Salt-sensitive hypertension in endothelin-B receptor-deficient rats

Abstract

The role of the endothelin-B receptor (ET(B)) in vascular homeostasis is controversial because the receptor has both pressor and depressor effects in vivo. Spotting lethal (sl) rats carry a naturally occurring deletion in the ET(B) gene that completely abrogates functional receptor expression. Rats homozygous for this mutation die shortly after birth due to congenital distal intestinal aganglionosis. Genetic rescue of ET(B)(sl/sl) rats from this developmental defect using a dopamine--hydroxylase (DBH)-ET(B) transgene results in ET(B)-deficient adult rats. On a sodium-deficient diet, DBH-ET(B);ET(B)(sl/sl) and DBH-ET(B);ET(B)(+/+) rats both exhibit a normal arterial blood pressure, but on a high-sodium diet, the former are severely hypertensive. We find no difference in plasma renin activity or plasma aldosterone concentration between salt-fed wild-type, DBH-ET(B);ET(B)(+/+) or DBH-ET(B);ET(B)(sl/sl) rats, and acute responses to intravenous L-NAME and indomethacin are similar between DBH-ET(B);ET(B)(sl/sl) and DBH-ET(B);ET(B)(+/+) rats. Irrespective of diet, DBH-ET(B);ET(B)(sl/sl) rats exhibit increased circulating ET-1, and, on a high-sodium diet, they show increased but incomplete hypotensive responses to acute treatment an ET(A)-antagonist. Normal pressure is restored in salt-fed DBH-ET(B);ET(B)(sl/sl) rats when the epithelial sodium channel is blocked with amiloride. We conclude that DBH-ET(B);ET(B)(sl/sl) rats are a novel single-locus genetic model of severe salt-sensitive hypertension. Our results suggest that DBH-ET(B);ET(B)(sl/sl) rats are hypertensive because they lack the normal tonic inhibition of the renal epithelial sodium channel.

Figure 1

In situ hybridization for ET_B mRNA in the kidney of wild-type and DBH-ET_B;ET_Bsl/sl adult rats. Low-power (a and c) and high-power (b and d) magnification of cross-sections of the renal cortex demonstrate strong hybridization signal for ET_B mRNA in the glomeruli and the associated cortical tubule and vascular structures in wild-type rats (a and b). This signal is absent in the DBH-ET_B;ET_Bsl/sl rats (c and d). Arrows indicate representative glomeruli. Findings were similar, though less dramatic, in the renal medulla (data not shown).

Figure 2

Arterial pressure response to exogenous ET-1 in DBH-ET_B;ET_Bsl/sl and DBH-ET_B;ET_B+/+ rats. Significant differences (P < 0.001 by paired t test) in the arterial blood pressure responses to acute injection of ET-1, 1 nmol/kg intravenously, are demonstrated in DBH-ET_B;ET_B+/+ and DBH-ET_B;ET_Bsl/sl rats. Measurements were made under ketamine and xylazine anesthesia in the abdominal aorta via a catheter inserted through the femoral artery. ET-1 was given intravenously (i.v.) in the tail. DBH-ET_B;ET_Bsl/sl rats were fed a high-sodium diet for 3 weeks and were treated with amiloride for 3 days before the study. Time of ET-1 injection is indicated by the arrow at the bottom of the figure. ET_B+/+ rats show the prototypic transient depressor response followed by sustained pressor response. The depressor response is absent in DBH-ET_B;ET_Bsl/sl rats. Similar absence of the transient depressor response was also observed in DBH-ET_B;ET_Bsl/sl fed a normal sodium diet before study (data not shown). Crosses indicate DBH-ET_B;ET_Bsl/sl animals that died during the study. Both of the 2 surviving DBH-ET_B;ET_Bsl/sl animals developed retinal hemorrhages seconds after ET-1 injection.

Figure 3

Effect of DBH-ET_B transgene and ET_B-genotype on MAP in rats. The DBH-ET_B transgene has no effect on arterial blood pressure (a). MAP is shown in nontransgenic ET_B+/+ (107 ± 2 mmHg) and DBH-ET_B;ET_B+/+ (110 ± 3 mmHg) rats. All rats were maintained on a high-sodium diet (8% NaCl) for at least 3 weeks before measurement and were 8- to 10-week-old males. No difference in pulse rate was found (data not shown). (b) MAP of DBH-ET_B;ET_B+/+, DBH-ET_B;ET_B+/sl, and DBH-ET_B;ET_Bsl/sl rats on a normal rodent chow diet reveals a significant elevation in MAP in DBH-ET_B;ET_Bsl/sl (124 ± 3 mmHg) compared with DBH-ET_B;ET_B+/+ (113 ± 2 mmHg) and DBH-ET_B;ET_B +/sl (106 ± 3 mmHg) rats. Systolic and diastolic pressures in DBH-ET_B;ET_Bsl/sl rats were increased. No significant difference in pulse rate was observed (data not shown). All rats were fed a normal rodent chow (0.8% NaCl) and were 8- to 10-week-old males. Blood pressure was measured in acclimated, conscious, unrestrained rats via a femoral artery catheter 24 hours after surgery.

Figure 4

Effect of dietary sodium and amiloride on blood pressure in DBH-ET_B;ET_B+/+ and DBH-ET_B;ET_Bsl/sl rats. MAP in DBH-ET_B;ET_Bsl/sl rats (127 ± 4 mmHg) was not significantly different from that of DBH-ET_B;ET_B+/+ rats (120 ± 2 mmHg) on a sodium-deficient diet (a). However, on a high-sodium diet, DBH-ET_B;ET_Bsl/sl rats exhibit severe hypertension (MAP = 168 ± 7 mmHg). This is a statistically significant increase over DBH-ET_B;ET_Bsl/sl rats on a sodium-deficient diet (a; P < 0.001) and DBH-ET_B;ET_B+/+ rats on a high-sodium diet (first bar b; P < 0.001). DBH-ET_B;ET_B+/+ rats exhibited no increase in blood pressure in response to increased dietary sodium. b illustrates the dramatic blood pressure response to amiloride in salt-fed DBH-ET_B;ET_Bsl/sl rats. The MAP in amiloride-treated, DBH-ET_B;ET_Bsl/sl rats on a high-sodium diet was not significantly different from the MAP in DBH-ET_B;ET_Bsl/sl rats fed a sodium-deficient diet (124 ± 5 vs. 127 ± 4 mmHg). DBH-ET_B;ET_B+/+ rats showed no response to amiloride treatment. All animals were maintained on the designated diet for 3 weeks before study and were 8- to 10-week old males.

Figure 5

Circulating ET-1 levels and blood pressure response to ET_A blockade in DBH-ET_B;ET_B+/+ and DBH-ET_B;ET_Bsl/sl rats. DBH-ET_B;ET_Bsl/sl rats exhibited increased circulating ET-1 levels as measured by sandwich-type enzyme immunoassay (a). The level of dietary sodium did not significantly affect circulating ET-1 levels in DBH-ET_B;ET_B+/+ rats. However, ET_B genotype significantly affected plasma ET-1 levels. a shows a significant increase in plasma ET-1 in DBH-ET_B;ET_Bsl/sl rats (13.2 ± 2.8 pg/mL) compared with DBH-ET_B;ET_B+/+ rats (2.1 ± 1.8 pg/mL; P < 0.02) on a sodium-deficient deficient diet. On a high-sodium diet, the plasma ET-1 level in DBH-ET_B;ET_Bsl/sl rats (23.9 ± 4.2 pg/mL) was also significantly increased compared with DBH-ET_B;ET_B+/+ rats (4.4 ± 1.4 pg/ml; P < 0.001). DBH-ET_B;ET_Bsl/sl rats on a high-sodium diet exhibited a significantly increased plasma ET-1 level compared with DBH-ET_B;ET_Bsl/sl rats on a sodium-deficient diet, although diet was not an independent variable affecting plasma ET-1 concentration by 2-way ANOVA. (b) DBH-ET_B;ET_Bsl/sl rats exhibit an increased acute depressor response to ET_A blockade with FR139317 (10 mg/kg, intra-arterially) compared with ET_B+/+ rats on a high-sodium diet. DBH-ET_B;ET_Bsl/sl rats are significantly hypertensive compared with DBH-ET_B;ET_B+/+ rats before and after treatment with FR139317. However, the change in MAP in DBH-ET_B;ET_Bsl/sl rats in response to FR139317 (–15 ± 5 mmHg) is significantly greater than in DBH-ET_B;ET_B+/+ rats (–3 ± 2 mmHg; AP = 0.01).