This study was designed to demonstrate the characteristic pattern of <em>angiotensin</em> II-induced JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) activation in cultured rat cardiomyocytes by comparing it with leukemia inhibitory factor (LIF)-induced activation. <em>Angiotensin</em> II (<em>1</em>0(-<em>7</em>) mol/L) induced rapid phosphorylation of JAK2 and Tyk2, but not JAK<em>1</em>, and phosphorylated STAT<em>1</em> and STAT2, but not STAT3, in the early stage up to 30 minutes. The time course of JAK/STAT activation by <em>angiotensin</em> II was apparently slower than that by LIF. Interestingly, <em>angiotensin</em> II phosphorylated STAT3 and rephosphorylated STAT<em>1</em> in the late stage at <em>1</em>20 minutes. We also found that <em>angiotensin</em> II induced the formation of interferon-stimulating gene factor (ISGF) complexes biphasically, in the early stage at <em>1</em>5 to 30 minutes and in the late stage at <em>1</em>20 minutes, and that <em>angiotensin</em> II induced delayed activation of the sis-inducing factor (SIF) complex at <em>1</em>20 minutes. Formation of ISGF and SIF complexes in response to <em>angiotensin</em> II paralleled the phosphorylation pattern of STAT<em>1</em> and STAT3 and was quite different from those obtained in response to LIF. The phosphorylation of STAT<em>1</em> was suppressed by pretreatment with the <em>angiotensin</em> II type-<em>1</em> (AT<em>1</em>) receptor antagonist CV<em>1</em><em>1</em>9<em>7</em>4, but the delayed addition of CV<em>1</em><em>1</em>9<em>7</em>4 failed to suppress phosphorylation of STAT3 at <em>1</em>20 minutes. In conclusion, <em>angiotensin</em> II-induced JAK/STAT activation in rat cardiomyocytes is biphasic and entirely different from LIF-induced activation.

BACKGROUND

Results of randomized trials of angiotensin-converting enzyme inhibitors in patients with coronary artery disease (CAD) and preserved left ventricular function are conflicting. We undertook this study to determine whether long-term prescription of angiotensin-converting enzyme inhibitors decreases major cardiovascular events and mortality in patients who have CAD and no evidence of left ventricular systolic dysfunction.

METHODS

We searched MEDLINE, EMBASE, and IPA databases, the Cochrane Controlled Trials Register (1990-2004), and reports from scientific meetings (2003-2004), and we reviewed secondary sources. Search terms included angiotensin-converting enzyme inhibitors, coronary artery disease, randomi(s)zed controlled trials, clinical trials, and myocardial infarction. Eligible studies included randomized controlled trials in patients who had CAD and no heart failure or left ventricular dysfunction, with follow-up omicronf 2 years or longer. Of 1146 publications screened, 7 met our selection criteria and included a total of 33 960 patients followed up for a mean of 4.4 years.

RESULTS

Five trials included only patients with documented CAD. One trial included patients with documented CAD (80%) or patients who had diabetes mellitus and 1 or more additional risk factors, and another trial included patients who had CAD, a history of transient ischemic attack, or intermittent claudication. Treatment with angiotensin-converting enzyme inhibitors decreased overall mortality (odds ratio, 0.86; 95% confidence interval, 0.79-0.93), cardiovascular mortality (odds ratio, 0.81; 95% confidence interval, 0.73-0.90), myocardial infarction (odds ratio, 0.82; 95% confidence interval, 0.75-0.89), and stroke (odds ratio, 0.77; 95% confidence interval, 0.66-0.88). Other end points, including resuscitation after cardiac arrest, myocardial revascularization, and hospitalization because of heart failure, were also reduced.

CONCLUSIONS

Angiotensin-converting enzyme inhibitors reduce total mortality and major cardiovascular end points in patients who have CAD and no left ventricular systolic dysfunction or heart failure.

The possible role of calcineurin in the attenuation of cardiac hypertrophy and fibrosis by blockade of the <em>angiotensin</em> II type <em>1</em> (AT<em>1</em>) receptor was investigated in Dahl salt-sensitive (DS) rats. The effect of the calcineurin inhibitor FK506 was also studied. DS rats progressively developed severe hypertension when fed a diet containing 8% NaCl from <em>7</em> weeks of age. In addition, marked cardiac hypertrophy and fibrosis were apparent and the activity of calcineurin and its mRNA expression in the myocardium was increased in these animals at <em>1</em>2 weeks in comparison with age-matched Dahl salt-resistant rats. The abundance of <em>angiotensin</em>-converting enzyme (ACE) and transforming growth factor (TGF)-beta<em>1</em> mRNAs was also increased in the hearts of DS rats at <em>1</em>2 weeks. Treatment of DS rats with a non-antihypertensive dose of the selective AT<em>1</em> receptor blocker candesartan (<em>1</em> mg/kg per day) or FK506 (0.<em>1</em> mg/kg per day) from <em>7</em> to <em>1</em>2 weeks attenuated both calcineurin activity and its mRNA expression in the heart, as well as the development of cardiac hypertrophy and fibrosis, without affecting cardiac function. Treatment with candesartan, but not FK506, prevented the upregulation of ACE and TGF-beta<em>1</em> gene expression. Both candesartan and FK506 prevented the load-induced induction of fetal-type cardiac genes. These results demonstrate that AT<em>1</em> receptor blockade attenuates the development of cardiac hypertrophy and fibrosis as well as the activation of calcineurin, without an antihypertensive effect, in rats with salt-sensitive hypertension. Calcineurin may be downstream from TGF-beta<em>1</em> in AT<em>1</em> receptor-mediated <em>angiotensin</em> II signaling in vivo.

<em>Angiotensin</em> II (Ang II) promotes vascular smooth muscle growth and may be involved in the initiation and progression of atherosclerosis. To examine whether Ang II receptor expression in vascular tissues is altered in atherosclerosis, male New Zealand White rabbits were fed a high-cholesterol diet (<em>1</em>% cholesterol + 4% coconut oil mixed with regular chow; hypercholesterolemic group, n=<em>1</em>2) or regular chow (control group, n=8) for <em>1</em>0 weeks. At the end of this period, the serum cholesterol level in the rabbits fed the high-cholesterol diet was higher than that in the control group (36<em>1</em>6 +/- <em>1</em>44 versus 30 +/- <em>1</em> mg/dL, P<0.00<em>1</em>). There was no atherosclerosis in the aortas of the control group, whereas 5<em>1</em> +/- 6% of the aorta was covered with atherosclerosis in the hypercholesterolemic group. Total Ang II receptor expression in the atherosclerotic aortic tissues was increased 5-fold in the hypercholesterolemic rabbits (292 +/- 28 versus 5<em>1</em> +/- 32 fmol/mg tissue, mean +/- SE, P<0.00<em>1</em>), and the increased Ang II receptor expression was entirely due to enhanced Ang II type <em>1</em> (AT<em>1</em>) receptor expression (289 +/- 38 versus 38 +/- <em>1</em>8 fmol/mg, P<0.00<em>1</em>), as Ang II type 2 receptor expression was unaltered (<em>7</em> +/- 5 versus 3 +/- 2 fmol/mg, P=NS). AT<em>1</em> receptors were localized primarily in the media and to some extent in the intima of the atherosclerotic aorta, as determined by immunohistochemistry with specific monoclonal and polyclonal AT<em>1</em> receptor antibodies. Increased synthesis of AT<em>1</em> receptor mRNA in atherosclerotic tissues was confirmed by reverse transcription-polymerase chain reaction. To evaluate the functional significance of increased AT<em>1</em> receptor expression, the constrictor response of aortic rings to Ang II was examined and found to be markedly enhanced in atherosclerotic aortic rings (P<0.0<em>1</em> versus control aortic rings). The endothelium-dependent relaxation of aortic rings from hypercholesterolemic rabbits was markedly attenuated (P<0.00<em>1</em>). This study shows that hypercholesterolemia in rabbits results in atherosclerosis, loss of endothelium-dependent relaxation, and increased Ang II receptor (entirely AT<em>1</em> receptor) expression in aortic tissues, which may result in altered vasoreactivity.

OBJECTIVE

To determine whether polymorphisms in the renin-angiotensin system can predict blood pressure-lowering response to antihypertensive treatment; more specifically, in response to treatment with irbesartan or atenolol.

METHODS

Eighty-six patients with hypertension were randomized to double-blind treatment with either the angiotensin II type 1 receptor antagonist irbesartan or the beta1 adrenergic receptor blocker atenolol and followed for 3 months. We analysed angiotensinogen T174M and M235T, angiotensin converting enzyme (ACE) I/D and angiotensin II type 1 receptor A1166C polymorphisms and related them to blood pressure reduction.

RESULTS

The mean reductions in blood pressure were similar for both treatments. In the irbesartan group, individuals homozygous for the ACE gene I allele showed a greater reduction in diastolic blood pressure, exceeding those with the D allele (-18 +/- 11 SD versus -7 +/- 10 mmHg, P = 0.0096). This was not the case during treatment with atenolol, and the interaction term between type of treatment and ACE II genotype was significant (P = 0.0176). The angiotensinogen and angiotensin II type 1 receptor polymorhisms were not related to the response to treatment.

CONCLUSIONS

ACE genotyping predicted the blood pressure-lowering response to antihypertensive treatment with irbesartan but not atenolol. Thus, specific genotypes might predict the response to specific antihypertensive treatment.

BACKGROUND

<em>Angiotensin</em>-(<em>1</em>-<em>1</em>2) [Ang-(<em>1</em>-<em>1</em>2)] functions as an endogenous substrate for the productions of Ang II and Ang-(<em>1</em>-<em>7</em>) by a non-renin dependent mechanism. This study evaluated whether Ang-(<em>1</em>-<em>1</em>2) is incorporated by neonatal cardiac myocytes and the enzymatic pathways of ¹²⁵I-Ang-(<em>1</em>-<em>1</em>2) metabolism in the cardiac myocyte medium from WKY and SHR rats.

RESULTS

The degradation of ¹²⁵I-Ang-(<em>1</em>-<em>1</em>2) (<em>1</em> nmol/L) in the cultured medium of these cardiac myocytes was evaluated in the presence and absence of inhibitors for angiotensin converting enzymes <em>1</em> and 2, neprilysin and chymase. In both strains uptake of ¹²⁵I-Ang-(<em>1</em>-<em>1</em>2) by myocytes occurred in a time-dependent fashion. Uptake of intact Ang-(<em>1</em>-<em>1</em>2) was significantly greater in cardiac myocytes of SHR as compared to WKY. In the absence of renin angiotensin system (RAS) enzymes inhibitors the hydrolysis of labeled Ang-(<em>1</em>-<em>1</em>2) and the subsequent generation of smaller Ang peptides from Ang-(<em>1</em>-<em>1</em>2) was significantly greater in SHR compared to WKY controls. ¹²⁵I-Ang-(<em>1</em>-<em>1</em>2) degradation into smaller Ang peptides fragments was significantly inhibited (90% in WKY and <em>7</em><em>1</em>% in SHR) in the presence of all RAS enzymes inhibitors. Further analysis of peptide fractions generated through the incubation of Ang-(<em>1</em>-<em>1</em>2) in the myocyte medium demonstrated a predominant hydrolytic effect of angiotensin converting enzyme and neprilysin in WKY and an additional role for chymase in SHR.

CONCLUSIONS

These studies demonstrate that neonatal myocytes sequester angiotensin-(<em>1</em>-<em>1</em>2) and revealed the enzymes involved in the conversion of the dodecapeptide substrate to biologically active angiotensin peptides.

Discovery of the unexpected intercellular messenger and transmitter nitric oxide (NO) was the highlight of highly competitive investigations to identify the nature of endothelium-derived relaxing factor. This labile, gaseous molecule plays obligatory roles as one of the most promising physiological regulators in cardiovascular function. Its biological effects include vasodilatation, increased regional blood perfusion, lowering of systemic blood pressure, and antithrombosis and anti-atherosclerosis effects, which counteract the vascular actions of endogenous <em>angiotensin</em> (ANG) II. Interactions of these vasodilator and vasoconstrictor substances in the circulation have been a topic that has drawn the special interest of both cardiovascular researchers and clinicians. Therapeutic agents that inhibit the synthesis and action of ANG II are widely accepted to be essential in treating circulatory and metabolic dysfunctions, including hypertension and diabetes mellitus, and increased availability of NO is one of the most important pharmacological mechanisms underlying their beneficial actions. ANG II provokes vascular actions through various receptor subtypes (AT<em>1</em>, AT2, and AT4), which are differently involved in NO synthesis and actions. ANG II and its derivatives, ANG III, ANG IV, and ANG-(<em>1</em>-<em>7</em>), alter vascular contractility with different mechanisms of action in relation to NO. This review article summarizes information concerning advances in research on interactions between NO and ANG in reference to ANG receptor subtypes, radical oxygen species, particularly superoxide anions, ANG-converting enzyme inhibitors, and ANG receptor blockers in patients with cardiovascular disease, healthy individuals, and experimental animals. Interactions of ANG and endothelium-derived relaxing factor other than NO, such as prostaglandin I2 and endothelium-derived hyperpolarizing factor, are also described.

BACKGROUND

Blockade of type <em>1</em> <em>angiotensin</em> (Ang) II receptors combined with ACE inhibition may amplify the efficacy of the renin-<em>angiotensin</em> system blockade because ACE inhibitors do not completely and permanently suppress Ang II production.

RESULTS

Enalapril or losartan (<em>1</em>, 3, <em>1</em>0, and 30 mg/kg) or their combination was administered for 2 to 4 weeks to spontaneously hypertensive rats. The combination of low doses of each agent induced greater reductions in blood pressure (BP) and left ventricular weight/body weight (LVW/ BW) ratio than monotherapy with the same or higher doses. When approximately equipotent regimens of enalapril, losartan, and their combination, as judged by BP fall, were compared, there were similar increases in plasma and renal renin and in plasma Ang-(<em>1</em>-7) and Ang I and similar reductions in plasma <em>angiotensin</em>ogen. Enalapril alone reduced plasma Ang II levels, and losartan alone increased Ang II levels. The combination of enalapril with losartan prevented or reduced the increase in Ang II levels observed with losartan alone.

CONCLUSIONS

These findings show that the synergistic interaction between the effects of low doses of enalapril and losartan on BP and LVW/BW ratio is due to more effective inhibition of the renin-angiotensin system by their combination than by either agent alone. When both drugs are given together, the ACE inhibitor-induced fall in plasma Ang II results in modulation of the Ang II antagonist-induced reactive rise in Ang II, thereby lowering the plasma Ang II concentration, which competes with the antagonist for the Ang II receptors.

Cells having morphological and histochemical properties of collecting tubules were isolated from rabbit renal papillae. Confluent monolayer cultures of these renal papillary collecting tubule (RPCT) cells formed hemicysts and adhered with morphological asymmetry to Millipore filters. Cultures of <em>1</em>-day-old RPCT cells synthesized cAMP in response to arginine vasopressin (AVP) (half-maximal response to <em>1</em>0(-<em>1</em>0) M), oxytocin, and parathyroid hormone (half-maximal responses at 5 X <em>1</em>0(-9) M) but not to adrenergic agents. After <em>1</em>0 days of growth (fourfold increase in cell number) RPCT cells retained the same pattern of histochemical and hormonal responses as <em>1</em>-day-old cells. Hormones were tested for their influence on the release of immunoreactive prostaglandins (iPG) by RPCT cells; the major product under both basal and stimulated conditions was iPGE2. At very low concentrations (greater than or equal to <em>1</em>0(-<em>1</em>0) M), bradykinin, lysyl-bradykinin, and methionyl-lysyl-bradykinin caused four- to sixfold increases in the rate of iPGE2 formation within 3 min; smaller (less than twofold) increases were observed with relatively high concentrations of epinephrine (<em>1</em>0(-5) M), norepinephrine (<em>1</em>0(-5) M), and <em>angiotensin</em> II (<em>1</em>0(-<em>7</em>) M), but only after longer incubations. Significantly, neither AVP (<em>1</em>0(-<em>7</em>) M) nor [deamino]AVP (<em>1</em>0(-<em>7</em>) M) caused prostaglandin release by RPCT cells. Our results indicate that kinins can act directly on the collecting tubule to elicit PGE2 formation; furthermore, this effect of kinins may be natriuretic, since PGE2 has been shown to inhibit Na+ resorption by the medullary collecting tubule and thick ascending limb.

To evaluate the safety and efficacy of sirolimus in treating patients with focal segmental glomerulosclerosis (FSGS), we performed a phase 2, open-label clinical trial. Inclusion criteria were adults and children <em>1</em>3 years and older with biopsy-proven idiopathic FSGS, proteinuria with protein of 3.5 g/d or greater while on <em>angiotensin</em> antagonist therapy, glomerular filtration rate (GFR) of 30 mL/min/<em>1</em>.<em>7</em>3 m(2) or greater >>or=0.50 mL/s), and failure to achieve sustained remission with at least <em>1</em> immunosuppressive agent. Eligible patients received sirolimus doses adjusted to achieve trough levels of 5 to <em>1</em>5 ng/mL during the first 4 months and <em>1</em>0 to 20 ng/mL for the subsequent 8 months. The primary outcome was decrease in proteinuria, expressed as complete remission (protein < 0.3 g/d) or partial remission (protein>>or= 50% decrease and <3.5 g/d). Six adult patients with FSGS were enrolled in the study; they had median disease duration of 4.0 years, mean age of 39 +/- <em>1</em><em>1</em> years, mean baseline Modification of Diet in Renal Disease-estimated GFR of 52 +/- <em>1</em>5 mL/min/<em>1</em>.<em>7</em>3 m(2) (0.8<em>7</em> +/- 0.25 mL/s), and median baseline proteinuria with protein of 6.6 g/d (interquartile range, 4.2 to 9.4). Five patients had received cyclosporine. No patient experienced a complete or partial remission. Sirolimus therapy was stopped prematurely in 5 patients for the following reasons: (<em>1</em>) precipitous decrease in GFR in 4 patients after <em>7</em> to 9 months of therapy with a greater than 2-fold increase in proteinuria in 3 patients and (2) hypertriglyceridemia with triglyceride levels greater than <em>1</em>,600 mg/dL >><em>1</em>8 mmol/L) at 5 months in <em>1</em> patient. Because of a rapid decrease in GFR with worsening proteinuria, the protocol was closed to further recruitment. We conclude that sirolimus may be associated with nephrotoxicity in some patients with FSGS, particularly those with prolonged disease duration and prior cyclosporine therapy.

The beneficial effects of <em>angiotensin</em>-converting enzyme inhibitors on ameliorating cardiac fibrosis have been partially attributed to their ability to prevent the degradation of kinins. The potential role of bradykinin and the related signaling molecule nitric oxide (NO) in modulating extracellular matrix (ECM) production was examined in primary cultures of adult rat cardiac fibroblasts. Treatment of fibroblasts with 5 nM bradykinin for 24 h led to a reduction in steady-state mRNA levels for fibronectin (34 +/- <em>7</em>%) and collagens type I (<em>1</em>9 +/- 8%) and type III (48 +/- 4%), as determined by Northern blot analysis. The NO synthase inhibitor L-NAME attenuated the reduction observed in fibronectin and collagen mRNA levels in response to bradykinin. The NO donor DETA NONOate (<em>1</em>00 microM) mimicked the effects of bradykinin on ECM mRNA levels. Protein levels of soluble fibronectin, assessed in conditioned medium by ELISA, were decreased by <em>1</em>4 +/- 4% and 2<em>1</em> +/- 4% after 48 h treatment with <em>1</em> microM bradykinin and <em>1</em>00 microM DETA NONOate, respectively. Bradykinin stimulated intracellular cGMP accumulation <em>7</em>3.<em>7</em> +/- <em>1</em>0.3% after <em>1</em>0 min of treatment. Cell proliferation rates at 48 h were unaffected by bradykinin, but were reduced by 26 +/- <em>1</em>2% by <em>1</em>00 microM DETA NONOate. These data indicate that bradykinin downregulates ECM protein production in cardiac fibroblasts and suggest that NO and the related signaling molecule cGMP may play an important role in mediating this response.

The enormous benefits of inhibition of ACE demonstrate that manipulation of the metabolism of peptide hormones is a valuable therapeutic strategy for cardiovascular disease. Recent attempts to expand these benefits have combined ACE inhibition with inhibition of other peptidases such as neutral endopeptidase (NEP) in a single molecule, a strategy known as vasopeptidase inhibition. NEP metabolizes natriuretic peptides, and NEP inhibition offers the prospect of combining the benefits of increased natriuretic peptide levels with those of ACE inhibition. However, peptidases such as ACE and NEP have many different substrates, and there are complex interactions between ACE inhibition and NEP inhibition. Both ACE and NEP metabolize the kinin peptides bradykinin and kallidin, and NEP also converts <em>angiotensin</em> (Ang) I to Ang-(<em>1</em>-<em>7</em>) and metabolizes Ang II and endothelin. Addition of NEP inhibition to ACE inhibition potentiates the ACE inhibitor-induced increase in kinin levels, increases Ang II levels, reduces Ang-(<em>1</em>-<em>7</em>) levels, and may increase endothelin levels. These additional consequences of combined ACE/NEP inhibition increase the risk of angioedema and may counteract any benefit of ACE inhibition that depends on reduced Ang II levels and increased Ang-(<em>1</em>-<em>7</em>) levels. Further considerations are that the ratio of ACE and NEP inhibition is fixed for vasopeptidase inhibitors, and there is uncertainty how these drugs should be compared with ACE inhibitors. Vasopeptidase inhibitors will therefore require careful evaluation before they are introduced to patient care.

Endogenous <em>angiotensin</em> (ANG) II and ANG-(<em>1</em>-<em>7</em>) act at the nucleus tractus solitarius (NTS) to differentially modulate neural control of the circulation. The role of these peptides endogenous to NTS on cardiovascular reflex function was investigated in transgenic rats with low brain <em>angiotensin</em>ogen (Aogen) due to glial overexpression of an antisense to Aogen (ASrAOGEN) and in Sprague-Dawley (SD) rats. Arterial baroreceptor reflex sensitivity (BRS) for control of heart rate (HR) in response to increases in mean arterial pressure (MAP) was tested before and after bilateral microinjection of the <em>angiotensin</em> type <em>1</em> (AT(<em>1</em>)) receptor blocker candesartan or the ANG-(<em>1</em>-<em>7</em>) receptor blocker (d-Ala(<em>7</em>))-ANG-(<em>1</em>-<em>7</em>) into the NTS of urethane-chloralose-anesthetized ASrAOGEN and SD rats. Baseline MAP was higher in ASrAOGEN than in SD rats under anesthesia (P < 0.0<em>1</em>). Injection of candesartan or (d-Ala(<em>7</em>))-ANG-(<em>1</em>-<em>7</em>) decreased MAP (P < 0.0<em>1</em>) and HR (P < 0.05) in ASrAOGEN, but not SD, rats. The BRS at baseline was similar in ASrAOGEN and SD rats. Candesartan increased BRS by 4<em>1</em>% in SD rats (P < 0.0<em>1</em>) but was without effect in ASrAOGEN rats. In contrast, the reduction in BRS after (d-Ala(<em>7</em>))-ANG-(<em>1</em>-<em>7</em>) administration was comparable in SD (3<em>1</em>%) and ASrAOGEN rats (34%). These findings indicate that the absence of glia-derived Aogen is associated with <em>1</em>) an increase in MAP under anesthesia mediated via AT(<em>1</em>) and ANG-(<em>1</em>-<em>7</em>) receptors within the NTS, 2) the absence of an endogenous ANG II contribution to tonic inhibition of BRS, and 3) a continued contribution of endogenous ANG-(<em>1</em>-<em>7</em>) to tonic enhancement of BRS.

BACKGROUND

We have previously shown that cyclooxygenase-2 (COX-2) expression is low in the renal cortex of adult rats, but is increased in macula densa/cortical thick ascending limb and in glomerular podocytes after subtotal renal ablation.

METHODS

To evaluate the functional consequences of this increased COX-2 expression, male rats were subjected to subtotal renal ablation and divided into four groups: (<em>1</em>) treatment with the selective COX-2 inhibitor SC58236, (2) treatment with vehicle, (3) treatment with the <em>angiotensin</em>-converting enzyme inhibitor enalapril, and (4) treatment with enalapril + SC58236. The administration of drugs was begun on the third day after ablation and continued for 6 to <em>1</em>0 weeks.

RESULTS

Within one week after ablation, vehicle-treated rats developed hypertension. Although enalapril led to significant reductions in blood pressure, either alone or in combination with the COX-2 inhibitor, SC58236 alone did not significantly alter ablation-induced hypertension. However, the SC58236-treated animals exhibited levels of proteinuria at six weeks after ablation that were comparable to those seen with enalapril (vehicle, 47 +/- 4; enalapril, 27 +/- 2; SC58236, 30 +/- 2 mg/day; N = 7, P < 0.0<em>1</em>, each group compared with vehicle), and continued SC58236 treatment led to persistent reductions in proteinuria at <em>1</em>0 weeks after renal ablation (vehicle, 77 +/- 4; SC58236, 50 +/- 4 mg/day; N = 6, P < 0. 0<em>1</em>). SC58236 treatment also significantly reduced the percentage of glomeruli exhibiting segmental or global sclerosis at <em>1</em>0 weeks (32.6 +/- 7.8% vs. <em>1</em>0.9 +/- 2.8%, N = 6, P < 0.03). Furthermore, SC58236 treatment partially inhibited increases in transforming growth factor-beta<em>1</em> mRNA expression and increases in collagen III and collagen IV mRNA expression.

CONCLUSIONS

These studies indicate that chronic treatment with a specific COX-2 inhibitor may retard the progression of progressive renal injury, and suggest that such compounds can be used in combination with angiotensin-converting enzyme inhibitors. Further studies are required to determine the mechanism by which COX-2 inhibition is renoprotective.

Endothelium-dependent vasodilation is impaired in patients with essential hypertension. The objective of this study was to determine whether long-term treatment with <em>angiotensin</em>-converting enzyme inhibitors improves endothelium-dependent vasodilation in forearm resistance vessels of patients with hypertension. Furthermore, since tissue thiols may be relevant to nitric oxide-mediated vasodilation, we queried whether an <em>angiotensin</em>-converting enzyme inhibitor with a sulfhydryl group preferentially augments endothelium-dependent vasodilation in these individuals. The study included 24 patients with essential hypertension (mean age, 45 +/- 2 years) and 20 normotensive subjects (mean age, 4<em>7</em> +/- <em>1</em> years). Methacholine chloride (0.3 to <em>1</em>0 micrograms/min) was infused via the brachial artery to assess endothelium-dependent vasodilation in forearm resistance vessels. Nitroglycerin (<em>1</em> to 30 micrograms/min) was administered to evaluate endothelium-independent vasodilation. Forearm blood flow was determined by venous occlusion strain-gauge plethysmography. Forearm vascular function studies were performed in hypertensive patients before and <em>7</em> to 8 weeks after randomization to either captopril or enalapril, <em>angiotensin</em>-converting enzyme inhibitors with and without a sulfhydryl moiety, respectively. Normotensive subjects were studied on only one occasion. Before treatment, the forearm vasodilative response to methacholine was attenuated in hypertensive compared with normotensive subjects (P < .0<em>1</em>). The effects of nitroglycerin on forearm blood flow did not differ significantly between the two groups. Both captopril and enalapril reduced mean blood pressure in the hypertensive subjects (<em>1</em>2 +/- 2 versus <em>1</em>5 +/- 3 mm Hg, respectively; P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

We tested the effect of <em>angiotensin</em> I converting enzyme inhibitor (ACEI) on established glomerular sclerosis. Starting eight weeks after subtotal nephrectomy (sNPX), rats were given enalapril for four weeks in a dose of 50 (Group II, N = 5) or 200 mg/liter drinking water (Group III, N = 5). A third group of sNPX rats not given ACEI served as control (Group I, N = <em>1</em>0). Glomerular sclerosis index (S<em>1</em>, 0 to 4 scale) was assessed three-dimensionally on serial thin sections for individual glomeruli at biopsy (Bx, 8 weeks), and divided into four different ranks of severity and compared to autopsy (Ax, <em>1</em>2 weeks). In Group I control rats, 48% of the glomeruli at Bx had SI between 0 and <em>1</em> (rank <em>1</em>, average: 0.49 +/- 0.06), 36% between <em>1</em> and 2 (rank 2, average: <em>1</em>.53 +/- 0.06), 9% between 2 and 3 (rank 3, average: 2.45 +/- 0.<em>1</em>2) and <em>7</em>% between 3 and 4 (rank 4, average: 3.54 +/- 0.<em>1</em>0). Glomeruli of the same rats at Ax were ranked according to severity of sclerosis, and then divided into percentile groups, corresponding to the percent of distribution at Bx. The 48% least sclerotic glomeruli at Ax had average SI of 0.69 +/- 0.08, the next 36% 2.58 +/- 0.<em>1</em><em>1</em>, and next 9% 3.9<em>7</em> +/- 0.02 and the most sclerotic <em>7</em>% 4.00 +/- 0.00. Thus, sclerosis advanced during the last four weeks after biopsy in all glomeruli, with more accelerated progression occurring toward later stages of sclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent evidence suggests that mineralocorticoid receptor (MR) antagonism has beneficial effects on tissue oxidative stress and insulin metabolic signaling as well as reducing proteinuria. However, the mechanisms by which MR antagonism corrects both renin-<em>angiotensin</em>-aldosterone system (RAAS) impairments in renal insulin metabolic signaling and filtration barrier/podocyte injury remain unknown. To explore this potential beneficial interactive effect of MR antagonism we used young transgenic (mRen2)2<em>7</em> (Ren2) rats with increased tissue RAAS activity and elevated serum aldosterone levels. Ren2 and age-matched Sprague-Dawley (SD) control rats (age 6-<em>7</em> wk) were implanted with a low dose of the MR antagonist spironolactone (0.24 mg/day) or vehicle, both delivered over 2<em>1</em> days. Albuminuria, podocyte-specific proteins (synaptopodin, nephrin, and podocin), and ultrastructural analysis of the glomerular filtration barrier were measured in relation to RAAS activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, reactive oxygen species (ROS), and the redox-sensitive Rho kinase (ROK). Insulin metabolic signaling was determined via measurement of insulin receptor substrate-<em>1</em> (IRS-<em>1</em>) phosphorylation, IRS-<em>1</em> ubiquitin/proteasomal degradation, and phosphorylation of Akt. Ren2 rats exhibited albuminuria, loss of podocyte-specific proteins, and podocyte foot process effacement contemporaneous with reduced renal IRS-<em>1</em> and protein kinase B/Akt phosphorylation compared with SD control rats (each P < 0.05). Ren2 kidneys also manifested increased NADPH oxidase/ROS/ROK in conjunction with enhanced renal tissue levels of <em>angiotensin</em> II (ANG II), ANG-(<em>1</em>-<em>1</em>2), and <em>angiotensin</em> type <em>1</em> receptor. Low-dose spironolactone treatment reduced albuminuria and tissue RAAS activity and improved podocyte structural and protein integrity with improvements in IRS-<em>1</em>/Akt phosphorylation. Thus, in this model of RAAS activation, MR antagonism attenuates glomerular/podocyte remodeling and albuminuria, in part through reductions in redox-mediated impairment of insulin metabolic signaling.

The peptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is known to enhance water transport in rat inner medullary collecting duct (IMCD). The aim of this study was to determine the mechanism of the Ang-(<em>1</em>-<em>7</em>) effect on osmotic water permeability (Pf). Pf was measured in the normal rat IMCD perfused in vitro in presence of agonists [Ang-(<em>1</em>-<em>7</em>), arginine vasopressin (AVP) and Ang-(3-8)], and antagonists of the <em>angiotensin</em> and the vasopressin cascade. Ang-(<em>1</em>-<em>7</em>), but not Ang-(3-8), increased Pf significantly. The effect of Ang-(<em>1</em>-<em>7</em>) on Pf was abolished by its selective antagonist, A-<em>7</em><em>7</em>9, added before or after Ang-(<em>1</em>-<em>7</em>). Prostaglandin E2 and the protein kinase A inhibitor H8 also blocked the Ang-(<em>1</em>-<em>7</em>) effect. Blockade of vasopressin V<em>1</em> receptors by antagonists did not change the Ang-(<em>1</em>-<em>7</em>) effect, but pre-treatment with a V2 antagonist abolished the effect of Ang-(<em>1</em>-<em>7</em>) on Pf. Similarly, pre-treatment with A-<em>7</em><em>7</em>9 inhibited AVP's effect on Pf. Forskolin-stimulated Pf was blocked both by A-<em>7</em><em>7</em>9 and by the V2 antagonist. Finally, Ang-(<em>1</em>-<em>7</em>) increased cAMP levels in fresh IMCD cell suspensions whilst the forskolin-stimulated cAMP synthesis was decreased by A-<em>7</em><em>7</em>9 and the V2 antagonist. These data provide evidence that Ang-(<em>1</em>-<em>7</em>) interacts via its receptor with the AVP V2 system through a mechanism involving adenylate-cyclase activation.

Evidence that <em>angiotensin</em>(<em>1</em>-<em>7</em>) (Ang(<em>1</em>-<em>7</em>)) is biologically active and can be synthesized by the kidney prompted us to examine its actions in the rat, isolated kidney. Ang(<em>1</em>-<em>7</em>) had three major effects producing, (<em>1</em>) a substantial natriuresis and diuresis, (2) an increase in urinary sodium concentration associated with a fall in potassium concentration and (3) an increase in glomerular filtration rate without affecting renal vascular resistance. Thus, Ang(<em>1</em>-<em>7</em>) may participate in the renal effects of the renin-<em>angiotensin</em> system.

BACKGROUND

The purpose of the present study was to assess the inhibitory effect of DuP 753, an orally active angiotensin II receptor antagonist, on the pressor action of exogenous angiotensin I and II in healthy male volunteers.

RESULTS

In the first study (single-dose study), eight volunteers were included in a 2-day protocol repeated four times at 1-week intervals. In each phase, a different dose of drug (2.5, 5, 10, 20, or 40 mg) or placebo was given. The peak systolic blood pressure response to a test-dose of angiotensin I was determined serially before and after oral administration of DuP 753 by continuously monitoring finger blood pressure using a photoplethysmographic method. DuP 753 reduced the systolic blood pressure response to angiotensin I in a dose-dependent fashion. Three, 6, and 13 hours after the 40-mg dose, blood pressure response decreased to 31 +/- 5%, 37 +/- 6%, and 45 +/- 3% of the control values (mean +/- SEM, n = 7), respectively. In the second study, 29 volunteers were treated for 8 days with either a placebo or DuP 753 (5, 10, 20, or 40 mg p.o. q.d.) and challenged on the first, fourth, and eighth days with bolus injections of angiotensin II. Again, the inhibitory effect on the systolic blood pressure response to angiotensin II was clearly dose dependent. Six hours after 40 mg DuP 753, the systolic blood pressure response to the test-dose of angiotensin II was reduced to 37 +/- 7%, 40 +/- 4%, and 38 +/- 6% of baseline values (mean +/- SEM, n = 6) on days 1, 4, and 8, respectively. With this latter dose, there was still a blocking effect detectable 24 hours after the drug. Similar to angiotensin converting enzyme and renin inhibitors, DuP 753 induced a dose-dependent increase in plasma renin that was more pronounced on the eighth than on the first day of drug administration. In these normal volunteers, no consistent clinically significant side effects were observed. There was no evidence for an agonist effect.

CONCLUSIONS

DuP 753 appears to be a well-tolerated, orally active, potent, and long-lasting antagonist of angiotensin II in men.

Intraglomerular renin-<em>angiotensin</em> system enzyme activities have been examined previously using glomerular lysates and immune-based assays. However, preparation of glomerular extracts compromises the integrity of their anatomic architecture. In addition, antibody-based assays focus on <em>angiotensin</em> (Ang) II detection, ignoring the generation of other Ang I-derived metabolites, some of which may cross-react with Ang II. Therefore, our aim was to examine the metabolism of Ang I in freshly isolated intact glomeruli using matrix-assisted laser desorption ionization time of flight mass spectrometry as an analytic method. Glomeruli from male Sprague-Dawley rats were isolated by sieving and incubated in Krebs buffer in the presence of <em>1</em> micromol/L of Ang I for <em>1</em>5 to 90 minutes, with or without various peptidase inhibitors. Peptide sequences were confirmed by matrix-assisted laser desorption ionization time of flight tandem mass spectrometry or linear-trap-quadrupole mass spectrometry. Peaks were quantified using customized valine-(<em>1</em>3)C(.<em>1</em>5)N-labeled peptides as standards. The most prominent peaks resulting from Ang I cleavage were 899 and <em>1</em><em>1</em>8<em>1</em> m/z, corresponding with Ang (<em>1</em>-<em>7</em>) and Ang (2-<em>1</em>0), respectively. Smaller peaks for Ang II, Ang (<em>1</em>-9), and Ang (3-<em>1</em>0) also were detected. The disappearance of Ang I was significantly reduced during inhibition of aminopeptidase A or neprilysin. In contrast, captopril did not alter Ang I degradation. Furthermore, during simultaneous inhibition of aminopeptidase A and neprilysin, the disappearance of Ang I was markedly attenuated compared with all of the other conditions. These results suggest that there is prominent intraglomerular conversion of Ang I to Ang (2-<em>1</em>0) and Ang (<em>1</em>-<em>7</em>), mediated by aminopeptidase A and neprilysin, respectively. Formation of these alternative Ang peptides may be critical to counterbalance the local actions of Ang II. Enhancement of these enzymatic activities may constitute potential therapeutic targets for Ang II-mediated glomerular diseases.

OBJECTIVE

Epidemiological data indicate a high incidence of cerebral aneurysms in postmenopausal women. To elucidate the pathogenesis of cerebral aneurysms, we focused on the contribution of endothelial damage in rats.

METHODS

We induced estradiol deficiency by oophorectomy (OVX), hypertension, or both, and hemodynamic stress in <em>7</em>-week-old female Sprague-Dawley rats. They were then given hormone-replacement therapy with <em>1</em><em>7</em>beta-estradiol or an <em>angiotensin</em> II type <em>1</em> receptor blocker (ARB). The effects of estradiol, <em>angiotensin</em> II type <em>1</em> receptor blocker, or both on cultured endothelial cells were also examined.

RESULTS

The number of anomalously shaped endothelial cells was higher in OVX than hypertensive rats (P < 0.05). Rats subjected to hypertension and OVX exhibited a marked increase in the incidence of saccular cerebral aneurysms. Estradiol or <em>angiotensin</em> II type <em>1</em> receptor blocker treatment reduced this incidence (P < 0.05). The endothelial nitric oxide synthase (eNOS) mRNA level in the intracranial artery of OVX and hypertensive and OVX rats was low (P < 0.05). Immunohistochemically, the expression of eNOS and estrogen receptor alpha (ERalpha) in the vascular wall of hypertensive and OVX rats was decreased; <em>angiotensin</em> II and the nicotinamide adenine dinucleotide phosphate oxidase subunits nicotinamide adenine dinucleotide phosphate oxidase 4 and p22phox were strongly expressed in cerebral aneurysms. In the absence of estradiol, eNOS was downregulated and nicotinamide adenine dinucleotide phosphate oxidase expression was increased in endothelial cells; <em>angiotensin</em> II augmented these phenomena. The regulation of eNOS was mediated by ERalpha. These results suggest that estrogen deficiency induces endothelial dysfunction and reactive oxygen species generation, triggering endothelial damage that leads to cerebral aneurysms and that hypertension is an additional risk factor.

CONCLUSIONS

A therapy targeted at the endothelium and management of hypertension may help to prevent cerebral aneurysms.

The localization of two type <em>1</em> <em>angiotensin</em> II receptor subtype mRNA, AT<em>1</em>A and AT<em>1</em>B, was determined by reverse transcription-PCR on microdissected glomeruli and nephron segments. The coupling sensitivity of these two receptor subtypes was evaluated by measuring variations in intracellular calcium ([Ca2+]i) elicited by <em>angiotensin</em> II (Ang II) in structures expressing either AT<em>1</em>A or AT<em>1</em>B mRNA, using Fura-2 fluorescence. The highest expression of AT<em>1</em> mRNA was found in glomerulus, proximal tubule, and thick ascending limb. In glomerulus, AT<em>1</em>A and AT<em>1</em>B mRNA were similarly expressed, whereas in all nephron segments AT<em>1</em>A mRNA expression was dominant (approximately 84%). The increase in [Ca2+]i elicited by <em>1</em>0(-<em>7</em>) mol/L Ang II was highest in proximal segments (delta [Ca2+]i is approximately equivalent to 300 to 400 nmol/L) and thick ascending limb (delta [Ca2+]i is approximately equivalent to 200 nmol/L). In glomerulus and collecting duct, the response was lower (delta < <em>1</em>00 nmol/L). The median effective concentrations for Ang II were of the same order of magnitude in glomerulus (<em>1</em>2.2 nmol/L), in which both AT<em>1</em>A and AT<em>1</em>B are expressed, and in cortical thick ascending limb (<em>1</em>0.3 nmol/ L), in which AT<em>1</em>A is almost exclusively expressed. The Ang II-induced calcium responses were totally abolished by the AT<em>1</em> receptor antagonist losartan (<em>1</em> mumol/L) but not by the AT2 antagonist PD <em>1</em>233<em>1</em>9 (<em>1</em> mumol/L). In the absence of external Ca2+, the peak phase of the response induced by <em>1</em>0(-<em>7</em>) mol/L Ang II was reduced and shortened, suggesting that a part of the [Ca2+]i increase originated from the mobilization of the intracellular Ca2+ pool. In conclusion, these results demonstrate that in the rat kidney: (<em>1</em>) AT<em>1</em>A is the predominant AT<em>1</em> receptor subtype expressed in the nephron segments, (2) glomerulus is the only structure with a relatively high AT<em>1</em>B mRNA content, and (3) AT<em>1</em>A and AT<em>1</em>B receptor subtypes do not differ in their efficiency for the activation of calcium second-messenger system.

<em>Angiotensin</em> II receptors are the specific receptors of <em>angiotensin</em> II of the renin-<em>angiotensin</em> system. The existence and role of the receptors in the skin have not been determined. We immunohistochemically studied the expression of <em>angiotensin</em> receptors in the human skin. The results demonstrated the expression of <em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>) in the normal human suprabasal epidermis. The expression pattern suggests the role of AT<em>1</em> in association with differentiation. In addition, we studied the expression of AT<em>1</em> in squamous cell carcinoma (SCC) of the skin, SCC of the lip, and keratoacanthoma (KA). Our experiments showed that high, intermediate, and low levels of AT<em>1</em> were observed in 3<em>7</em> (<em>7</em>4.0%), <em>7</em> (<em>1</em>4.0%), and 2 (4.0%) of 50 cases of SCC of the skin, respectively, and the negative periphery pattern was observed in <em>1</em><em>7</em> (<em>7</em><em>7</em>.3%) of 22 cases of KA. These observations suggest that the immunohistochemical study of AT<em>1</em> is useful to distinguish SCC from KA. Studying the role and distribution of AT<em>1</em> may help in understanding the pathophysiology of the skin.