(Hypertension. 1996;28:379-385.)
© 1996 American Heart Association, Inc.
Articles |
Endothelin Antagonism in End-Organ Damage of Spontaneously Hypertensive Rats
Comparison With Angiotensin-Converting Enzyme Inhibition and Calcium Antagonism
the Pharma Division, Preclinical Research, F Hoffmann–La Roche Ltd, Basel, Switzerland (H.K., B.-M.L., M.C., J.-P.C.); INSERM U430, Hopital Broussais (D.H., P.B.); and INSERM U367 (M.-F.G.), Paris, France.
Correspondence to Dr J.-P. Clozel, Pharma Division, Preclinical Research, F Hoffmann–La Roche Ltd, Grenzacherstrasse, 124, CH-4002 Basel, Switzerland. E-mail jeanpaul.clozel@roche.com.
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Abstract |
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High blood pressure results in cardiac hypertrophy and fibrosis, increased thickness and stiffness of large artery walls, and decreased renal function. The objective of our study was to assess the role of endothelin, angiotensin II, and high blood pressure in the end-organ damage observed in spontaneously hypertensive rats (SHR). For this purpose, SHR were treated for 10 weeks with either a mixed endothelin-A and endothelin-B receptor antagonist, bosentan (100 mg/kg per day), an angiotensin-converting enzyme inhibitor, enalapril (10 mg/kg per day), or a long-acting calcium antagonist, mibefradil (20 mg/kg per day). A group of SHR was left untreated, and a group of normotensive Wistar rats was used as control. At the end of treatment, maximal coronary blood flow was measured in isolated perfused hearts. Cardiac hypertrophy and fibrosis, aortic medial thickness, and extracellular matrix content were evaluated by quantitative morphometry. Proteinuria and urea and creatinine clearances were measured, and renal histopathology was assessed. SHR exhibited cardiac hypertrophy, perivascular fibrosis, and decreased maximal coronary blood flow. Aortic medial thickness was increased, whereas elastin density was decreased. Finally, SHR showed decreased urinary excretion and decreased urea and creatinine clearances. No renal histological lesions were observed. Although bosentan did not affect blood pressure, it normalized renal function and slightly decreased left ventricular hypertrophy and fibrosis. Enalapril and mibefradil were both effective in significantly decreasing blood pressure, left ventricular hypertrophy, and aortic medial thickness and improving coronary blood flow, but in contrast to bosentan, they did not improve creatinine clearance. We conclude that in SHR, high blood pressure plays a major role in end-organ damage and that endothelin may partly mediate renal dysfunction and cardiac remodeling independently of a direct hemodynamic effect.
Key Words: angiotensin II • endothelin • kidney • mibefradil • bosentan • enalapril
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Introduction |
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The heart, kidney, and aorta are major target organs of hypertension, but the mechanisms responsible for the damages observed in these organs during the development and maintenance of chronic hypertension are not fully understood. The cardiac remodeling observed in this pathological situation comprises myocardial hypertrophy,1 a decrease in coronary vascular reserve,2 and interstitial and perivascular fibrosis.3 4 Hypertension is also associated with morphological and functional alterations of large capacitance arteries, inducing increased thickness and stiffness of the media of these arteries5 and increased medial extracellular matrix (ie, elastin and collagen).6 As far as the kidney is concerned, the experimental results vary depending on the differences in the intraglomerular hemodynamics observed in the experimental models.7 The alterations in renal function observed in the SHR include decreased renal excretion, renal plasma flow, and glomerular filtration rate.8
In addition to high BP, several humoral factors seem to be involved in the end-organ damage occurring in hypertension. Indeed, antihypertensive drugs such as nonspecific arterial vasodilators normalize BP but are unable to reverse cardiac hypertrophy.9 ACE inhibition or Ang II receptor blockade has been shown to prevent cardiac hypertrophy10 and fibrosis11 and aortic medial hypertrophy12 as well as to limit glomerular injury and proteinuria.13 14 On the other hand, experiments have shown that ET-1 induces hypertrophy of cardiomyocytes,15 promotes cardiac fibroblast collagen synthesis,16 and plays a role in renal hemodynamic and excretory functions.17 Endothelin antagonism has also been shown to improve renal function in SHR.18
SHR, an experimental model of genetic hypertension,19 have normal plasma renin20 characterized by an increased afferent arteriolar resistance.21 The aim of this study was to evaluate the role of endothelin versus Ang II and high BP in the end-organ damage observed in this model. For this purpose, rats were treated with bosentan, a new mixed ETA and ETB receptor antagonist,22 enalapril, an ACE inhibitor, or mibefradil, a new long-acting nondihydropyridine calcium antagonist23 able to block both L- and T-type calcium channels.24 25 Moreover, mibefradil has been shown to decrease arterial pressure for 24 hours in SHR.26
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Methods |
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Study Design
Five groups of rats (Fullinsdorf, Switzerland) were compared. At 6 weeks of age, SHR were randomly allocated to four groups and treated for 10 weeks with oral doses of mibefradil (20 mg/kg per day as food admix, n=18 to 22), enalapril (10 mg/kg per day as food admix, n=18 to 22), or bosentan (100 mg/kg per day as food admix, n=18 to 22). The fourth SHR group was left untreated (n=18 to 22). Finally, one control group of normotensive Wistar rats received no treatment (n=18 to 22). The doses of mibefradil and enalapril chosen have been previously tested with a telemetry system and have shown to give a maximal antihypertensive effect. The dose of bosentan was the higher effective dose in deoxycorticosterone acetate hypertensive rats (unpublished data, 1995). The groups were divided into two subgroups with equivalent SABP values. One subgroup was used for morphometry (n=8 to 12) and the other for coronary blood flow measurement (n=8 to 12). SABP was measured indirectly by the tail-cuff method every 2 weeks. The rats were killed after 10 weeks of treatment.
Biochemical Measurements
The rats were placed in individual metabolic cages, and two 24-hour urine collections were performed consecutively. The following urinary data are therefore the mean value of the two measurements for each rat. Urine volumes were determined gravimetrically. Urinary creatinine and urea nitrogen were measured by a centrifugal analyzer (Roche-Cobas Fara II). Urinary protein concentrations were determined after precipitation with 0.2 mol/L trichloroacetic acid. Turbidity was then determined by measurement of absorbance at a wavelength of 450 nm with the centrifugal analyzer. Plasma urea and creatinine were measured by the centrifugal analyzer.
Plasma renin activity was measured by radioimmunoassay of the Ang I generated by the incubation of plasma with an excess of angiotensinogen provided by renin-free plasma obtained from rats binephrectomized 24 hours previously.27
Immunoreactive ET-1 was measured by radioimmunoassay as described.28 It was shown that the cross-reactivity with ET-3 and big endothelin was 14% and 6%, respectively.28 Briefly, duplicates of 200-µL aliquots of plasma were extracted on Sep-Pak Vac C18 cartridges (Waters Associates) after previous conditioning with 3 mL of methanol, followed by 3 mL of 0.2 mol/L phosphate–citric acid, pH 7. Cartridges were eluted with 2 mL of methanol/water (90:10, vol/vol). The eluates were dried and reconstituted in assay buffer (20 mmol/L borate-HCl, 0.1% [wt/vol] bovine serum albumin, 0.1% [wt/vol] NaN3, pH 7.4). Radioimmunoassay was then performed. Free and bound tracers were separated by adsorption at 25°C for 15 minutes on 250 mL Amerlex-M magnetobeads (Amersham) supplemented with 0.1% (wt/vol) Tween 20.
Measurements of MCBF in Isolated Perfused Hearts
Rats previously heparinized were anesthetized with 100 mg/kg sodium hexobarbital IP and killed by cervical dislocation. The hearts were isolated, cannulated from the aorta, and retrogradely perfused in a Langendorff apparatus with a modified Krebs-Henseleit solution of the following composition (mmol/L): NaCl 114.7, KCl 4.7, MgSO4 1.2, KH2PO4 1.5, NaHCO3 25, CaCl2 2.5, and glucose 11.1. The solution was gassed with 95% O2/5% CO2, and pH was adjusted to 7.3. Coronary arterial flow was measured at the outflow from the right atrium with an electromagnetic flowmeter (Narcomatic RT 500, Narco BioSystems Inc). During the preparation, the heart was beating but did not work.
Maximal coronary vasodilation was obtained by addition of adenosine (10-5 mol/L) to the perfusion solution. In each rat, abolition of reactive hyperemia by adenosine was verified. During maximal coronary vasodilation, coronary blood flow was measured at perfusion pressures of 90, 80, 70, 60, and 50 mm Hg. After perfusion, the hearts were blotted and weighed.
Morphometric Analysis
Quantitative morphometry was performed for quantification of cardiac hypertrophy (left ventricular wall thickness and area) and fibrosis (interstitial and perivascular collagen contents and density) as well as aortic medial hypertrophy and extracellular matrix modifications (elastin and collagen contents and density). The measurements were performed with a microscope, with a video camera (Sony) connected to an image-analysis processor (Nachet 1500) and a microcomputer (Macintosh II, Apple). The investigator responsible for the analysis was blinded as to each experimental group.
The second subgroup of rats was heparinized and anesthetized with 100 mg/kg sodium hexobarbital IP. The chest was opened, and the hearts were arrested in diastole with a saturated KCl solution and then fixed under a perfusion pressure of 80 mm Hg with 4% paraformaldehyde. The left and right ventricles were dissected, blotted, and weighed. Left ventricles were dehydrated with ethanol and xylol and embedded in paraffin. Two 4-µm-thick coronal sections, taken at the equator of the heart, were mounted on glass slides and colored with the collagen-specific stain Sirius red F3BA (Pfaltz and Bauer Inc). After fixation by immersion, transversal sections of the aortas were embedded in paraffin. Three successive 4-µm-thick sections were colored with Sirius red F3BA or with the elastin-specific stain orcein.
Cardiac Changes
Both myocardial sections were analyzed macroscopically (a complete section per field) with a x2.5 objective, giving a final calibration of 6.7x10-3 mm per pixel. Total surface area and left ventricular wall thickness were averaged over both sections per rat.
For measurement of collagen in the interstitium, a study of the running mean and running variance allowed us to determine that measurement of 20 fields in the subendocardium and 20 fields in the subepicardium was suitable to get a convergent estimation of the variables. Systematic field displacement over subepicardial and subendocardial regions was done measuring one field over three. For each field analyzed, the interstitial collagen density was determined as the ratio of collagen surface area over myocardial surface area. Microscopic scars (thick collagen fibers located in areas previously occupied by myocytes) and all fields containing coronary artery (diameter >10 µm) were excluded from the analysis. Perivascular collagen was measured separately around every coronary artery (diameter >10 µm) visible in the analyzed field. Only collagen surrounding and connected to the coronary wall was considered as perivascular collagen. The total perivascular collagen surface area and the perimeter of the coronary lumen were measured for each selected field. The perivascular collagen area per unit of lumen perimeter length was calculated for normalization of the amount of collagen with respect to the vessel size.
Aortic Changes
The thicknesses of the media and medial cross-sectional areas were measured. Medial elastin network and collagen matrix were analyzed. Elastin and collagen measurements are expressed as content and density (total elastin or collagen amount normalized for cross-sectional medial area). Then, the total cellular surface area was calculated as the total surface area of the media minus collagen and elastin surface areas. Repetitive measurements were performed and averaged for all the quantified variables in the corresponding stained section of the aortic wall media of each rat. For measurement of elastin and collagen, a study of the running mean and running variance determined that measurement of 20 fields was sufficient to get a convergent estimate of each variable. Systematically, one field out of two was analyzed.
Histopathologic Analysis
Half the kidneys were fixed in alcoholic Bouin's solution and embedded in paraffin. Sections 4 µm thick were stained with Masson's trichrome and examined under light microscopy. Two investigators blinded to the experimental groups assessed the severity of the morphological changes, ie, the presence of infarction, glomerulosclerosis, and tubulointerstitial and vascular lesions. Each type of lesion was graded semiquantitatively as previously described.29
Statistical Analysis
All results are expressed as mean±SE. All variables were compared by one-way ANOVA. Where a significant F value was obtained, the data were further analyzed with Fisher's protected least significant difference test. A value of P<.05 was considered significant.
The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office, London, UK.
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Results |
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Hemodynamic Variables and Body Weights
At the beginning of the treatment period, SABP values were comparable in the untreated and enalapril-, mibefradil-, and bosentan-treated groups and were significantly higher than SABP in the control group (Fig 1
). During the 10-week treatment period, the SABP of the control group remained unchanged and elevated in the untreated SHR. Bosentan had no effect on SABP, whereas enalapril and mibefradil decreased it by 40 mm Hg each (P<.01), without, however, normalizing it. (Fig 1, Table 1). Bosentan and enalapril had no effect on heart rate. Mibefradil decreased heart rate significantly (Table 1). Before treatment, the four SHR groups had equivalent baseline body weights. At the end of the treatment period, body weights were unchanged in the four groups (Table 1).
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Humoral Changes
The ET-1 plasma concentration was slightly increased (10%) in the untreated SHR versus the control group (P<.05). Bosentan did not affect immunoreactive ET-1. Enalapril and mibefradil decreased it by 29% and 27% (P<.001), respectively (Fig 2A). Plasma renin activity was not different in the untreated SHR (11.50±2.57 ng Ang I/mL per hour) compared with the control group (14.40±3.07 ng Ang I/mL per hour). Neither bosentan nor mibefradil affected it. Enalapril, however, dramatically increased plasma renin activity (227.84±48.57 ng Ang I/mL per hour, P<.001) compared with the untreated SHR (Fig 2B).
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Maximal Coronary Blood Flow
During adenosine infusion, coronary autoregulation was abolished and MCBF was linearly related to perfusion pressure (Fig 3). MCBF measured at 90 mm Hg was decreased by 45% in untreated SHR. Bosentan had no effect on MCBF, whereas enalapril and mibefradil improved it significantly (P<.001) by 38% and 30%, respectively, compared with the untreated SHR (Table 2, Fig 3).
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Cardiac Hypertrophy
Untreated SHR showed left ventricular hypertrophy, with a significant increase of 62% in the ratio of left ventricular weight to body weight compared with the control group. Morphometric measurements showed an increase of 41% in the ratio of left ventricular area to body weight in the untreated SHR. Bosentan, enalapril, and mibefradil improved it significantly by 7% (P<.05), 13% (P<.001), and 11% (P<.01), respectively (Table 2
). The ratio of right ventricular weight to body weight (x1000) was significantly increased in the untreated SHR compared with the control group (0.55±0.01 versus 0.45±0.01, P<.001). Only enalapril significantly (P<.05) decreased this ratio by 7%.
Left Ventricular Fibrosis
Interstitial collagen (subepicardial and subendocardial) density was similar in all five rat groups (Table 3). However, the perivascular collagen (total area and area over pericoronary lumen) was increased in the untreated SHR by 143% (P<.001) and 103% (P<.01), respectively, compared with the control group (Table 3). Bosentan decreased the ratio of perivascular collagen area to pericoronary lumen by 35% (P<.05). Enalapril decreased significantly the perivascular collagen area by 29% (P<.05) and the ratio of area to pericoronary lumen by 36% (P<.01). Mibefradil decreased the perivascular collagen area by 29% (P<.05) (Table 3).
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Aortic Changes
Aortic medial thickness in SHR increased by 24% compared with the control normotensive rats. Bosentan had no effect on medial thickness. Enalapril and mibefradil decreased it by 16% (P<.001) and 11% (P<.01), respectively (Table 4). Elastin density in the aortic wall of SHR decreased by 18% compared with that in control rats (P<.001). Bosentan had no effect on elastin density, whereas treatment with enalapril and mibefradil almost completely prevented this reduction (P<.01) (Table 4). The collagen density and the ratio of collagen density to elastin density were similar in the five experimental groups (Table 4).
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Renal Function
Urea and creatinine clearances decreased in the untreated SHR compared with the control group (Table 5). Proteinuria was not increased in the untreated SHR, whereas diuresis decreased significantly in this group (Table 5). Bosentan (P<.05) and mibefradil (P<.01) increased proteinuria slightly and did not affect urine volume. Enalapril had no effect on proteinuria and normalized diuresis. Urea clearance was improved with the three treatments (P<.01), whereas only bosentan significantly increased creatinine clearance (P<.05) (Table 5).
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Renal Morphology
Histopathologic evaluation of the kidneys showed no lesions in the five rat groups. Glomeruli, tubulointerstitial, and vascular compartments were normal, and the scores were extremely low and similar in all the groups (data not shown).
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Discussion |
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The present study shows that in SHR, high BP is a major determinant of end-organ damage, as shown by the striking effect of a calcium antagonist. However, hormonal factors such as endothelin seem to play an additional role at the renal and cardiac levels.
In our study, plasma endothelin level was slightly increased (12%) in the untreated SHR compared with the control group, as has been previously observed.30 In contrast with what has been observed in deoxycorticosterone acetate hypertensive rats,31 bosentan did not increase plasma endothelin levels. The explanation proposed for this previously described increase of endothelin by bosentan was a displacement of ET-1 from the ETB receptors,32 which may play a role in endothelin clearance from the circulation.33 Since vascular production of ET-1 is decreased in SHR,34 the ETB receptors in the endothelium might have been less occupied and more able to buffer the rise of endothelin in plasma.
In the present study, the main end-organ lesions described in SHR21 35 were observed. Renal dysfunction resulted in a decrease in creatinine clearance. However, we did not observe renal histopathologic changes. This is most likely because of the relatively young age of the rats. Indeed, the absence of an effect of enalapril is probably due to the fact that in SHR, proteinuria starts to increase at 5 months of age and rapidly accelerates after 10 months of age.36 Since in our study the rats were evaluated at 4 months of age, no abnormal proteinuria was measured. The same explanation holds true for the absence of detectable glomerulosclerosis. We also observed left ventricular hypertrophy associated with cardiac perivascular fibrosis and a decrease of coronary vascular reserve. Finally, aortic medial thickness was markedly increased.
The most striking effects of bosentan were the improvement of renal function and decrease of perivascular cardiac fibrosis. Since bosentan did not significantly decrease arterial BP, these results suggest that endothelin has tissular effects dissociated from its hemodynamic systemic effects. In SHR, the kidney seems to play an important role in the development of hypertension.37 Renal vascular resistance is increased in SHR because of a predominant preglomerular vasoconstriction.21 The striking effect of bosentan suggests that ET-1 plays an important role in the renal functional changes of SHR. Indeed, in rats, ET-1 can constrict both afferent and efferent arterioles38 and decrease glomerular filtration rate.39 Moreover, the vasoconstrictive effect of ET-1 on isolated renal arteries is greater in the SHR than in the Wistar-Kyoto rat.40
Bosentan was also able to decrease perivascular fibrosis and to a smaller extent left ventricular hypertrophy. These effects are also likely due to the previously described cardiac effects of endothelin. In vitro, ET-1 induces hypertrophy of cultured rat cardiomyocytes that are able to synthesize and secrete ET-115 41 and express ETA and ETB receptors.42 In vitro, collagen synthesis can be induced by ET-1 and ET-3 via ETA and ETB receptors.43 Moreover, ETA and ETB receptors are present on rat cardiac fibroblasts, with a predominance of ETB receptors.44 Finally, a recent study showed that cardiac fibroblasts express preproET-1 mRNA, which is upregulated by ET-1 and Ang II and that both ET-1 and Ang II stimulate the DNA synthesis by cardiac fibroblasts.45 Despite an effect on perivascular fibrosis, bosentan did not affect MCBF, showing that in this model, MCBF is independent of perivascular fibrosis.
Interestingly, both enalapril and mibefradil decreased plasma endothelin levels. This effect of enalapril might be due to either the decrease of Ang II or the accumulation of bradykinin. Captopril has already been shown to inhibit ET-1 secretion by endothelial cells via the accumulation of bradykinin.46 The decrease of endothelin by mibefradil might also be a direct endothelial effect. Indeed, incubation of human umbilical artery endothelial cells with the calcium antagonist nisoldipine resulted in a dose-dependent reduction in endothelin levels in the conditioned medium.47 Therefore, taking into account these results, we cannot exclude the fact that part of the effects of enalapril and mibefradil were related to a decrease in endothelin production.
Bosentan had no effect on coronary vascular reserve and aortic medial hypertrophy. In contrast, both mibefradil and enalapril increased coronary vascular reserve to the same extent and normalized aortic medial thickness. This decrease of aortic medial thickness was also associated with an increase in elastin content. Since the effects of mibefradil and enalapril were surprisingly similar and since both drugs decreased arterial pressure to the same extent, it is tempting to conclude that these changes were mainly related to the BP decrease. However, we cannot exclude the possibility that the decrease of Ang II caused by enalapril on the one hand and the decrease of intracellular calcium caused by mibefradil on the other contributed to these changes. Indeed, mibefradil is a selective T-type calcium channel blocker,25 and it has been shown that these channels are increased in proliferating cells.48
The cardiac10 and aortic49 effects of ACE inhibition have been previously described in SHR. They might be due to the trophic effects of Ang II, which have been shown on both smooth muscle cells12 and cardiomyocytes.50 The decrease of cardiac fibrosis might also be due to the decrease of Ang II or the accumulation of bradykinin. Indeed, Ang II is able to induce fibrosis,51 and bradykinin receptors have been shown to be present in the myocardial fibrotic tissue, where they are colocalized with ACE.52
Since enalapril not only decreases Ang II but also decreases arterial pressure, it is not possible to dissociate these effects. In deoxycorticosterone acetate hypertensive rats, ACE inhibitors are known to be ineffective on BP levels as well as on cardiac remodeling.53 In the present study, mibefradil had the same effect as enalapril. Again, this suggests that high BP is the main determinant of the cardiac and aortic changes.
This is clearly not the case for the renal damage. Indeed, at this level, the effects of the three drugs were quite different. As mentioned before, bosentan was the most efficient in increasing creatinine clearance, but like mibefradil, it showed a trend for an increase in proteinuria. In contrast, enalapril had no effect on proteinuria. It is clear that these differences are likely to be due to the different locations of the renal vasodilation induced by the three drugs. Enalapril, as has been previously shown with other blockers of the renin-angiotensin system, has a predominant postglomerular effect,54 whereas calcium antagonists have a predominant preglomerular effect, explaining the slight increase in proteinuria.55 Endothelin potently constricts both afferent and efferent arterioles,56 and the preglomerular effect of bosentan would also explain the slight proteinuria observed in this group. Our results obtained with chronic bosentan confirm previous results obtained with acute administration of endothelin receptor antagonists showing improvement in the renal plasma flow of SHR.57
In conclusion, our study shows that in genetic spontaneous hypertension, the mechanisms explaining the end-organ damage are different among organs. In addition to elevated BP, hormonal factors such as endothelin and Ang II seem to be involved. Clinical studies are needed to evaluate whether the differences observed among the three drugs are also observed in humans.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We wish to thank Patrick Hess, Andree Roeckel-Tschaegle, Jean-Paul Maire, and Marie-France Belair for their technical assistance.
Received January 19, 1996;
first decision March 11, 1996; first decision April 19, 1996;
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