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Angiotensin-(1-12) [ANG(1-12)] is a newly identified peptide detected in a variety of rat tissues, including the brain. To determine whether brain ANG-(1-12) participates in blood pressure regulation, we treated male adult (mRen2)27 hypertensive rats (24-28 wk of age) with Anti-ANG-(1-12) IgG or Preimmune IgG via an intracerebroventricular cannula for 14 days. Immunoneutralization of brain ANG-(1-12) lowered systolic blood pressure (-43 [+ or -] 8 mmHg on day 3 and -26 [+ or -] 7 mmHg on day 10 from baseline, P < 0.05). Water intake was lower on intracereroventricular day 6 in the Anti-ANG-(1-12) IgG group, accompanied by higher plasma osmolality on day 13, but there were no differences in urine volume, food intake, or body weight during the 2-wk treatment. In Preimmune IgG-treated animals, there were no significant changes in these variables over the 2-wk period. The antihypertensive effects produced by endogenous neutralization of brain ANG-(1-12) suggest that ANG-(1-12) is functionally active in brain pathways regulating blood pressure. hypertension; transgenic rats; renin-angiotensin system; brain; sympathetic nerve; antibody

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1[beta] and TNF[alpha] acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase 'recognition motif.' Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface. a disintegrin and metalloprotease 17; severe acute respiratory syndrome; coronavirus

Expression of nuclear angiotensin II type 1 ([AT.sub.1]) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist [sup.125]I-[[Sar.sup.1],[Thr.sup.8]]-ANG II ([sup.125]I-sarthran) with the [AT.sub.1] antagonist losartan (LOS) or the [AT.sub.2] antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC ([greater than or equal to] 70%) and PM ([greater than or equal to]80%) sites while LOS competition predominated in medullary NUC ([greater than or equal to]75%) and PM ([greater than or equal to]70%). Immunodetection with an [AT.sub.2] antibody revealed a single ~42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized [AT.sub.2] in the tubulointerstitium, [AT.sub.1] in the medulla and vasa recta, and both [AT.sub.1] and [AT.sub.2] in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-L-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear [AT.sub.2] receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure. nucleus; kidney; intracellular renin angiotensin system

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of angiotensin-(1-12) [ANG-(1-12)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(1-12) being an alternate renin-independent, angiotensin-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac angiotensins. Significant decreases in plasma ANG-(1-12), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(1-12), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(1-12) decreased 34% following nephrectomy, which accompanied 78 and 66% decreases in plasma ANG I and ANG II, respectively (P < 0.05 vs. controls). Contrastingly, cardiac ANG-(1-12) in anephric rats averaged 276 [+ or -] 24 fmol/mg compared with 144 [+ or -] 20 fmol/mg in controls (P < 0.005). Cardiac ANG I and ANG II values were 300 [+ or -] 15 and 62 [+ or -] 7 fmol/mg, respectively, in anephric rats compared with 172 [+ or -] 8 fmol/mg for ANG I and 42 [+ or -] 4 fmol/mg for ANG II in controls (P < 0.001). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue angiotensinogen, ANG I, ANG II, and [AT.sub.1] receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in [p47.sup.phox], Rac1, and Nox4 isoform expression. In conclusion, ANG-(1-12) may be a functional precursor for angiotensin peptide formation in the absence of circulating renin. renin; angiotensinogen; angiotensin II; blood pressure

Exposure to clinically relevant doses of glucocorticoids during fetal life increases blood pressure in adult male and female sheep. The purpose of this study was to evaluate the effects of prenatal exposure to betamethasone at 80-81 days of gestation on renal function in ewes and rams at 1.5 yr of age. In prenatal betamethasone-exposed males, compared with the vehicle-exposed animals, basal glomerular filtration rate (GFR) (1.93 [+ or -] 0.08 vs. 2.27 [+ or -] 0.10 ml * [min.sup.-1] * kg body [wt.sup. 1]) and the ability to excrete an acute [Na.sup.+] load (37.1 [+ or -] 4.4 vs. 53.7 [+ or -] 9.7%) were reduced. (P < 0.03 and P = 0.03, respectively). In contrast, prenatal betamethasone exposure had no effect on basal GFR, [Na.sup.+] excretion, or the percentage of the [Na.sup.+] load excreted during the experiment in females. Systemic infusions of ANG-(1-7) at 9 ng * [min.sup.-1] * [kg.sup.-1] for 2 h had minimal effects on basal GFR, renal plasma flow, and [Na.sup.+] excretion in males but increased [Na.sup.+] excretion in females. However, the percentage of [Na.sup.+] load excreted during ANG-(1-7) infusion did not change in prenatal betamethasone-exposed females (113.1 [+ or -] 14.2 vs. 98.1 [+ or -] 12.2%) compared with the significant increase in vehicle females (139.2 [+ or -] 22.3 vs. 92.2 [+ or -] 7.5%) (P = 0.01). The data indicate that antenatal betamethasone exposure produces gender-specific alternations in renal function and thus suggest that different mechanisms underlie the antenatal steroid-induced elevations in blood pressure in male and female offspring. prenatal steroid exposure; sodium load; glomerular filtration rate; sodium excretion; angiotensin-(1-7)

Sex differences in blood pressure are evident in experimental models and human subjects, yet the mechanisms underlying this disparity remain equivocal. The current study sought to define the extent of male-female differences in the circulating and tissue renin-angiotensin aldosterone systems (RAASs) of congenic mRen(2).Lewis and control Lewis rats. Male congenics exhibited higher systolic blood pressure than females [200 [+ or -] 4 vs. 146 [+ or -] 7 mmHg, P < 0.01] or Lewis males and females [113 [+ or -] 2 vs. 112 [+ or -] 2 mmHg, P > 0.05]. Plasma ANG II levels were twofold higher in male congenics [47 [+ or -] 3 vs. 19 [+ or -] 3 pM, P < 0.01] and fivefold higher than in male or female Lewis rats [6 [+ or -] 1 vs. 6 [+ or -] 1 pM]. ANG I levels were also highest in the males; however, plasma ANG-(1-7) was higher in female congenics. Male congenics exhibited greater circulating renin and angiotensin-converting enzyme (ACE) activities, as well as angiotensinogen, than female littermates. Renal cortical and medullary ANG II levels were also higher in the male congenics versus all the other groups; ANG I was lower in the males. Cortical ACE2 activity was higher in male congenics, yet neprilysin activity and protein were greater in the females, which may contribute to reduced renal levels of ANG II. These data reveal that sex differences in both the circulating and renal RAAS are apparent primarily in the hypertensive group. The enhanced activity of the RAAS in male congenics may contribute to the higher pressure and tissue injury evident in the strain. angiotensin II; angiotensin-converting enzyme; angiotensin-(1-7), estrogen; neprilysin; cardiac hypertrophy; proteinuria; renin-angiotensin aldosterone system

A low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II on tissues. The recent identification of angiotensin(1-12) in an array of rat organs suggests this propeptide may be an alternate substrate for local angiotensin production. To test this hypothesis, tissues from 11-wk-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (n = 14) were stained with purified antibodies directed to the COOH terminus of angiotensin-(112). Robust angiotensin-(1-12) staining was predominantly found in ventricular myocytes with less staining found in the medial layer of intracoronary arteries and vascular endothelium. In addition, angiotensin-(1-12) immunoreactivity was present in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains. Preadsorption of the antibody with angiotensin-(1-12) abolished staining in both tissues. Corresponding tissue measurements by radioimmunoassay showed 47% higher levels of angiotensin-(l-12) in the heart of SHR compared with WKY rats (P < 0.05). In contrast, renal angiotensin-(1-12) levels were 16.5% lower in SHR compared with the WKY rats (P < 0.05). This study shows for first time the localization of angiotensin-(l-12) in both cardiac myocytes and renal tubular components of WKY and SHR. In addition, we show that increased cardiac angiotensin-(l-12) concentrations in SHR is associated with a small, but statistically significant, reduction in renal angiotensin-(1-12) levels. angiotensinogen; angiotensin I; angiotensin II; hypertension; renin

Identification of angiotensin-(112) as an intermediate precursor derived directly from angiotensinogen led us to explore whether the heart has the capacity to process angiotensin-(1-12) into biologically active angiotensin peptides. The generation of angiotensin 1, angiotensin II, and angiotensin-(1-7) from exogenous angiotensin-(1-12) was evaluated in the effluent of isolated perfused hearts mounted on a Langendorff apparatus in three normotensive and two hypertensive strains: Sprague-Dawley, Lewis, congenic mRen2.Lewis, Wistar-Kyoto, and spontaneously hypertensive rats. Hearts were perfused with Krebs solution for 60 min before and after the addition of angiotensin-(1-12) (10 nmol/l). Angiotensin-(112) caused the rapid appearance of both angiotensin I and angiotensin II in the perfusate that peaked between 30 and 60 min of recirculation. Production of angiotensin-(1-7) from exogenous angiotensin-(1-12) rose steadily over the course of the 60-min experiment. These data directly demonstrate that angiotensin-(1-12) is a substrate for the formation of angiotensin peptides in cardiac tissue. This finding further suggests that this angiotensinogen-derived product is a previously unrecognized important precursor peptide to the renin-angiotensin system cascade. angiotensinogen; angiotensin II; angiotensin-(1-7); renin: hypertension

This study determined the contribution of chronic salt loading and early loss of ovarian hormones on diastolic function in the hypertensive female mRen(2). Lewis rat, a monogenetic strain that expresses the mouse renin-2 gene in various tissues. Estrogen-intact mRen2 rats fed a high salt (HS) (8% sodium chloride) diet exhibited early diastolic dysfunction when compared to normal salt-fed (NS) (1% sodium chloride) rats. In contrast, ovariectomized (OVX) rats on either NS or HS diets showed impaired relaxation with evidence of elevated left ventricular filling pressures (E/e') or pseudonormalization. This more advanced stage of diastolic dysfunction was associated with increases in interstitial cardiac fibrosis and high circulating levels of aldosterone, two factors leading to reduced ventricular compliance. These findings may explain the preponderance of diastolic dysfunction and diastolic heart failure in postmenopausal women and provide a potential animal model for evaluating prevention and treatment interventions for this disorder. Key Words: Salt--Hormone, ovarian--Diastolic function--Rat.

Studies in experimental animals and younger women suggest a protective role for estrogen; however, clinical trials may not substantiate this effect in older females. Therefore, the present study assessed the outcome of ovariectomy in older mRen2.Lewis rats subjected to a high-salt diet for 4 wk. Intact or ovariectomized (OVX, 15 wk of age) mRen2.Lewis rats were aged to 60 wk and then placed on a high-salt (HS, 8% sodium chloride) diet for 4 wk. Systolic blood pressures were similar between groups [OVX 169 [+ or -] 6 vs. Intact 182 [+ or -] 7 mmHg; P = 0.22] after the 4-wk diet; however, proteinuria [OVX 0.8 [+ or -] 0.2 vs. Intact 11.5 [+ or -] 2.6 mg/mg creatinine; P < 0.002, n = 6], renal interstitial fibrosis, glomerular sclerosis, and tubular casts were lower in OVX vs. Intact rats. Kidney injury molecule-1 mRNA, a marker of tubular damage, was 53% lower in the OVX HS group. Independent from blood pressure, OVX HS rats exhibited significantly lower cardiac (24%) and renal (32%) hypertrophy as well as lower C-reactive protein (28%). Circulating insulin-like growth factor-I (IGF-I) levels were not different between the Intact and OVX groups; however, renal cortical IGF-I mRNA and protein were attenuated in OVX rats [P < 0.05, n = 6]. We conclude that ovariectomy in the older female mRen2.Lewis rat conveys protection against salt-dependent increase in renal injury. estrogen: aging; female rat; renal injury: insulin-like growth factor-I; kidney injury molecule-1

Angiotensin-converting enzyme-2 (ACE2) converts angiotensin II (ANG II) to angiotensin-(1-7) [ANG-(1-7)], and this enzyme may serve as a key regulatory juncture in various tissues. Although the heart expresses ACE2, the extent that the enzyme participates in the cardiac processing of ANG II and ANG-(1-7) is equivocal. Therefore, we utilized the Langendorff preparation to characterize the ACE2 pathway in isolated hearts from male normotensive Sprague-Dawley [[Tg.sup.(-)]] and hypertensive [mRen2]27 [[Tg.sup.(+)]] rats. During a 60-rain recirculation period with 10 nM ANG II, the presence of ANG-(1-7) was assessed in the cardiac effluent. ANG-(1-7) generation from ANG II was similar in both the normal and hypertensive hearts [[Tg.sup(-)]: 510 [+ or -] 55 pM, n = 20 vs. [Tg.sup.(+)]: 497 [+ or -] 63 pM, n = 14] with peak levels occurring at 30 rain after administration of the peptide. ACE2 inhibition (MLN-4760, 1 [micro]M) significantly reduced ANG-(1-7) production by 83% (57 [+ or -] 19 pM, P < 0.01, n = 7) in the [Tg.sup.(-)] rats, whereas the inhibitor had no significant effect in the [Tg.sup.(-)] rats (285 [+ or -] 53 pM, P > 0.05, n = 10). ACE2 activity was found in the effluent of perfused [Tg.sup.(-)] and [Tg.sup.(+)] hearts, and it was highly associated with ACE2 protein expression (r = 0.78). This study is the first demonstration for a direct role of ACE2 in the metabolism of cardiac ANG II in the hypertrophic heart of hypertensive rats. We conclude that predominant expression of cardiac ACE2 activity in the [Tg.sup.(+)] may be a compensatory response to the extensive cardiac remodeling in this strain. angiotensin II; hypertension; isolated heart doi:10.1152/ajpheart.01198.2006.

Estrogen depletion markedly exacerbates hypertension in female congenic mRen2.Lewis rats, a model of tissue renin overexpression. Because estrogen influences nitric oxide synthase (NOS) and NO may exert differential effects on blood pressure, the present study investigated the functional expression of NOS isoforms in the kidney of ovariectomized (OVX) mRen2.Lewis rats. OVX-mRen2.Lewis exhibited an increase in systolic blood pressure (SBP) of 171 [+ or -] 5 vs. 141 [+ or -] 7 mmHg (P < 0.01) for intact littermates. Renal cortical mRNA and protein levels for endothelial NOS (eNOS) were reduced 50-60% (P < 0.05) and negatively correlated with blood pressure. In contrast, cortical neuronal NOS (nNOS) mRNA and protein levels increased 100 to 300% (P < 0.05). In the OVX kidney, nNOS immunostaining was more evident in the macula densa, cortical tubules, and the medullary collecting ducts compared with the intact group. To determine whether the increase in renal nNOS expression constitutes a compensatory response to the reduction in renal eNOS, we treated both intact and OVX mRen2.Lewis rats with the selective nNOS inhibitor L-VNIO from 11 to 15 wk of age. The nNOS inhibitor reduced blood pressure in the OVX group (185 [+ or -] 3 vs. 151 [+ or -] 8 mmHg, P < 0.05), but pressure was not altered in the intact group (146 [+ or -] 4 vs. 151 [+ or -] 4 mmHg). In summary, exacerbation of blood pressure in the OVX mRen2.Lewis rats was associated with the discoordinate regulation of renal NOS isoforms. Estrogen sensitivity in this congenic strain may involve the influence of NO through the regulation of both eNOS and nNOS. endothelial nitric oxide synthase; estrogen; hypertension; aldosterone; angiotensin II; renin

Despite the evidence that angiotensin-converting enzyme (ACE)2 is a component of the reninangiotensin system (RAS), the influence of ACE2 on angiotensin metabolism within the kidney is not well known, particularly in experimental models other than rats or mice. Therefore, we investigated the metabolism of the angiotensins in isolated proximal tubules, urine, and serum from sheep. Radiolabeled [[sup.125]I]ANG I was hydrolyzed primarily to ANG II and ANG-(1-7) by ACE and neprilysin, respectively, in sheep proximal tubules. The ACE2 product ANG-(1-9) from ANG I was not detected in the absence or presence of ACE and neprilysin inhibition. In contrast, the proximal tubules contained robust ACE2 activity that converted ANG II to ANG-(1-7). Immunoblots utilizing an [NH.sub.2] terminal-directed ACE2 antibody revealed a single 120-kDa band in proximal tubule membranes. ANG-(1-7) was not a stable product in the tubule preparation and was rapidly hydrolyzed to ANG-(1-5) and ANG-(1-4) by ACE and neprilysin, respectively. Comparison of activities in the proximal tubules with nonsaturating concentrations of substrate revealed equivalent activities for ACE (ANG I to ANG II: 248 [+ or -] 17 fmol*[mg.sup.-1]*[min.sup.-1]) and ACE2 [ANG II to ANG-(1-7): 253 [+ or -] 11 fmol*[mg.sup.-1]*[min.sup.-1]], but lower neprilysin activity [ANG II to ANG-(1-4): 119 [+ or -] 24 fmol*[mg.sup.-1]*[min.sup.-1]; p < 0.05 vs. ACE or ACE2]. Urinary metabolism of ANG I and ANG II was similar to the proximal tubules; soluble ACE2 activity was also detectable in sheep serum. In conclusion, sheep tissues contain abundant ACE2 activity that converts ANG II to ANG-(1-7) but does not participate in the processing of ANG I into ANG-(1-9). ANG-(1-7); ANG-(1-5); neprilysin; ACE; ANG-(1-9)

The present study determined whether early loss of estrogen influences salt-sensitive changes in blood pressure, renal injury, and cardiac hypertrophy as well as the effects on the circulating renin-angiotensinaldosterone system (RAAS) in the hypertensive female mRen(2).Lewis strain. Ovariectomy (OVX) of heterozygous mRen(2).Lewis rats on a normal salt (NS) diet (0.5% sodium) increased systolic blood pressure from 137 [+ or -] 3 to 177 [+ or -] 5 mmHg (P < 0.01) by 15 wk but did not show any changes in cardiac-to-body weight index (CI), proteinuria, or creatinine clearance. Maintenance with a high-sodium (HS) diet (4%) increased blood pressure (203 [+ or -] 4 mmHg, P < 0.01), proteinuria (3.5 [+ or -] 0.3 vs. 6.4 [+ or -] 0.7 mg/day, P < 0.05), and CI (4.0 [+ or -] 0.1 vs. 5.2 [+ or -] 0.1 mg/kg, P < 0.01) but decreased creatinine clearance (0.89 [+ or -] 0.15 vs. 0.54 [+ or -] 0.06 m1/min, P < 0.05). OVX exacerbated the effects of salt on the degree of hypertension (230 [+ or -] 5 mmHg), CI (5.6 [+ or -] 0.2 mg/kg), and proteinuria (13 [+ or -] 3.0 mg/day). OVX increased the urinary excretion of aldosterone approximately twofold in animals on the NS diet (3.8 [+ or -] 0.5 vs. 6.6 [+ or -] 0.5 ng x mg [creatinine.sup.-1] x [day.sup.-1], P < 0.05) and HS diet (1.4 [+ or -] 0.2 vs. 4.5 [+ or -] 1.0 ng x mg [creatinine.sup.-1] x [day.sup.-1], p < 0.05). Circulating renin, angiotensin-converting enzyme, and angiotensin II were also significantly increased in the OVX group fed a HS diet. These results reveal that the protective effects of estrogen apart from the increase in blood pressure were only manifested in the setting of a chronic HS diet and suggest that the underlying sodium status may have an important influence on the overall effect of reduced estrogen. angiotensin II; angiotensin-coverting enzyme; aldosterone; hypertension; cardiac hypertrophy; proteinuria

The generation of the Lew.Tg(mRen2) congenic hypertensive rat strain, developed through a backcross of the hypertensive (mRen2)27 transgenic rat with normotensive Lewis rats, provides a new model by which primary hypertension can be studied without the genetic variability found in the original strain. The purpose of this study was to characterize the Lew.Tg(mRen2) rats by dually investigating the effects of type 1 angiotensin II (ANG II) receptor (ATe) blockade and angiotensin-converting enzyme (ACE) activity inhibition on the ANG-(1-7)/ACE2 axis of the renin-angiotensin system in this new hypertensive model. The control of blood pressure elicited by 12-day administration of either lisinopril (mean difference change = 92 [+ or -] 2, P < 0.05) or losartan (mean difference change = 69 [+ or -] 2, P < 0.05) was associated with 54% and 33% increases in cardiac ACE2 mRNA and 54% and 43% increases in cardiac ACE mRNA, respectively. Lisinopril induced a 3.1-fold (P < 0.05) increase in renal cortical expression of ACE2, whereas losartan increased ACE2 mRNA 3.5-fold (P < 0.05). Both treatment regimens increased renal ACE mRNA 2.6-fold (P < 0.05). The two therapies augmented ACE2 protein activity, as well as increased cardiac and renal [AT.sub.1] receptor mRNAs. ACE inhibition reduced plasma ANG 13 levels (81%, P < 0.05) and increased plasma ANG-(1-7) (265%, P < 0.05), whereas losartan had no effect on the peptides. In contrast with what had been shown in normotensive rats, ACE inhibition decreased renal ANG II excretion and transiently decreased ANG-(1-7) excretion, whereas losartan treatment was associated with a consistent decrease in ANG-(1-7) urinary excretion rates. In response to the treatments, the expression of both renal cortical renin and angiotensinogen mRNAs was significantly augmented. The paradoxical effects of blockade of ANG II synthesis and activity on urinary excretion rates of the peptides and plasma angiotensins levels suggest that, in Lew.Tg(mRen2) congenic rats, a failure of compensatory ACE2 and ANG-(1-7)-dependent vasodepressor mechanisms may contribute both to the development and progression of hypertension driven by increased formation of endogenous ANG II. lisinopril; losartan; angiotensin-converting enzyme 2; angiotensin-(1-7)

We established a new congenic model of hypertension, the mRen(2).Lewis rat and assessed the intracellular expression of angiotensin peptides and receptors in the kidney. The congenic strain was established from the backcross of the (mRen2)27 transgenic rat that expresses the mouse renin 2 gene onto the Lewis strain. The 20-wk-old male congenic rats were markedly hypertensive compared with the Lewis controls (systolic blood pressure: 195 [+ or -] 2 vs. 107 [+ or -] 2 mmHg, P < 0.01). Although plasma ANG II levels were not different between strains, circulating levels of ANG-(1-7) were 270% higher and ANG I concentrations were 40% lower in the mRen2.Lewis rats. In contrast, both cortical (CORT) and medullary (MED) ANG II concentrations were 60% higher in the mRen2.Lewis rats, whereas tissue ANG I was 66 and 84% lower in CORT and MED. For both strains, MED ANG II, ANG I, and ANG-(1-7) were significantly higher than CORT levels. Intracellular ANG II binding distinguished nuclear (NUC) and plasma membrane (PM) receptor using the ANG II radioligand [sup.125]I-sarthran. Isolated CORT nuclei exhibited a high density (B.sub.max] >200 fmol/mg protein) and affinity for the sarthran ligand ([K.sub.D]95%) were the A[T.sub.1] receptor subtype. CORT ANG II receptor [B.sub.max] and [K.sub.D] values in nuclei were 75 and 50% lower, respectively, for the mRen2.Lewis vs. the Lewis rats. In the MED, the PM receptor density (Lewis: 50 [+ or -] 4 vs. mRen2.Lewis: 21 [+ or -] 5 fmol/mg protein) and affinity (Lewis: 0.31 [+ or -] 0.1 vs. 0.69 [+ or -] 0.1 nM) were lower in the mRen2.Lewis rats. In summary, the hypertensive mRen2.Lewis rats exhibit higher ANG II in both CORT and MED regions of the kidney. Evaluation of intracellular ANG II receptors revealed lower CORT NUC and MED PM A[T.sub.1] sites in the mRen2.Lewis. The downregulation of A[T.sub.1] sites in the mRen2.Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II. ANG II type 1 receptor; hypertension; intrarenal angiotensin; renal cortex; renal medulla; ANG II

Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE that preferentially forms angiotensin-(1-7) [ANG-(1-7)] from angiotensin II (ANG II). Incubation of neonatal rat cerebellar or medullary astrocytes with ANG II reduced ACE2 mRNA by ~60%, suggesting transcriptional regulation of the enzyme. In contrast, ANG II had no effect on ACE mRNA in astrocytes isolated from either brain region, demonstrating a differential regulation of the two enzymes by ANG II. The ANG II-mediated reduction in ACE2 mRNA was blocked by the angiotensin type 1 (A[T.sub.1]) receptor antagonists losartan or valsartan; the angiotensin type 2 (A[T.sub.2]) antagonist PD123319 was ineffective. The reduction in ACE2 mRNA by ANG II also was associated with a 50% decrease in cerebellar and medullary ACE2 protein, which was blocked by losartan. Treatment of medullary astrocytes with ANG(1-7), the product of ACE2 hydrolysis of ANG II, did not affect ACE2 mRNA; however, ANG-(1-7) prevented the ANG II-mediated reduction in ACE2 mRNA. The addition of [D-[Ala.sup.7]]-ANG-(1-7), a selective A[T.sub.(1-7)] receptor antagonist, blocked the inhibitory actions of ANG-(1-7). These data are the first to demonstrate transcriptional regulation of ACE2 by ANG II and ANG-(1-7). Because ACE2 preferentially converts ANG II to ANG-(1-7), downregulation of the enzyme by ANG II constitutes a novel positive feed-forward system within the brain that may favor ANG II-mediated neural responses. Furthermore, the modulatory role of ANG-(1-7) in the transcriptional regulation of ACE2 by ANG II suggests a complex interplay between these peptides that is mediated by distinct receptor systems. central nervous system; glia; angiotensin receptor

We tested the hypothesis that long-term growth hormone (GH) replacement in aged rats would preserve diastolic function and attenuate left ventricular remodeling associated with normal aging. Male Brown Norway x F344 rats were randomized to receive twice daily injections of porcine GH (200 [micro]g/injection, subcutaneous) or saline from 24 to 30 months of age. Adult rats (6- to 9-months old) received saline injections throughout the study. Thirty-month-old, saline-treated rats exhibited low levels of insulin-like growth factor 1 (IGF-1), impaired diastolic left ventricular filling (Doppler), increased cardiac angiotensin II (Ang II), reduced plasma Ang II, and increased cardiac collagen. GH administration in old rats restored IGF-1 and diastolic indices to values comparable to those of adults. These effects were associated with reduced cardiac Ang II and attenuations in cardiac collagen. Age-related decreases in GH and IGF-1 may contribute to the decline in diastolic function of aging, in part through alterations in renin-angiotensin system-mediated ventricular remodeling.

This study evaluated the contribution of angiotensin peptides acting at various receptor subtypes to the arterial pressure and heart rate of adult 9-wk-old male conscious salt-depleted spontaneously hypertensive rats (SHR). Plasma ANG II and ANG I in salt-depleted SHR were elevated sevenfold compared with peptide levels measured in sodium-replete SHR, whereas plasma ANG-(1-7) was twofold greater in salt-depleted SHR compared with salt-replete SHR. Losartan (32.5 [micro]mol/kg), PD-123319 (0.12 [micro]mol.[kg.sup.-1] [min.sup.-1]), [D-[Ala.sup.7]]ANG-(1-7) (10 and 100 [micro]mol/min), and a polyclonal ANG II antibody (0.08 mg/min) were infused intravenously alone or in combination. Combined blockade of A[T.sub.2] and A[T.sub.(1-7)] receptors significantly increased the blood pressure of losartan-treated SHR (+ 15 [+ or -] 1 mmHg; P < 0.01); this change did not differ from the blood pressure elevation produced by the sole blockade of A[T.sub.(1-7)] receptors (15 [+ or -] 4 mmHg). On the other hand, sole blockade of A[T.sub.2] receptors in losartan-treated SHR increased mean arterial pressure by 8 [+ or -] 1 mmHg (P < 0.05 vs. 5% dextrose in water as vehicle), and this increase was less than the pressor response produced by blockade of A[T.sub.(1-7)] receptors alone or combined blockade of A[T.sub.(1-7)] and A[T.sub.2] receptors. The ANG II antibody increased blood pressure to the greatest extent in salt-depleted SHR pretreated with only losartan (+ 11 [+ or -] 2 mmHg) and to the least extent in salt-depleted SHR previously treated with the combination of losartan, PD-123319, and [D-[Ala.sup.7]]ANG-(1-7) (+7 [+ or -] 1 mmHg; P < 0.01). Losartan significantly increased heart rate, whereas other combinations of receptor antagonists or the ANG II antibody did not alter heart rate. Our results demonstrate that ANG II and ANG-(1-7) act through non-A[T.sub.1] receptors to oppose the vasoconstrictor actions of ANG II in salt-depleted SHR. Combined blockade of A[T.sub.2] and A[T.sub.(1-7)] receptors and ANG II neutralization by the ANG II antibody reversed as much as 67% of the blood pressure-lowering effect of losartan. angiotensin II; angiotensin-(1-7); angiotensin receptor antagonists; hypertension; losartan; PD-123319; salt depletion; spontaneously hypertensive rats

Author(s): Michael A. Crackower [1, 2, 3]; Renu Sarao [1, 4, 5]; Gavin Y. Oudit [3, 5, 6, 7]; Chana Yagil [8]; Ivona Kozieradzki [1, 4]; Sam E. Scanga [9]; [...]

The renin-angiotensin system (RAS) is fundamental to COVID-19 pathobiology, due to the interaction between the SARS-CoV-2 virus and the angiotensin-converting enzyme 2 (ACE2) coreceptor for cellular entry. The prevailing hypothesis is that SARS-CoV-2-ACE2 interactions lead to an imbalance of the RAS, favoring proinflammatory angiotensin II (ANG II)-related signaling at the expense of the anti-inflammatory ANG-(1–7)-mediated alternative pathway. Indeed, multiple clinical trials targeting this pathway in COVID-19 are underway. Therefore, precise measurement of circulating RAS components is critical to understand the interplay of the RAS on COVID-19 outcomes. Multiple challenges exist in measuring the RAS in COVID-19, including improper patient controls, ex vivo degradation and low concentrations of angiotensins, and unvalidated laboratory assays. Here, we conducted a prospective pilot study to enroll 33 patients with moderate and severe COVID-19 and physiologically matched COVID-19-negative controls to quantify the circulating RAS. Our enrollment strategy led to physiological matching of COVID-19-negative and COVID-19-positive moderate hypoxic respiratory failure cohorts, in contrast to the severe COVID-19 cohort, which had increased severity of illness, prolonged intensive care unit (ICU) stay, and increased mortality. Circulating ANG II and ANG-(1–7) levels were measured in the low picomolar (pM) range. We found no significant differences in circulating RAS peptides or peptidases between these three cohorts. The combined moderate and severe COVID-19-positive cohorts demonstrated a mild reduction in ACE activity compared with COVID-19-negative controls (2.2 ± 0.9 × 105 vs. 2.9 ± 0.8 × 105 RFU/mL, P = 0.03). These methods may be useful in designing larger studies to physiologically match patients and quantify the RAS in COVID-19 RAS augmenting clinical trials. [ABSTRACT FROM AUTHOR]