Endogenous Renin-angiotensin System Research Articles

Effects of intrarenal angiotensin 1-7 infusion on renal haemodynamic and excretory function in anaesthetised two-kidney one-clip and deoxycorticosterone acetate-salt hypertensive rats.

What is the central question of this study? Are renal functional responses to intrarenal angiotensin 1-7 (Ang (1-7)) infusion dependent on the level of the endogenous renin-angiotensin system (RAS) in the two-kidney one-clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt animal models of hypertension? What is the main finding and its importance? The renal actions of Ang (1-7) are dependent on the relative endogenous levels of each arm of the classical angiotensin II-angiotensin II type 1 receptor (AT1 R) axis and those of the Ang (1-7)-Mas receptor axis. These findings support the hypothesis that a balance exists between the intrarenal classical and novel arms of the RAS, and in particular the relative abundance of AT1 R to Mas receptor, which may to a large extent determine the renal excretory response to Ang (1-7) infusion. This study investigated the action of angiotensin 1-7 (Ang (1-7)) on renal haemodynamic and excretory function in the two-kidney one-clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt rat models of hypertension, in which the endogenous renin-angiotensin system (RAS) activity was likely to be raised or lowered, respectively. Rats were anaesthetised and prepared for the measurement of mean arterial pressure and kidney function during renal interstitial infusion of Ang (1-7) or saline. Kidney tissue concentrations of angiotensin II (Ang II) and Ang (1-7) were determined. Intrarenal infusion of Ang (1-7) into the clipped kidney of 2K1C rats increased urine flow (UV), absolute (UNa V) and fractional sodium (FENa ) excretions by 110%, 214% and 147%, respectively. Renal Ang II concentrations of the clipped kidney were increased with no major changes in Ang (1-7) concentration. By contrast, Ang (1-7) infusion decreased UV, UNa V, and FENa by 27%, 24% and 21%, respectively in the non-clipped kidney in which tissue Ang (1-7) concentrations were increased, but renal Ang II concentrations were unchanged compared to sham animals. Ang (1-7) infusion in DOCA-salt rats had minimal effects on glomerular filtration rate but significantly decreased UV, UNa V and FENa by ∼30%. Renal Ang (1-7) concentrations were higher and Ang II concentrations were lower in DOCA-salt rats compared to sham rats. These findings demonstrate that the intrarenal infusion of exogenous Ang (1-7) elicits different renal excretory responses the magnitude of which is dependent on the balance between the endogenous renal Ang II-AT1 receptor axis and Ang (1-7)-Mas receptor axis.

Melanocortin MC4R receptor is required for energy expenditure but not blood pressure effects of angiotensin II within the mouse brain.

The brain renin-angiotensin system (RAS) is implicated in control of blood pressure (BP), fluid intake, and energy expenditure (EE). Angiotensin II (ANG II) within the arcuate nucleus of the hypothalamus contributes to control of resting metabolic rate (RMR) and thereby EE through its actions on Agouti-related peptide (AgRP) neurons, which also contribute to EE control by leptin. First, we determined that although leptin stimulates EE in control littermates, mice with transgenic activation of the brain RAS (sRA) exhibit increased EE and leptin has no additive effect to exaggerate EE in these mice. These findings led us to hypothesize that leptin and ANG II in the brain stimulate EE through a shared mechanism. Because AgRP signaling to the melanocortin MC4R receptor contributes to the metabolic effects of leptin, we performed a series of studies examining RMR, fluid intake, and BP responses to ANG II in mice rendered deficient for expression of MC4R via a transcriptional block (Mc4r-TB). These mice were resistant to stimulation of RMR in response to activation of the endogenous brain RAS via chronic deoxycorticosterone acetate (DOCA)-salt treatment, whereas fluid and electrolyte effects remained intact. These mice were also resistant to stimulation of RMR via acute intracerebroventricular (ICV) injection of ANG II, whereas BP responses to ICV ANG II remained intact. Collectively, these data demonstrate that the effects of ANG II within the brain to control RMR and EE are dependent on MC4R signaling, whereas fluid homeostasis and BP responses are independent of MC4R signaling.

Critical role of the endogenous renin-angiotensin system in maintaining self-renewal and regeneration potential of epidermal stem cells

A tightly controlled activity of renin-angiotensin system (RAS) including renin, angiotensin-converting enzymes (ACEs), and angiotensin II (Ang II) receptors is critical not only for maintaining systemic hemodynamics and blood volume but also for controlling cell proliferation, differentiation, and tissue remodeling in target organs. ACE inhibitors or Ang II receptor type 1 (AT1R) blockers are widely used as first line drugs for the treatment of cardiovascular diseases that are caused by chronical activation of RAS. However, about 15% of patients using ACE inhibitors develop side effects in the skin and the underlying mechanisms have been poorly understood or even neglected. Herein we show an endogenous RAS in maintaining self-renewal and regeneration potential of epidermal stem cells (ESCs) thereby contributing to wound healing. Firstly, we found that ESCs may express ACE, and its members in wound edges were positively associated with wound healing in Captopril-treated rats. Secondly, we demonstrated that human ESCs had a functional RAS including ACE1, ACE2, Ang II, AT1R, and AT2R. ACE-Ang II axis maintains human ESC function via activation of both AT1R and AT2R, which are negatively regulated by each other. Ang II-induced activation of extracellular signal-regulated kinase (ERK) and signal transducers and activators of transcription (STAT)1 and STAT3 was mediated by the negative cross-talk between AT1R and AT2R in human ESCs. These results suggest that Ang II is a critical regulator of ESC function and ESC-mediated epidermal regeneration. Inappropriate interruption of Ang II-operated signaling may prejudice ESC function leading to impaired skin wound healing or even disease.

Interference With Endothelial PPAR (Peroxisome Proliferator-Activated Receptor)-γ Causes Accelerated Cerebral Vascular Dysfunction in Response to Endogenous Renin-Angiotensin System Activation.

Low-salt diet is beneficial in salt-sensitive hypertension but may provoke cardiovascular risk in patients with heart failure, diabetes mellitus, or other cardiovascular abnormalities because of endogenous renin-angiotensin system activation. PPAR (peroxisome proliferator-activated receptor)-γ is a transcription factor which promotes an antioxidant pathway in the endothelium. We studied transgenic mice expressing a dominant-negative mutation in PPAR-γ selectively in the endothelium (E-V290M) to test the hypothesis that endothelial PPAR-γ plays a protective role in response to low salt-mediated renin-angiotensin system activation. Plasma renin and Ang II (angiotensin II) were significantly and equally increased in all mice fed low salt for 6 weeks. Vasorelaxation to acetylcholine was not affected in basilar artery from E-V290M at baseline but was significantly and selectively impaired in E-V290M after low salt. Unlike basilar artery, low salt was not sufficient to induce vascular dysfunction in carotid artery or aorta. Endothelial dysfunction in the basilar artery from E-V290M mice fed low salt was attenuated by scavengers of superoxide, inhibitors of NADPH oxidase, or blockade of the Ang II AT1 (angiotensin type-1) receptor. Simultaneous AT1 and AT2 receptor blockade revealed that the restoration of endothelial function after AT1 receptor blockade was not a consequence of AT2 receptor activation. We conclude that interference with PPAR-γ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to low salt-mediated activation of the endogenous renin-angiotensin system, mediated at least in part, through AT1 receptor activation and perturbed redox homeostasis. Moreover, our data suggest that the cerebral circulation may be particularly sensitive to inhibition of PPAR-γ activity and renin-angiotensin system activation.

Abstract 036: Interference With PPARγ in the Endothelium Produces Endothelial Dysfunction in the Cerebral Circulation in Response to Activation of the Endogenous Renin-Angiotensin System

Low salt diet (LSD) is beneficial in salt-sensitive hypertension but may provoke cardiovascular risk in patients with heart failure, diabetes, or other cardiovascular abnormalities because of renin-angiotensin system (RAS) activation. PPARγ is a transcription factor which promotes an anti-oxidant pathway in the endothelium. We studied transgenic mice expressing a dominant-negative mutation in PPARγ selectively in the endothelium (E-DN) to test the hypothesis that endothelial PPARγ plays a protective role in response to LSD-mediated RAS activation. Plasma renin and angiotensin were significantly and equally increased in all mice fed LSD for 6-weeks (Renin - NT: 39±7 vs 20±1 ng/ml; E-DN: 34±1 vs 16±4 ng/ml; Ang - NT: 257±54 vs 47±6 pg/ml; E-DN: 294±69 vs 63±14 pg/ml p<0.05, n=5). Vasorelaxation to acetylcholine was not affected in basilar artery from E-DN at baseline, but was significantly and selectively impaired in E-DN after LSD (33±5 vs 69±2%, p<0.05, n=6). Unlike basilar artery, LSD was not sufficient to induce vascular dysfunction in carotid artery (carotid artery: 86±4 vs 92±3%, n=5). Endothelial dysfunction in the basilar artery from E-DN mice fed LSD was attenuated by scavengers of superoxide (improved from 29±5% to 55±7%, n=6), inhibitors of NADPH oxidase (improved from 23±3% to 54±7%, p<0.05, n=6), or blockade of the angiotensin-II AT1 receptor (improved from 31±5% to 64±9%, p<0.05, n=5). Gene expression levels of Nox2 was elevated (2.1±0.3 vs 0.4±0.1, p<0.05, n=7) while those of antioxidant enzymes catalase and SOD3 were blunted in cerebral vessels of E-DN mice on a LSD (catalase: 0.5±0.1 vs 2.5±0.2; SOD3: 0.2±0.1 vs 1.1±0.1, p<0.05, n=7). Simultaneous AT1 and AT2 receptor blockade revealed the restoration of endothelial function after AT1 receptor blockade was not a consequence of AT2 receptor activation (59±10 vs 48±2, p<0.05, n=4). We conclude that interference with PPARγ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to LSD-mediated activation of the endogenous RAS, mediated at least in part, through AT1 receptor activation and perturbed redox homeostasis. Moreover, our data suggest that the cerebral circulation may be particularly sensitive to inhibition of PPARγ activity and RAS activation.

Endogenous Renin‐Angiotensin System Activation Causes Accelerated Cerebral Vascular Dysfunction in Mice Expressing Dominant‐Negative Mutations in PPARγ in Endothelium

Abnormal activation of the renin‐angiotensin system (RAS) has been implicated in cardiovascular (CV) disease. Whereas low salt diet (LSD) may be beneficial in salt‐sensitive hypertension, it has been proposed to induce CV risk due to RAS activation. PPARγ is a ligand‐activated transcription factor which regulates the actions of angiotensin II (ANG) in the vasculature and promotes anti‐oxidant pathways. We hypothesized that endothelial PPARγ plays a protective role in the vasculature in response to RAS activation. Transgenic mice specifically expressing dominant‐negative mutation in PPARγ in the endothelium (E‐DN) were fed a LSD. Plasma renin and ANG were significantly increased in both non‐transgenic (NT) and E‐DN mice fed a LSD for 6 weeks compared with normal chow (Renin ‐ NT: 39±7 vs 20±1 ng/ml; E‐DN: 34±1 vs 16±4 ng/ml; ANG ‐ NT: 257±54 vs 47±6 pg/ml; E‐DN: 294±69 vs 63±14 pg/ml p<0.05, n=5). At baseline, vasorelaxation to acetylcholine (Ach) was not affected in E‐DN compared to non‐transgenic (NT) controls. Six weeks of LSD significantly impaired acetylcholine‐mediated relaxation in the basilar artery of E‐DN but not in NT (33±5 vs 69±2%, p<0.05, n=6). Unlike basilar artery, 6 weeks of LSD was not sufficient to induce vascular dysfunction in carotid artery or aorta of E‐DN (carotid artery: 92±6 vs 86±9%, n=5; aorta: 87±6 vs 81±5%, n=3). The endothelial dysfunction in the basilar artery of E‐DN was attenuated upon treatment with inhibitors of superoxide (improved from 29±5% to 55±6%, p<0.05, n=7) and NADPH oxidase (improved from 24±4% to 65±6%, p<0.05, n=3). Consistent with this, gene expression levels of Nox2 was elevated (2.1±0.3 vs 0.4±0.1, p<0.05, n=7) while those of antioxidant enzymes catalase and SOD3 were significantly blunted in cerebral vessels of E‐DN mice on a LSD (catalase: 0.5±0.1 vs 2.5±0.2; SOD3: 0.2±0.1 vs 1.1±0.1, p<0.05, n=7). Further, blockade of AT1 receptor using Losartan (0.6g/L drinking water for the last 3 weeks of LSD regimen) restored the endothelial function in the basilar artery of E‐DN mice fed LSD (improved from 31±5% to 64±9%, p<0.05, n=5). Simultaneous blockade of AT1 and AT2 (PD123319 10 mg/kg/day sc) receptors did not affect the improvement in ACh‐mediated relaxation (53±3 vs 23±3, p<0.05, n=4), suggesting that the restoration of endothelial function in the basilar artery of E‐DN mice upon losartan administration was not a consequence of AT2 receptor activation. Thus, interference with PPARγ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to LSD‐mediated activation of the endogenous RAS and this dysfunction is mediated, at least in part, through AT1 receptor activation and perturbed redox homeostasis. Moreover, our data suggest that the basilar artery and perhaps cerebral circulation is particularly sensitive to inhibition of PPARγ activity and RAS activation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 140: Endogenous Renin-angiotensin System Activation Causes Accelerated Cerebral Vascular Dysfunction in Mice Expressing Dominant-negative Mutations in PPARγ in Endothelium

Abnormal activation of the renin-angiotensin system (RAS) has been implicated in cardiovascular (CV) disease. Whereas low salt diet (LSD) may be beneficial in salt-sensitive hypertension, it has been proposed to induce CV risk due to RAS activation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor which regulates the actions of angiotensin II (ANG) in the vasculature and promotes anti-oxidant pathways. We hypothesize that endothelial PPARγ plays a protective role in the vasculature in response to RAS activation. Transgenic mice specifically expressing dominant-negative (DN) mutations in PPARγ in the endothelium (E-DN) were fed a LSD and endothelial function was measured. Plasma renin and ANG were significantly increased in both non-transgenic (NT) and E-DN mice fed a LSD for 6 weeks compared with normal chow (Renin - NT: 39±7 vs 20±1 ng/ml; E-DN: 34±1 vs 16±4 ng/ml; AngII - NT: 257±54 vs 47±6 pg/ml; E-DN: 294±69 vs 63±14 pg/ml p<0.05, n=5). At baseline, vasorelaxation to acetylcholine (ACh) was not affected in E-DN compared to NT (basilar artery: 66±12 vs 64±4%; carotid artery: 93±4 vs 91±4%, n=5). Six weeks of LSD significantly impaired ACh-mediated relaxation in basilar artery of E-DN but not in NT (42±8 vs 74±5%, p<0.05, n=5). Unlike basilar artery, 6 weeks of LSD was not sufficient to induce vascular dysfunction in carotid artery of E-DN (carotid artery: 86±4 vs 92±3%, n=5). The endothelial dysfunction observed in the basilar artery of E-DN was attenuated upon in vitro incubation with tempol (improved from 29±5% to 55±7%, n=6). Further, administration of the AT1 receptor blocker, Losartan (0.6g/L drinking water) for the last 2 weeks of LSD blunted the endothelial dysfunction observed in the basilar artery of E-DN (improved from 24±2% to 64±9%, n=5). We conclude that interference with PPARγ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to LSD-mediated activation of the endogenous RAS and this dysfunction is mediated, at least in part, through AT1 receptor activation and ROS signaling pathways. Moreover, our data suggest that the basilar artery and perhaps cerebral circulation is particularly sensitive to inhibition of PPARγ activity and activation of the RAS.

Pressure-overload-induced angiotensin-mediated early remodeling in mouse heart.

Our previous work on angiotensin II-mediated electrical-remodeling in canine left ventricle, in connection with a long history of other studies, suggested the hypothesis: increases in mechanical load induce autocrine secretion of angiotensin II (A2), which coherently regulates a coterie of membrane ion transporters in a manner that increases contractility. However, the relation between load and A2 secretion was correlative. We subsequently showed a similar or identical system was present in murine heart. To investigate whether the relation between mechanical load and A2-mediated electrical remodeling was causal, we employed transverse aortic constriction in mice to subject the left ventricle to pressure overload for short-term (1 to 2 days) or long-term (1 to 2 weeks) periods. Heart-to-body weight ratios and cell capacitance measurements were used to determine hypertrophy. Whole-cell patch clamp recordings of the predominant repolarization currents Ito,fast and IK,slow were used to assess electrical remodeling. Hearts or myocytes subjected to long-term load displayed significant hypertrophy, which was not evident in short-term load. However, short-term load induced significant reductions in Ito,fast and IK,slow. Incubation of these myocytes with the angiotensin II type 1 receptor inhibitor saralasin for 2 hours restored Ito,fast and IK,slow to control levels. The number of Ito.fast or IK,slow channels did not change with A2 or long-term load, however the hypertrophic increase in membrane area reduced the current densities for both channels. For Ito,fast but not IK,slow there was an additional reduction that was reversed by inhibition of angiotensin receptors. These results suggest increased load activates an endogenous renin angiotensin system that initially reduces Ito,fast and IK,slow prior to the onset of hypertrophic growth. However, there are functional interactions between electrical and anatomical remodeling. First, hypertrophy tends to reduce all current densities. Second, the hypertrophic program can modify signaling between the angiotensin receptor and target current.

C-C Motif Chemokine 5 Attenuates Angiotensin II–Dependent Kidney Injury by Limiting Renal Macrophage Infiltration

Inappropriate activation of the renin angiotensin system (RAS) is a key contributor to the pathogenesis of essential hypertension. During RAS activation, infiltration of immune cells into the kidney exacerbates hypertension and renal injury. However, the mechanisms underpinning the accumulation of mononuclear cells in the kidney after RAS stimulation remain unclear. C-C motif chemokine 5 (CCL5) drives recruitment of macrophages and T lymphocytes into injured tissues, and we have found that RAS activation induces CCL5 expression in the kidney during the pathogenesis of hypertension and renal fibrosis. We therefore evaluated the contribution of CCL5 to renal damage and fibrosis in hypertensive and normotensive models of RAS stimulation. Surprisingly, during angiotensin II-induced hypertension, CCL5-deficient (knockout, KO) mice exhibited markedly augmented kidney damage, macrophage infiltration, and expression of proinflammatory macrophage cytokines compared with wild-type controls. When subjected to the normotensive unilateral ureteral obstruction model of endogenous RAS activation, CCL5 KO mice similarly developed more severe renal fibrosis and greater accumulation of macrophages in the kidney, congruent with enhanced renal expression of the macrophage chemokine CCL2. In turn, pharmacologic inhibition of CCL2 abrogated the differences between CCL5 KO and wild-type mice in kidney fibrosis and macrophage infiltration after unilateral ureteral obstruction. These data indicate that CCL5 paradoxically limits macrophage accumulation in the injured kidney during RAS activation by constraining the proinflammatory actions of CCL2.

Abstract P205: Endothelium-specific Interference with PPARG Causes Cerebral Vascular Dysfunction in Response to Endogenous Renin-angiotensin System Activation

Abnormal activation of the endogenous renin-angiotensin system (RAS) has been implicated in various cardiovascular (CV) disorders including hypertension, atherosclerosis and stroke. Whereas a low salt diet may be beneficial in salt-sensitive hypertension, it has been proposed to also cause CV risk due to activation of the RAS. The molecular mechanism by which RAS activation mediates vascular dysfunction remains undefined. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor which activates anti-oxidant and anti-inflammatory processes and can regulate the actions of angiotensin II (AngII) in the vasculature. We examined endothelial function in transgenic mice specifically expressing dominant-negative (DN) mutations in PPARγ in the endothelium (E-V290M) fed a low salt diet to test the hypothesis that endothelial PPARγ plays a protective role in the vasculature in response to endogenous RAS activation. Circulating levels of renin were significantly increased in both non-transgenic (NT) and E-V290M mice fed a low-salt diet for 6 weeks compared to standard chow (NT: 39.3±7.4 vs 19.8±1.3 ng/ml; E-V290M: 34.3±0.8 vs 16.0±3.8 ng/ml, p<0.05, n=5). Under baseline conditions, responses to endothelium-dependent agonist acetylcholine were not affected in E-V290M mice compared to NT (basilar artery: 66.1±11.8 vs 63.5±3.7%; carotid artery: 93.3±3.6 vs 91.1±4.2%, n=5). Six weeks of low-salt diet significantly impaired acetylcholine-mediated dilation in the basilar artery of E-V290M mice but not in NT (41.7±7.7 vs 74.2±5.0%, p<0.05, n=5). Unlike basilar artery, 6 weeks of low salt diet was not sufficient to induce vascular dysfunction in carotid artery or aorta of E-V290M mice (carotid artery: 85.6±4.4 vs 91.9±2.5%, n=5; aorta: 80.8±5.4 vs 87.0±5.6%, n=3). The responses to endothelium-independent vasodilator sodium nitroprusside (SNP) were not different in E-V290M mice compared to NT controls. We conclude that endothelial-specific interference with PPARγ causes endothelial dysfunction in response to endogenous RAS activation induced by a low-salt diet. Moreover, the cerebral circulation is particularly susceptible to low salt diet-induced dysfunction in conjunction with PPARγ impairment.

Sex-linked differences in the course of chronic kidney disease and congestive heart failure: a study in 5/6 nephrectomized Ren-2 transgenic hypertensive rats with volume overload induced using aorto-caval fistula.

The role of hypertension and the renin-angiotensin system (RAS) in sex-related differences in the course of chronic kidney disease (CKD) and congestive heart failure (CHF) remain unclear, especially when the two diseases are combined. In male and female Ren-2 transgenic rats (TGR), a model of hypertension with activation of endogenous RAS, CKD was induced by 5/6 renal mass reduction (5/6 NX) and CHF was elicited by volume overload achieved by creation of an aorto-caval fistula (ACF). The primary aim of the study was to examine long-term CKD- and CHF-related mortality, especially in animals with CKD and CHF combined, with particular interest in the potential sex-related differences. The follow-up period was 23weeks after the first intervention (5/6 NX). We found, first, that TGR did not exhibit sexual dimorphism in the course of 5/6 NX-induced CKD. Second, in contrast, TGR exhibited important sex-related differences in the course of ACF-induced CHF-related mortality: intact female TGR showed higher survival rate than male TGR. This situation is reversed in the course of combined 5/6 NX-induced CKD and ACF-induced CHF-related mortality: intact female TGR exhibited poorer survival than male TGR. Third, the survival rate in animals with combined 5/6 NX-induced CKD and ACF-induced CHF was significantly worsened as compared with rat groups that were exposed to 'single organ disease'. Collectively, our present results clearly show that CKD aggravates long-term mortality of animals with CHF. In addition, TGR exhibit remarkable sexual dimorphism with respect to CKD- and CHF-related mortality, especially in animals with combined CKD and CHF.

Exogenous and endogenous angiotensin-II decrease renal cortical oxygen tension in conscious rats by limiting renal blood flow.

Key points Our understanding of the mechanisms underlying the role of hypoxia in the initiation and progression of renal disease remains rudimentary.We have developed a method that allows wireless measurement of renal tissue oxygen tension in unrestrained rats.This method provides stable and continuous measurements of cortical tissue oxygen tension (PO2) for more than 2 weeks and can reproducibly detect acute changes in cortical oxygenation.Exogenous angiotensin‐II reduced renal cortical tissue PO2 more than equi‐pressor doses of phenylephrine, probably because it reduced renal oxygen delivery more than did phenylephrine.Activation of the endogenous renin–angiotensin system in transgenic Cyp1a1Ren2 rats reduced cortical tissue PO2; in this model renal hypoxia precedes the development of structural pathology and can be reversed acutely by an angiotensin‐II receptor type 1 antagonist.Angiotensin‐II promotes renal hypoxia, which may in turn contribute to its pathological effects during development of chronic kidney disease. We hypothesised that both exogenous and endogenous angiotensin‐II (AngII) can decrease the partial pressure of oxygen (PO2) in the renal cortex of unrestrained rats, which might in turn contribute to the progression of chronic kidney disease. Rats were instrumented with telemeters equipped with a carbon paste electrode for continuous measurement of renal cortical tissue PO2. The method reproducibly detected acute changes in cortical oxygenation induced by systemic hyperoxia and hypoxia. In conscious rats, renal cortical PO2 was dose‐dependently reduced by intravenous AngII. Reductions in PO2 were significantly greater than those induced by equi‐pressor doses of phenylephrine. In anaesthetised rats, renal oxygen consumption was not affected, and filtration fraction was increased only in the AngII infused animals. Oxygen delivery decreased by 50% after infusion of AngII and renal blood flow (RBF) fell by 3.3 ml min−1. Equi‐pressor infusion of phenylephrine did not significantly reduce RBF or renal oxygen delivery. Activation of the endogenous renin–angiotensin system in Cyp1a1Ren2 transgenic rats reduced cortical tissue PO2. This could be reversed within minutes by pharmacological angiotensin‐II receptor type 1 (AT1R) blockade. Thus AngII is an important modulator of renal cortical oxygenation via AT1 receptors. AngII had a greater influence on cortical oxygenation than did phenylephrine. This phenomenon appears to be attributable to the profound impact of AngII on renal oxygen delivery. We conclude that the ability of AngII to promote renal cortical hypoxia may contribute to its influence on initiation and progression of chronic kidney disease.

Activation of the Renin-Angiotensin System Promotes Colitis Development.

The renin-angiotensin system (RAS) plays pathogenic roles in renal and cardiovascular disorders, but whether it is involved in colitis is unclear. Here we show that RenTgMK mice that overexpress active renin from the liver developed more severe colitis than wild-type controls. More than 50% RenTgMK mice died whereas all wild-type mice recovered. RenTgMK mice exhibited more robust mucosal TH17 and TH1/TH17 responses and more profound colonic epithelial cell apoptosis compared to wild-type controls. Treatment with aliskiren (a renin inhibitor), but not hydralazine (a smooth muscle relaxant), ameliorated colitis in RenTgMK mice, although both drugs normalized blood pressure. Chronic infusion of angiotensin II into wild-type mice mimicked the severe colitic phenotype of RenTgMK mice, and treatment with losartan [an angiotensin type 1 receptor blocker (ARB)] ameliorated colitis in wild-type mice, confirming a colitogenic role for the endogenous RAS. In human biopsies, pro-inflammatory cytokines were suppressed in patients with inflammatory bowel disease who were on ARB therapy compared to patients not receiving ARB therapy. These observations demonstrate that activation of the RAS promotes colitis in a blood pressure independent manner. Angiotensin II appears to drive colonic mucosal inflammation by promoting intestinal epithelial cell apoptosis and mucosal TH17 responses in colitis development.

THE RELATION BETWEEN FIBRINOLYTIC SYSTEM AND THE RENIN ANGIOTENSIN SYSTEM

Several lines of evidence point to an interrelation of the renin-angiotensin system (RAS) with the endogenous fibrinolytic system. This study was planned to examine the effect of salt depletion as a method of activation of the endogenous RAS on plasma fibrinolytic balance in 10 healthy human subjects in the presence and absence of angiotensin converting enzyme inhibitor-ACEi (captopril). Activation of the RAS during low salt intake was documented by a significant increase in serum aldosterone concentration. The data suggest that activation of the RAS results in increased plasminogen activator inhibitors (PAI-I) antigen and that interruption of the RAS with the ACE inhibitor captopril significantly lowers PAI-I antigen without lowering tissue-type plasminogen activator (t-PA) antigen. In conclusion, this study provides an evidence of a direct functional link between the RAS and the fibrinolytic system in humans and these findings may help to elucidate possible mechanisms by which ACE inhibition exerts vasculo protective effects and reduces the risk of atherothrombotic events.

Sex-Specific Effect of Endothelin in the Blood Pressure Response to Acute Angiotensin II in Growth-Restricted Rats.

The renal endothelin system contributes to sex differences in blood pressure with males demonstrating greater endothelin type-A receptor-mediated responses relative to females. Intrauterine growth restriction programs hypertension and enhance renal sensitivity to acute angiotensin II in male growth-restricted rats. Endothelin is reported to work synergistically with angiotensin II. Thus, this study tested the hypothesis that endothelin augments the blood pressure response to acute angiotensin II in male growth-restricted rats. Systemic and renal hemodynamics were determined in response to acute angiotensin II (100 mg/kg per minute for 30 minutes) with and without the endothelin type-A receptor antagonist, Atrasentan (ABT-627; 10 ng/kg per minute for 30 minutes), in rats pretreated with enalapril (250 mg/L for 1 week) to normalize the endogenous renin-angiotensin system. Endothelin type-A receptor blockade reduced angiotensin II-mediated increases in blood pressure in male control and male growth-restricted rats. Endothelin type-A receptor blockade also abolished hyper-responsiveness to acute angiotensin II in male growth-restricted rats. Yet, blood pressure remained significantly elevated above baseline after endothelin type-A receptor blockade, suggesting that factors in addition to endothelin contribute to the basic angiotensin II-induced pressor response in male rats. We also determined sex-specific effects of endothelin on acute angiotensin II-mediated hemodynamic responses. Endothelin type-A receptor blockade did not reduce acute angiotensin II-mediated increases in blood pressure in female control or growth-restricted rats, intact or ovariectomized. Thus, these data suggest that endothelin type-A receptor blockade contributes to hypersensitivity to acute angiotensin II in male growth-restricted rats and further supports the sex-specific effect of endothelin on blood pressure.

Angiotensin(1-7) and ACE2, "The Hot Spots" of Renin-Angiotensin System, Detected in the Human Aqueous Humor.

Background: The main purpose of the study was to establish whether essential components of the renin-angiotensin system (RAS) exist in the human aqueous humor.Methods: Forty-five patients ≥ 60 (74±7) years of age undergoing cataract surgery at Tampere University Hospital were randomly selected for the prospective study. The exclusion criterion was the use of oral antihypertensive medicine acting via renin-angiotensin system. Aqueous humor samples were taken at the beginning of normal cataract extraction. The samples were frozen and stored at -80 °C. The concentrations of intraocular endogenous RAS components Ang(1-7), ACE2, and ACE1 were measured using ELISA.Results: Concentration medians of Ang(1-7), ACE2, and ACE1 in the aqueous humor were: Ang(1-7) 4.08 ng/ml, ACE2 2.32 ng/ml and ACE1 0.35 ng/ml. The concentrations were significantly higher in glaucomatous than in non-glaucomatous eyes, ACE1 (p=0.014) and Ang(1-7) (p=0.026) vs non-glaucomatous eyes.Conclusions: Ang(1-7), ACE2 and ACE1 are found in the human aqueous humor. The observations are consistent with the conception that local tissue-RAS exists in the human eye and it might have a role in the control of intraocular pressure.

Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells.

The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1-7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS.

Combined suppression of the intrarenal and circulating vasoconstrictor renin-ACE-ANG II axis and augmentation of the vasodilator ACE2-ANG 1-7-Mas axis attenuates the systemic hypertension in Ren-2 transgenic rats exposed to chronic hypoxia.

The aim of the present study was to test the hypothesis that chronic hypoxia would aggravate hypertension in Ren-2 transgenic rats (TGR), a well-defined monogenetic model of hypertension with increased activity of endogenous renin-angiotensin system (RAS). Systolic blood pressure (SBP) in conscious rats and mean arterial pressure (MAP) in anesthetized TGR and normotensive Hannover Sprague-Dawley (HanSD) rats were determined under normoxia that was either continuous or interrupted by two weeks´ hypoxia. Expression, activities and concentrations of individual components of RAS were studied in plasma and kidney of TGR and HanSD rats under normoxic conditions and after exposure to chronic hypoxia. In HanSD rats two weeks´ exposure to chronic hypoxia did not alter SBP and MAP. Surprisingly, in TGR it decreased markedly SBP and MAP; this was associated with substantial reduction in plasma and kidney renin activities and also of angiotensin II (ANG II) levels, without altering angiotensin-converting enzyme (ACE) activities. Simultaneously, in TGR the exposure to hypoxia increased kidney ACE type 2 (ACE2) activity and angiotensin 1-7 (ANG 1-7) concentrations as compared with TGR under continuous normoxia. Based on these results, we propose that suppression of the hypertensiogenic ACE-ANG II axis in the circulation and kidney tissue, combined with augmentation of the intrarenal vasodilator ACE2-ANG 1-7 axis, is the main mechanism responsible for the blood pressure-lowering effects of chronic hypoxia in TGR.

Abstract 314: Evidence for the Expression of Renin Angiotensin System (RAS) and a Disintegrin and Metalloproteinase (ADAM) 17-mediated shedding of ACE2 in COS-7 cells

The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal system. COS-7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aim of this study was to 1) demonstrate the presence of an endogenous and functional RAS in COS7 cells and 2) to investigate the role of ADAM17 in the ectodomain shedding of ACE2. COS-7 cells were grown to 90% confluency followed by incubation in serum-free media for 24 hours. Western blot, immunohistochemistry and mass spectrometry (MS)-based enzyme assays were used to study RAS protein expression and activity in COS7 cells. Western blot and immunostaining confirmed endogenous expression of ACE (195 kDa), ACE2 (75-70 kDa), AT1R (43 kDa), renin (41 kDa) and ADAM17 (130 kDa) in COS7 cells. A sensitive and selective MS approach was used to determine endogenous RAS enzymatic activity in COS-7 lysate and media using the natural RAS substrate Ang II (m/z 1046). MS analysis detected Ang-(1-7) formation (m/z 899) in lysate and media of COS7 cells. While Ang-(1-7) formation in media was completely blocked by the ACE2-specific inhibitor MLN-4760, Ang-(1-7) formation in the lysate was significantly inhibited by the prolyl carboxypeptidase-specific inhibitor Z-pro-prolinal. Using short-hairpin (sh) RNA-mediated technology, ADAM17 protein expression and activity was significantly reduced in silenced cells compared to normal cells. Western blot analysis showed a significant increase of ACE2 protein expression in the lysate of silenced cells compared to normal cell and ACE2 shedding into the media was significantly reduced in silenced cells. This is the first study to demonstrate endogenous expression of the RAS and ADAM17 in COS-7 cells. Use of COS-7 could enhance our understanding of the cross talk between different arms of the RAS and dissect the direct effects of ACE inhibitors and Ang II receptor blockers. The transfectable nature of this cell line makes it an attractive in vitro cell model for studying the molecular, functional and pharmacological properties of the renal RAS. The data support the utility of COS-7 for the study of ectodomain shedding of ACE2 in vitro.

Role of vascular smooth muscle PPARγ in regulating AT1 receptor signaling and angiotensin II-dependent hypertension.

Peroxisome proliferator activated receptor γ (PPARγ) has been reported to play a protective role in the vasculature; however, the underlying mechanisms involved are not entirely known. We previously showed that vascular smooth muscle-specific overexpression of a dominant negative human PPARγ mutation in mice (S-P467L) leads to enhanced myogenic tone and increased angiotensin-II-dependent vasoconstriction. S-P467L mice also exhibit increased arterial blood pressure. Here we tested the hypotheses that a) mesenteric smooth muscle cells isolated from S-P467L mice exhibit enhanced angiotensin-II AT1 receptor signaling, and b) the increased arterial pressure of S-P467L mice is angiotensin-II AT1 receptor dependent. Phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) was robustly increased in mesenteric artery smooth muscle cell cultures from S-P467L in response to angiotensin-II. The increase in ERK1/2 activation by angiotensin-II was blocked by losartan, a blocker of AT1 receptors. Angiotensin-II-induced ERK1/2 activation was also blocked by Tempol, a scavenger of reactive oxygen species, and correlated with increased Nox4 protein expression. To investigate whether endogenous renin-angiotensin system activity contributes to the elevated arterial pressure in S-P467L, non-transgenic and S-P467L mice were treated with the AT1 receptor blocker, losartan (30 mg/kg per day), for 14-days and arterial pressure was assessed by radiotelemetry. At baseline S-P467L mice showed a significant increase of systolic arterial pressure (142.0±10.2 vs 129.1±3.0 mmHg, p<0.05). Treatment with losartan lowered systolic arterial pressure in S-P467L (132.2±6.9 mmHg) to a level similar to untreated non-transgenic mice. Losartan also lowered arterial pressure in non-transgenic (113.0±3.9 mmHg) mice, such that there was no difference in the losartan-induced depressor response between groups (−13.53±1.39 in S-P467L vs −16.16±3.14 mmHg in non-transgenic). Our results suggest that interference with PPARγ in smooth muscle: a) causes enhanced angiotensin-II AT1 receptor-mediated ERK1/2 activation in resistance vessels, b) and may elevate arterial pressure through both angiotensin-II AT1 receptor-dependent and -independent mechanisms.