Differential effects of angiotensin II type-1 receptor antisense oligonucleotides on renal function in spontaneously hypertensive rats

The effect of selectively decreasing renal angiotensin II type 1 (AT1) receptor expression on renal function and blood pressure has not been determined. Therefore, we studied the consequences of selective renal inhibition of AT1 receptor expression in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) in vivo. Vehicle, AT1 receptor antisense oligodeoxynucleotides (AS-ODN), or scrambled oligodeoxynucleotides were infused chronically into the cortex of the remaining kidney of conscious, uninephrectomized WKY and SHR on a 4% NaCl intake. Basal renal cortical membrane AT1 receptor protein was greater in SHR than in WKY. In WKY and SHR, AS-ODN decreased renal but not cardiac AT1 receptors. AT1 receptor AS-ODN treatment increased plasma renin activity to a greater extent in WKY than in SHR. However, plasma angiotensin II and aldosterone were increased by AS-ODN to a similar degree in both rat strains. In SHR, sodium excretion was increased and sodium balance was decreased by AS-ODN but had only a transient ameliorating effect on blood pressure. Urinary protein and glomerular sclerosis were markedly reduced by AS-ODN-treated SHR. In WKY, AS-ODN had no effect on sodium excretion, blood pressure, or renal histology but also modestly decreased proteinuria. The major consequence of decreasing renal AT1 receptor protein in the SHR is a decrease in proteinuria, probably as a result of the amelioration in glomerular pathology but independent of systemic blood pressure and circulating angiotensin II levels.     

Altered AT1 receptor regulation of ETB receptors in renal proximal tubule cells of spontaneously hypertensive rats

The renin-angiotensin and endothelin systems regulate blood pressure, in part, by affecting renal tubular sodium transport. In rodents, ETB receptors decrease proximal tubular reabsorption, whereas AT1 receptors produce the opposite effect. We hypothesize that ETB and AT1 receptors interact at the receptor level, and that the interaction is altered in spontaneously hypertensive rats (SHRs). In immortalized renal proximal tubule (RPT) cells from Wistar-Kyoto (WKY) rats, angiotensin II, via AT1 receptors, increased ETB receptor protein in a time- and concentration-dependent manner. In contrast, in SHR RPT cells, angiotensin II (10(-8) M/24 hours) had no effect on ETB receptor protein. AT1/ETB receptors colocalized and co-immunoprecipitated in both rat strains but long-term angiotensin II (10(-8) M/24 hours) treatment increased AT1/ETB co-immunoprecipitation in WKY but not in SHR cells. Short-term angiotensin II (10(-8) M/15 minutes) treatment decreased ETB receptor phosphorylation in both WKY and SHR cells, and increased ETB receptors in RPT cell surface membranes of RPT cells in WKY but not SHRs. Basal cell surface membrane ETB receptor expression was also higher in WKY than in SHRs. We conclude that AT1 receptors regulate ETB receptors by receptor interaction and modulation of receptor expression. The altered AT1 receptor regulation of ETB receptors in SHRs may play a role in the pathogenesis of hypertension.     

Differential regulation of cardiac adrenomedullin and natriuretic peptide gene expression by AT1 receptor antagonism and ACE inhibition in normotensive and hypertensive rats

OBJECTIVE: To study the effects of long-term treatment with the type 1 angiotensin (AT1) receptor antagonist losartan and the angiotensin-converting enzyme (ACE) inhibitor enalapril, on cardiac adrenomedullin (ADM), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression. METHODS: Spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were given losartan (15 mg/kg per day) or enalapril (4 mg/kg per day) orally for 10 weeks. The effects of drugs on systolic blood pressure, cardiac hypertrophy, ANP, BNP and ADM mRNA and immunoreactive-ANP (IR)-ANP, IR-BNP and IR-ADM levels in the left ventricle and atria were compared. RESULTS: Losartan and enalapril treatments completely inhibited the increase of systolic blood pressure occurring with ageing in SHR. The ratio of heart to body weight was reduced in both losartan- and enalapril-treated SHR and WKY rats. Treatment with losartan or enalapril reduced left ventricular ANP mRNA and IR-ANP in both strains, and ventricular BNP mRNA levels in SHR rats. Inhibition of ACE, AT1 receptor antagonism, changes in blood pressure or cardiac mass had no effect on left ventricular ADM gene expression in SHR and WKY rats. In addition, atrial IR-ANP and IR-ADM levels increased in SHR whereas IR-BNP levels decreased in WKY and SHR rats in response to drug treatments. CONCLUSIONS: Our results show that ventricular ADM synthesis is an insensitive marker of changes in haemodynamic load or cardiac hypertrophy. Furthermore, the expression of ADM, ANP and BNP genes is differently regulated both in the left ventricle and atria in response to AT1 receptor antagonism and ACE inhibition.     

Renal renin-angiotensin system activity in naturally reared and cross-fostered spontaneously hypertensive rats

BACKGROUND: Young (4 week) spontaneously hypertensive rats (SHR) exhibit greater renal responses to angiotensin II (Ang II) than normotensive Wistar Kyoto (WKY) rats. SHR pups cross-fostering to a WKY dam at birth (SHRX) are less sensitive to Ang II and have lower adult blood pressure. The aim of this study was to compare renal renin-angiotensin system activity in young naturally reared and cross-fostered SHR pups. METHODS: SHR and WKY rats were reared either by their natural mothers or by a foster mother of the opposite strain. At 5, 10, and 15 days of age, renal tissue renin activity and Ang II concentration were measured by radioimmunoassay. Renin-secreting cells were identified by in situ hybridization and AT(1) receptor expression was compared using Western blots. Ang II-mediated cAMP generation was measured in isolated proximal tubules. CONCLUSIONS: Tissue renin activity and numbers of renin-secreting cells did not differ, but Ang II was higher in SHRX. The AT(1) receptor expression was significantly lower in SHRX compared with SHR. Basal and Ang II-stimulated cAMP was lower in SHR tubules compared with WKY and SHRX tubules.Cross-fostering reversed the increased renal sensitivity of the SHR to Ang II. These data suggest that renal AT(1) receptor expression can be manipulated during the postnatal period and that this may affect adult blood pressure.     

高血压左心室肥厚与血管紧张素Ⅱ受体的关系

目的探讨自发性高血压大鼠(SHR)左心室肥厚和血管紧张素Ⅱ(AngⅡ)受体的关系。 方法雄性SHR自10周龄始,给予依那普利［enalapril20mg/(kg     

Rat strain effects of AT1 receptor activation on D1 dopamine receptors in immortalized renal proximal tubule cells

The dopaminergic and renin-angiotensin systems regulate blood pressure, in part, by affecting sodium transport in renal proximal tubules (RPTs). We have reported that activation of a D1-like receptor decreases AT1 receptor expression in the mouse kidney and in immortalized RPT cells from Wistar-Kyoto (WKY) rats. The current studies were designed to test the hypothesis that activation of the AT1 receptor can also regulate the D1 receptor in RPT cells, and this regulation is aberrant in spontaneously hypertensive rats (SHRs). Long-term (24 hours) stimulation of RPT cells with angiotensin II, via AT1 receptors increased total cellular D1 receptor protein in a time- and concentration-dependent manner in WKY but not in SHR cells. Short-term stimulation (15 minutes) with angiotensin II did not affect total cellular D1 receptor protein in either rat strain. However, in the short-term experiments, angiotensin II decreased cell surface membrane D1 receptor protein in WKY but not in SHR cells. D1 and AT1 receptors colocalized (confocal microscopy) and their coimmunoprecipitation was greater in WKY than in SHRs. However, AT1/D1 receptor coimmunoprecipitation was decreased by angiotensin II (10(-8) M/24 hours) to a similar extent in WKY (-22+/-8%) and SHRs (-22+/-12%). In summary, these studies show that AT1 and D1 receptors interact differently in RPT cells from WKY and SHRs. It is possible that an angiotensin II-mediated increase in D1 receptors and dissociation of AT1 from D1 receptors serve to counter regulate the long-term action of angiotensin II in WKY rats; different effects are seen in SHRs.     

Upregulation of angiotensin-converting enzyme 2 by all-trans retinoic acid in spontaneously hypertensive rats

There is increasing evidence that all-trans retinoic acid (atRA) influences gene expression of components of renin-angiotensin system (RAS), which plays a pivotal role in the pathophysiology of essential hypertension. To further validate effects of atRA on the RAS and to assess the possibility that atRA affects the activity of angiotensin-converting enzyme 2 (ACE2), gene, and protein expression of ACE2 have been examined by real-time polymerase chain reaction and Western blot methods in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Rats were treated with atRA (10 or 20 mg x kg(-1) x day(-1)) or placebo given as daily intraperitoneal injection for 1 month. ACE2 expression was markedly decreased in placebo-treated SHR when compared with WKY rats. However, in atRA-treated SHR, a significant upregulation of ACE2 expression was observed in heart and kidney. In conclusion, chronic atRA treatment increases gene and protein expressions of ACE2, resulting in the reduction of blood pressure and the attenuation of myocardial damage in SHR, which suggests that atRA may be an attractive candidate for the potential prevention and treatment of human essential hypertension.     

Effect of AT2 receptor on expression of AT1 and TGF-beta receptors in VSMCs from SHR

We recently reported that overexpression of the angiotensin II type 2 (AT2) receptor downregulates the AT1a receptor through the bradykinin/NO pathway in a ligand-independent manner in vascular smooth muscle cells (VSMCs). In the present study, we investigated the effect of AT2 receptor overexpression on the expression of the AT1a receptor and transforming growth factor-beta (TGF-beta) receptor subtypes in VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Transfection of the AT2 receptor gene downregulated expression of the AT1a receptor in VSMCs from WKY, but did not affect expression of the AT1a receptor in VSMCs from SHR. Transfection of the AT2 receptor abolished DNA synthesis in response to angiotensin II in VSMCs from WKY; in VSMCs from SHR, basal DNA synthesis was suppressed, but DNA synthesis in response to Ang II was not altered. The NO substrate L-arginine augmented downregulation of the AT1a receptor in VSMCs from WKY, whereas it did not affect expression of the AT1a receptor in VSMCs from SHR. In response to AT2 receptor transfection, expression of TGF-beta type I receptor mRNA was suppressed significantly in VSMCs from WKY, whereas expression of TGF-beta type I receptor was not altered in VSMCs from SHR. These results suggest that the AT2 receptor downregulates AT1a and TGF-beta type I receptors in normal VSMCs, but not in SHR-derived VSMCs. The lack of downregulation of the AT1a receptor may contribute, in part, to the exaggerated growth of VSMCs from SHR.     

Activation of D3 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D(3) dopamine receptor gene in mice produces renin-dependent hypertension. In rats, D(2)-like receptors reduce angiotensin II binding sites in renal proximal tubules (RPTs). Because the major D(2)-like receptor in RPTs is the D(3) receptor, we examined whether D(3) receptors regulate angiotensin II type 1 (AT(1)) receptors in rat RPT cells. The effect of D(3) receptors on AT(1) receptors was studied in vitro and in vivo. The D(3) receptor agonist PD128907 decreased AT(1) receptor protein and mRNA in WKY RPT cells and increased it in SHR cells. PD128907 increased D(3) receptors in WKY cells but had no effect in SHR cells. D(3)/AT(1) receptors colocalized in RPT cells; D(3) receptor stimulation decreased the percent amount of D(3) receptors that coimmunoprecipitated with AT(1) receptors to a greater extent in WKY than in SHR cells. However, D(3) receptor stimulation did not change the percent amount of AT(1) receptors that coimmunoprecipitated with D(3) receptors in WKY cells and markedly decreased the coimmunoprecipitation in SHR cells. The D(3) receptor also regulated the AT(1) receptor in vivo because AT(1) receptor expression was increased in kidneys of D(3) receptor-null mice compared with wild type littermates. D(3) receptors may regulate AT(1) receptor function by direct interaction with and regulation of AT(1) receptor expression. One mechanism of hypertension may be related to increased renal expression of AT(1) receptors due decreased D(3) receptor regulation.     

Alterations in sarcoplasmic reticulum and angiotensin II receptor type 1 gene expression in spontaneously hypertensive rat hearts

Left ventricular hypertrophy (LVH) is an adaptive change in response to hypertensive pressure overload. Some evidence indicates that the decrease in sarcoplasmic reticulum (SR) Ca2+-ATPase mRNA expression, which may contribute to a diastolic dysfunction of the heart, occurs in the experimental pressure overload model. Also, recent studies have demonstrated that angiotensin II (Ang II) and angiotensin II receptor type 1 (AT1) play important roles in LVH. The purpose of this study was to investigate the function of the SR and the role of AT1 in genetic hypertension in spontaneously hypertensive rats (SHR) at ages 10 and 18 weeks. In SHR, cardiac hypertrophy has already developed at 10 weeks of age. SR Ca2+-ATPase activity and mRNA expression were significantly lower in SHR than in Wistar-Kyoto rats (WKY). Plasma renin activity in SHR was unchanged compared with WKY, whereas the Ang II concentration in SHR was significantly higher than that in WKY. AT1 mRNA expression in SHR was similar to that in WKY. These results suggest that in the early stage of hypertension in SHR Ang II may stimulate hypertrophy in the cardiomyocytes through the AT1, which is not downregulated by a high concentration of Ang II.     

AT(2) receptor stimulation enhances antihypertensive effect of AT(1) receptor antagonist in hypertensive rats

In the present study, we investigated the role of the angiotensin type 2 (AT(2)) receptor in the regulation of blood pressure in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We tested the hypothesis that AT(2) receptor activation may contribute to the antihypertensive effects of angiotensin type 1 (AT(1)) receptor antagonists. Mean arterial pressure (MAP) and heart rate were measured over a 4-day protocol in various groups of rats that received the following drug combinations: the AT(1) receptor antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, the AT(2) receptor agonist CGP42112 (1 microg/kg per minute) alone, and candesartan plus CGP42112. In both SHR and WKY, 4-hour infusions of saline and CGP42112 alone did not alter MAP. In WKY, both doses of candesartan alone caused small decreases in MAP, which were similar when combined with CGP42112. In SHR, candesartan (0.1 mg/kg) caused an immediate, marked decrease in MAP, which was unaffected when combined with CGP42112. By contrast, in separate SHR, a 10-fold lower dose of candesartan (0.01 mg/kg) caused a slower-onset depressor response, which was enhanced when combined with CGP42112. The involvement of AT(2) receptors was confirmed in another group of SHR, since this facilitation of the antihypertensive effect of candesartan by CGP42112 was abolished by the coinfusion of the AT(2) receptor antagonist PD123319 (50 microg/kg per minute) with the candesartan/CGP42112 combination. Collectively, these data suggest that in SHR, AT(2) receptor activation can facilitate the initial depressor response caused by an AT(1) receptor antagonist.     

Perturbation of D1 dopamine and AT1 receptor interaction in spontaneously hypertensive rats

The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Because this interaction may be perturbed in genetic hypertension, we studied D1 dopamine and AT1 angiotensin receptors in immortalized renal proximal tubule (RPT) and A10 aortic vascular smooth muscle cells. In normotensive Wistar-Kyoto (WKY) rats, the D1-like agonist fenoldopam increased D1 receptors but decreased AT1 receptors. These effects were blocked by the D1-like antagonist SCH 23390 (10(-7) mol/L per 24 hours). In spontaneously hypertensive rat (SHR) RPT cells, fenoldopam also decreased AT1 receptors but no longer stimulated D1 receptor expression. Basal levels of AT1/D1 receptor coimmunoprecipitation were greater in WKY RPT cells (29+/-2 density units, DU) than in SHR RPT cells (21+/-2 DU, n=7 per group, P<0.05). The coimmunoprecipitation of D1 and AT1 receptors was increased by fenoldopam (10(-7) mol/L per 24 hours) in WKY RPT cells but decreased in SHR RPT cells. The effects of fenoldopam in RPT cells from WKY rats were similar in aortic vascular smooth muscle cells from normotensive BD IX rats, that is, fenoldopam decreased AT1 receptors and increased D1 receptors. Our studies show differential regulation of the expression of D1 and AT1 receptors in RPT cells from WKY and SHR. This regulation and D1/AT1 receptor interaction are different in RPT cells of WKY and SHR. An altered interaction of D1 and AT1 receptors may play a role in the impaired sodium excretion and enhanced vasoconstriction in hypertension.     

Role of chymase-dependent angiotensin II formation in regulating blood pressure in spontaneously hypertensive rats.

Vascular smooth muscle cells in spontaneously hypertensive rats (SHR) express angiotensin II-forming chymase (rat vascular chymase [RVCH]), which may contribute to blood pressure regulation. In this study, we studied whether chymase-dependent angiotensin II formation contributes to the regulation of blood pressure in SHR. The systolic blood pressure in 16-week-old Wistar-Kyoto (WKY) rats was 113 +/- 9 mmHg, compared to 172 +/- 3 mmHg in SHR. Using synthetic substrates for measuring angiotensin-converting enzyme (ACE) and chymase activities, it was found that both ACE and chymase activities in extracts from SHR aortas were significantly higher than in those from WKY rat aortas. Using angiotensin I as a substrate, angiotensin II formation in SHR was found to be significantly higher than that in WKY rats, and its formation was completely suppressed by an ACE inhibitor, but not by a chymase inhibitor. RVCH mRNA expression could not be detected in aorta extracts from either WKY rats or SHR. In carotid arteries isolated from WKY rats and SHR, angiotensin I-induced vasoconstriction was completely suppressed by an ACE inhibitor, but not by a chymase inhibitor. Angiotensin I-induced pressor responses in both WKY rats and SHR were also completely inhibited by an ACE inhibitor, but they were not affected by a chymase inhibitor. In SHR, an ACE inhibitor and an angiotensin II receptor blocker showed equipotent hypotensive effects, but a chymase inhibitor did not have a hypotensive effect. These results indicated that chymase-dependent angiotensin II did not regulate blood pressure in SHR in the present study.     

The Mechanism of Distinct Diurnal Variations of Renin-Angiotensin System in Aorta and Heart of Spontaneously Hypertensive Rats

Diurnal variations in plasminogen activator inhibitor-1 mRNA expression are different between the spontaneously hypertensive rats (SHRs) and the Wistar-Kyoto (WKY) rats, and between the aorta and the heart. To elucidate the mechanisms, we examined diurnal changes in the circulating renin-angiotensin system in the SHR and WKY rats. Diurnal variations in plasma renin activity (PRA), plasma angiotensin I, and aldosterone concentrations were similar between the SHR and WKY rats. On the other hand, plasma angiotensin II (Ang II) concentration in the SHR was lower than that in the WKY rats at most time points, but increased to the level of the WKY rats in the late light phase. Treatment with AT1 receptor antagonist candesartan increased plasma Ang II concentration except at ZT 8 and lessened its diurnal variation in the SHR. At the peak in plasma Ang II in the SHR, Ang II regulated genes such as transforming growth factor-β1 and p22phox were upregulated in the aorta. On the other hand, these genes were upregulated throughout the day in the heart of SHR. Candesartan treatment increased AT1a receptor mRNA expression in the heart but not in the aorta of SHR. These findings suggest that an AT1 receptor-mediated mechanism might cause a surge in plasma Ang II concentration at the late light phase in the SHR. Homologous down-regulation of AT1a receptor by Ang II may dampen the effect of a surge in plasma Ang II concentration in the heart of SHR.     

全反式维甲酸对高血压大鼠心脏氧化应激水平的影响

目的:探讨全反式维甲酸(atRA)对高血压心脏还原型辅酶I氧化酶P22亚单位(p22phox)表达以及氧化应激水平的影响。方法:采用12周龄雄性自发性高血压大鼠(SHR)及其同源对照WKY大鼠,经腹腔注射at-RA,为期1月。分别采用免疫印迹、硫代巴比妥酸比色以及透射电镜技术测定atRA治疗后SHR心脏p22phox的表达、丙二醛(MDA)含量以及心肌超微结构情况。结果:与WKY对照组相比,SHR心脏组织中p22phox蛋白表达与MDA含量明显升高(P均<0.01)。而atRA治疗后SHR(低、高剂量atRA组)大鼠心脏组织中p22phox蛋白表达与MDA水平出现下调(P均<0.05),同时伴有心肌损伤减轻。结论:长期atRA治疗可降低SHR大鼠心脏组织中p22phox表达与MDA水平,提示atRA在高血压病中具有一定的抗氧化效应。     

Pressor response induced by central angiotensin II is mediated by activation of Rho/Rho-kinase pathway via AT1 receptors

OBJECTIVES: The brain renin-angiotensin system plays an important role in cardiovascular regulation and the pathogenesis of hypertension. Angiotensin II activates the Rho/Rho-kinase pathway in vascular smooth muscle cells and cardiomyocytes in vitro. The aim of the present study was to determine whether angiotensin II in the brainstem activates the Rho/Rho-kinase pathway, and, if so, whether this mechanism is involved in the central pressor action of angiotensin II. METHODS AND RESULTS: Angiotensin II infused intracisternally for 7 days in Wistar-Kyoto rats (WKY) increased systolic blood pressure (SBP) and urinary norepinephrine excretion. These responses were abolished by the co-infusion of Y-27632, a specific Rho-kinase inhibitor, or valsartan. The intracisternal infusion of Y-27632 or valsartan also reduced SBP and norepinephrine excretion in spontaneously hypertensive rats (SHR). Western blot analysis was performed to examine the expression levels of membranous RhoA and phosphorylated ezrin, radixin, and moesin (p-ERM), which reflects Rho/Rho-kinase activity. The expression levels of membranous RhoA and p-ERM in the brainstem were significantly greater in both angiotensin II-treated WKY and SHR than in vehicle-treated WKY. Valsartan reduced the expression levels of membranous RhoA and p-ERM in angiotensin II-treated WKY and SHR. Y-27632 reduced the expression levels of p-ERM in angiotensin II-treated WKY and SHR. CONCLUSIONS: These results suggest that the pressor response induced by intracisternally infused angiotensin II is substantially mediated by the activation of the Rho/Rho-kinase pathway via AT1 receptors of the brainstem in WKY, and that this pathway might be involved in the hypertensive mechanisms of SHR.