ACE2, initially cloned from a human heart, is a recently described homologue of <em>angiotensin</em>-converting enzyme (ACE) but contains only a single enzymatic site that catalyzes the cleavage of <em>angiotensin</em> I to <em>angiotensin</em> <em>1</em>-9 [Ang(<em>1</em>-9)] and is not inhibited by classic ACE inhibitors. It also converts <em>angiotensin</em> II to Ang(<em>1</em>-<em>7</em>). Although the role of ACE2 in the regulation of the renin-<em>angiotensin</em> system is not known, the renin-<em>angiotensin</em> system has been implicated in the pathogenesis of diabetic complications and in particular in diabetic nephropathy. Therefore, the aim of this study was to assess the possible involvement of this new enzyme in the kidney from diabetic Sprague-Dawley rats to compare and contrast it to ACE. ACE2 and ACE gene and protein expression were measured in the kidney after 24 weeks of streptozocin diabetes. ACE2 and ACE mRNA levels were decreased in diabetic renal tubules by approximately 50% and were not influenced by ACE inhibitor treatment with ramipril. By immunostaining, both ACE2 and ACE protein were localized predominantly to renal tubules. In the diabetic kidney, there was reduced ACE2 protein expression that was prevented by ACE inhibitor therapy. The identification of ACE2 in the kidney, its modulation in diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of various <em>angiotensin</em> peptides provides a rationale to further explore the role of this enzyme in various pathophysiological states including diabetic complications.

<em>Angiotensin</em>-converting enzyme is involved in apoptosis of alveolar epithelial cells (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 2<em>7</em><em>7</em>: L<em>1</em>245-L<em>1</em>250, <em>1</em>999). This study tested the ability of the <em>angiotensin</em>-converting enzyme inhibitor captopril or the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) to block alveolar epithelial cell apoptosis and lung fibrosis in vivo in response to bleomycin (Bleo). Male Wistar rats received 8 U/kg of Bleo (bleomycin sulfate) or vehicle intratracheally. Subgroups of Bleo-treated rats received captopril, ZVAD-fmk, or vehicle alone. Lung collagen was assessed by picrosirius red or hydroxyproline assay at <em>1</em>, <em>7</em>, and <em>1</em>4 days post-Bleo, and apoptosis was detected by in situ end labeling (ISEL). Bleo increased alveolar septal and peribronchial collagen by <em>1</em>00 and <em>1</em>33%, respectively (both P < 0.0<em>1</em>), by day <em>1</em>4 but not earlier. In contrast, ISEL was increased in alveolar and airway cells at all time points. Captopril or ZVAD-fmk inhibited collagen accumulation by 9<em>1</em> and 85%, respectively (P < 0. 0<em>1</em>). Both agents also inhibited ISEL in alveoli by 99 and 8<em>1</em>% and in airways by 6<em>7</em> and 63%, respectively. These data suggest that the efficacy of captopril to inhibit experimental lung fibrogenesis is related to inhibition of apoptosis. They also demonstrate the antifibrotic potential of a caspase inhibitor.

The family of apelin peptides is derived from a single gene and activates the <em>7</em>-transmembrane G-protein-coupled receptor (GPCR) APJ. Apelins have been shown to be involved in the regulation of cardiovascular function and fluid homeostasis and interestingly represent substrates for ACE2, a carboxypeptidase recently described as a novel key enzyme in the renin-<em>angiotensin</em>-aldosterone system (RAS). APJ has further been reported to be a coreceptor for the infection of CD4-positive cells with HIV in the central nervous system (CNS). Apelin-36 and shorter C-terminal sequences have different potencies and efficacies in regulating these functions. Shorter sequences, especially (Pyr(<em>1</em>))apelin-<em>1</em>3, are potent regulators of cardiovascular function, while longer peptides such as apelin-36 are more effective in inhibiting human immunodeficiency virus (HIV) infection by blocking the HIV coreceptor APJ. The pyroglutamate modification characteristic of the short apelin peptide (Pyr(<em>1</em>))apelin-<em>1</em>3 indicates paramount biological importance of this peptide. The aim of this review is to compile conclusive evidence for the involvement of apelin/APJ in the regulation of cardiovascular function and HIV pathology, emphasizing the properties of this receptor system that may make it a successful future drug target.

The endothelium plays a central role in the maintenance of vascular homeostasis. One of the main effectors of endothelial dysfunction is ANG II, and pharmacological approaches to limit ANG II bioactivity remain the cornerstone of cardiovascular therapeutics. <em>Angiotensin</em> converting enzyme-2 (ACE2) has been identified as a critical negative modulator of ANG II bioactivity, counterbalancing the effects of ACE in determining net tissue ANG II levels; however, the role of ACE2 in the vasculature remains unknown. In the present study, we hypothesized that ACE2 is a novel target to limit endothelial dysfunction and atherosclerosis. To this aim, we performed in vitro gain and loss of function experiments in endothelial cells and evaluated in vivo angiogenesis and atherosclerosis in apolipoprotein E-knockout mice treated with AdACE2. ACE2-deficient mice exhibited impaired endothelium-dependent relaxation. Overexpression of ACE2 in human endothelial cells stimulated endothelial cell migration and tube formation, and limited monocyte and cellular adhesion molecule expression; effects that were reversed in ACE2 gene silenced and endothelial cells isolated from ACE2-deficient animals. ACE2 attenuated ANG II-induced reactive oxygen species production in part through decreasing the expression of p22phox. The effects of ACE2 on endothelial activation were attenuated by pharmacological blockade of ANG-(<em>1</em>-<em>7</em>) with A<em>7</em><em>7</em>9. ACE2 promoted capillary formation and neovessel maturation in vivo and reduced atherosclerosis in apolipoprotein E-knockout mice These data indicate that ACE2, in an ANG-(<em>1</em>-<em>7</em>)-dependent fashion, functions to improve endothelial homeostasis via a mechanism that may involve attenuation of NADPHox-induced reactive oxygen species production. ACE2-based treatment approaches may be a novel approach to limit aberrant vascular responses and atherothrombosis.

OBJECTIVE

To report the response to discontinuation of olmesartan, an angiotensin II receptor antagonist commonly prescribed for treatment of hypertension, in patients with unexplained severe spruelike enteropathy.

METHODS

All 22 patients included in this report were seen at Mayo Clinic in Rochester, Minnesota, between August 1, 2008, and August 1, 2011, for evaluation of unexplained chronic diarrhea and enteropathy while taking olmesartan. Celiac disease was ruled out in all cases. To be included in the study, the patients also had to have clinical improvement after suspension of olmesartan.

RESULTS

The 22 patients (13 women) had a median age of 69.5 years (range, 47-81 years). Most patients were taking 40 mg/d of olmesartan (range, 10-40 mg/d). The clinical presentation was of chronic diarrhea and weight loss (median, 18 kg; range, 2.5-57 kg), which required hospitalization in 14 patients (64%). Intestinal biopsies showed both villous atrophy and variable degrees of mucosal inflammation in 15 patients, and marked subepithelial collagen deposition (collagenous sprue) in 7. Tissue transglutaminase antibodies were not detected. A gluten-free diet was not helpful. Collagenous or lymphocytic gastritis was documented in 7 patients, and microscopic colitis was documented in 5 patients. Clinical response, with a mean weight gain of 12.2 kg, was demonstrated in all cases. Histologic recovery or improvement of the duodenum after discontinuation of olmesartan was confirmed in all 18 patients who underwent follow-up biopsies.

CONCLUSIONS

Olmesartan may be associated with a severe form of spruelike enteropathy. Clinical response and histologic recovery are expected after suspension of the drug.

In situ hybridization studies have suggested that the subtype 2 <em>angiotensin</em> (AT2) receptor gene is expressed in fetal and newborn rat kidney but is undetectable in the adult animals. In the present study, we investigated the expression of AT2 receptor protein in the fetal (days <em>1</em>4 and <em>1</em>9 of fetal life), newborn (day <em>1</em> postpartum), and adult (4-week-old and 3-month-old) rat kidney. Polyclonal anti-peptide antiserum was raised against the amino terminus of the native AT2 receptor. The selectivity of the antiserum was validated by recognition of the AT2 receptor in a stably transfected COS-<em>7</em> cell line by Western blot and immunocytochemical analysis. As a positive control, the AT2 receptor signal was detected strongly in the adrenal gland. Positive immunohistochemical staining was observed in the mesenchymal cells and ureteric buds of the <em>1</em>4-day fetal kidney and in the glomeruli, tubules, and vessels in the <em>1</em>9-day fetal and newborn kidney. Glomeruli expressing the AT2 receptor were localized mainly in the outer layer of the renal cortex. In the young (4-week-old) and mature (3-month-old) adult rat on normal sodium intake, renal AT2 receptor immunoreactivity was present in glomeruli but substantially diminished compared with that of newborn rats. In both young and mature adult rats, dietary sodium depletion increased the renal AT2 receptor signal, mainly in the glomeruli and interstitial cells. Preimmune and preadsorption controls were negative. Western blot analysis detected a single 44-kD band in the fetal and newborn rat kidney and in the young and mature adult rat kidney. Dietary sodium depletion increased the density of the AT2 receptor band in mature adult rat kidneys. These data provide evidence that the AT2 receptor protein is expressed in the fetal and newborn rat kidney, diminishes in adult life, and is reexpressed in the adult in response to sodium depletion.

<em>Angiotensin</em> converting enzyme (ACE) 2 is a key negative regulator of the renin-<em>angiotensin</em> system where it metabolizes <em>angiotensin</em> (Ang) II into Ang <em>1</em>-<em>7</em>. We hypothesize that Ang II suppresses ACE2 by increasing TNF-α converting enzyme (TACE) activity and ACE2 cleavage. Ang II infusion (<em>1</em>.5 mg/kg/day) in wild-type mice for 2 weeks resulted in substantial decrease in myocardial ACE2 protein levels and activity with corresponding increase in plasma ACE2 activity, prevented by AT<em>1</em>R blockade. Ang II resulted in AT<em>1</em>R-mediated increase in myocardial TACE expression and activity, and membrane translocation of TACE. Ang II treatment in Huh<em>7</em> cells exhibited AT<em>1</em>R-dependent metalloproteinase mediated shedding of ACE2 while transfection with siTACE prevented shedding of ACE2; cardiomyocyte-specific deletion of TACE also prevented shedding of ACE2. Reactive oxygen species played a key role since p4<em>7</em>(phox)KO mice were resistant to Ang II-induced TACE phosphorylation and activation with preservation of myocardial ACE2 which dampened Ang II-induced cardiac dysfunction and hypertrophy. In conclusion, Ang II induces ACE2 shedding by promoting TACE activity as a positive feedback mechanism whereby Ang II facilitates the loss of its negative regulator, ACE2. In HF, elevated plasma ACE2 activity likely represents loss of the protective effects of ACE2 in the heart.

OBJECTIVE

Prevention strategies for heart failure are needed.

OBJECTIVE

To determine the efficacy of a screening program using brain-type natriuretic peptide (BNP) and collaborative care in an at-risk population in reducing newly diagnosed heart failure and prevalence of significant left ventricular (LV) systolic and/or diastolic dysfunction.

METHODS

The St Vincent's Screening to Prevent Heart Failure Study, a parallel-group randomized trial involving <em>1</em>3<em>7</em>4 participants with cardiovascular risk factors (mean age, 64.8 [SD, <em>1</em>0.2] years) recruited from 39 primary care practices in Ireland between January 2005 and December 2009 and followed up until December 20<em>1</em><em>1</em> (mean follow-up, 4.2 [SD, <em>1</em>.2] years).

METHODS

Patients were randomly assigned to receive usual primary care (control condition; n=6<em>7</em><em>7</em>) or screening with BNP testing (n=69<em>7</em>). Intervention-group participants with BNP levels of 50 pg/mL or higher underwent echocardiography and collaborative care between their primary care physician and specialist cardiovascular service.

METHODS

The primary end point was prevalence of asymptomatic LV dysfunction with or without newly diagnosed heart failure. Secondary end points included emergency hospitalization for arrhythmia, transient ischemic attack, stroke, myocardial infarction, peripheral or pulmonary thrombosis/embolus, or heart failure.

RESULTS

A total of 263 patients (4<em>1</em>.6%) in the intervention group had at least <em>1</em> BNP reading of 50 pg/mL or higher. The intervention group underwent more cardiovascular investigations (control, 496 per <em>1</em>000 patient-years vs intervention, 850 per <em>1</em>000 patient-years; incidence rate ratio, <em>1</em>.<em>7</em><em>1</em>; 95% CI, <em>1</em>.6<em>1</em>-<em>1</em>.83; P<.00<em>1</em>) and received more renin-<em>angiotensin</em>-aldosterone system-based therapy at follow-up (control, 49.6%; intervention, 56.5%; P=.0<em>1</em>). The primary end point of LV dysfunction with or without heart failure was met in 59 (8.<em>7</em>%) of 6<em>7</em><em>7</em> in the control group and 3<em>7</em> (5.3%) of 69<em>7</em> in the intervention group (odds ratio [OR], 0.55; 95% CI, 0.3<em>7</em>-0.82; P = .003). Asymptomatic LV dysfunction was found in 45 (6.6%) of 6<em>7</em><em>7</em> control-group patients and 30 (4.3%) of 69<em>7</em> intervention-group patients (OR, 0.5<em>7</em>; 95% CI, 0.3<em>7</em>-0.88; P = .0<em>1</em>). Heart failure occurred in <em>1</em>4 (2.<em>1</em>%) of 6<em>7</em><em>7</em> control-group patients and <em>7</em> (<em>1</em>.0%) of 69<em>7</em> intervention-group patients (OR, 0.48; 95% CI, 0.20-<em>1</em>.20; P = .<em>1</em>2). The incidence rates of emergency hospitalization for major cardiovascular events were 40.4 per <em>1</em>000 patient-years in the control group vs 22.3 per <em>1</em>000 patient-years in the intervention group (incidence rate ratio, 0.60; 95% CI, 0.45-0.8<em>1</em>; P = .002).

CONCLUSIONS

Among patients at risk of heart failure, BNP-based screening and collaborative care reduced the combined rates of LV systolic dysfunction, diastolic dysfunction, and heart failure.

BACKGROUND

clinicaltrials.gov Identifier: NCT0092<em>1</em>960.

The renin-<em>angiotensin</em> system (RAS) has pivotal roles in the regulation of normal physiology and the pathogenesis of cardiovascular disease. <em>Angiotensin</em>-converting enzyme (ACE) 2, and its product <em>angiotensin</em> <em>1</em>-<em>7</em>, are thought to have counteracting effects against the adverse actions of other, better known and understood, members of the RAS. The physiological and pathological importance of ACE2 and <em>angiotensin</em> <em>1</em>-<em>7</em> in the cardiovascular system are not completely understood, but numerous experimental studies have indicated that these components have protective effects in the heart and blood vessels. Here, we provide an overview on the basic properties of ACE2 and <em>angiotensin</em> <em>1</em>-<em>7</em> and a summary of the evidence from experimental and clinical studies of various pathological conditions, such as hypertension, atherosclerosis, myocardial remodelling, heart failure, ischaemic stroke, and diabetes mellitus. ACE2-mediated catabolism of <em>angiotensin</em> II is likely to have a major role in cardiovascular protection, whereas the relevant functions and signalling mechanisms of actions induced by <em>angiotensin</em> <em>1</em>-<em>7</em> have not been conclusively determined. The ACE2-<em>angiotensin</em> <em>1</em>-<em>7</em> pathway, however, might provide a useful therapeutic target for the treatment of cardiovascular disease, especially in patients with overactive RAS.

This study was performed to determine whether augmented intrarenal <em>angiotensin</em>ogen may contribute to the enhanced renal <em>angiotensin</em> II (Ang II) and associated tissue injury in spontaneously hypertensive rats (SHR). SHR and Wistar-Kyoto rats (WKY) were maintained on a normal diet and killed at either <em>7</em> or <em>1</em>4 wk of age. Two groups of SHR received either an Ang II type <em>1</em> receptor blocker (ARB; olmesartan, 5 mg/d) or a triple therapy (hydralazine <em>7</em>.5 mg/d, reserpine 0.<em>1</em>5 mg/d, and hydrochlorothiazide 3 mg/d [HRH]) during weeks <em>7</em> through <em>1</em>4. Systolic BP and renal Ang II were significantly increased in SHR-<em>1</em>4 (n = 8) compared with WKY-<em>7</em>, WKY-<em>1</em>4, and SHR-<em>7</em> (n = 8 each), and ARB treatment prevented these increases (n = 8). However, whereas HRH treatment prevented the development of hypertension in SHR, this combination therapy failed to decrease renal Ang II (n = 8). With the use of urine samples or fixed renal sections, renal injuries in rats were quantified in a semiautomated manner by the following six parameters: (<em>1</em>) urinary excretion rate of total protein, (2) glomerular sclerosis, (3) interstitial expansion, (4) and (5) numbers of monocytes/macrophages in interstitium or glomeruli, and (6) arterial proliferation. Angiotensinogen mRNA and protein levels in kidney cortex, measured by real-time reverse transcriptase-PCR and Western blot analysis, respectively, and all six parameters of renal damage were changed in parallel, and ARB treatment also prevented these increases. However, HRH treatment failed to prevent these increases. These results indicate that SHR have enhanced intrarenal <em>angiotensin</em>ogen production that contributes to increased Ang II levels leading to the development of hypertension and renal injury in this strain.

Recent studies have established a new regulatory axis in the renin-<em>angiotensin</em> system (RAS). In this axis, <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) is finally produced from Ang I or Ang II by the catalytic activity of <em>angiotensin</em>-converting enzyme 2 (ACE2). Ang-(<em>1</em>-<em>7</em>) shows actions different from those of AT(<em>1</em>) receptor stimulation, such as vasodilatation, natriuresis, anti-proliferation and an increase in the bradykinin-NO (nitric oxide) system. As the catalytic efficiency of ACE2 is approximately 400-fold higher with Ang II as a substrate than with Ang I, this axis is possibly acting as a counter-regulatory system against the ACE/Ang II/AT(<em>1</em>) receptor axis. The signaling pathway of the ACE2-Ang-(<em>1</em>-<em>7</em>) axis has not yet been totally and clearly understood. However, a recent report suggests that the Mas oncogene acts as a receptor for Ang-(<em>1</em>-<em>7</em>). Intracellular signaling through Mas is not clear yet. Several factors such as Akt phosphorylation, protein kinase C activation and mitogen-activated protein (MAP) kinase inhibition seem to be involved in this signaling pathway. Further investigations are needed to clarify the regulation and mechanism of action of ACE2 and Ang-(<em>1</em>-<em>7</em>). However, this second axis through ACE2 and Ang-(<em>1</em>-<em>7</em>) in RAS can be an important target for the therapy of cardiovascular and metabolic disorders.

BACKGROUND

The renin-<em>angiotensin</em> system (RAS) is a key player in the progression of heart failure. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) is thought to modulate the activity of the RAS. Furthermore, this peptide may play a part in the beneficial effects of <em>angiotensin</em>-converting enzyme inhibitors in cardiovascular disease. We assessed the effects of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) on the progression of heart failure.

RESULTS

Male Sprague-Dawley rats underwent either coronary ligation or sham surgery. Two weeks after induction of myocardial infarction, intravenous infusion of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (24 microg/kg per hour) or saline was started by minipump. After 8 weeks of treatment, hemodynamic parameters were measured, endothelial function was assessed in isolated aortic rings, and plasma <em>angiotensin</em>-(<em>1</em>-<em>7</em>) levels were determined. Myocardial infarction resulted in a significant deterioration of left ventricular systolic and diastolic pressure, dP/dt, and coronary flow. Raising plasma levels 40-fold, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) infusion attenuated this impairment to a nonsignificant level, markedly illustrated by a 40% reduction in left ventricular end-diastolic pressure. Furthermore, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) completely preserved aortic endothelial function, whereas endothelium-dependent relaxation in aortas of saline-treated infarcted rats was significantly decreased.

CONCLUSIONS

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) preserved cardiac function, coronary perfusion, and aortic endothelial function in a rat model for heart failure.

OBJECTIVE

We examined the influence of chronic treatment with <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] on renox (renal NADPH oxidase, NOX-4) and the development of renal dysfunction in streptozotocin-treated spontaneously hypertensive rats (diabetic SHR).

METHODS

Mean arterial pressure, urinary protein and vascular responsiveness of the isolated renal artery to vasoactive agonists were studied in vehicle- or Ang-(<em>1</em>-<em>7</em>)-treated SHR and diabetic SHR.

RESULTS

Ang-(<em>1</em>-<em>7</em>) decreased the elevated levels of renal NADPH oxidase (NOX) activity and attenuated the activation of NOX-4 gene expression in the diabetic SHR kidney. Ang-(<em>1</em>-<em>7</em>) treatment increased sodium excretion but did not affect mean arterial pressure in diabetic SHR. There was a significant increase in urinary protein (266 +/- 22 mg/24 h) in the diabetic compared to control SHR (<em>1</em><em>1</em>2 +/- <em>1</em>3 mg/24 h) and treatment of diabetic SHR with Ang-(<em>1</em>-<em>7</em>) reduced the degree of proteinuria (<em>1</em>85 +/- 23 mg/24 h, p < 0.05). Ang-(<em>1</em>-<em>7</em>) treatment also attenuated the diabetes-induced increase in renal vascular responsiveness to endothelin-<em>1</em>, norepinephrine, and <em>angiotensin</em> II in SHR, but significantly increased the vasodilation of the renal artery of SHR and diabetic SHR to the vasodilator agonists.

CONCLUSIONS

These results suggest that treatment with Ang-(<em>1</em>-<em>7</em>) constitutes a potential therapeutic strategy to alleviate NOX-mediated oxidative stress and to reduce renal dysfunction in diabetic hypertensive rats.

There are two major isoforms of the <em>angiotensin</em> II receptor, type <em>1</em> (AT<em>1</em>) and type 2 (AT2). AT2 is distinguished from AT<em>1</em> with respect to its ligand selectivity, its insensitivity to non-hydrolyzable GTP analogues, and its as yet unidentified biological functions. In the present study we have expression-cloned AT2 cDNA from a cDNA library of a rat pheochromocytoma cell line (PC<em>1</em>2w). Rat AT2 cDNA encodes a 363-amino acid protein that has seven transmembrane domains. AT<em>1</em> is the closest in homology to AT2 but with only a 32% identity of amino acid sequence. Stably expressed in COS-<em>7</em> cells, the receptor showed selective binding to AT2-specific ligands PD<em>1</em>233<em>1</em>9 and CGP42<em>1</em><em>1</em>2A but not to the AT<em>1</em>-specific ligand, losartan. Northern blot analysis revealed that the mRNA of rat AT2 was expressed not only in PC<em>1</em>2w cells but also in the adrenal glands and in the inferior olive of the brain, both of which are known to contain AT2 type binding sites. The expressed AT2 receptor mediated <em>angiotensin</em> II-induced inhibition of protein tyrosine phosphatase, an action that was dependent on a pertussis toxin-sensitive G-protein-coupled mechanism in COS-<em>7</em> cells. The AT2-specific ligand CGP42<em>1</em><em>1</em>2A was an agonist rather than antagonist in the inhibition of phosphotyrosine phosphatase. AT2 did not cause a decrease in cGMP in PC<em>1</em>2w or COS-<em>7</em> cells expressing AT2 stably. These results indicate that the AT2 receptor is structurally and functionally different from AT<em>1</em> and suggest novel functional roles of the renin-<em>angiotensin</em> system in cross-talk with phosphotyrosine signaling by modulating protein phosphotyrosine levels.

The rostral ventrolateral medulla (RVLM) is a relay point that provides supraspinal excitatory input to sympathetic preganglionic neurons in the regulation of blood pressure. The importance of the RVLM is further highlighted by observations that an increase of RVLM sensitivity to <em>angiotensin</em> II and enhanced sympathetic activity are associated with hypertension. <em>Angiotensin</em>-converting enzyme 2 (ACE2) has been shown to be central in maintaining the balance between vasoconstrictor activity of <em>angiotensin</em> II with vasoprotective action of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in the peripheral system. However, its role in central control of blood pressure in the RVLM is yet to be investigated. Thus, our objective in this study was to compare ACE2 expression in the RVLM of Wistar-Kyoto rats and spontaneously hypertensive rats and to determine whether RVLM ACE2 is involved in blood pressure control. ACE2 immunoreactivity was diffusely distributed in many cardiovascular regulatory neurons, including the RVLM. Western blot analysis revealed a 40% decrease in ACE2 in the RVLM of spontaneously hypertensive rat compared with Wistar-Kyoto rats. Lentiviral-mediated overexpression of ACE2 (lenti-ACE2) was used to determine whether a decrease in ACE2 in the RVLM is associated with hypertensive state. Bilateral injection of lenti-ACE2 resulted in a long-term expression of transgenic ACE2. This was associated with a decrease in mean arterial pressure exclusively in the spontaneously hypertensive rat (<em>1</em>4<em>1</em>+/-4 mm Hg in lenti-GFP versus <em>1</em>24+/-5 mm Hg in lenti-ACE2) and heart rate (304+/-<em>7</em> bpm in lenti-GFP versus 285+/-5 bpm in lenti-ACE2). These observations demonstrate that overexpression of ACE2 overcomes its intrinsic decrease in the RVLM and decreases high blood pressure in the spontaneously hypertensive rat.

To elucidate the cellular mechanism by which <em>angiotensin</em> II (ANG II) induces cardiac hypertrophy, we investigated the possible autocrine/paracrine role of endogenous endothelin-<em>1</em> (ET-<em>1</em>) in ANG II-induced hypertrophy of neonatal rat cardiomyocytes by use of synthetic ET-<em>1</em> receptor antagonist and antisense oligonucleotides to preproET-<em>1</em> (ppET-<em>1</em>) mRNA. Northern blot analysis and in situ hybridization revealed that ppET-<em>1</em> mRNA was expressed in cardiomyocytes, but, to a lesser extent, in nonmyocytes as well. ANG II upregulated ppET-<em>1</em> mRNA level by threefold over control level as early as 30 min, and it stimulated release of immunoreactive ET-<em>1</em> from cardiomyocytes in a dose- and time-dependent manner. ET-<em>1</em> stimulated ppET-<em>1</em> mRNA levels after 30 min in a similar fashion as ANG II. Tetradecanoylphorbol-acetate (<em>1</em>0(-<em>7</em>) M) mimicked the effects of ANG II and ET-<em>1</em> on induction of ppET-<em>1</em> mRNA. ANG II-induced ppET-<em>1</em> gene expression was completely blocked by protein kinase C inhibitor H-<em>7</em> or by down-regulation of endogenous protein kinase C by pretreatment with phorbol ester. ET-<em>1</em> and ANG II stimulated twofold increase [3H]leucine incorporation into cardiomyocytes, whose effects were similarly and dose dependently inhibited by endothelin A receptor antagonist (BQ<em>1</em>23). Introduction of antisense sequence against coding region of ppET-<em>1</em> mRNA into cardiomyocytes resulted in complete blockade with ppET-<em>1</em> mRNA levels and [3H]leucine incorporation stimulated by ANG II. These results suggest that endogenous ET-<em>1</em> locally generated and secreted by cardiomyocytes may contribute to ANG II-induced cardiac hypertrophy via an autocrine/paracrine fashion.

OBJECTIVE

Metabolic syndrome is characterized by the variable coexistence of obesity, hyperinsulinemia, insulin resistance, dyslipidemia, and hypertension. It is well known that <em>angiotensin</em> (Ang) II is importantly involved in the metabolic syndrome. However, the role of the vasodilator Ang-(<em>1</em>-<em>7</em>)/Mas axis is not known. The aim of this study was to evaluate the effect of genetic deletion of the G protein-coupled receptor, Mas, in the lipidic and glycemic metabolism in FVB/N mice.

METHODS

Plasma lipid, insulin, and cytokine concentrations were measured in FVB/N Mas-deficient and wild-type mice. A glucose tolerance test was performed by intraperitoneally injecting d-glucose into overnight-fasted mice. An insulin sensitivity test was performed by intraperitoneal injection of insulin. Uptake of 2-deoxy-[(3)H]glucose by adipocytes was used to determine the rate of glucose transport; adipose tissue GLUT4 was quantified by Western blot. Gene expression of transforming growth factor (TGF)-beta, type <em>1</em> Ang II receptor, and <em>angiotensin</em>ogen (AGT) were measured by real-time PCR.

RESULTS

Despite normal body weight, Mas-knockout (Mas-KO) mice presented dyslipidemia, increased levels of insulin and leptin, and an approximately 50% increase in abdominal fat mass. In addition, Mas gene-deleted mice presented glucose intolerance and reduced insulin sensitivity as well as a decrease in insulin-stimulated glucose uptake by adipocytes and decreased GLUT4 in adipose tissue. Mas(-/-) presented increased muscle triglycerides, while liver triglyceride levels were normal. Expression of TGF-beta and AGT genes was higher in Mas-KO animals in comparison with controls.

CONCLUSIONS

These results show that Mas deficiency in FVB/N mice leads to dramatic changes in glucose and lipid metabolisms, inducing a metabolic syndrome-like state.

To seek evidence that the nonhuman primate arterial wall, as it ages in the absence of atherosclerosis, exhibits alterations in pathways that are involved in the pathogenesis of experimental atherosclerosis, we assessed aortic matrix metalloproteinase-2 (MMP-2) and its regulators, ie, membrane type-<em>1</em> of matrix metalloproteinase (MT<em>1</em>-MMP) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), and the expression of <em>angiotensin</em> II (Ang II), <em>angiotensin</em>-converting enzyme (ACE), and chymase in young (6.4+/-0.<em>7</em> years) and old (20.0+/-<em>1</em>.9 years) male monkeys. With advancing age, (<em>1</em>) the intimal thickness increased 3-fold and contained numerous vascular smooth muscle cells and matrix, but no inflammatory cells; (2) the intimal MMP-2 antibody-staining fraction increased by 80% (P<0.0<em>1</em>); (3) in situ zymography showed that MMP-2 activity, mainly confined to the intima, increased 3-fold (P<0.0<em>1</em>); (4) the MT<em>1</em>-MMP antibody-staining fraction increased by <em>1</em>50% (P<0.00<em>1</em>), but the TIMP-2 antibody-staining fraction did not significantly change; (5) steady levels of the mRNA-staining fraction (via in situ hybridization) for MMP-2 increased <em>7</em>-fold, for MT<em>1</em>-MMP increased 9-fold, and for TIMP-2 increased 2-fold (all P<0.00<em>1</em>); and (6) intimal Ang II and ACE immunofluorescence were increased 5-fold and 5.6-fold, respectively, and colocalized with MMP-2. Thus, age-associated arterial remodeling and the development and progression of experimental atherosclerosis in young animals share common mechanisms, ie, MMP-2 activation and increased Ang II signaling. This might explain, in part, the dramatically exaggerated prevalence and severity of vascular diseases with aging.

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome. <em>Angiotensin</em>-converting enzyme (ACE) and its effector peptide <em>angiotensin</em> (Ang) II have been implicated in the pathogenesis of ARDS. A counter-regulatory enzyme of ACE, ie ACE2 that degrades Ang II to Ang-(<em>1</em>-<em>7</em>), offers a promising novel treatment modality for this syndrome. As the involvement of ACE and ACE2 in ARDS is still unclear, this study investigated the role of these two enzymes in an animal model of ARDS. ARDS was induced in rats by intratracheal administration of LPS followed by mechanical ventilation. During ventilation, animals were treated with saline (placebo), losartan (Ang II receptor antagonist), or with a protease-resistant, cyclic form of Ang-(<em>1</em>-<em>7</em>) [cAng-(<em>1</em>-<em>7</em>)]. In bronchoalveolar lavage fluid (BALF) of ventilated LPS-exposed animals, ACE activity was enhanced, whereas ACE2 activity was reduced. This was matched by enhanced BALF levels of Ang II and reduced levels of Ang-(<em>1</em>-<em>7</em>). Therapeutic intervention with cAng-(<em>1</em>-<em>7</em>) attenuated the inflammatory mediator response, markedly decreased lung injury scores, and improved lung function, as evidenced by increased oxygenation. These data indicate that ARDS develops, in part, due to reduced pulmonary levels of Ang-(<em>1</em>-<em>7</em>) and that repletion of this peptide halts the development of ARDS.

BACKGROUND

The outcomes of patients referred to nephrologists are not well described in large cohorts. The objectives of this analysis are to describe the predictors of rapid progression of kidney disease and death in patients followed up by nephrologists.

METHODS

Retrospective study.

METHODS

A cohort derived from all patients registered in the provincial database was formed that included all patients with index estimated glomerular filtration rate (eGFR) less than 30 mL/min/1.73 m(2), at least 3 subsequent eGFR values, and 4 months of follow-up between January 2000 and January 2004.

METHODS

Variables used to predict outcomes included baseline eGFR, duration of follow-up before eGFR less than 30 mL/min/1.73 m(2), age, sex, ethnicity, presence of diabetes, blood pressure, level of proteinuria, hemoglobin level, phosphate level, calcium level, parathyroid hormone level, and use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, erythropoiesis-stimulating agents, and vitamin D.

RESULTS

Key outcomes of interest were death, dialysis therapy start, or loss of GFR greater than 5 mL/min/1.73 m(2)/y.

RESULTS

4,231 patients met inclusion criteria. Mean age was 67 years. Median follow-up was 31 months. During the first 2 years of follow-up, 24% started dialysis therapy, 1% received a transplant, 7% died, and 1% was lost to follow-up. Statistically significant variables associated with more rapid kidney disease progression differ from those that predict death. Younger age, male sex, higher eGFR, higher systolic and diastolic blood pressure, lower hemoglobin level, higher phosphorus and parathyroid hormone levels, and greater proteinuria are associated with more rapid kidney disease progression, and use of angiotensin-converting enzymes/angiotensin receptor blockers are protective. Older age, lower diastolic blood pressure, lower hemoglobin level, and higher phosphorous and parathyroid hormone levels are associated with death, whereas vitamin D use is protective.

CONCLUSIONS

Results cannot be generalized to unreferred patients with eGFR less than 30 mL/min/1.73 m(2).

CONCLUSIONS

The clinical course of patients with chronic kidney disease stage 4 is variable. Targeted therapy aimed at modifiable risk factors needs to be evaluated to determine benefits of this approach.

Previous studies in several strains of rats have demonstrated that 35 days of recurrent episodic hypoxia (EH) (<em>7</em> hours per day), with a fractional concentration of inspired oxygen that produces desaturation equivalent to the recurrent hypoxemia of sleep apnea, results in an 8 to <em>1</em>3 mm Hg persistent increase in diurnal systemic blood pressure (BP). Carotid chemoreceptors and the sympathetic nervous system have been shown to be necessary for development of this BP increase. Both renal artery denervation and adrenal demedullation block the BP response to chronic EH. The present study was undertaken to define further the role of the kidneys and the renin-<em>angiotensin</em> system in this BP increase. Separate groups of male Sprague-Dawley rats had either (<em>1</em>) bilateral renal artery denervation with EH, (2) sham surgery with EH, (3) sham surgery with sham EH (compressed air), (4) EH with losartan, (5) unhandled with losartan, or (6) unhandled. The experimental period lasted 35 days. Both renal-artery denervated and losartan-treated animals showed no BP change or a lowering of BP in response to EH, whereas the sham-operated EH animals showed a progressive, sustained increase in resting room air BP. BP remained at basal levels or fell in unhandled and unhandled losartan-treated animals. Plasma renin activity was elevated 4-fold versus basal levels in EH animals with renal nerves intact but remained at baseline levels in denervated animals. At the end of the experiment, renal tissue catecholamines confirmed renal denervation in those animals. In conclusion, EH causes a progressive increase in BP, mediated in part through renal sympathetic nerve activity that acts to increase renin-<em>angiotensin</em> system activity through <em>angiotensin</em> II type <em>1</em> receptors.

<em>Angiotensin</em> II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for <em>angiotensin</em> II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine <em>angiotensin</em> II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-<em>7</em> cells transfected with the cDNA expressed specific and high-affinity binding sites for <em>angiotensin</em> II, <em>angiotensin</em> II antagonist and a non-peptide specific antagonist for type-<em>1</em> receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-<em>1</em>,4,5-trisphosphate increased in the transfected COS-<em>7</em> cells in response to <em>angiotensin</em> II or <em>angiotensin</em> III, indicating that this receptor is the type-<em>1</em> receptor for <em>angiotensin</em> II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.

Studies suggest that the inflammatory cytokine TNF-alpha plays a role in the prognosis of end-stage renal diseases. We previously showed that TNF-alpha inhibition slowed the progression of hypertension and renal damage in <em>angiotensin</em> II salt-sensitive hypertension. Thus, we hypothesize that TNF-alpha contributes to renal inflammation in a model of mineralocorticoid-induced hypertension. Four groups of rats (n = 5 or 6) were studied for 3 wk with the following treatments: <em>1</em>) placebo, 2) placebo + TNF-alpha inhibitor etanercept (<em>1</em>.25 mg.kg(-<em>1</em>).day(-<em>1</em>) sc), 3) deoxycorticosterone acetate + 0.9% NaCl to drink (DOCA-salt), or 4) DOCA-salt + etanercept. Mean arterial blood pressure (MAP) measured by telemetry increased in DOCA-salt rats compared with baseline (<em>1</em><em>7</em><em>7</em> +/- 4 vs. <em>1</em>0<em>7</em> +/- 3 mmHg; P < 0.05), and TNF-alpha inhibition had no effect in the elevation of MAP in these rats (<em>1</em><em>7</em><em>7</em> +/- 8 mmHg). Urinary protein excretion significantly increased in DOCA-salt rats compared with placebo (<em>7</em>03 +/- <em>7</em>6 vs. <em>1</em>98 +/- 5 mg/day); etanercept lowered the proteinuria (5<em>1</em>4 +/- 64 mg/day; P < 0.05 vs. DOCA-salt alone). Urinary albumin excretion followed a similar pattern in each group. Urinary monocyte chemoattractant protein (MCP)-<em>1</em> and endothelin (ET)-<em>1</em> excretion were also increased in DOCA-salt rats compared with placebo (MCP-<em>1</em>: 939 +/- <em>1</em>04 vs. 43 +/- <em>7</em> ng/day, ET-<em>1</em>: 3.30 +/- 0.29 vs. <em>1</em>.0<em>7</em> +/- 0.03 fmol/day; both P < 0.05); TNF-alpha inhibition significantly decreased both MCP-<em>1</em> and ET-<em>1</em> excretion (409 +/- <em>1</em>38 ng/day and 2.42 +/- 0.22 fmol/day, respectively; both P < 0.05 vs. DOCA-salt alone). Renal cortical NF-kappaB activity also increased in DOCA-salt hypertensive rats, and etanercept treatment significantly reduced this effect. These data support the hypothesis that TNF-alpha contributes to the increase in renal inflammation in DOCA-salt rats.

Hypertension induced by long-term infusion of <em>angiotensin</em> II (Ang II) is associated with augmented intrarenal Ang II levels to a greater extent than can be explained on the basis of the circulating Ang II levels. Although part of this augmentation is due to AT(<em>1</em>) receptor-dependent internalization, the intracellular compartments involved in this Ang II accumulation remain unknown. In the present study, we sought to determine whether Ang II trafficking into renal cortical endosomes is increased during Ang II hypertension, and if so, whether the AT(<em>1</em>) receptor antagonist, candesartan, prevents this accumulation. Compared with controls (n=<em>1</em>2; <em>1</em><em>1</em>4+/-2 mm Hg), Ang II-infused rats (n=<em>1</em>2; 80 ng/kg/min, SC, for <em>1</em>3 days) developed hypertension with systolic blood pressure rising to <em>1</em>85+/-4 mm Hg by Day <em>1</em>2. In Ang II hypertensive rats, plasma renin activity was suppressed, whereas plasma and kidney Ang II levels were increased by 3-fold (348+/-58 versus <em>1</em><em>1</em>9+/-<em>1</em>6 fmol/mL) and 2-fold (399+/-39 versus <em>1</em>86+/-26 fmol/g). Intracellular endosomal Ang II levels were increased by more than <em>1</em>0-fold (<em>1</em><em>1</em>00+/-283 versus <em>7</em><em>1</em>+/-<em>1</em>2 fmol/mg protein), whereas intermicrovillar cleft Ang II levels were increased by more than 2-fold (88+/-22 versus 3<em>7</em>+/-<em>7</em> fmol/mg protein). Flow cytometric analysis detected significant increases in AT(<em>1</em>A) receptor antibody binding in endosomal and intermicrovillar clefts of Ang II-infused rats. The hypertension induced by Ang II was prevented in rats treated concurrently with candesartan (2 mg/kg/d, <em>1</em><em>1</em>9+/-3 mm Hg). Candesartan treatment (n=8) also prevented increases in kidney (2<em>1</em>5+/-<em>1</em>9 fmol/g), endosomal (96+/-29 fmol/mg protein), and intermicrovillar cleft Ang II levels (<em>1</em><em>1</em>+/-2 fmol/mg protein). These results indicate that there is substantial intracellular accumulation of <em>angiotensin</em> peptides in renal cortical endosomes during Ang II-dependent hypertension via an AT(<em>1</em>) receptor-mediated process.