Hypertension is characterized by high values of systolic or diastolic arterial pressure or both, caused by enhanced total peripheral resistance (TPR) with normal cardiac output (CO; 1). In addition, certain structural and functional changes of the cardiovascular and other organs appear. Although they have been extensively described elsewhere (2,3), the most important changes at the cardiovascular level are (a) left ventricular hypertrophy, (b) hypertrophy and sclerosis of the vascular wall associated with hemodynamic (lumen encroachment) and functional alterations [in relation to the mass (1) geometric effects, such as the geometric amplifier (4,5) and others], and (c) endothelial dysfunction with impaired capacity to produce smooth-muscle relaxation (6).
In the long term, the kidney is probably the main regulator of blood pressure and is responsible for establishing the current levels on which many other factors, very effective in the short term, produce transitory variations. Through its infinite-gain pressure-natriuresis-diuresis mechanism, the kidney inversely compensates CO and TPR to maintain blood pressure. This organ also exerts strong control at the periphery, not only by regulating blood pressure, but also by synthesizing substances (e.g., renin and medullipin) that have intrarenal and, when incorporated into the circulation, systemic actions on the structure and functionality of target organs (7,8).
In spontaneous hypertension, cardiovascular remodeling and functionality are subject to the influence of pressure and perhaps to certain other non-pressure-related mechanisms that are genetically or circumstantially determined (or both), because alterations in vascular structure and functionality have been reported at the prehypertensive stage (5,9-11). In this sense, although unilateral nephrectomy (UNX) in the spontaneously hypertensive rat (SHR) does not elicit any change in blood pressure (12), it has been reported that nephron mass is critically involved in the initiation and progression of experimentally induced hypertensive-renal disorders (13). Whether this involvement reaches the systemic vascular beds is not known. We performed our study to elucidate whether a calcium antagonist (verapamil) and a converting-enzyme inhibitor (trandolapril), either alone or in combination, are able to exert their antihypertensive actions and restorative effects on vascular structure and function in a model of half-renal-mass ablation that might correspond to hypertensive human subjects undergoing a similar degree of nephrectomy for some cause or another.
Currently the antihypertensive effect of a drug is evaluated not only in terms of blood pressure reduction, but also for its ability to restore the remodeling and functionality of the affected organs (2).
Animals and experimental design
Female spontaneously hypertensive rats (SHRs) of ∼3 months old and weighing ∼200 g (PANLAB, Barcelona, Spain) were operated on, and the left kidney was removed under ether anesthesia. Henceforth, the animals are referred as to SHR-UNX. All animals were housed six per cage under controlled environmental conditions, fed with regular chow, and were allowed to drink ad libitum.
One week after nephrectomy, animals were randomly divided into four groups for treatment over a 6-month period.
- Control: rats without treatment (C).
- Verapamil: (20 mg/kg/day) (V)
- Trandolapril: (0.7 mg/kg/day) (T)
- Verapamil (20 mg/kg/day) plus trandolapril (0.7 mg/kg/day) (veratran, VT).
All drugs were administered in the drinking water.
Arterial pressure was measured in conscious animals by using the bloodless tail-cuff method (14) with a electrosphygmomanometer (Digital Pressure Meter LE 5000; Letica, S.A., Barcelona, Spain) before and after nephrectomy, at the end of each of the first 5 weeks, and at the end of each month of treatment. Systolic and diastolic values were converted into mean arterial pressure [MAP; MAP = (2 diastolic pressure + systolic pressure)/3]. After 6 months, animals were weighed, lightly anesthetized with ether, and killed. The thoracic aorta and heart were removed and placed in physiological salt solution (Krebs-like; PSS) kept at 37°C and continuously gassed with a carbogen mixture of 95% O2 and 5% CO2. The PSS had the following composition (in mM): NaCl, 118; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2.5; KH2PO4, 1.2; KCl, 4.75; and glucose, 11.
Hearts were repeatedly rinsed in PSS, squeezed with drying paper, and weighed. This value was referred to total body weight as the cardiac hypertrophy index.
After excess fat and connective tissue had been dissected, the aortae were cut into rings (3 mm in length). One ring was assigned to morphologic studies and the rest to contractility assays.
The rings were placed between stainless steel hooks and set up in organ chambers filled with 5 ml of PSS gassed with carbogen, and kept at 37°C. One of the hooks was fixed and the other connected to an isometric force transducer (UF1; Harvard Apparatus Inc., South Natick, MA, U.S.A.). Force was registered on a polygraph (79E; Grass Instruments Co., Quincy, MA, U.S.A.).
All rings were allowed to equilibrate for 1 h with a resting tension of 2 g. During this period, the PSS was periodically changed, and tension was reset at 2 g.
After this equilibration period, a reference contraction was performed with a 105 mM concentration of potassium chloride (KCl). After repeated rinses and a 30-min equilibration period under resting conditions, the following tests were carried out in different rings:
- Cumulative concentration-response curves to potassium chloride (KCl, 25-125 mM) and noradrenaline (NA, 10−9-10−4M) were obtained, allowing an intermediate interval of 45 min under resting conditions between each curve.
- After precontraction with 1 μM NA, and at the steady maximal contraction, cumulative concentration-response curves to acetylcholine (ACh, 10−6-5 × 10−4M) or sodium nitroprusside (NTP, 10−9-5 × 10−7M) were obtained.
One aortic ring was fixed in buffered 4% formaldehyde, dehydrated in a graded series of ethanol concentrations (75-100%) and toluene (100%), and immersed in paraplast at 56°C for a minimal period of 2 h. Then individual blocks of this material containing the tissue (usefully oriented) inside were sculpted. Fine slices (5-10 μm width) of the cross section of the tissue were obtained with a microtome and placed on slides. The paraplast was removed by heating at 56°C overnight. The tissue was stained with hematoxylin-eosin and photographed under light microscopy. After this, by means of an image analyzer, the internal and external perimeters of the lamina media were converted into internal and external radii (Ri and Ro, respectively), lamina media width (w), and lamina media cross-sectional area (Am).
Verapamil (Knoll, S.A. Laboratorios, Madrid, Spain), trandolapril (Knoll, S.A. Laboratorios, Madrid, Spain), KCl (Panreac, Madrid, Spain), norepinephrine bitartrate (Sigma-Aldrich, Madrid, Spain), acetylcholine chloride (Sigma-Aldrich, Madrid, Spain), and sodium nitroprusside (Sigma-Aldrich, Madrid, Spain) were used. All drugs were soluble in water. Trandolapril needed a few drops of methanol to dissolve. Stock solutions were frozen for storage and converted into those used in the experiments immediately beforehand. Before the experiments, samples were protected from light at all times.
Data analysis and statistics
All data are expressed as the mean ± SEM. pD2 refers to the negative logarithm of concentrations that produce 50% of the maximal response. pI25 and pI50 are the negative logarithms of concentrations that produce 25 and 50% (respectively) reduction in 1 μM NA-evoked contractions. Emax represents the maximal effect exerted by the tissue in response to a given stimulus, and Ed-max refers to the effect obtained with the maximal concentration used.
Statistical analysis to compare the different groups was carried out by using two-way analysis of variance (ANOVA) for contractility and pressure experiments, and one-way ANOVA followed by the Newman-Keuls test for morphologic studies. A value of p < 0.05 was considered statistically significant.
Mean arterial pressure
The evolution of MAP during the 6 months of treatment is shown in Fig. 1. The untreated SHR-UNX animals (control group) showed severe hypertension, which persisted over time. Treatment with verapamil (group V) induced a moderate but significant reduction in MAP that reached a maximum during the second month, thereafter decreasing. The reductions in MAP caused by trandolapril (T) and verapamil plus trandolapril (VT) were similar and much greater than that elicited by verapamil alone, returning MAP to normal values and maintaining it at those levels until the end of treatment.
Responses to KCl and noradrenaline. Potassium chloride (KCl) caused concentration-dependent contractions that were attenuated across the whole range of concentrations by all three treatments (V, T, and VT) with respect to the control group (C). The maximal effect (Emax) decreased in all groups (more effectively in the VT than in the V and T groups), whereas pD2 had similar values (Fig. 2 and Table 1).
The concentration-dependent contractions induced by NA were significantly reduced in all treatment groups as compared with controls (Fig. 2, Table 1), although no differences were observed among them. Reduction affected Emax but not pD2.
Responses to acetylcholine and sodium nitroprusside. In aortic rings precontracted with 1 μM NA, the endothelium-dependent relaxation occurring in response to ACh had statistically significant increases in Emax and similar pD2 values in the V, T, and VT groups in relation to C (Fig. 3 and Table 2).
The effect of SNP on aortic rings precontracted with 1 μM NA was similar in all four groups, with a slight but significant increase in Ed-max in the T and VT groups (Fig. 3 and Table 2).
Aortae. All numerical results are shown in Table 3. In all three treatment groups (V, T, and VT), the Rj/w ratio decreased in a similar manner as compared with the C group. In the V group, this was due to a decrease in lamina media width, with no changes in lumen diameter, which led to a reduction in lamina media cross-sectional area (Am). In the T group, because lumen diameter increased even though w decreased, there was a much less pronounced reduction in Am. Finally, the VT group showed the greatest reduction in w as well as a slight decrease in Ri, which also caused the greatest decrease in Am.
Hearts.Figure 4 shows the effects of the different treatments on cardiac versus body weight. Trandolapril and veratran were more effective than verapamil at reducing cardiac mass. This effect was significantly different only between the control and veratran groups.
We based selection of the drug doses to be used in this study on our previous experience (data not shown). High doses of verapamil were able to reduce blood pressure to only a slightly higher extent than were lower doses and were accompanied by increasing reflex tachycardia as the doses were increased. The trandolapril dose used here proved to be effective at controlling pressure. Administration of the combination aimed at establishing the additive effect of verapamil and trandolapril as regards reversing structural and functional cardiovascular alterations with no further reduction in arterial pressure (otherwise harmful).
As mentioned, in the long term, the kidney is the main organ responsible for regulating arterial pressure (15). In this sense, the main action of antihypertensive drugs in reducing arterial pressure must lie in their capacity to displace the pressure-natriuresis curve toward the left. These experiments demonstrate that the combination of verapamil and trandolapril (VT) is able to decrease arterial pressure in SHR-UNX. Trandolapril and veratran had comparable effects that were more effective and appeared earlier than when verapamil alone was administered (Fig. 1). It is curious that verapamil requires between 1 and 2 months of administration before significant effects are observed, whereas trandolapril and the combination (allegedly through the action of T) exert important effects as soon as 1 week after the beginning of administration (Fig. 1). In terms of time courses, the response to trandolapril is well correlated with possible effects on renal functionality, most likely through a blockade of the hypertensive actions of angiotensin II, whose synthesis would be blocked by the drug. Theoretically, if verapamil also acted at renal functional level, its action would also be expected to elicit a similar effect over time, regardless of its efficiency. This, however, does not occur. Although direct and indirect diuretic effects have been described for calcium antagonists (16,17), for some reason in our experiments, such effects were either counterbalanced by neurohumoral responses or else did not occur. The issue thus arises as to the nature of the late antihypertensive effect that requires between 1 and 2 months to become apparent. The answer is complex and probably involves structural modifications with functional consequences that take longer to appear than direct functional adaptations. In this sense, an action on the trophic status of the mesangium, the walls, and architecture of renal vessels or other renal structures could be invoked.
If V does indeed reverse certain structural alterations, some sclerotic or hypertrophied structures or both must exist on which this effect can be exerted. It is well known that because of their autoregulation, renal afferent arteries are quite well protected from hypertensive aggression (18-20). Although one early consequence of partial nephrectomy is a loss of autoregulatory capacity in the remnant renal mass (18,21,22), reversal of the structural alterations caused by pressure after nephrectomy can be ruled out as the antihypertensive mechanism of verapamil because hypertension has already become established and is not affected by renal ablation. Alternative possibilities would contemplate other renal pressure-sensitive structures involved in regulation of the pressure-natriuresis curve, which may be constitutively altered in genetic hypertension.
The cardiovascular system is able to adapt to the prevailing conditions of blood flow and pressure so that its structural tension (Laplace law) and hemodynamic shear rate (5,23,24) can remain constant. When pressure increases, the vascular walls undergo adaptive changes that allow them to maintain tension with minimal energy expenditure. The fact that events appear earlier [mass increase (hyperplasia or hypertrophy or both), remodeling of the vascular geometric architecture, endothelial dysfunction] or later (sclerosis, phenotypic changes) depends on the intensity and duration of the pressure increase. At this point, it should be stressed that once the pressure increase has ceased, morphohistologic alterations take longer to return to basal conditions (2). This has therapeutic implications for patients with chronic hypertension who are suddenly subjected to severe antihypertensive treatments that do not repair tissue alterations in parallel. In these situations, cardiovascular incompetence and tissue ischemia might be important. In this sense, angiotensin-converting enzyme inhibitors such as trandolapril reverse the structural changes twofold more than do any other antihypertensive drug per millimeter of pressure reduction (25-30).
In our experiments, in the verapamil and veratran treatment groups, the cross-sectional area of the aortic wall decreased, indicating tissue loss, thus reinforcing the previously mentioned hypothesis on verapamil antihypertensive mechanism. Because functional studies also pointed to reductions in contraction capacity (response to KCl) and in the response to NA that were similar for both groups, and because no important changes in the pD2 values of the contraction response were observed, we are inclined toward the possibility that these treatments would elicit a loss of smooth-muscle mass, leading to the observed decline in contractility. A caveat should be included here, however, because many other factors regulate the slope of the mass-to-force-generation capacity relation that governs whether the treated aortae will produce the same, more, or less force with the same, more, or less mass. This could be the case in the trandolapril group, in which decreased contractility but no tissue loss was observed.
Interestingly, in the case of aortae from rats treated with verapamil, structural weakening seems to occur because the w/Ri ratio decreased to the same extent as in the other two treatment groups (trandolapril and veratran), even though MAP underwent a much more moderate reduction. In vivo, the aortae from the verapamil group are probably subject to greater tension than those from the trandolapril or veratran groups. This further points to the ability of verapamil to reduce vascular (but not cardiac) hypertrophy, regardless of the reduction in MAP. Regarding trandolapril, in the light of the results obtained here, we are unable to separate its pressure-dependent from its pressure-independent restorative action on both cardiac and aortic structures.
The response of medial smooth muscle to nitric oxide was very similar in all treatment groups, as evidenced by the nitroprusside relaxation curves. However, the endothelium-dependent relaxations caused by ACh were substantially improved in the trandolapril and veratran groups, and to a lesser extent in the verapamil group, with respect to the controls. The relation between pressure and endothelial dysfunction is controversial. Although in almost all models of hypertension (3,6,30-35), this relation has been well documented, some authors reported that pressure reduction suffices to restore endothelial function (3,36), whereas others (30,34,37,38) reject this notion. A possible explanation could be the time required for the restorative action to appear after pressure has been reversed to normal values. In this respect, we have no data. Wall tension (rather than pressure itself) could be involved in the effect, which would be consistent with the results found by us for the verapamil group.
Some authors (39) reported better improvements in endothelial dysfunction after shorter treatment periods (2 months) by using the same agents at the same doses in stroke-prone SHRs with intact renal mass. In any case, we believe that reductions in renal mass, different treatment lengths, and the evolution of structural alterations are all potentially involved in the effects described here.
These results may be of relevance in the development of clinical studies on the long-term treatment of hypertensive patients with a deficit in renal mass. In such subjects, if our results are confirmed, the combined treatment (veratran) is more efficient than the individual treatments, at least according to the criteria currently demanded for antihypertensive drugs. Veratran combines the efficiency in the reduction of arterial pressure of trandolapril with the reversal of the vascular hypertrophy, deriving from verapamil. This could introduce a modification in the treatment of these patients because, as has been mentioned, the results obtained to date have indicated that angiotensin-converting enzyme inhibitors are the most efficient drugs in reversing hypertrophy per millimeter decrease in arterial pressure. Future studies aimed at elucidating the mechanism of action of the beneficial effects exerted on the aorta, their extension to other vascular territories and to resistance arteries, and the effects of these treatments on renal function and histocellular structure may offer a better understanding and suggest better uses for these compounds in this experimental model.
Acknowledgment: This study was supported in part by a grant from Knoll, S.A. We thank J. Villoria and A. Pascua for technical assistance.
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