Endothelin-1 Gene Expression in Blood Vessels and Kidney of Spontaneously Hypertensive Rats (SHR), L-NAME-Treated SHR, and Renovascular Hypertensive Rats

Abstract

Spontaneously hypertensive rats (SHR), renovascular hypertensive rats [two-kidney one clip Goldblatt (2-K 1C) and 1-K 1C], and SHR treated with the nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME), do not respond to endothelin (ET) receptor antagonists with a decrease in blood pressure. However, treatment with ET receptor antagonists has shown some beneficial renal and coronary effects in SHR. In this study we examined tissues from SHR, L-NAME-treated SHR, 2-K 1C, and 1-K 1C, using in situ hybridization with a specific rat preproET-1 cRNA probe to evaluate preproET-1 mRNA abundance in blood vessels, heart, and kidneys. Grain density was similar in SHR and Wistar-Kyoto (WKY) rats in all tissues examined. L-NAME-treated SHR showed increased grain density vs. SHR in endothelium of aorta and of small coronary arteries and in kidney glomeruli, but not in renal or mesenteric arteries. 2-K 1C presented increased grain density in coronary arteries and in glomeruli of the unclipped but not the clipped kidney vs. glomeruli of control rats. 1-K 1C rats exhibited increases in preproET-1 mRNA relative to unilaterally nephrectomized control rats in endothelium of aorta and in mesenteric and coronary arteries, but not in renal arteries or glomeruli. None of the hypertensive models studied showed detectable evidence of myocardial overexpression of preproET-1 mRNA. Therefore, tissue-specific enhancement of ET-1 expression may underlie ET-dependent functional alterations and may explain the beneficial effects of ET receptor antagonists in the coronary circulation or in the kidney in some hypertensive models, but not in SHR.

Endothelin-1(ET-1) expression in the endothelium of blood vessels is increased in deoxycorticosterone acetate (DOCA)-salt hypertensive rats and in spontaneously hypertensive rats (SHR) treated with DOCA and salt, both of which groups respond to ET antagonism with a decrease in blood pressure (reviewed in ref. ¹). In SHR ⁽²⁾ and in SHR treated with the nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME), which develop malignant hypertension⁽³⁾, no increase in ET-1 expression has been noted, except in the aorta in L-NAME-treated SHR, and blood pressure is unaffected by ET antagonism. Two-kidney one clip (2-K 1C) and 1-K 1C Goldblatt hypertensive rats do not respond to ET receptor antagonism ⁽⁴⁾, and only the latter (1-K 1C) exhibit some degree of overexpression of ET-1 in blood vessels ⁽⁵⁾. Therefore, only hypertensive rats that have important overexpression of ET-1 in the endothelium of small arteries respond with a blood pressure decrease to ET receptor antagonism. However, SHR show beneficial changes in the coronary circulation ⁽⁶⁾ and in renal function ^(6,7) after ET receptor antagonists. It is possible that localized overexpression of ET-1 occurs in kidneys or in coronary vessels of SHR to explain these findings. In situ hybridization was therefore applied to tissues from SHR, L-NAME-treated SHR, and renovascular hypertensive rats to study tissue-specific preproET-1 mRNA expression.

MATERIALS AND METHODS

Animal experiments

Wistar-Kyoto (WKY) rats and SHR, aged 16 weeks, from Taconic Farms (Germantown, NY, U.S.A.) were investigated. SHR received tapwater or water containing L-NAME 30 mg/kg/day for 4 weeks. Renovascular hypertension was induced in Sprague-Dawley rats (Charles River, St. Constant, Québec, Canada), weighing 150 g at age 5-6 weeks, by applying a silver clip with a 0.2-mm lumen to the left renal artery under pentobarbital anesthesia (40 mg/kg) (Somnotol; MTS Pharmaceuticals, Cambridge, Ontario, Canada) with/without a right nephrectomy as previously described ^(4,5). Rats were killed by decapitation. The heart, kidney, thoracic aorta, and the mesenteric vasculature were frozen in isopentane cooled to −35°C and were stored at −80°C until processing.

In situ hybridization

Frozen sections (10 μm thick) of tissues were obtained on a Bright-Hacker cryostat and thaw-mounted on polylysine-coated glass slides for in situ hybridization, which was performed as previously described in detail ^(8-10). Specificity of labeling was established with the sense strand preproET-1 RNA probe of the same size and specific activity as the anti-sense strand probe. Tissues from three or four different animals were sectioned and multiple sections for each group were examined (minimum of 12 tissue sections). The rat preproET-1 sense and anti-sense RNA probes of 392 bp were prepared by the RNA transcription reaction using T7 or SP6 RNA polymerase, respectively, and a cDNA plasmid construct previously described in detail ^(8-10). Radiolabeled riboprobes were prepared using [³⁵S]UTP and [³⁵S]CTP (1,250 Ci/mmole; Amersham, Arlington Hills, IL, U.S.A.) in the same labeling reaction.

RESULTS

Table 1 shows systolic blood pressures and body weights of rats. In situ hybridization quantification of preproET-1 mRNA is shown in the figures. Grain density with the anti-sense preproET-1 riboprobe was similar in all tissues examined from WKY rats and SHR (Fig. 1). L-NAME-treated SHR showed increased grain density vs. SHR in endothelium of the aorta and of small coronary arteries and in kidney glomeruli when the anti-sense riboprobe was used, but not in renal or mesenteric arteries (Fig. 1). 2-K 1C showed increased grain density with the ET-1 anti-sense riboprobe in coronary arteries and in glomeruli of the unclipped but not the clipped kidney vs. glomeruli of control rats (Fig. 2). The aorta and the coronary and mesenteric arteries did not exhibit increased expression of preproET-1 mRNA. 1-K 1C hypertensive rats exhibited increases in preproET-1 mRNA relative to unilaterally nephrectomized control rats in endothelium of the aorta and in mesenteric and coronary arteries, but not in renal arteries or glomeruli (Fig. 3). None of the hypertensive models studied showed detectable evidence of myocardial overexpression of preproET-1 mRNA. The sense preproET-1 riboprobe did not show specific increases in hybridization in any tissue of control or hypertensive models.

TABLE 1: Blood pressure and body weight

FIG. 1: Bar graph depicts abundance of preproET-1 mRNA in vessels and kidneys as means ± SEM of grains resulting hybridization of the ET-1 riboprobe with preproET-1 mRNA, counted in three to five vessels of equivalent lumen and glomeruli per section, in two sections from three age-matched WKY, SHR, and L-NAME-treated SHR. *p < 0.05 vs. SHR and WKY.

FIG. 2: Bar graph depicts abundance of preproET-1 mRNA in vessels and kidneys as means ± SEM of grains as in Fig. 1, counted in tissues from three control and 2-K 1C hypertensive rats. *p < 0.05. C, clipped kidney; U, unclipped kidney.

FIG. 3: Bar graph depicts abundance of preproET-1 mRNA in vessels and kidneys as means± SEM of grains, as in Fig. 1, counted in tissues from three (Uni-Nx) control and 1-K 1C hypertensive rats. *p < 0.05.

DISCUSSION

The present results demonstrate that localized enhancement of preproET-1 mRNA expression may occur in a tissue-specific manner in the heart, kidney, and in some vascular beds of some models of experimental hypertension. In L-NAME-treated SHR there was increased abundance of preproET-1 mRNA in glomeruli and, as previously shown, in endothelium of the aorta. This may contribute to deterioration of renal function⁽¹¹⁾ and aortic hypertrophy ⁽³⁾ in the absence of cardiac ⁽¹²⁾ or small artery hypertrophy ⁽³⁾. In 2-K 1C hypertensive rats, vessels did not exhibit increased density of grains, confirming previous data from Northern blot analysis ⁽⁵⁾ and in agreement with the absence of significant hypertropic remodeling of small arteries in this model ⁽⁴⁾. Glomeruli of the clipped kidney did not exhibit enhanced expression of preproET-1, whereas those of the kidney exposed to elevated blood pressure did, which suggests that blood pressure plays a role in this increase in ET-1 production. Therefore, ET-1 overexpression may contribute to reducing the function of the unclipped kidney. Because chronic angiotensin II infusion generates an ET-dependent form of hypertension, with overexpression of ET-1 and a blood pressure-lowering response to ET antagonism ⁽¹³⁾, the localized expression in tissues exposed to elevated blood pressure with no generalized overexpression of ET-1 in 2-K 1C hypertensive rats (a high plasma renin and therefore a circulating angiotensin II model) suggests that increased endogenous angiotensin II is less able to stimulate the expression of ET-1 than exogenous angiotensin II. In 1-K 1C hypertensive rats, the increase in preproET-1 mRNA in endothelium of extrarenal arteries suggests that ET may participate in the severe vascular growth characteristic of this hypertensive model ⁽⁴⁾. This may not be enough to cause ET-1-dependent hypertension, as demonstrated by the absence of blood pressure lowering by ET antagonists ⁽⁴⁾. Absence of enhancement in renal arteries and glomeruli may result from protection by the clip against elevated blood pressure, and could also contribute to the absence of response to ET receptor antagonists. Finally, in SHR, there was no evidence of localized preproET-1 mRNA overexpression relative to WKY tissues.

Tissue-specific enhancement of ET-1 expression therefore appears to occur in some hypertensive models(such as L-NAME-treated SHR, 1-K 1C, and 2-K 1C), but not in SHR. In the kidney and in coronary arteries, increased expression of ET-1 may play a pathophysiologic role and underlie the beneficial effects of ET receptor antagonism in these models of experimental hypertension, but may not explain those reported in SHR. These results suggest that blood pressure elevation plays a role in the expression of ET-1 by glomeruli in the kidney. In addition, they indicate that coronary endothelium may be particularly vulnerable to damage in hypertension resulting in ET-1 overexpression.

Acknowledgments: This work was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension and by grants from the Fondation des Maladies du Coeur du Québec.