Uninephrectomized Control Rats Research Articles

Methanol extract of Caesalpinia benthamiana normalizes blood pressure and attenuates oxidative stress in uninephrectomized hypertensive rats.

Hypertension is the number one risk factor and primary contributor of cardiovascular diseases. Caesalpinia benthamiana is a valuable medicinal plant withunvalidated anti-hypertensive activity. This study was carried out to explore the antihypertensive effect of C.benthamiana on uninephrectomized hypertensive rats. Fifty rats were grouped into five groups, each containing 10 animals: Group A-normal control (normotensive); B-uninephrectomized control; C-uninephrectomized treated with 50mg/kg C.benthamiana extract; D-uninephrectomized treated with 100mg/kg C.benthamiana; and E- uninephrectomized treated with 10mg/kg of Lisinopril. Significant increases were observed in systolic,diastolic and mean blood pressure of uninephrectomized control rats. Furthermore, markers ofoxidative stress (malondialdehyde, hydrogen peroxide, protein carbonyl, myeloperoxidase and the advancedoxidative protein products) increased significantly while antioxidant status (reduced glutathione, glutathione peroxidase, glutathione S-transferase andsuperoxidedismutase), reduced significantly in uninephrectomized hypertensive rats. Histopathology revealed thrombosis and occlusion of coronary vessels in the heart, and congestion in the kidney. However, the observed high blood pressure parameters were remarkably normalized together with reduction in markers of oxidative stress and improvement in antioxidant defence system of uninephrectomized hypertensive rats treated with C.benthamiana extract similar to normotensive rats. C.benthamiana extract exhibited antihypertensive action, strong antioxidant ability, attenuated oxidative stress-mediated hypertension and lessened the development of cardiac and renal damage associated with hypertension induced by uninephrectomy and high dietary intake of salt. Together, C.benthamiana extract might be useful in the management of hypertension due to volume overload in the cardiovascular system.

Pentosan polysulfate preserves renal microvascular P2X1 receptor reactivity and autoregulatory behavior in DOCA-salt hypertensive rats.

Inflammation contributes to ANG II-associated impairment of renal autoregulation and microvascular P2X1 receptor signaling, but its role in renal autoregulation in mineralocorticoid-induced hypertension is unknown. Autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. Hypertension was induced in uninephrectomized control rats (UNx) by subcutaneous implantation of a DOCA pellet plus administration of 1% NaCl in the drinking water (DOCA-salt) for 3 wk. DOCA-salt rats developed hypertension that was unaltered by anti-inflammatory treatment with pentosan polysulfate (DOCA-salt+PPS) but was suppressed with "triple therapy" (hydrochlorothiazide, hydralazine, and reserpine; DOCA-salt+TTx). Baseline arteriolar diameters were similar across all groups. UNx rats exhibited pressure-dependent vasoconstriction with diameters declining to 69 ± 2% of control at 170 mmHg, indicating intact autoregulation. DOCA-salt treatment significantly blunted this pressure-mediated vasoconstriction. Diameters remained between 91 ± 4 and 98 ± 3% of control over 65-170 mmHg, indicating impaired autoregulation. In contrast, pressure-mediated vasoconstriction was preserved in DOCA-salt+PPS and DOCA-salt+TTx rats, reaching 77 ± 7 and 75 ± 3% of control at 170 mmHg, respectively. ATP is required for autoregulation via P2X1 receptor activation. ATP- and β,γ-methylene ATP (P2X1 receptor agonist)-mediated vasoconstriction were markedly attenuated in DOCA-salt rats compared with UNx (P < 0.05), but significantly improved by PPS or TTx (P < 0.05 vs. DOCA-salt) treatment. Arteriolar responses to adenosine and UTP (P2Y2 receptor agonist) were unaffected by DOCA-salt treatment. PPS and TTx significantly reduced MCP-1 and protein excretion in DOCA-salt rats. These results support the hypothesis that hypertension triggers inflammatory cascades but anti-inflammatory treatment preserves renal autoregulation in DOCA-salt rats, most likely by normalizing renal microvascular reactivity to P2X1 receptor activation.

Flow- and acetylcholine-induced dilatation in small arteries from rats with renovascular hypertension — effect of tempol treatment

We investigated whether renovascular hypertension alters vasodilatation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) and the influence of the superoxide dismutase mimetic tempol on vasodilatation. One-kidney one-clip hypertensive Sprague–Dawley rats, treated with either vehicle or tempol (from weeks 5 to 10 after placement of the clip), and uninephrectomized control rats were investigated. In renal hypertensive rats systolic blood pressure increased to 171 ± 6 mmHg ( n = 10), while in tempol-treated rats systolic blood pressure remained normal (139 ± 7 mmHg, n = 5). In isolated pressurized mesenteric small arteries NO-mediated dilatation was obtained by increasing flow rate and EDHF-mediated dilatation by acetylcholine. In arteries from hypertensive rats, flow-induced dilatation was blunted, as compared to normotensive and tempol-treated rats, while acetylcholine-induced dilatation remained normal. Measured by dihydroethidium staining there was an increased amount of superoxide in arteries from vehicle-treated rats, but not from tempol-treated rats. Expression by immunoblotting of endothelial NO synthase and the NAD(P)H oxidase subunit p47phox remained unaffected by high blood pressure and tempol treatment. Simultaneous measurements of NO-concentration and relaxation were performed in isolated coronary arteries from the same animals. As compared to vehicle-treated rats, both acetylcholine-induced relaxation and NO-concentration increased in arteries from tempol-treated animals, while only the relaxation was improved by the NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In conclusion renovascular hypertension selectively inhibits flow-induced NO-mediated vasodilatation, while EDHF-type vasodilatation remains unaffected, suggesting that high blood pressure leads to increased generation of superoxide contributing to decreased NO bioavailability. Furthermore, the abnormal endothelium function can be corrected by tempol treatment, but this seems to involve mechanisms partly independent of NO.

Mechanisms for Renal Blood Flow Control Early in Diabetes as Revealed by Chronic Flow Measurement and Transfer Function Analysis

The purpose of this study was to establish the roles of the myogenic response and the TGF mechanism in renal blood flow (RBF) control at the very earliest stages of diabetes. Mean arterial pressure (MAP) and RBF were measured continuously, 18 h/d, in uninephrectomized control and diabetic rats, and transfer function analysis was used to determine the dynamic autoregulatory efficiency of the renal vasculature. During the control period, MAP averaged 91 +/- 0.5 and 89 +/- 0.4 mmHg, and RBF averaged 8.0 +/- 0.1 and 7.8 +/- 0.1 ml/min in the control and diabetic groups, respectively. Induction of diabetes with streptozotocin caused a marked and progressive increase in RBF in the diabetic rats, averaging 10 +/- 6% above control on day 1 of diabetes and 22 +/- 3 and 34 +/- 1% above control by the end of diabetes weeks 1 and 2. MAP increased approximately 9 mmHg during the 2 wk in the diabetic rats, and renal vascular resistance decreased. Transfer function analysis revealed significant increases in gain to positive values over the frequency ranges of both the TGF and myogenic mechanisms, beginning on day 1 of diabetes and continuing through day 14. These very rapid increases in RBF and transfer function gain suggest that autoregulation is impaired at the very onset of hyperglycemia in streptozotocin-induced type 1 diabetes and may play an important role in the increase in RBF and GFR in diabetes. Together with previous reports of decreases in chronically measured cardiac output and hindquarter blood flow, this suggests that there may be differential effects of diabetes on RBF versus nonrenal BF control.

Effects of long-term inhibition of neuronal nitric oxide synthase (NOS1) in uninephrectomized diabetic rats

Nitric oxide (NO) has been implicated in the pathogenesis of renal hemodynamic changes in diabetes mellitus (DM). However, the role of NO in the pathophysiology of diabetic nephropathy remains controversial. Renal hemodynamic changes in experimental DM can be acutely normalized by selective inhibition of neuronal NO synthase (nNOS). This observation suggests a nephroprotective potential of nNOS inhibition in DM. To explore this issue we assessed the long-term effects (12 weeks) of selective nNOS inhibition with the specific inhibitor S-methyl- l-thiocitrulline (SMTC) in uninephrectomized control and streptozotocin-diabetic rats. No beneficial effects of SMTC were observed in nondiabetic controls. In contrast, SMTC delayed the development of proteinuria (32 ± 8 vs. 53 ± 9 mg/24 h, week 8, p < 0.05) and glomerulosclerosis (GS, 0.30 ± 0.08 vs. 0.57 ± 0.05, p < 0.05) in diabetic rats. These effects coincided with early effects of treatment on the glomerular filtration rate, and were associated with lower renal expression of nNOS. Furthermore, SMTC-treated diabetic rats demonstrated reduced weight gain and urinary sodium excretion as compared to vehicle-treated counterparts, despite similar metabolic control and blood pressure. In summary, long-term nNOS inhibition had modest nephroprotective effects in uninephrectomized diabetic rats. These effects may be mediated by renal hemodynamic mechanisms, as well as by lower food (protein) intake.

Renoprotective effects of chronic candesartan treatment in uninephrectomized rat.

The authors recently observed an age-dependent reduction in the diuretic and natriuretic responses to plasma volume expansion in uninephrectomized control and glucose-intolerant rats. To determine the involvement of angiotensin II AT receptors in this phenomenon, the authors tested the hypothesis that chronic candesartan treatment preserves renal excretory function in the uninephrectomized rat. Control and glucose-intolerant rats underwent right nephrectomy at 4 weeks of age. Two weeks later, the animals in each group were further subdivided and maintained on tap water containing either candesartan cilexetil (10 mg. kg(-1). day(-1) ) or vehicle. Renal excretory responses to acute extracellular fluid volume expansion (5% of body weight over 30 min) were determined in the 9-month-old conscious animal. Candesartan treatment markedly reduced the mean arterial pressure of controls and glucose-intolerant rats. Nonetheless, the baseline rates of fluid and electrolyte excretion, as well as the saline volume-induced diuretic, natriuretic, and kaliuretic responses, were greater in the candesartan-treated rats than in their vehicle-treated counterparts. The augmented baseline rates of fluid and sodium excretion in candesartan-treated rats were caused by a reduction in tubular reabsorption activity and an increase in glomerular filtration rate. However, the candesartan-mediated enhancement in saline volume-induced diuresis and natriuresis was caused by a reduction in tubular reabsorption activity. In addition to improving renal function, candesartan treatment reduced proteinuria in both groups. In conclusion, chronic blockade of the angiotensin II receptors exerts hypotensive and renoprotective effects in the aging uninephrectomized rat.

Central Bromocriptine‐Induced Tachycardia is Reversed to Bradycardia in Conscious, Deoxycorticosterone Acetate‐Salt Hypertensive Rats

A central dopaminergic origin has been demonstrated for the bromocriptine-induced tachycardia in conscious, normotensive rats. The present study investigated the effect of bromocriptine on heart rate and the principal site of action of this agonist in conscious, deoxycorticosterone acetate-salt hypertensive rats, in which altered central dopaminergic activity has been previously reported. Intravenous administration of bromocriptine (150 microg/kg) increased heart rate (49+/-5 beats/min.) in uninephrectomized control rats, while it induced a significant bradycardia (50+/-6 beats/min.) in deoxycorticosterone acetate-salt hypertensive rats. In the latter animals, intravenous (500 microg/kg) or intrathecal (40 microg/rat at T9-T10) pretreatment with domperidone, a selective dopamine D2 receptor antagonist that does not cross the blood-brain barrier, reduced partially, but significantly, the bradycardiac responses to bromocriptine (reduction of about 44% and 48% of the maximal effect, respectively). In contrast, the bromocriptine-induced bradycardia was fully abolished by intravenous pretreatment with metoclopramide (300 microg/kg), a dopamine D2 receptor antagonist that crosses the blood-brain barrier, or by combined pretreatment with intravenous and intrathecal domperidone. These results indicate that, in deoxycorticosterone acetate-salt hypertensive rats, bromocriptine decreases rather than increases heart rate, an effect that is mediated partly through a peripheral D2 dopaminergic mechanism and partly through stimulation of spinal dopamine D2 receptors. They further support the concept that, in normotensive, conscious rats, the central tachycardia of bromocriptine appears to predominate and to mask the bradycardia of this agonist at both peripheral and spinal dopamine D2 receptors.

Benidipine, a Long-Acting Calcium-Channel Blocker, Prevents the Progression to End-Stage Renal Failure in a Rat Mesangioproliferative Glomerulonephritis

Background: Although the renoprotective effect of calcium-channel blockers (CCBs) has been examined in several models of hypertensive nephropathy, it remains unclear. It also remains to be clarified whether CCBs prevent the progression to end-stage renal failure in chronic progressive glomerulonephritis (GN). A new rat model of progressive mesangioproliferative GN was used to study the effect of benidipine hydrochloride, a long-acting dihydropyridine CCB, on the clinical features and morphological lesions. Methods: This animal model of progressive GN was induced by a single intravenous injection of anti-Thy-1 monoclonal antibody (MoAb 1-22-3) two weeks after unilateral nephrectomy. After 10 weeks of treatment with benidipine (1, 3, and 5 mg/kg body weight, p.o.) or hydralazine (5 mg/kg body weight, p.o.), systolic blood pressure (SBP), urinary protein excretion, creatinine clearance, glomerulosclerosis index, tubulointerstitial lesion index, glomerular cross-sectional area, and glomerular expression of transforming growth factor-β (TGF-β) and α-smooth muscle actin (α-SMA) were measured. Results: Untreated rats developed hypertension, massive proteinuria, renal dysfunction, severe glomerular and tubulointerstitial injury, higher glomerular size, and marked glomerular staining for TGF-β and α-SMA, while uninephrectomized control rats did not. Each dose of benidipine and hydralazine equally reduced SBP to uninephrectomized control levels. Three and five mg/kg/day of benidipine increased creatinine clearance, ameliorated glomerular and tubulointerstitial injury, and reduced glomerular staining for TGF-β and α-SMA, but 1 mg/kg/day of benidipine and hydralazine failed. Only a dose of 5 mg/kg/day of benidipine reduced glomerular size, although it did not reduce the size to control levels. Conclusion: These results indicate that in a rat model of progressive mesangioproliferative GN, benidipine prevents the progression to end-stage renal failure in a dose-dependent manner. This renoprotective action is associated with the suppression of glomerular expression of TGF-β and α-SMA.

Cardiovascular responses to intrathecal dopamine receptor agonists in conscious DOCA-salt hypertensive rats.

Previous studies have demonstrated that in conscious deoxycorticosterone acetate (DOCA)-salt hypertensive rats, the hypotensive action of intravenous (i.v.) bromocriptine, a selective dopamine D2 receptor agonist, was mediated partly by peripheral and partly by spinal dopamine D2 receptor stimulation, and that this effect was greater and longer-lasting than that in uninephrectomized control rats. To determine whether this amplification results partly from a putative spinal hypersensitivity phenomenon, cardiovascular responses to intrathecal (i.t.) administration of apomorphine and quinpirole were studied in conscious, 4-week DOCA-salt hypertensive rats and compared with those in uninephrectomized control rats. In both groups, upper thoracic (T2-T4) i.t. injections of apomorphine (9.1, 45.5 and 91.1 microg/rat) induced immediate and dose-dependent decreases in mean aortic pressure (MAP) and heart rate (HR), while i.t. quinpirole (38.4 microg/rat) induced only bradycardia. Neither magnitude nor duration of these responses was enhanced in DOCA-salt hypertensive rats when compared to control rats. In DOCA-salt hypertensive rats, apomorphine-induced hypotension and bradycardia remained unaffected by i.v. (500 microg/kg) pretreatment with domperidone, a selective dopamine D2 receptor antagonist that does not cross the blood-brain barrier. However, i.t. (40 microg/rat at T2-T4) pretreatment with domperidone significantly reduced apomorphine-induced hypotension, but fully suppressed bradycardia elicited by either apomorphine or quinpirole. These results demonstrated that in conscious DOCA-salt hypertensive rats, intrathecally-injected apomorphine or quinpirole decreased MAP and/or HR through a spinal D2 dopaminergic mechanism, as previously demonstrated in normotensive intact rats. Since both magnitude and duration of these responses were unchanged with respect to uninephrectomized control rats, enhancement of the hypotensive effect of intravenously-administered bromocriptine in DOCA-salt hypertensive rats does not appear to involve spinal dopamine D2 receptors.

Blocking angiotensin II ameliorates proteinuria and glomerular lesions in progressive mesangioproliferative glomerulonephritis

The renin-angiotensin system is thought to be involved in the progression of glomerulonephritis (GN) into end-stage renal failure (ESRF) because of the observed renoprotective effects of angiotensin-converting enzyme inhibitors (ACEIs). However, ACEIs have pharmacological effects other than ACE inhibition that may help lower blood pressure and preserve glomerular structure. We previously reported a new animal model of progressive glomerulosclerosis induced by a single intravenous injection of an anti-Thy-1 monoclonal antibody, MoAb 1-22-3, in uninephrectomized rats. Using this new model of progressive GN, we examined the hypothesis that ACEIs prevent the progression to ESRF by modulating the effects of angiotensin II (Ang II) on the production of transforming growth factor-beta (TGF-beta) and extracellular matrix components. We studied the effect of an ACEI (cilazapril) and an Ang II type 1 receptor antagonist (candesartan) on the clinical features and morphological lesions in the rat model previously reported. After 10 weeks of treatment with equihypotensive doses of cilazapril, cilazapril plus Hoe 140 (a bradykinin receptor B2 antagonist), candesartan, and hydralazine, we examined systolic blood pressure, urinary protein excretion, creatinine clearance, the glomerulosclerosis index, and the tubulointerstitial lesion index. We performed a semiquantitative evaluation of glomerular immunostaining for TGF-beta and collagen types I and III by immunofluorescence study and of these cortical mRNA levels by Northern blot analysis. Untreated rats developed massive proteinuria, renal dysfunction, and severe glomerular and tubulointerstitial injury, whereas uninephrectomized control rats did not. There was a significant increase in the levels of glomerular protein and cortical mRNA for TGF-beta and collagen types I and III in untreated rats. Cilazapril and candesartan prevented massive proteinuria, increased creatinine clearance, and ameliorated glomerular and tubulointerstitial injury. These drugs also reduced levels of glomerular protein and cortical mRNA for TGF-beta and collagen types I and III. Hoe 140 failed to blunt the renoprotective effect of cilazapril. Hydralazine did not exhibit a renoprotective effect. These results indicate that ACEIs prevent the progression to ESRF by modulating the effects of Ang II via Ang II type 1 receptor on the production of TGF-beta and collagen types I and III, as well as on intrarenal hemodynamics, but not by either increasing bradykinin activity or reducing blood pressure in this rat model of mesangial proliferative GN.

Different contributions of endothelin-A and endothelin-B receptors in the pathogenesis of deoxycorticosterone acetate-salt-induced hypertension in rats.

We investigated the involvement of actions mediated by endothelin-A (ETA) and endothelin-B (ETB) receptors in the pathogenesis of deoxycorticosterone acetate (DOCA)-salt-induced hypertension in rats. Two weeks after the start of DOCA-salt treatment, rats were given ABT-627 (10 [mg/kg]/d), a selective ETA receptor antagonist; A-192621 (30 [mg/kg]/d), a selective ETB receptor antagonist; or their vehicle for 2 weeks. Uninephrectomized rats without DOCA-salt treatment served as controls. Treatment with DOCA and salt for 2 weeks led to a mild but significant hypertension; in vehicle-treated DOCA-salt rats, systolic blood pressure increased markedly after 3 to 4 weeks. Daily administration of ABT-627 for 2 weeks almost abolished any further increases in blood pressure, whereas A-192621 did not affect the development of DOCA-salt-induced hypertension. When the degree of vascular hypertrophy of the aorta was histochemically evaluated at 4 weeks, there were significant increases in wall thickness, wall area, and wall-to-lumen ratio in vehicle-treated DOCA-salt rats compared with uninephrectomized control rats. The development of vascular hypertrophy was markedly suppressed by ABT-627. In contrast, treatment with A-192621 significantly exaggerated these vascular changes. In vehicle-treated DOCA-salt rats, renal blood flow and creatinine clearance decreased, and urinary excretion of protein, blood urea nitrogen, fractional excretion of sodium, and urinary N-acetyl-beta-glucosaminidase activity increased. Such damage was overcome by treatment with ABT-627 but not with A-192621; indeed, the latter agent led to worsening of the renal dysfunction. Histopathologic examination of the kidney in vehicle-treated DOCA-salt rats revealed tubular dilatation and atrophy as well as thickening of small arteries. Such damage was reduced in animals given ABT-627, whereas more severe histopathologic changes were observed in A-192621-treated animals. These results strongly support the view that ETA receptor-mediated action plays an important role in the pathogenesis of DOCA-salt-induced hypertension. On the other hand, it seems likely that the ETB receptor-mediated action protects against vascular and renal injuries in this model of hypertension. A selective ETA receptor antagonist is likely to be useful for treatment of subjects with mineralocorticoid-dependent hypertension, whereas ETB-selective antagonism alone is detrimental to such cases.

Hypotensive action of bromocriptine in the DOCA-salt hypertensive rat: contribution of spinal dopamine receptors.

To assess the role of spinal dopamine receptors in mediation of hypotension induced by systemic administration of the dopamine D2 receptor agonist, bromocriptine, conscious deoxycorticosterone acetate (DOCA)-salt hypertensive rats were pretreated with either intravenous (i.v.; 500 micrograms/kg) or intrathecal (i.t.; 40 micrograms/rat at T9-T10) domperidone, a selective dopamine D2 receptor antagonist that does not cross the blood-brain barrier. In DOCA-salt hypertensive rats, i.v. administration of a sub-maximal dose of bromocriptine (150 micrograms/kg) induced a significant decrease in mean aortic pressure (MAP) which was greater and longer lasting than that in uninephrectomized control rats. Intravenous or i.t. pretreatment with domperidone reduced partially, but significantly, the hypotensive effect of bromocriptine (reduction of about 57% and 45% of the maximal effect, respectively). The remaining responses observed during the 60 min postinjection period were still statistically significant as compared with vehicle injection. In contrast, the bromocriptine-induced hypotension was fully abolished by i.v. pretreatment with metoclopramide (300 micrograms/kg), a dopamine D2 receptor antagonist that crosses the blood-brain barrier, or by combined pretreatment with i.v. and i.t. domperidone. These results suggest that, in DOCA-salt hypertensive rats, the hypotension induced by i.v. bromocriptine is mediated partly through a peripheral D2 dopaminergic mechanism and partly through stimulation of spinal dopamine D2 receptors, has been demonstrated in conscious normotensive rats.

Effects of endothelin-1 on norepinephrine-induced vasoconstriction in deoxycorticosterone acetate-salt hypertensive rats.

We investigated the effects of endothelin-1 on pressor responses to norepinephrine in perfused mesenteric arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The response to norepinephrine (10(-6) M) was significantly increased in DOCA-salt rats compared with that in uninephrectomized control rats. Perfusion of the arteries with subpressor dose of endothelin-1 (3 x 10(-10) M) for 15 min markedly enhanced the pressor responses to norepinephrine (10(-6) and 3 x 10(-6) M) in control rats and this effect was significantly prevented by BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1-methoxycarbonyl-tryptophanyl-D-norleucine] (10(-6) M), but not by FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-+ ++methylpentanoyl]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]ami no-3-(2-pyridyl)propionic acid) (10(-6) M). In DOCA-salt hypertensive rats, increased pressor response to norepinephrine was declined to the level of control rats by BQ788, but not by FR139317. In contrast to the case seen with control rat, exogenous endothelin-1 had little effect on pressor responses to norepinephrine in the arteries of DOCA-salt hypertensive rats. Total immunoreactive endothelin content in the arteries of DOCA-salt hypertensive rats was significantly higher than that of uninephrectomized control rats. These results suggest that endothelin-1 enhances contractile responses to norepinephrine through endothelin ETB receptor. Moreover, this phenomenon is stimulated tonically by endogenous endothelin-1 in DOCA-salt hypertensive rats and may contribute to the maintenance of hypertension in DOCA-salt rats.

Endothelin-1 expression in blood vessels of DOCA-salt hypertensive rats treated with the combined ETA/ETB endothelin receptor antagonist bosentan

In previous studies it has been shown that blood vessels of deoxycorticosterone acetate (DOCA) salt hypertensive rats present significantly higher immunoreactive ET-1 (ir-ET-1) content and increased ET-1 gene expression. DOCA-salt hypertensive rats respond to treatment with the combined ETA/ETB endothelin receptor antagonist bosentan with lowering of blood pressure. In the present study, we investigated the ir-ET-1 levels and the expression of the ET-1 gene in blood vessels of DOCA-salt hypertensive rats treated or not treated with bosentan. Blood pressure was significantly lower in bosentan-treated rats (185 +/- 6 mmHg; 1 mmHg = 133.3 Pa) compared with DOCA-salt hypertensive rats (203 +/- 4 mmHg; p < 0.01). Plasma ir-ET-1 concentration was slightly but significantly elevated (p < 0.01) in DOCA-salt hypertensive rats compared with uninephrectomized control rats, and was further increased (p < 0.01) in bosentan-treated rats. The tissue wet weight and ir-ET-1 content of segments of thoracic aorta were significantly increased (p < 0.01) in DOCA-salt hypertensive rats in comparison with control rats, but were similar in bosentan-treated DOCA-salt rats. The abundance of ET-1 mRNA measured by Northern blot analysis in thoracic aorta and the ir-ET-1 content were attenuated by bosentan treatment. Tissue wet weight and ir-ET-1 content in the mesenteric vascular bed were similar in bosentan-treated and -untreated DOCA-salt rats, and were significantly higher in both groups than in control rats (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

In situ hybridization shows increased endothelin-1 mRNA levels in endothelial cells of blood vessels of deoxycorticosterone acetate-salt hypertensive rats

Endothelin-1 is a potent vasoconstrictor peptide produced in blood vessels and other tissues that may play an important role in the control of local blood flow and could be involved in the pathogenesis of hypertension. Our previous studies have documented increases in endothelin-1 peptide content and gene expression in mesenteric arteries and thoracic aorta of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Although changes in endothelin-1 were observed in the blood vessels of hypertensive rats, the exact cellular sites of these changes were not identified clearly. In the present study we investigated endothelin-1 gene expression in DOCA-salt hypertensive rats by in situ hybridization histochemistry using a high specific activity 35S-labeled complementary RNA probe. Robust increases in endothelin-1 mRNA levels were observed in both mesenteric blood vessels and aorta of DOCA-salt hypertensive rats as compared with the vessels from the uninephrectomized control rats. In both cases it was shown clearly that these increased endothelin-1 mRNA levels only originated in the endothelial cell layer, not in the underlying smooth muscle cells. Higher expression levels of endothelin-1 mRNA by the endothelial cells of DOCA-salt hypertensive rats may play an important role in vascular hypertrophy and in the maintenance of elevated blood pressure in this and perhaps other models of experimental hypertension.

Bradykinin receptor and tissue ACE binding in myocardial fibrosis: Response to chronic angiotensin II or aldosterone administration in rats

Y. Sun, A. Ratajska and K. T. Weber. Bradykinin Receptor and Tissue ACE Binding in Myocardial Fibrosis: Response to Chronic Angiotensin II or Aldosterone Administration in Rats. Journal of Molecular and Cellular Cardiology (1995) 27, 813–822. High density angiotensin converting enzyme (ACE) binding is present in the perivascular fibrosis involving intramyocardial coronary arteries and the microscopic scarring of the myocardium that accompanies chronic elevations in circulating angiotensin II (AngII) and/or aldosterone (ALDO). As a kininase II, ACE degrades bradykinin. Herein we sought to determine whether bradykinin (BK) receptor binding was associated with ACE binding in each of these experimental models. BK receptor binding was localized and quantified by in vitro quantitative autoradiography, using [ 125I-Tyr 8]BK. In serial sections of the same heart hematoxylin and eosin (H&E) and picrosirius red (PSR) staining were utilized to address cardiac myocyte injury and fibrosis respectively. Four experimental groups were examined: unoperated, untreated, age/sex matched controls; age/sex matched uninephrectomized control rats receiving a high sodium diet; animals that received AngII (9 μg/h sc) for 2, 4 or 6 weeks; and uninephrectomized rats on a high sodium diet that received ALDO (0.75 μg/h sc) for similar periods of time. We found: (a) myocardial fibrosis, including perivascular fibrosis and microscopic scarring, at week 2 of AngII, but not until week 4 or more of ALDO treatment; (b) low BK receptor binding in normal ventricles that was increased in scars and markedly increased in perivascular fibrosis at week 2 of AngII and each increased further at week 4 and 6 of AngII; (c) low BK receptor binding at week 2 and 4 weeks of ALDO treatment which became markedly increased at fibrous tissue sites at week 6. BK receptor and ACE binding were anatomically coincident and localized to each site of fibrosis in both models. The co-location of BK receptor and ACE binding in these models raises the prospect that fibrous tissue ACE may utilize BK as substrate and BK, in turn, may play a role in fibrous tissue formation.

Fibrosis and myocardial ACE: Possible substrate and independence from circulating angiotensin II

In order to determine the relation between myocardial fibrosis and (1) angiotensin converting enzyme (ACE) binding density, (2) receptor binding of ACE-related peptides, angiotensin II (AngII) and bradykinin (BK), and (3) the regulation of myocardial ACE by circulating Ang II, we used in vitro quantitative autoradiography to localize and assess ACE ([ 125I]351A), AngII receptor ( 125I[Sar 1, IIe 8]AngII), and BK receptor ([ 125I]Tyr 8) binding densities in the rat myocardium. The experimental groups were (1) AngII (9 μg/h subcutaneously) or aldosterone (0.75 μg/h subcutaneously in uninephrectomized rats on a high-sodium diet) administered by implanted minipump to chronically increase or decrease circulating AngII, respectively, (2) unoperated, untreated age- and sex-matched control rats, and (3) age- and sex-matched uninephrectomized control rats on a high-sodium diet. In the same heart studied at 2, 4, and 6 weeks of hormone treatment, serial sections were assessed for myocardial (hematoxylin and eosin) and fibrillar collagen (picrosirius red) morphology. The authors found that (1) marked ACE binding was coincident with sites of fibrous tissue that appeared in both ventricles as either a perivascular fibrosis of intramyocardial coronary arterioles or microscopic scarring in response to AngII or aldosterone infusion, (2) ACE binding was independent of circulating AngII, (3) BK, not AngII, receptor binding density was markedly increased at fibrous tissue sites, and (4) high-density ACE and BK receptor binding were anatomically coincident. Thus, in these experimental models, ACE is related to fibrous tissue formation where it may use BK, not angiotensin I, as a substrate, and its binding density is independent of circulating AngII.

Effect of a nonselective endothelin antagonist on vascular remodeling in deoxycorticosterone acetate-salt hypertensive rats. Evidence for a role of endothelin in vascular hypertrophy.

We have previously shown that the endothelin content in arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats is increased. We designed this study to examine, using the new orally active nonselective endothelin receptor antagonist bosentan, whether this increase in vascular endothelin may contribute to elevated blood pressure and vascular hypertrophy in DOCA-salt hypertensive rats. Rats received bosentan (100 mg/kg body wt per day) for 3 weeks mixed with their food. Systolic blood pressure of DOCA-salt hypertensive rats rose to 197 +/- 5 mm Hg, and that of bosentan-treated DOCA-salt hypertensive rats was 177 +/- 4 mm Hg (P < .01). Mesenteric resistance arteries were studied on a wire myograph. The media width, ratio of media width to lumen diameter, and cross-sectional area of the media of resistance arteries of bosentan-treated DOCA-salt hypertensive rats were significantly smaller than those of untreated DOCA-salt hypertensive rats. The lumen diameter and cross-sectional area of the media of vessels of bosentan-treated rats were not different from those of uninephrectomized control rats. Vasoconstrictor responses, which were altered in DOCA-salt hypertensive rats, approached control in the bosentan-treated rats. We conclude that these results with a nonselective endothelin receptor antagonist may suggest a role for endothelin in the elevation of blood pressure and vascular hypertrophy and remodeling in DOCA-salt hypertensive rats.

Increased expression of endothelin-1 gene in blood vessels of deoxycorticosterone acetate-salt hypertensive rats.

We have recently shown that the content of immunoreactive endothelin-1 is increased in acid extracts from blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats compared with uninephrectomized control rats. We have also found by immunohistochemistry a significant increase in immunoreactive endothelin-1 in endothelial cells of aorta and mesenteric arteries of DOCA-salt hypertensive rats. In the present study, we investigated preproendothelin-1 gene expression in blood vessels of DOCA-salt hypertensive rats and uninephrectomized control rats. Northern blot analysis using a specific 32P-labeled complementary RNA probe for rat preproendothelin-1 of 319 base pairs revealed a fourfold to fivefold increase in abundance of preproendothelin-1 messenger RNA transcripts in both aorta and mesenteric arteries from DOCA-salt hypertensive rats. Thus, increased immunoreactive endothelin-1 content in blood vessels of DOCA-salt hypertensive rats is secondary to increased preproendothelin-1 gene expression. Exaggerated expression of the preproendothelin-1 gene in mineralocorticoid hypertension may contribute to the maintenance of elevated blood pressure.

Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats.

Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide produced in the endothelium of blood vessels that may play an important role in the control of local blood flow and could be involved in the pathogenesis of hypertension. We investigated immunoreactive ET-1 (ir-ET-1) levels in acid extracts from blood vessels of deoxycorticosterone acetate (DOCA)-salt and spontaneously hypertensive rats. We found that segments of thoracic aorta and the mesenteric vascular bed contain significantly more ir-ET-1 (11.84 +/- 0.84 and 17.30 +/- 1.89 fmol, respectively) than uninephrectomized control rats (1.78 +/- 0.20 and 9.19 +/- 0.63 fmol, respectively; p < 0.001). High performance liquid chromatography showed that ir-ET-1 of blood vessels of DOCA-salt hypertensive rats eluted in the same position as synthetic ET-1. Significantly increased ir-ET-1 was localized by immunohistochemistry in endothelial cells of aorta and large and small mesenteric arteries of DOCA-salt hypertensive rats. In contrast to the latter, in spontaneously hypertensive rats, vascular content of ir-ET-1 was similar to that of blood vessels of Wistar-Kyoto control rats, at both 6 and 16 weeks of age. High levels of vascular ET-1 may explain the downregulation of vascular endothelin receptors previously described in DOCA-salt hypertensive rats. Furthermore, this suggests that ET-1 may be involved in the maintenance of high blood pressure in mineralocorticoid hypertension.