastolic pressures as well as the first derivatives of pressure over time (dP/dtmax and dP/dtmin), as indexes of global contractility and relaxation, were recorded. After LV ventricular hemodynamics had been recorded, the Millar catheter was withdrawn, and a jugular vein, femoral artery, and the LV were cannulated with polyethylene catheters (PE-50) for measurements of systemic, coronary, and renal hemodynamics (reference standard microsphere method) as we have previously described (39). After the regional hemodynamics had been measured, rats were killed with an overdose of pentobarbital, and their hearts, aortae, and kidneys were removed and weighed. As an estimate of ventricular collagen content, hydroxyproline concentrations of the LV samples were determined and expressed as milligrams per gram of dry weight (1, 38).
After rats had received their respective diets for 8 wk, they were killed with a pentobarbital overdose, and their hearts were removed, fixed in 10% formalin solution, dehydrated by routine methods, and embedded in paraffin for subsequent histological study (40). The myocardial volume fraction occupied by fibrillar collagen (CVF) was determined by quantitative morphometry (40) using an automated image-analysis system (AnalySYS, Soft Imaging System) in sections stained with collagen-specific picrosirius red. Total and perivascular CVF were measured, and interstitial CVF was calculated as the subtraction of perivascular CVF from total CVF.
All values are expressed as means ± SE. Data were analyzed with the use of ANOVA, followed by the Bonferroni test for multiple comparisons. A value of P < 0.05 was considered to be of statistical significance.
Body masses were significantly lower in salt-loaded SHRs than their controls, and candesartan attenuated this effect (Table 1). Compared with rats on control diet, untreated salt-loaded SHRs expectedly had higher mean arterial pressure and increased LV mass. Importantly, AT1 receptor blockade failed to prevent the salt-induced pressure increase, whereas it attenuated the increased LV mass (Table 1). However, without affecting the increased pulse pressure with salt excess, AT1 receptor antagonism failed to prevent the arterial stiffening and hypertrophy as reflected by the increased PWV and aortic mass index (Table 2).
Echocardiographic examination of the LV confirmed our previous findings showing that the increased LV mass in the untreated salt-loaded group mirrored increased septal and posterior wall thicknesses (Table 3). There were no differences in LV diameters among the groups. Prolonged treatment with candesartan ameliorated that LV remodeling. Diastolic indexes demonstrated impaired LV relaxation in untreated salt-loaded SHRs compared with their controls, as manifested by decreases in the early to late filling wave ratio measured by pulse and tissue Doppler and decreased LV dP/dtmin normalized for maximal LV pressure (LVPmax) developed in the LV (Table 3). Thus, candesartan prevented salt-induced impaired LV relaxation. There were no differences between the groups with respect to LV end-diastolic pressure and contractility (dP/dtmax/LVPmax) (Table 3).
There were no differences between baseline coronary blood flows and vascular resistances in the untreated salt-loaded group compared with controls (Table 3). However, as we reported previously, minimal coronary vascular resistance after dipyridamole infusion was significantly higher in these salt-loaded SHRs, and the coronary flow reserve was correspondingly reduced (Table 3). AT1 receptor antagonism by candesartan prevented the foregoing coronary hemodynamic impairment.
LV hydroxyproline concentrations and CVF are shown in Table 1. Candesartan ameliorated the increased LV hydroxyproline concentrations and CVF resulting from high salt intake and was paralleled by histological findings of decreased interstitial and perivascular fibrosis compared with untreated counterparts (Fig. 1).
Finally, AT1 receptor antagonism effectively prevented the alterations in renal hemodynamics that were associated with the prevention of excessive urinary protein excretion in untreated salt-loaded rats (Table 4).
The present data demonstrated that AT1 receptor antagonism by candesartan effectively prevented salt-induced target organ structural and functional damage in SHR. Most notably, these beneficial effects were achieved without an antihypertensive effect. Furthermore, despite that failure to decrease pressure, candesartan prevented the salt-induced deterioration in coronary and renal hemodynamics, thereby resulting in the prevention of cardiac and renal functional impairment. Therefore, these data provide strong support to the concept that in addition to its hemodynamic effects, dietary salt excess is responsible for nonpressure-related target organ injury.
The role of high salt intake in exacerbating hypertension and further increasing LV mass in patients with essential hypertension has been demonstrated in many major epidemiological, interventional, and experimental studies (5–8, 24, 32, 42). However, the greater risk for adverse cardiovascular events intrinsic to hypertensive LVH does not depend specifically on ventricular mass itself, since increased cardiac mass found in trained athletes is not associated with adverse cardiovascular outcomes (20). It is likely that other factors, including ventricular fibrosis, dysfunction, and ischemia, participate in the increased vulnerability and risk of the hypertrophied LV to the adverse events of salt excess (9, 10, 41). Earlier reports from our laboratory linked salt-related collagen deposition to impaired LV coronary hemodynamic and ventricular function in SHRs (1, 13, 38). The present study provides evidence that angiotensin II (via AT1 receptors) mediated salt-induced collagen deposition within the ventricles (interstitially and perivascularly) that seems to be independent of pressure. Angiotensin II, in addition to increasing ventricular load, participated in the development and progression of LVH via its nonhemodynamic (mitogenic and growth promoting) effects on cardiac myocytes and fibroblasts. Most of these effects are mediated through the stimulation of AT1 receptors (9, 41). Although the present study was not designed to address the fundamental biological mechanism of the beneficial effects of AT1 receptor blockade, our previous report (39) suggested a significant contribution of AT2 receptor to the antifibrotic effects of AT1 receptor antagonism.
Thus, our data confirm previous findings in reports demonstrating the cardioprotective action of RAS inhibition in hypertensive rats on a salt excess diet (14, 18, 33). It reflected decreased LV collagen deposition in treated salt-loaded rats. We extended those observations and demonstrated beneficial functional consequences of RAS blockade despite no reduction in arterial pressure with salt load. These results emphasize two important points. First, we provide strong evidence that LV fibrosis was linked with cardiac functional deterioration in the SHR subjected to salt excess. Second, in the view of the reports that plasma renin activity should be suppressed in response to high salt dietary intake (43), our study implies that the beneficial effects of AT1 blockade on salt-related target organ injuries were independent of its hemodynamic effects and could be associated with inhibition of local tissue angiotensin II function. In general, there is an agreement that all components of the RAS are expressed in heart tissue including cardiac myocytes, fibroblasts, endothelial cells, and vascular smooth muscle cells (29). Moreover, higher angiotensin-converting enzyme (ACE) and AT1 binding have been recognized at the sites of cardiac injury, suggesting a contributory role of the local “de novo” angiotensin II synthesis (34, 35). In addition, angiotensin II initiates and enhances inflammation and oxidative stress (17), and ACE has been detected in activated macrophages accounted for the initiation of the inflammatory response to tissue damage (15, 19). Colocalization of inflammatory cells invading intramural coronary arteries with perivascular fibrosis and high ACE binding in an animal model of aldosterone/NaCl-induced cardiac injury support the reactive profibrotic role of locally produced angiotensin II (35). We recently demonstrated that salt induced considerable interstitial and perivascular fibrosis that was associated with diminished coronary flow reserve (CFR) and LV diastolic dysfunction (38). In the present study, we report that AT1 receptor antagonism without pressure-reducing action did prevent salt-induced decreased CFR in parallel with reduced LV mass and interstitial and perivascular fibrosis. This important finding as well as reports demonstrating that salt loading induced cardiac ACE mRNA expression and activity (21) and increased cardiac AT1 receptor expression (36, 45) support the role of the local RAS in salt-related cardiovascular injury.
The arterial stiffening associated with high dietary salt intake was not prevented by RAS blockade, thereby confirming a previous report (22) on the vascular effects of valsartan. These findings support the concept that dietary salt excess participates in the pathogenesis of reduced arterial distensibility and suggest that different mechanisms may operate in mediating the adverse effects of salt excess on conduit as well as resistance vessels. We emphasize that AT1 receptor antagonism in the present study reduced, but did not normalize, LV mass and that reduced large arterial compliance continued to impose a significant hemodynamic burden on the LV.
In this report, we found that AT1 receptor blockade improved renal hemodynamics and prevented renal functional decline mirrored by excessive urinary protein excretion. Our recent study demonstrated that salt excess, independent of the increased arterial pressure, produced severe glomerular dysfunction and consequently significant nephrosclerosis (26). It therefore seems likely that angiotensin II (via AT1 receptors), at least in part, participates importantly in pressure-independent effects of salt excess. Direct effects of angiotensin II indeed, at least in part, drove glomerular sclerosis, interstitial fibrosis, and tubular necrosis in angiotensin II-infused Sprague-Dawly rats (27). Moreover, the role of the local intrarenal RAS has been implicated in renal injury of Dahl salt-sensitive rats (28). The data from this study, therefore, provide further support to the concept that in addition to its hemodynamic effects, dietary salt excess may exert independent RAS-mediated target organ injury.
The significant body weight loss in untreated salt-loaded rats draws some attention as well. Progressive muscle, fat, and bone tissue lost may be accounted for observed end-stage cachexia. Stimulation of the sympathetic nervous system may have a deleterious catabolic effect, and an increased concentration of plasma norepinephrine was detected in SHRs fed a high-salt diet (23). In addition, it is intriguing to speculate on the possibility that secondary hyperparathyroidism due to calciuric effect of the high salt intake may contribute to bone resorption (4). Furthermore, intracellular calcium overloading, frequently driven by secondary hyperparathyroidism, has been implied in the progressive degeneration of skeletal muscle (2). Clearly, further studies are necessary to explain how blockade of the RAS prevented the observed cachectic response related to the high dietary salt excess. On the other hand, it is also important to note that intracellular calcium overloading of peripheral blood mononuclear cells associated with aldosteronism has been implicated in the activation of immune cells and induction of oxidative stress (3) and may explain the consecutive development of perivascular fibrosis around coronary vessels in this experimental model. Importantly, an enhanced local cardiac production of aldosterone along with AT1 receptor overexpression was observed in stroke-prone SHRs fed a high-salt diet (36). Moreover, increased cytosolic calcium may initiate cardiac myocyte apoptosis or necrosis, leading to reparative interstitial fibrosis. Indeed, numerous reports with respect to cardiac complications in chronic renal failure (31, 44) implied the role of altered intracellular calcium homeostasis in cardiac myocytes that was driven by secondary hyperparathyroidism. Consequently, it remains to be determined whether cellular calcium toxicity indeed is involved in salt-related cardiovascular damage.
Finally, our results are in striking contrast with Maitland et al.'s study (25) on the effects of RAS blockade on end-organ damages in salt-sensitive Dahl rats. In that study, serious concern was raised over the increased tissue injury after candesartan treatment in Dahl salt-sensitive rats on a high-salt diet despite its antihypertensive effects. Our most recent study (35a) demonstrated the renal protective role of losartan in salt-loaded SHRs, suggesting that the protection stemmed from AT1 receptor antagonism properties and is not specific for candesartan only. Differences in rat strains, AT1 receptor blockers, and the starting point of the respective treatment provide some reasoning to explain the findings of the two studies.
In summary, the present data clearly demonstrated that angiotensin II (via AT1 receptors), at least in part, participated importantly in the pressure-independent effects of salt excess on target organ damage. These findings provide support for large epidemiological studies indicating that dietary salt excess is a strong and even independent contributor to the increased cardiovascular risk and mortality associated with hypertensive disease (11, 16, 37).
This study was supported by American Heart Association Mid-Atlantic Affiliate Grant 0765308U, the Wake Forest Univerisity School of Medicine Venture Fund, and National Heart, Lung, and Blood Institute Grant HL-51952.
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- Copyright © 2008 by the American Physiological Society