BACKGROUND

Hypertension is a common comorbidity in patients with type 2 diabetes mellitus and a major risk factor for microvascular and macrovascular disease. Although the blood pressure-lowering effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors are already established, guidance is needed on how to use these drugs in patients already receiving antihypertensive therapy. We aimed to compare blood pressure and glycaemic effects of the SGLT2 inhibitor dapagliflozin with placebo in patients with inadequately controlled type 2 diabetes mellitus and hypertension.

METHODS

In this double-blind, placebo-controlled, phase 3 study we enrolled patients from 3<em>1</em><em>1</em> centres in <em>1</em>6 countries across five continents. Patients had uncontrolled type 2 diabetes (HbA<em>1</em>c <em>7</em>·0%-<em>1</em>0·5%; 53-9<em>1</em> mmol/mol) and hypertension (systolic <em>1</em>40-<em>1</em>65 mm Hg and diastolic 85-<em>1</em>05 mm Hg at both enrolment and randomisation, and a mean 24 h blood pressure of ≥<em>1</em>30/80 mm Hg by ambulatory monitoring within <em>1</em> week of randomisation) and were receiving oral antihyperglycaemic drugs, insulin, or both, plus a renin-<em>angiotensin</em> system blocker and an additional antihypertensive drug. Using an interactive voice-response system, we randomly assigned (<em>1</em>:<em>1</em>) patients to dapagliflozin <em>1</em>0 mg once a day or to placebo, with randomisation stratified by additional antihypertensive drug use and insulin use at baseline, in a block size of two. The co-primary endpoints were changes in seated systolic blood pressure and HbA<em>1</em>c measured in the full analysis set, which included all patients who received at least one dose of study drug and had both a baseline and at least one post-baseline measurement of efficacy. This trial is registered with ClinicalTrials.gov, number NCT0<em>1</em><em>1</em>95662.

RESULTS

Between Oct 29, 20<em>1</em>0, and Oct 4, 20<em>1</em>2, we randomly assigned 225 patients to dapagliflozin and 224 to placebo. Seated systolic blood pressure was significantly reduced in the group assigned to dapagliflozin (adjusted mean change from baseline -<em>1</em><em>1</em>·90 mm Hg [95% CI -<em>1</em>3·9<em>7</em> to -9·82]) compared with those assigned to placebo (-<em>7</em>·62 mm Hg [-9·<em>7</em>2 to -5·5<em>1</em>]; placebo-adjusted difference for dapagliflozin -4·28 mm Hg [-6·54 to -2·02]; p=0·0002). Reductions in HbA<em>1</em>c concentrations were also significantly greater in patients assigned to dapagliflozin (adjusted mean change from baseline -0·63% [95% CI -0·<em>7</em>6 to -0·50]) than in those assigned to placebo (-0·02% [-0·<em>1</em>5 to 0·<em>1</em>2]; placebo-adjusted difference -0·6<em>1</em>% [-0·<em>7</em>6 to -0·46,]; p<0·000<em>1</em>). In a post-hoc analysis, we found difference in blood pressure versus placebo was greater in patients receiving a β blocker (-5·<em>7</em>6 mm Hg [95% CI -<em>1</em>0·28 to -<em>1</em>·23]) or a calcium-channel blocker (-5·<em>1</em>3 mm Hg, [-9·4<em>7</em> to -0·<em>7</em>9]) as their additional antihypertensive drug than in those receiving a thiazide diuretic (-2·38 mm Hg [-6·<em>1</em>6 to <em>1</em>·40]). Adverse events were similar in the dapagliflozin and placebo groups (98 [44%] patients vs 93 [42%], respectively, had at least one adverse event), with few adverse events related to renal function (<em>1</em>% vs (<em>1</em>%) or volume depletion ((<em>1</em>% vs 0%).

CONCLUSIONS

Dapagliflozin <em>1</em>0 mg significantly improved blood pressure and HbA<em>1</em>c and was tolerated similarly to placebo. Its blood pressure-lowering properties were particularly favourable in patients already receiving a β blocker or calcium-channel blocker. Dapagliflozin could benefit patients with type 2 diabetes who need a diuretic-like effect to optimise control of blood pressure, adding meaningful efficacy to antihypertensive drug regimens.

BACKGROUND

Bristol-Myers Squibb, AstraZeneca.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) degrades <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After <em>1</em>6 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by <em>1</em>6 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at <em>1</em>6 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β<em>1</em> was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

Hypertension is associated with vascular changes characterised by remodelling, endothelial dysfunction and hyperreactivity. Cellular processes underlying these perturbations include altered vascular smooth muscle cell growth and apoptosis, fibrosis, hypercontractility and calcification. Inflammation, associated with macrophage infiltration and increased expression of redox-sensitive pro-inflammatory genes, also contributes to vascular remodelling. Many of these features occur with ageing, and the vascular phenotype in hypertension is considered a phenomenon of 'premature vascular ageing'. Among the many factors involved in the hypertensive vascular phenotype, <em>angiotensin</em> II (Ang II) is especially important. Ang II, previously thought to be the sole effector of the renin-<em>angiotensin</em> system (RAS), is converted to smaller peptides [Ang III, Ang IV, Ang-(<em>1</em>-<em>7</em>)] that are biologically active in the vascular system. Another new component of the RAS is the (pro)renin receptor, which signals through Ang-II-independent mechanisms and might influence vascular function. Ang II mediates effects through complex signalling pathways on binding to its G-protein-coupled receptors (GPCRs) AT<em>1</em>R and AT2R. These receptors are regulated by the GPCR-interacting proteins ATRAP, ARAP<em>1</em> and ATIP. AT<em>1</em>R activation induces effects through the phospholipase C pathway, mitogen-activated protein kinases, tyrosine kinases/phosphatases, RhoA/Rhokinase and NAD(P)H-oxidase-derived reactive oxygen species. Here we focus on recent developments and new research trends related to Ang II and the RAS and involvement in the hypertensive vascular phenotype.

OBJECTIVE

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] has anti-inflammatory effects in peripheral organs, but its effects in ischaemic stroke are unclear as yet. We investigated whether its anti-inflammatory effect contributes to the neuroprotection induced by Ang-(<em>1</em>-<em>7</em>) in a rat model of permanent middle cerebral artery occlusion (pMCAO).

METHODS

We infused Ang-(<em>1</em>-<em>7</em>), Mas receptor antagonist A-<em>7</em><em>7</em>9, angiotensin II type 2 receptor antagonist PD<em>1</em>233<em>1</em>9 or artificial CSF into the right lateral ventricle of male Sprague-Dawley rats from 48 h before onset of pMCAO until the rats were killed. Twenty-four hours after pMCAO, the neuroprotective effect of Ang-(<em>1</em>-<em>7</em>) was analysed by evaluating infarct volume and neurological deficits. The levels of oxidative stress were detected by spectrophotometric assay. The activation of NF-κB was assessed by Western blot and immunohistochemistry analysis. The level of COX-2 was tested by Western blot analysis and concentrations of pro-inflammatory cytokines were measured by elisa.

RESULTS

Infusion of Ang-(<em>1</em>-<em>7</em>), i.c.v., significantly reduced infarct volume and improved neurological deficits. It decreased the levels of oxidative stress and suppressed NF-κB activity, which was accompanied by a reduction of pro-inflammatory cytokines and COX-2 in the peri-infarct regions. These effects of Ang-(<em>1</em>-<em>7</em>) were reversed by A-<em>7</em><em>7</em>9 but not by PD<em>1</em>233<em>1</em>9. Additionally, infusion of A-<em>7</em><em>7</em>9 alone increased oxidative stress levels and enhanced NF-κB activity, which was accompanied by an up-regulation of pro-inflammatory cytokines and COX-2.

CONCLUSIONS

Our findings indicate that suppressing NF-κB dependent pathway via Mas receptor may represent one mechanism that contributes to the anti-inflammatory effects of Ang-(<em>1</em>-<em>7</em>) in rats with pMCAO.

Aldosterone plays an important role in the pathophysiology of congestive heart failure (CHF), and spironolactone improves cardiovascular function and survival rates in patients with CHF. We hypothesized that the mineralocorticoid receptor blockade (MRB) exerted its beneficial effects by reducing oxidative stress and changing the balance between the counter-acting enzymes <em>angiotensin</em>-converting enzyme (ACE) and ACE2. Monocyte-derived macrophages were obtained from <em>1</em>0 patients with CHF before and after <em>1</em> month of treatment with spironolactone (25 mg/d). Spironolactone therapy significantly (P<0.005) reduced oxidative stress, as expressed by reduced lipid peroxide content, superoxide ion release, and low-density lipoprotein oxidation by 28%, 53%, and <em>7</em>0%, respectively. Although spironolactone significantly (P<0.0<em>1</em>) reduced macrophage ACE activity by 4<em>7</em>% and mRNA expression by 53%, ACE2 activity and mRNA expression increased by 300% and 654%, respectively. In mice treated for 2 weeks with eplerenone (200 mg.kg(-<em>1</em>).d(-<em>1</em>)), cardiac ACE2 activity significantly (P<0.05) increased by 2-fold and was paralleled by increased ACE2 activity in macrophages. The mechanism of aldosterone antagonist action was studied in mouse peritoneal macrophages (MPMs) in vitro. Although ACE activity and mRNA were significantly increased by 250 nmol/L aldosterone, ACE2 was significantly reduced. Cotreatment with eplerenone (2 micromol/L) attenuated these effects. In MPM obtained from p4<em>7</em> knockout mice, where NADPH oxidase is inactive, as well as in control MPMs treated with NADPH oxidase inhibitor, aldosterone did not increase ACE or decrease ACE2. MRB reduced oxidative stress, decreased ACE activity, and increased ACE2 activity, suggesting a protective role for MRB by possibly increasing generation of <em>angiotensin</em> (<em>1</em>-<em>7</em>) and decreasing formation of <em>angiotensin</em> II. These effects are mediated, at least in part, by NADPH oxidase.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr<em>1</em>068, Tyr<em>1</em><em>1</em>48 or Tyr<em>1</em><em>1</em><em>7</em>3) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) <em>1</em>-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-<em>7</em> microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A43<em>1</em> cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr<em>1</em><em>1</em><em>7</em>3 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of <em>1</em><em>1</em>0-<em>1</em>30 microM, and the peptide inhibited autophosphorylation with a Ki of <em>1</em>50 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of <em>7</em>0-<em>1</em>00 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates <em>angiotensin</em> II and a peptide derived from c-src. The Km of <em>angiotensin</em> II phosphorylation was reduced from <em>1</em><em>1</em>00 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from <em>1</em>0<em>1</em>0 microM to <em>7</em><em>7</em>0 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr<em>1</em><em>1</em><em>7</em>3 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-<em>7</em>50 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolylcarboxypeptidase was purified from human kidney <em>1</em>200-fold with <em>1</em>8% yield. The enzyme had no cathepsin A activity and appeared to be homogeneous in gel electrophoresis. The molecular weight of prolylcarboxypeptidase was estimated to be <em>1</em><em>1</em>5,000 by gel filtration. Under denaturing conditions the enzyme dissociated into subunits of 45,000 and 66,500 molecular weight. The enzyme cleaved benzyloxycarbonyl (Cbz)-Pro-Phe, representing the COOH-terminal end of <em>angiotensin</em> II and des-Asp<em>1</em>-<em>angiotensin</em> II (<em>angiotensin</em> III), at a rate of 3<em>1</em> micronmol/h/mg of protein. The rate of hydrolysis increased when phenylalanine in the N-protected dipeptide was replaced with alanine, valine, or leucine or when the octapeptide <em>angiotensin</em> II or the heptapeptide <em>angiotensin</em> III were the substrates. The enzyme also cleaved the <em>angiotensin</em> II antagonist saralasin (Sar<em>1</em>-Ala8-<em>angiotensin</em> II). The Km values were <em>1</em> mM, 2mM, and 0.<em>7</em><em>7</em> mM with Cbz-Pro-Phe, <em>angiotensin</em> II, and <em>angiotensin</em> III, respectively. The enzyme had an acid pH optimum (4.5 to 5.5), but hydrolyzed <em>angiotensin</em> III at pH <em>7</em> at 50% of the optimal rate. Prolylcarboxypeptidase was inhibited by diisopropyl phosphorofluoridate and pepstatin, but not by sequestering agents or -SH reagents.

BACKGROUND AND PURPOSE <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] has anti-inflammatory effects in models of cardiovascular disease and arthritis, but its effects in asthma are unknown. We investigated whether Ang-(<em>1</em>-<em>7</em>) has anti-inflammatory actions in a murine model of asthma. EXPERIMENTAL APPROACH The effects of Ang-(<em>1</em>-<em>7</em>) alone or in combination with the MAS<em>1</em> receptor antagonist, A<em>7</em><em>7</em>9, were evaluated over a 4 day period in an ovalbumin-challenged mouse model of allergic asthma. On day 5, bronchoalveolar lavage was performed, and lungs were sectioned and assessed histologically for quantification of goblet cells, perivascular and peribronchial inflammation and fibrosis. Biochemical analysis of the pro-inflammatory ERK<em>1</em>/2 and IκB-α was assessed. In addition, the effect of Ang-(<em>1</em>-<em>7</em>) on proliferation of human peripheral blood mononuclear cells (HPBMC) was investigated. KEY RESULTS Ang-(<em>1</em>-<em>7</em>) attenuated ovalbumin-induced increases in total cell counts, eosinophils, lymphocytes and neutrophils. Ang-(<em>1</em>-<em>7</em>) also decreased the ovalbumin-induced perivascular and peribronchial inflammation, fibrosis and goblet cell hyper/metaplasia. Additionally, Ang-(<em>1</em>-<em>7</em>) reduced the ovalbumin-induced increase in the phosphorylation of ERK<em>1</em>/2 and IκB-α. These effects of Ang-(<em>1</em>-<em>7</em>) were reversed by the MAS<em>1</em> receptor antagonist A<em>7</em><em>7</em>9. Furthermore, Ang-(<em>1</em>-<em>7</em>) inhibited phytohaemagglutinin (PHA)-induced HPBMC proliferation. CONCLUSION AND IMPLICATIONS Ang-(<em>1</em>-<em>7</em>), via its MAS<em>1</em> receptor, acts as an anti-inflammatory pathway in allergic asthma, implying that activation of the MAS<em>1</em> receptor may represent a novel approach to asthma therapy.

Despite recent evidence indicating sustained activation of the baroreflex during chronic infusion of <em>angiotensin</em> II (Ang II), sinoaortic denervation does not exacerbate the severity of the hypertension. Therefore, to determine whether Ang II hypertension is relatively resistant to the blood pressure-lowering effects of the baroreflex, the carotid baroreflex was electrically activated bilaterally for <em>7</em> days in 5 dogs both in the presence and absence of a continuous infusion of Ang II (5 ng/kg per minute) producing high physiological plasma levels of the peptide. Under control conditions, basal values for mean arterial pressure (MAP) and plasma norepinephrine concentration (NE) were 93+/-<em>1</em> mm Hg and 99+/-25 pg/mL, respectively. By day <em>7</em> of baroreflex activation, MAP and NE were reduced to <em>7</em>2+/-4 mm Hg (-2<em>1</em>+/-3 mm Hg) and 56+/-<em>1</em>5 pg/mL, respectively, but PRA was unchanged (control=0.4<em>1</em>+/-0.06 ng ANG I/mL per hour). All values returned to basal levels by the end of a <em>7</em>-day recovery period. After <em>7</em> days of Ang II infusion, MAP increased from 93+/-3 to <em>1</em>29+/-3 mm Hg, whereas NE fell from <em>1</em><em>1</em><em>7</em>+/-<em>1</em>5 to 86+/-23 pg/mL. During the next <em>7</em> days of baroreflex activation/Ang II infusion, further reductions in NE were not statistically significant, and on the final day of baroreflex activation, the reduction in MAP was only 5+/-<em>1</em> mm Hg, compared with 2<em>1</em>+/-3 mm Hg in the control normotensive state. These findings indicate that long-term baroreflex-mediated reductions in arterial pressure are markedly diminished, but not totally eliminated, in the presence of hypertension produced by chronic infusion of Ang II.

Chronic intermittent hypoxia (CIH) models repetitive bouts of arterial hypoxemia that occur in humans suffering from obstructive sleep apnea. CIH has been linked to persistent activation of arterial chemoreceptors and the renin-<em>angiotensin</em> system, which have been linked to chronic elevations of sympathetic nerve activity (SNA) and mean arterial pressure (MAP). Because Fos and FosB are transcription factors involved in activator protein (AP)-<em>1</em> driven central nervous system neuronal adaptations, this study determined if CIH causes increased Fos or FosB staining in brain regions that regulate SNA and autonomic function. Male Sprague Dawley rats were instrumented with telemetry transmitters for continuous recording of MAP and heart rate (HR). Rats were exposed to continuous normoxia (CON) or to CIH for 8 h/day for <em>7</em> days. CIH increased MAP by <em>7</em>-<em>1</em>0 mmHg without persistently affecting HR. A separate group of rats was killed <em>1</em> day after <em>7</em> days of CIH for immunohistochemistry. CIH did not increase Fos staining in any brain region examined. Staining for FosB/ΔFosB was increased in the organum vasculosum of the lamina terminalis (CON: 9 ± <em>1</em>; CIH: 34 ± 3 cells/section), subfornical organ (CON: <em>7</em> ± 2; CIH: 3<em>1</em> ± 3), median preoptic nucleus (CON <em>1</em>5 ± <em>1</em>; CIH: 38 ± 3), nucleus of the solitary tract (CON: 9 ± 2; CIH: 28 ± 4), A5 (CON: 3 ± <em>1</em>; CIH: <em>1</em>0 ± <em>1</em>), and rostral ventrolateral medulla (CON: 5 ± <em>1</em>; CIH: <em>1</em><em>7</em> ± 2). In the paraventricular nucleus, FosB/ΔFosB staining was located mainly in the dorsal and medial parvocellular subnuclei. CIH did not increase FosB/ΔFosB staining in caudal ventrolateral medulla or supraoptic nucleus. These data indicate that CIH induces an increase in FosB/ΔFosB in autonomic nuclei and suggest that AP-<em>1</em> transcriptional regulation may contribute to stable adaptive changes that support chronically elevated SNA.

<em>Angiotensin</em> II (Ang II)-induced activation of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase leads to increased production of reactive oxygen species (ROS), an important intracellular second messenger in renal disease. Recent findings suggest that Ang II induces mitochondrial depolarization and further amplifies mitochondrial generation of ROS. We examined the hypothesis that ROS injury mediated by Ang II-induced mitochondrial Nox4 plays a pivotal role in mitochondrial dysfunction in tubular cells and is related to cell survival. In addition, we assessed whether <em>angiotensin</em> (<em>1</em>-<em>7</em>) peptide (Ang-(<em>1</em>-<em>7</em>)) was able to counteract Ang II-induced ROS-mediated cellular injury. Cultured NRK-52E cells were stimulated with <em>1</em>0(-6) M Ang II for 24 h with or without Ang-(<em>1</em>-<em>7</em>) or apocynin. Ang II simulated mitochondrial Nox4 and resulted in the abrupt production of mitochondrial superoxide (O(2) (-)) and hydrogen peroxide (H(2)O(2)). Ang II also induced depolarization of the mitochondrial membrane potential, and cytosolic secretion of cytochrome C and apoptosis-inducing factor (AIF). Ang-(<em>1</em>-<em>7</em>) attenuated Ang II-induced mitochondrial Nox4 expression and apoptosis, and its effect was comparable to that of the NAD(P)H oxidase inhibitor. These findings suggest that Ang II-induced activation of mitochondrial Nox4 is an important endogenous source of ROS, and is related to cell survival. The ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas receptor axis should be investigated further as a novel target of Ang II-mediated ROS injury.

BACKGROUND

Patients with chronic kidney disease are at increased risk for cardiovascular (CV) events.

METHODS

We randomly assigned <em>1</em>,094 African Americans with hypertensive nephrosclerosis (glomerular filtration rate [GFR], 20 to 65 mL/min/<em>1</em>.73 m(2) [0.33 to <em>1</em>.08 mL/s]) to initial antihypertensive treatment with either: (<em>1</em>) a beta-blocker, metoprolol; (2) an <em>angiotensin</em>-converting enzyme inhibitor, ramipril; or (3) a dihydropyridine calcium channel blocker, amlodipine, and either a usual-blood pressure (BP) or low-BP treatment goal. Using a design powered to detect renal outcome differences, we compared the effect of treatment on the CV event rate (cardiac death, myocardial infarction, stroke, and heart failure) during a mean follow-up period of 4.<em>1</em> years and determined baseline factors that predict CV outcomes.

RESULTS

Thirty-one patients died of CV disease (0.7%/patient-year), and <em>1</em>49 patients experienced at least <em>1</em> CV outcome (3.3%/patient-year). Overall, 202 CV events (4.5%/patient-year) occurred. The CV outcome rate was not related significantly to randomized interventions. In multivariable analyses, 7 baseline risk factors remained independently associated with increased risk for the CV composite outcome after controlling for age, sex, baseline GFR, and baseline proteinuria group: pulse pressure, duration of hypertension, abnormal electrocardiogram result, non-high-density lipoprotein cholesterol level, serum urea nitrogen level, urine protein-creatinine ratio, urine sodium-potassium ratio, and annual income less than <em>1</em>5,000 dollars.

CONCLUSIONS

Neither randomized class of antihypertensive therapy nor BP level had a significant effect on the occurrence of CV events, possibly because of limited power. However, this analysis identifies unique and potentially modifiable CV risk factors in this high-risk cohort.

Reduced uterine perfusion initiated in late gestation in the rat results in intrauterine growth restriction (IUGR) and development of hypertension by 4 wk of age. We hypothesize that the renin <em>angiotensin</em> system (RAS), a regulatory system important in the long-term control of blood pressure, may be programmed by placental insufficiency and may contribute to the etiology of IUGR hypertension. We previously reported that RAS blockade abolished hypertension in adult IUGR offspring; however, the mechanisms responsible for the early phase of hypertension are unresolved. Therefore, the purpose of this study was to examine RAS involvement in early programmed hypertension and to determine whether temporal changes in RAS expression are observed in IUGR offspring. Renal renin and <em>angiotensin</em>ogen mRNA expression were significantly decreased at birth (80 and 60%, respectively); plasma and renal RAS did not differ in conjunction with hypertension (mean increase of <em>1</em>4 mmHg) in young IUGR offspring; however, hypertension (mean increase of 22 mmHg) in adult IUGR offspring was associated with marked increases in renal <em>angiotensin</em>-converting enzyme (ACE) activity (<em>1</em>22%) and renal renin and <em>angiotensin</em>ogen mRNA (<em>7</em>-fold and <em>7</em>.4-fold, respectively), but no change in renal ANG II or <em>angiotensin</em> type <em>1</em> receptor. ACE inhibition (enalapril, <em>1</em>0 mg x kg(-<em>1</em>) x day(-<em>1</em>), administered from 2 to 4 wk of age) abolished hypertension in IUGR at 4 wk of age (decrease of <em>1</em>5 mmHg, respectively) with no significant depressor effect in control offspring. Therefore, temporal alterations in renal RAS are observed in IUGR offspring and may play a key role in the etiology of IUGR hypertension.

BACKGROUND

cardiovascular risk factors are associated with dementia and cognitive decline. We investigated the effects of renin-angiotensin system blockade on cognitive function in patients aged 55 years and older with established atherosclerotic cardiovascular disease or diabetes with end-organ damage in two clinical trials.

METHODS

in the main study, ONTARGET, a double-blind, double-dummy, randomised controlled trial, the effects on cardiovascular outcomes of standard doses of an angiotensin-converting enzyme (ACE) inhibitor (ramipril), an angiotensin-receptor blocker (telmisartan), and a combination of the drugs were evaluated in 25 620 participants. In the parallel TRANSCEND trial, the effects of telmisartan were compared with those of placebo in 5926 participants intolerant to ACE inhibitors. Secondary outcomes included cognitive impairment (defined by investigator-reported diagnosis of dementia or significant cognitive dysfunction, or a score of ≤ 23 on the Mini-Mental State Examination [MMSE]) and cognitive decline (a decrease of ≤ 3 points on the MMSE from baseline during follow-up). Analyses were by intention to treat. We pooled data from these studies to identify baseline predictors of cognitive impairment and its frequency according to mean systolic blood pressure during follow-up. These studies were registered with ClinicalTrials.gov, number NCT00153101.

RESULTS

During a median duration of 56 months (IQR 51-64) of follow-up in ONTARGET, cognitive impairment occurred in 652 (8%) of 7865 patients allocated ramipril, 584 (7%) of 7797 allocated telmisartan, and 618 (8%) of 7807 allocated combination treatment (combination vs ramipril, odds ratio [OR] 0·95, 95% CI 0·85-1·07, p= 0·39; telmisartan vs ramipril, OR 0·90, 0·80-1·01, p = 0·06). Corresponding figures for cognitive decline were 1314 (17%), 1279 (17%), and 1240 (17%) in each of the groups, respectively (telmisartan vs ramipril, OR 0·97, 0·89-1·06, p= 0·53; combination vs ramipril, OR 0·95, 0·88-1·04, p=0·28). In TRANSCEND, cognitive impairment occurred in 239 (9%) of 2694 participants allocated telmisartan compared with 245 (9%) of 2689 allocated placebo (OR 0·97, 0·81-1·17, p= 0·76). The corresponding figures for cognitive decline were 454 (17%) and 412 (16%; OR 1·10, 0·95-1·27, p= 0·22).

CONCLUSIONS

In patients with cardiovascular disease or diabetes, different approaches to blocking of the renin-angiotensin system had no clear effects on cognitive outcomes. Although patients with the lowest systolic blood pressure had the greatest preservation of cognitive function, meta-regression analyses did not show any benefits of blood-pressure lowering on cognition over several years of treatment.

The reduced expression of <em>angiotensin</em>-converting enzyme (ACE) 2 in the kidneys of animal models and patients with diabetes suggests ACE2 involvement in diabetic nephrology. To explore the renoprotective effects of ACE2 overexpression, ACE inhibition (ACEI) or both on diabetic nephropathy and the potential mechanisms involved, 50 Wistar rats were randomly divided into a normal group that received an injection of sodium citrate buffer and a diabetic model group that received an injection of 60 mg/kg streptozotocin. Eight wks after streptozotocin injection, the diabetic rats were divided into no treatment group, adenoviral (Ad)-ACE2 group, Ad-green flurescent protein (GFP) group, ACEI group receiving benazepril and Ad-ACE2 + ACEI group. Four wks after treatment, physical, biochemical, and renal functional and morphological parameters were measured. An experiment in cultured glomerular mesangial cells was performed to examine the effects of ACE2 on cellular proliferation, oxidative stress and collagen IV synthesis. In comparison with the Ad-GFP group, the Ad-ACE2 group exhibited reduced systolic blood pressure, urinary albumin excretion, creatinine clearance, glomeruli sclerosis index and renal malondialdehyde level; downregulated transforming growth factor (TGF)-β<em>1</em>, vascular endothelial growth factor (VEGF) and collagen IV protein expression; and increased renal superoxide dismutase activity. Ad-ACE2 and ACEI had similar effects, whereas combined use of Ad-ACE2 and ACEI offered no additional benefits. ACE2 transfection attenuated <em>angiotensin</em> (Ang) II-induced glomerular mesangial cell proliferation, oxidative stress and collagen IV protein synthesis. In conclusion, ACE2 exerts a renoprotective effect similar to that of ACEI treatment. Decreased renal Ang II, increased renal Ang-(<em>1</em>-<em>7</em>) levels, and inhibited oxidative stress were the possible mechanisms involved.

A rat <em>angiotensin</em>, type <em>1</em>A (AT<em>1</em>A) receptor cDNA was cloned recently and shown to be a member of the <em>7</em>-transmembrane, G-protein coupled family of receptors. Here, we report the cloning, sequencing, and expression of a previously unsuspected second form of the type <em>1</em> receptor (AT<em>1</em>B) in the rat which exhibits high similarity with the AT<em>1</em>A receptor relative to amino acid sequence (95% identity), binding of <em>angiotensin</em> II analogs, and utilization of Ca+2 as its intracellular second messenger. The adrenal and pituitary gland express primarily AT<em>1</em>B mRNA whereas vascular smooth muscle and lung express primarily AT<em>1</em>A mRNA. Estrogen treatment suppressed AT<em>1</em>B but not AT<em>1</em>A mRNA levels in the pituitary gland. Thus, the unexpected existence of two putative AT<em>1</em> receptor genes appears to be related to the differential regulation of their expression rather than to different functional properties of the encoded receptor proteins.

AngII (<em>angiotensin</em> II), ACE (<em>angiotensin</em> I-converting enzyme) and the AT<em>1</em> receptor (AngII type <em>1</em> receptor) are associated with the inflammatory process and microvascular dysfunction of AKI (acute kidney injury) induced by renal I/R (ischaemia/reperfusion). However, Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)], ACE2 (<em>angiotensin</em> I-converting enzyme 2) and the Mas receptor also play a role in renal disease models. Therefore, in the present study, we have examined the renal profile of Ang-(<em>1</em>-<em>7</em>), ACE2 and the Mas receptor in renal I/R and compared them with that of AngII, ACE and the AT<em>1</em> receptor. Male Wistar rats were submitted to left nephrectomy and ischaemia (45 min) followed by reperfusion (2 or 4 h) in the right kidney. At 4 h of reperfusion, renal AngII was increased (P<0.0<em>1</em>) and renal Ang-(<em>1</em>-<em>7</em>) was decreased substantially (P<0.05), although plasma levels of both <em>angiotensins</em> were unchanged. In addition, renal I/R decreased the renal mRNA expression of renin (P<0.05), AT<em>1</em> receptors (P<0.00<em>1</em>) and ACE2 (P<0.05). At 2 and 4 h of reperfusion, renal ACE activity was reduced (P<0.05). On the other hand, renal expression of the Mas receptor was greatly increased at 4 h of reperfusion (P<0.0<em>1</em>), which was confirmed by immunohistochemical and Western blot analysis. In conclusion, increased renal expression of the Mas receptor associated with changes in the RAS (renin-<em>angiotensin</em> system)-related peptidases support an important role for the ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas axis in AKI.

Previous studies have shown that glucose increases <em>angiotensin</em> II (AngII) levels in rat glomerular mesangial cells and that AngII mediates the inhibitory effects of high glucose on matrix degradation in these cells. The present study addresses the following questions: (<em>1</em>) What are the mechanisms for the generation of AngII in mesangial cells? (2) What are the effects of glucose on AngII generation by these mechanisms? Experiments employed primary mesangial cells from normal Sprague-Dawley rats. The levels of immunoreactive <em>angiotensin</em>ogen (AGT), <em>angiotensin</em> I (AngI), and <em>angiotensin</em> II (AngII) were measured by ELISA. AGT mRNA expression was determined by Northern blot analysis. Incubation of cells for 24 h in high glucose (30 mM) increased AGT levels by <em>1</em>.5-fold and increased AGT mRNA expression; this was accompanied by a <em>1</em>.5-fold increment in AngI and <em>1</em>.<em>7</em>-fold increment in AngII levels. Renin activity (measured as AngI generation in the presence of excess AGT) and ACE levels and activity were not altered by high glucose. In further experiments, the effect of high glucose on formation of Ang peptides from exogenous AngI in mesangial cell extracts was examined using HPLC. Exogenous AngI was converted into various Ang peptides, including AngII, Ang(<em>1</em>-9), Ang(<em>1</em>-<em>7</em>), and Ang(3-8). A significant increase in formation of AngII from AngI was observed in cells incubated in high glucose. In addition, AngII production from exogenous Ang(<em>1</em>-9) in cell extracts was also stimulated by high glucose. These findings demonstrate that glucose increases mesangial AngII levels via an increase in AGT and AngI. In addition, this study provides new information that Ang(<em>1</em>-9) is produced by mesangial cells, can be converted to AngII, and that this conversion is also stimulated under high-glucose conditions.

The present study sought to determine whether individual neurones of the median preoptic nucleus (MnPO) with axonal projections to the hypothalamic paraventricular nucleus (MnPO-PVN) respond to osmotic, circulating <em>angiotensin</em> II (Ang II), and baroreceptor stimulation. Hypertonic NaCl (0.<em>7</em>5 or <em>1</em>.5 osmol l(-<em>1</em>)) or Ang II (<em>1</em>50 ng) was injected into the internal carotid artery (ICA). Baroreceptor stimulation was performed by i.v. injection of phenylephrine or sodium nitroprusside to increase or decrease arterial blood pressure, respectively. Of 65 MnPO neurones, 50 units were antidromically activated from the PVN with an average onset latency of <em>1</em><em>1</em>.3 +/- 0.<em>7</em> ms. Only 9.5% of MnPO-PVN neurones were antidromically activated from the PVN bilaterally. Type I MnPO-PVN neurones (n = <em>1</em>4) responded to osmotic but not Ang II stimulation. In <em>7</em>9% (<em>1</em><em>1</em>/<em>1</em>4) of these type I neurones, the response was an increase in cell discharge. Type II MnPO-PVN neurones (n = <em>7</em>) displayed a significant increase in cell discharge in response to ICA injection of Ang II but not hypertonic NaCl. Type III MnPO-PVN neurones (n = <em>1</em>6) responded to both ICA injection of hypertonic NaCl and Ang II. In 88% (<em>1</em>4/<em>1</em>6) of type III neurones, osmotic and Ang II stimulation each increased cell discharge. Type IV MnPO-PVN neurones (n = <em>1</em>3) displayed no change in cell discharge in response to ICA injection of hypertonic NaCl or Ang II. Baroreceptor stimulation altered the discharge in subpopulations of type I, II and III MnPO-PVN neurones (43-63% depending on neuronal type). Only one MnPO-PVN neurone responded solely to baroreceptor stimulation (type IV). In addition, a subset of type I, II and III neurones displayed a significant correlation with sympathetic nerve activity and/or the cardiac cycle. These findings suggest that a significant population of MnPO-PVN neurones respond to osmotic and circulating Ang II stimulation and thereby represents a neural substrate through which neurohumoral inputs are integrated within the forebrain lamina terminalis.

Vascular immunotargeting is a mean for a site-selective delivery of drugs and genes to endothelium. In this study, we compared recognition of pulmonary and systemic vessels in rats by candidate carrier monoclonal antibodies (MAbs) to endothelial antigens platelet endothelial cell adhesion molecule (PECAM)-<em>1</em> (CD3<em>1</em>), intercellular adhesion molecule (ICAM)-<em>1</em> (CD54), Thy-<em>1</em>.<em>1</em> (CD90.<em>1</em>), <em>angiotensin</em>-converting enzyme (ACE; CD<em>1</em>43), and OX-43. Tissue immunostaining showed that endothelial cells were Thy-<em>1</em>.<em>1</em> positive in capillaries but negative in large vessels. In the lung, anti-ACE MAb provided a positive staining in <em>1</em>00% capillaries vs. 5-20% capillaries in other organs. Other MAbs did not discriminate between pulmonary and systemic vessels. We determined tissue uptake after infusion of <em>1</em> microg of (<em>1</em>25)I-labeled MAbs in isolated perfused lungs (IPL) or intravenously in intact rats. Uptake in IPL attained 46% of the injected dose (ID) of anti-Thy-<em>1</em>.<em>1</em> and 20-25% ID of anti-ACE, anti-ICAM-<em>1</em>, and anti-OX-43 (vs. 0.5% ID of control IgG). However, after systemic injection at this dose, only anti-ACE MAb 9B9 displayed selective pulmonary uptake (<em>1</em>6 vs. <em>1</em>% ID/g in other organs). Anti-OX-43 displayed low pulmonary (0.5% ID/g) but significant splenic and cardiac uptake (<em>7</em> and 2% ID/g). Anti-Thy-<em>1</em>.<em>1</em> and anti-ICAM-<em>1</em> displayed moderate pulmonary (4 and 6% ID/g, respectively) and high splenic and hepatic uptake (e.g., <em>1</em>8% ID/g of anti-Thy-<em>1</em>.<em>1</em> in spleen). The lung-to-blood ratio was 5, <em>1</em>0, and <em>1</em>5 for anti-Thy-<em>1</em>.<em>1</em>, anti-ACE, and anti-ICAM-<em>1</em>, respectively. PECAM antibodies displayed low pulmonary uptake in perfusion (2% ID) and in vivo (3-4% ID/g). However, conjugation with streptavidin (SA) markedly augmented pulmonary uptake of anti-PECAM in perfusion (<em>1</em>0-54% ID, depending on an antibody clone) and in vivo (up to <em>1</em>5% ID/g). Therefore, ACE-, Thy-<em>1</em>.<em>1</em>-, ICAM-<em>1</em>-, and SA-conjugated PECAM MAbs are candidate carriers for pulmonary targeting. ACE MAb offers a high selectivity of pulmonary targeting in vivo, likely because of a high content of ACE-positive capillaries in the lungs.

The T allele of the C825T polymorphism of the gene encoding the beta(3)-subunit of G proteins has been associated with increased sodium-hydrogen exchange and low renin in patients with essential hypertension. To assess its association with blood pressure response to diuretic therapy, we measured the C825T polymorphism in <em>1</em>9<em>7</em> blacks (<em>1</em>34 men, 63 women) and <em>1</em>90 non-Hispanic whites (<em>7</em>6 men, <em>1</em><em>1</em>4 women) with essential hypertension (mean+/-SD age 48+/-<em>7</em> years), who underwent monotherapy with hydrochlorothiazide for 4 weeks. Mean declines in systolic and diastolic blood pressures were 6+/-2 (P:<0.00<em>1</em>) and 5+/-<em>1</em> (P:<0.00<em>1</em>) mm Hg greater, respectively, in TT than in CC homozygotes. Responses in heterozygotes were intermediate between the homozygous groups. Other univariate predictors of greater blood pressure responses included black race, female gender, higher pretreatment blood pressure, older age, lower waist-to-hip ratio, and measures of lower renin-<em>angiotensin</em>-aldosterone system activity. After the effects of the other predictors were considered, the TT genotype remained a significant predictor of greater declines in systolic and diastolic blood pressures. Thus, the C825T polymorphism of the G protein beta(3)-subunit may help identify patients with essential hypertension who are more responsive to diuretic therapy.

Prostaglandins may modulate renal function and play a role in the hyperreninism and <em>angiotensin</em> pressor resistance of chronic liver disease. To study this possibility, we evaluated <em>1</em>2 patients with alcoholic cirrhosis and ascites. Urine immunoassayable prostaglandin E in 5 female patients was 3.3 +/- 0.5 micrograms/day [normal, 0.3 +/- 0.<em>1</em> (SE)], renin was <em>1</em>4.6 +/- 3.<em>7</em> ng/ml.h, and aldosterone was <em>7</em>6 +/- <em>1</em>9 ng/dl. After either indomethacin (200 mg) or ibuprofen (2000 mg) for <em>1</em> day, urine immunoassayable prostaglandin E fell to 0.8 +/- 0.4 micrograms/day, renin to 8.0 +/- 2.4 ng/mol.h, and aldosterone to 54 +/- <em>1</em>4 ng/dl (all P less than 0.0<em>1</em>). Pressor sensitivity increased dramatically, and creatinine clearance transiently fell from <em>7</em>3 +/- <em>1</em>0 to 32 +/- <em>7</em> cc/min (P less than 0.0<em>1</em>). Because a primary effect on renin might explain the renal impairment, an additional study used propranolol to lower renin activity. Renal function was unaltered by propranolol. We conclude that prostaglandins play a supportive role in maintaining renal function and are involved in the hyperreninism and pressor resistance of patients with liver disease.

<em>Angiotensin</em> (Ang) II mediates pathophysiologial changes in the kidney. Ang-(<em>1</em>-<em>7</em>) by interacting with the G protein-coupled receptor Mas may also have important biological activities.In this study, renal deficiency for Mas diminished renal damage in models of renal insufficiency as unilateral ureteral obstruction and ischemia/reperfusion injury while the infusion of Ang-(<em>1</em>-<em>7</em>) to wild-type mice pronounced the pathological outcome by aggravating the inflammatory response. Mas deficiency inhibited NF-kappaB activation and thus the elevation of inflammation-stimulating cytokines, while Ang-(<em>1</em>-<em>7</em>) infusion had proinflammatory properties in experimental models of renal failure as well as under basal conditions. The Ang-(<em>1</em>-<em>7</em>)-mediated NF-kappaB activation was Mas dependent but did not involve Ang II receptors. Therefore, the blockade of the NF-kappaB-activating properties of the receptor Mas could be a new strategy in the therapy of failing kidney.

The renin-<em>angiotensin</em> system (RAS) is thought to regulate placentation, however, the expression and localization of RAS pathways in early gestation human placenta is not known. Here we describe the expression of prorenin (REN), (pro)renin receptor (ATP6AP2), <em>angiotensin</em>ogen (AGT), <em>angiotensin</em>-converting enzyme <em>1</em> and 2 (ACE; ACE2), <em>angiotensin</em> II type <em>1</em> and 2 receptors (AGTR<em>1</em>; AGTR2) and <em>angiotensin</em> <em>1</em>-<em>7</em> receptor (MAS<em>1</em>), as well as the angiogenic factor, vascular endothelial growth factor (VEGF), and transforming growth factor-β<em>1</em> (TGF-β<em>1</em>), in early gestation (6-<em>1</em>6 weeks) and term (>3<em>7</em> weeks) human placentae. We also describe the location of all of the key RAS proteins in the early gestation placentae. The highest levels of REN, ATP6AP2, AGT, AGTR<em>1</em> and ACE2 mRNAs were found in early gestation, whereas ACE<em>1</em> mRNA was highest at term. AGTR2 and MAS<em>1</em> mRNA expression were low to undetectable in all samples. REN, ATP6AP2 and AGTR<em>1</em> mRNA levels were correlated with VEGF expression, but not with TGF-β<em>1</em> mRNA. In early gestation placentae, prorenin, (pro)renin receptor and the <em>angiotensin</em> II type <em>1</em> receptor (AT(<em>1</em>)R) were localized to extravillous trophoblast cells, suggesting they play a key role in trophoblast migration. ACE2 in syncytiotrophoblasts could regulate release of Ang <em>1</em>-<em>7</em> into the maternal circulation contributing to the vasodilation of the maternal vasculature. ACE was only found in fetal vascular endothelium and may specifically target the growing fetal placental vessels. Because REN, ATP6AP2 and AGTR<em>1</em> show strong correlations with expression of VEGF this pathway is likely to be important in placental angiogenesis.