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The mRen(2).Lewis (mRen2) strain is an ANG If-dependent model of hypertension expressing marked sex differences in blood pressure and tissue injury that also exhibits estrogen and salt sensitivity. Because estrogen and salt influence angiotensinogen (AGT), circulating and renal expression of the protein were assessed in the mRen2 using a sensitive and specific ELISA. Hemizygous female and male mRen2 were placed on normal (1% NaCl, NS)- or high (8% NaCl, HS)-salt diets from 5 to 15 wk of age while a separate NS cohort was ovariectomized (OVX). The OVX mRen2 exhibited higher blood pressure (184 [+ or -] 6 vs. 149 [+ or -] 5 mmHg, n = 6), a 16-fold increase in urinary AGT (uAGT) (0.2 [+ or -] 0.02 vs. 0.01 [+ or -] 0.01 [micro]gx[kg.sup.-1]x[day.sup.-1], P < 0.01), but no change in proteinuria (PROT). Excretion of AGT was correlated with blood pressure and PROT in the female groups. The HS diet led to higher blood pressure (224 [+ or -] 8 mmHg), a 180-fold increase in uAGT (1.8 [+ or -] 0.2 [micro]gx[kg.sup.-1]x[day.sup.-1]), and increased PROT (98 [+ or -] 9 vs. 7 [+ or -] 1 mgx[kg.sup.-1]x[day.sup.-1]). Compared with females, NS males expressed higher excretion of uAGT (3.0 [+ or -] 0.4 [micro]gx[kg.sup-1]x[day.sup.-1]) and PROT (32 [+ or -] 5 mgx[kg.sup.-1]x[day.sup.-1]); both were increased eightfold with HS (uAGT: 23 [+ or -] 3 [micro]gx[kg.sup.-1]x[day.sup.-1]; PROT: 285 [+ or -] 28 mgx[kg.sup.-1]x[day.sup.-1]) without a change in blood pressure. Although uAGT was markedly higher in the OVX and HS groups, neither renal cortical AGT mRNA or protein expression was increased. Moreover, AGT release in cortical slices was similar for the NS and HS females. We conclude that the increase in uAGT with estrogen depletion or HS likely may be a biomarker for glomemlar damage reflecting filtration of the circulating protein in the mRen2. angiotensin II; normal salt diet; high-salt diet doi: 10.1152/ajprenal.00138.2010.

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

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

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

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 1-7 [ANG- (1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. JNeurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol.min-1-mg-1) compared with the tubules (96 ± 12. min-1-mg-1) and cortex (107 ± 9 fmol.min-1-mg-1). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercurycontaining compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala*]-ANG- (1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp*]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone. [ABSTRACT FROM AUTHOR]

The kidney is an important target for the actions of the renin-angiotensin system (RAS) and this tissue contains a complete local RAS that expresses the bioactive peptides angiotensin II (ANG II) and Ang-(1-7). We find both angiotensin type 1 (AT1R) and type 2 (AT2R) receptors expressed on renal nuclei that stimulate reactive oxygen species and nitric oxide (NO), respectively. Since Ang-(1-7) also exhibits actions within the kidney and the Ang-(1-7)/Mas receptor protein contains a nuclear localization sequence, we determined the expression of Ang-(1-7) receptors in nuclei isolated from the kidneys of young adult sheep. Binding studies with 125I-[Sar(1)Thr8]-ANG II revealed sites sensitive to the Ang-(1-7) antagonist [d-Ala7]-Ang-(1-7) (DALA, A779), as well as to AT2 and AT(1) antagonists. Incubation of Ang-(1-7) [10-15 to 10-9 M] with isolated cortical nuclei elicited a dose-dependent increase in the fluorescence of the NO indicator [4-amino-5-methylamino-2',7']-difluorofluorescein diacetate. The NO response to Ang-(1-7) was abolished by the NO inhibitor N-nitro-l-arginine methyl ester and DALA, but not the AT(1) antagonist losartan or the AT(2) blocker PD123319. Immunofluorescent studies utilizing the Ang-(1-7)/Mas receptor antibody revealed immunolabeling of the proximal tubules but not staining within the glomerulus in cortical sections of the sheep kidney. In the nuclear fraction of isolated proximal tubules, immunoblots revealed the precursor angiotensinogen and renin, as well as functional activity for ACE, ACE2, and neprilysin. We conclude that renal nuclei express Ang-(1-7)/Mas receptors that are functionally linked to NO formation. The marked sensitivity of the intracellular NO response to Ang-(1-7) implicates a functional role of the Ang-(1-7) axis within the nucleus. Moreover, evidence for the precursor and enzymatic components of the RAS within the nuclear compartment of the proximal tubules provides a potential pathway for the intracellular generation of Ang-(1-7). [ABSTRACT FROM AUTHOR]

Expression of nuclear angiotensin II type I (AT1) 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 251-[Sar1,Thr8]-ANG II (125I-sarthran) with the AT1 antagonist losartan (LOS) or the AT2 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 (⩾70%) and PM (⩾80%) sites while LOS competition predominated in medullary NUC (⩾75%) and PM (⩾ 70%). Immunodetection with an AT2 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 AT2 in the tubulointerstitium, AT1 in the medulla and vasa recta, and both AT1 and AT2 in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG 11(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 AT2 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. [ABSTRACT FROM AUTHOR]

The cannabinoids arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) are endogenous ligands for the cannabinoid receptors including CB1 and CB2 subtypes. The endocannabinoid system (ECS) is typically associated with regulation of metabolic balance and appetite, although CB1 receptors (CB1R) may contribute to diabetic renal injury while CB2R are protective. The hypertensive mRen2.Lewis (mRen2) females have less renal injury than the males; however, diabetic female mRen2 lack this protection and show comparable indices of injury to the diabetic males. The present study addressed whether female and male diabetic mRen2 exhibit similar alterations in the renal ECS. Diabetes was induced with a single dose of streptozotocin (STZ at 65 mg/kg; ip). Cortical content of AEA and 2-AG was quantified by UPLC MS/MS. Receptor levels and the metabolizing enzymes for AEA (FAAH) and 2-AG (MAGL) were quantified by protein immunoblot. Basal 2-AG content was higher in male versus female mRen2, and significantly increased in both diabetic females and males (figure, n=6) while AEA was unchanged. Cortical Ang II correlated to 2-AG [p<0.01; r = 0.89]. CB1R protein expression was higher in female diabetic mRen2 but unchanged in the males (figure, n=4). CB2R expression was lower in the male diabetics [p<0.01; n=4], but not changed in females. MAGL expression was >70% lower in female diabetics compared to controls [p<0.05; n=4] while FAAH was not changed. Elements of the renal ECS are differentially regulated in the diabetic mRen2. Higher expression of both 2-AG and CB1R, but reduced MAGL (females) and CB2R (males) may contribute to the development and progression of renal injury in this model. [ABSTRACT FROM AUTHOR]

Antenatal betamethasone (BM) therapy accelerates lung development in preterm infants but may induce early programming events with long-term cardiovascular consequences. To elucidate these programming events, we developed a model of programming whereby pregnant ewes are administered BM (2 doses of 0.17 mg/kg) or vehicle at the 80th day of gestation and offspring are delivered at term. The BM-exposed (BMX) offspring develop elevated blood pressure, decreased baroreflex sensitivity, and alterations in the circulating, renal, and brain renin-angiotensin systems (RAS) by 6-months of age. While components of the RAS are present in cerebrospinal fluid (CSF), it is not known if BM exposure influences the CSF RAS to the same extent as other tissue compartments. Therefore, we characterized the CSF RAS in 6-month old male sheep, and established the impact of antenatal exposure on the RAS. Consistent with our previous findings of predominant ACE2 activity in the choroid plexus, CSF Ang-(1-7) levels are significantly higher than Ang I (320.8 ± 96.0 vs. 19.3 ± 4.6 pM; p<0.01, N = 12) or Ang II (1.4 ± 0.5 pM; p<0.01, N = 12). Moreover, Ang-(1-7) was 70% lower in the CSF of the BMX group (97.3 ± 31.4 pM; p<0.05) without changes in Ang I or Ang II. We next assessed the metabolism of Ang-(1-7) in the CSF and demonstrate a trend for increased enzyme activity in BMX males. Further analysis revealed two peptidases that contribute to Ang-(1-7) degradation in CSF of both groups. ACE accounted for approximately 15% (85 out of 100) of the overall Ang-(1-7) metabolism that hydrolyzed the peptide to Ang-(1-5) in CSF and ACE was 1.4-fold higher in the BMX group (6.5 ± 0.5 vs. 8.8 ± 0.5 fmol/min/ml; p<0.02, N = 4). We also identified a novel thiol peptidase activity (Ki = 4 μM PCMB) as the predominant activity that metabolized Ang-(1-7) to Ang-(1-4) in CSF; kinetic analysis of this peptidase in pooled CSF revealed Km and Vmax constants of 5.4 μM and 54 nmol/min/mg for control, and 4 μM and 60 nmol/min/mg for BMX. In summary, CSF levels of Ang-(1-7) were markedly lower following antenatal exposure. The reduced expression of central Ang-(1-7) potentially through an enhanced metabolism pathway may contribute to the long-term increase in blood pressure and an attenuated baroreflex in this model of fetal programming. [ABSTRACT FROM AUTHOR]

Antenatal betamethasone (BM) therapy accelerates lung development in preterm infants but may induce early programming events with long-term cardiovascular consequences. To elucidate these events, we developed a model of programming whereby pregnant ewes are administered BM (2 doses of 0.17 mg/kg) or vehicle at the 80th day of gestation and offspring are delivered at term. BM-exposed (BMX) offspring develop elevated blood pressure; decreased baroreflex sensitivity; and alterations in the circulating, renal, and brain renin-angiotensin systems (RAS) by 6 mo of age. We compared components of the choroid plexus fourth ventricle (ChP4) and cerebral spinal fluid (CSF) RAS between control and BMX male offspring at 6 mo of age. In the choroid plexus, high-molecular-weight renin protein and ANG I-intact angiotensinogen were unchanged between BMX and control animals. Angiotensin-converting enzyme 2 (ACE2) activity was threefold higher than either neprilysin (NEP) or angiotensin 1-converting enzyme (ACE) in control and BMX animals. Moreover, all three enzymes were equally enriched by approximately 2.5-fold in ChP4 brush-border membrane preparations. CSF ANG-(1-7) levels were significantly lower in BMX animals (351.8 ± 76.8 vs. 77.5 ± 29.7 fmol/mg; P < 0.05) and ACE activity was significantly higher (6.6 ± 0.5 vs. 8.9 ± 0.5 fmol·min(-1)·ml(-1); P < 0.05), whereas ACE2 and NEP activities were below measurable limits. A thiol-sensitive peptidase contributed to the majority of ANG-(1-7) metabolism in the CSF, with higher activity in BMX animals. We conclude that in utero BM exposure alters CSF but not ChP RAS components, resulting in lower ANG-(1-7) levels in exposed animals.

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 +/- 4 vs. 146 +/- 7 mmHg, P < 0.01] or Lewis males and females [113 +/- 2 vs. 112 +/- 2 mmHg, P > 0.05]. Plasma ANG II levels were twofold higher in male congenics [47 +/- 3 vs. 19 +/- 3 pM, P < 0.01] and fivefold higher than in male or female Lewis rats [6 +/- 1 vs. 6 +/- 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.

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 +/- 6 vs. Intact 182 +/- 7 mmHg; P = 0.22] after the 4-wk diet; however, proteinuria [OVX 0.8 +/- 0.2 vs. Intact 11.5 +/- 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.