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 [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 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 Ϯ 17 fmol ⅐ mg Ϫ1 ⅐ min Ϫ1 ) and ACE2 [ANG II to ANG-(1-7): 253 Ϯ 11 fmol ⅐ mg Ϫ1 ⅐ min Ϫ1 ], but lower neprilysin activity [ANG II to ANG-(1-4): 119 Ϯ 24 fmol ⅐ mg Ϫ1 ⅐ min Ϫ1 ; P Ͻ 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 INFLUENCE OF the renin-angiotensin-aldosterone system (RAAS) on the kidney to regulate blood pressure, the development of hypertension, and the extent of renal injury is established. Approaches that block the RAAS comprising angiotensin-converting enzyme (ACE) inhibitors, AT 1 receptor, or aldosterone antagonists constitute powerful therapies to control hypertension and attenuate renal damage in experimental and clinical studies. The kidney is a key target for biologically active components derived from both the circulating and intrarenal RAAS systems (25). Within the kidney, the proximal tubular epithelium is the primary if not sole site for angiotensinogen synthesis and release into the tubular fluid and contributes to the enzymatic processing of angiotensin peptides (20,26). The proximal tubule and other tubular elements of the nephron are also target sites for the actions of angiotensin peptides and aldosterone to influence sodium and water transport. Indeed, Davisson et al. (9) suggest that renal angiotensinogen contributes to the development of hypertension in a mouse model of human renin and proximal tubule angiotensinogen that does not exhibit a significant increase in circulating ANG II.Although the mechanisms for the generation and secretion of angiotensins within the proximal tubule are yet to be defined, the ...

Abstract-The angiotensin (Ang) type 1 receptor (AT 1 R) is highly expressed on renal nuclei and stimulates reactive oxygen species (ROS). It is not known whether other functional components of the Ang system regulate the nuclear Ang II-AT 1 R ROS pathway. Therefore, we examined the expression of Ang receptors in nuclei isolated from the kidneys of young adult (1.5 years) and older adult (3.0 to 5.0 years) sheep. Binding studies in renal nuclei revealed the AT 2 R as the predominant receptor subtype (Ϸ80%) in young sheep, with the Ang-(1-7) (AT 7 R; Mas protein) and AT 1 R antagonists competing for the remaining sites. Conversely, in older sheep, the AT 1 R accounted for Ϸ85% of nuclear sites, whereas the Ang type 2 receptor and AT 7 R subtypes comprise Ϸ20% of remaining sites. Ang II increased nuclear ROS to a greater extent in older (97Ϯ22%; nϭ6) versus young animals (7Ϯ2%; Pϭ0.01; nϭ4), and this was abolished by an AT 1 R antagonist. The AT 7 R antagonist D-Ala 7 -Ang-(1-7) increased ROS formation to Ang II by Ϸ2-fold (174Ϯ5% versus 97Ϯ22%; PϽ0.05) in older adults. Immunoblots of renal nuclei revealed protein bands for the AT 7 R and Ang-converting enzyme 2 (ACE2), which metabolizes Ang II to Ang-(1-7). The ACE2 inhibitor MLN4760 also exacerbated the Ang II-dependent formation of ROS (156Ϯ15%) and abolished the generation of Ang-(1-7) from Ang II. We conclude that an ACE2-Ang-(1-7)-AT 7 R pathway modulates Ang II-dependent ROS formation within the nucleus, providing a unique protective mechanism against oxidative stress and cell damage.

Expression of nuclear angiotensin II type 1 (AT(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 (125)I-[Sar(1),Thr(8)]-ANG II ((125)I-sarthran) with the AT(1) antagonist losartan (LOS) or the AT(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 (> or =70%) and PM (> or =80%) sites while LOS competition predominated in medullary NUC (> or =75%) and PM (> or =70%). Immunodetection with an AT(2) antibody revealed a single approximately 42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT(2) in the tubulointerstitium, AT(1) in the medulla and vasa recta, and both AT(1) and AT(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(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.

lined ground squirrels and other circannual hibernators undergo profound physiological changes on an annual basis, transitioning from summer homeothermy [body temperature (Tb) ϳ37°C] to winter heterothermy (Tb cycling between 0°C and 37°C). We hypothesize that these physiological changes are reflected in biochemical changes that provide mechanistic insights into, and biomarkers for, hibernation states. Here we report the results of an NMR-based metabolomics analysis of liver extracts from ground squirrels in three distinct physiological states of circannual hibernation: summer active (SA), late torpor (LT), and reentering torpor (Ent) after one of the euthermic arousals. Of the 43 identified and quantified metabolites, 36 differed among these three states and fell into two patterns of variation: 1) SA differed from both of the two winter states; or 2) the two winter states differed from each other, but one of the two was not different from SA. Concentrations of hepatic glucose, lactate, alanine, succinate, ␤-hydroxybutyrate, glutamine, and betaine were identified as robust hepatic biomarkers that together distinguish among animals in these three states of the circannual hibernation rhythm. These data are consistent with a proposed two-switch model of hibernation, in which setting the summer-winter switch to winter enables expression of a distinct torpor-arousal switch. The summer-winter switch is characterized by the metabolites associated with the well-known switch from carbohydrate to lipid fuel utilization during hibernation. The torpor-arousal switch is characterized by the accumulation of metabolites of nitrogen (glutamine) and phospholipid (betaine) catabolism in LT with the capacity to act as protective osmolytes. metabolomics; nitrogen metabolism; osmolytes; Spermophilus tridecemlineatus; torpor MAMMALIAN HIBERNATORS are uniquely able to orchestrate and survive extended periods of extremely low body temperature (T b ) and metabolic, respiratory, and heart rates in a state called torpor. The deep, multiday periods of torpor that characterize hibernation alternate with periodic short arousals that reverse the dramatic physiological depressions associated with the torpid state (reviewed in Ref. 5). Thus hibernators, including 13-lined ground squirrels (Spermophilus tridecemlineatus), are homeothermic like most mammals in summer but switch to heterothermy in winter (Fig. 1). The biochemical consequences of periodic arousals from torpor are complex and come with tremendous costs. Although hibernation is a strategy that saves large amounts of energy over the winter compared with remaining euthermic (ϳ90%), most of the energy used in winter (Ͼ70%) is used to fuel these interbout arousals (Ref. 22 and references therein). Hence the arousals are an enigma-Why arouse when remaining torpid would save so much more energy? It has been suggested that hibernators must rewarm to restore or remove a metabolic imbalance (Ref. 27, chapter 6).Metabolomics seeks to identify and quantify the low-molecular-weight endogenous co...

Abstract-Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97Ϯ3 versus 83Ϯ2 mm Hg; PϽ0.05) and a 25% increase in serum ACE activity (48.4Ϯ7.0 versus 36.0Ϯ2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8Ϯ1.2 versus 31.4Ϯ4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming-induced hypertension. Key Words: fetal programming Ⅲ hypertension Ⅲ renin-angiotensin system Ⅲ sheep Ⅲ angiotensin-converting enzyme 2 Ⅲ proximal tubules and serum I n a landmark article, Liggins and Howie 1 demonstrated that antenatal glucocorticoid therapy administered to women at risk for preterm delivery reduced the incidence of respiratory distress syndrome and mortality in their offspring. The efficacy of antenatal glucocorticoid therapy has been confirmed subsequently by many randomized, controlled trials. 2,3 The reduction in the severity and incidence of respiratory distress syndrome has resulted in decreased requirements for surfactant therapy, lower concentrations of supplemental oxygen, and a decreased need for prolonged mechanical ventilation in the neonatal period. 4 Indeed, various organizations, including the National Institutes of Health 4 and the American College of Obstetricians and Gynecologists, have recommended antenatal glucocorticoid treatment for women at risk for preterm delivery before 34 weeks of gestation. However, a recent report showed that, at 14 years of age, blood pressure was higher in a group of children born preterm whose mothers received betamethasone before preterm delivery compared with a similar group who were not exposed to corticosteroids. 5 The mechanism for this increase in blood pressure is not fully known, but severa...