Antenatal betamethasone attenuates the angiotensin-(1-7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells.

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1-7) [Ang-(1-7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1-7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1-7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1-7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1-7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1-7). We conclude that the tubular Ang-(1-7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

Antenatal corticosteroids and the renin-angiotensin-aldosterone system in adolescents born preterm.

BACKGROUND: Antenatal corticosteroid (ANCS) treatment hastens fetal lung maturity and improves survival of premature infants, but the long-term effects of ANCS are not well-described. Animal models suggest that ANCS increases the risk of cardiovascular disease through programmed changes in the renin-angiotensin (Ang)-aldosterone system (RAAS). We hypothesized that ANCS exposure alters the RAAS in adolescents born prematurely. METHODS: A cohort of 173 adolescents born prematurely was evaluated, of whom 92 were exposed to ANCS. We measured plasma and urine Ang II and Ang-(1-7) and calculated Ang II/Ang-(1-7) ratios. We used general linear regression models to estimate the difference in the RAAS between the ANCS-exposed and unexposed groups, adjusting for confounding variables. RESULTS: In unadjusted analyses, and after adjustment for sex, race, and maternal hypertension, ANCS exposure was associated with increased urinary Ang II/Ang-(1-7) (estimate 0.27 (95% CI 0.03, 0.5), P = 0.03), increased plasma Ang-(1-7) (0.66 (0.26, 1.07), P = 0.002), and decreased plasma Ang II/Ang-(1-7) (-0.48 (-0.91, -0.06), P = 0.03). CONCLUSION: These alterations indicate an imbalance in the urinary RAAS, promoting the actions of Ang II at the expense of Ang-(1-7), which over time may increase the risk of renal inflammation and fibrosis and ultimately hypertension and renal disease.

Antenatal steroid exposure and heart rate variability in adolescents born with very low birth weight.

BACKGROUND: Reduced heart rate variability (HRV) suggests autonomic imbalance in the control of heart rate and is associated with unfavorable cardiometabolic outcomes. We examined whether antenatal corticosteroid (ANCS) exposure had long-term programming effects on HRV in adolescents born with very low birth weight (VLBW). METHODS: Follow-up study of a cohort of VLBW 14-y olds born between 1992 and 1996 with 50% exposed to ANCS. HRV in both the time and frequency domains using Nevrokard Software was determined from a 5-min electrocardiogram tracing. RESULTS: HRV data from 89 (35 male, 53 non-black) exposed (ANCS+) and 77 (28 male, 29 non-black) unexposed (ANCS-) adolescents were analyzed. HRV did not differ between ANCS+ and ANCS- black participants. However, in non-black participants, a significant interaction between ANCS and sex was observed, with ANCS- females having significantly greater HRV than ANCS+ females and males, and ANCS- males for both time and frequency domain variables. CONCLUSION: Among non-black adolescents born with VLBW, ANCS exposure is associated with reduced HRV with apparent sex-specificity. Reduced HRV has been associated with development of adverse cardiometabolic outcomes, thus supporting the need to monitor these outcomes in VLBW adolescents as they mature.

Angiotensinogen import in isolated proximal tubules: evidence for mitochondrial trafficking and uptake.

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min-1·mg protein-1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Sex-Specific Changes in Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 Gene Expression and Enzyme Activity at Birth and Over the First Year of Life.

OBJECTIVE: Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) are key enzymes of the renin-angiotensin system. We investigated developmental changes in renal ACE and ACE2 gene expression and activity in both male and female sheep. METHODS: Three groups of sheep (fetus, newborn, and adult) were used. Renal ACE and ACE2 activities, messenger RNA (mRNA), and protein expression were studied. RESULTS: Renal ACE and ACE2 activities increased at 1 year in males, while there were no changes throughout development in females. Renal ACE and ACE2 mRNA and protein showed no sex differences but increased by 1 year of age. CONCLUSION: There are sex-related differences in the development of renal-converting enzyme activities that may have functional implications in terms of the regulation of blood pressure and renal function in men and women. The difference in the patterns of gene expression and enzyme activity indicates that changes in gene expression may not accurately reflect changes in activity.

Evidence for a mitochondrial angiotensin-(1-7) system in the kidney.

Evidence for an intracellular renin-angiotensin system (RAS) in various cell organelles now includes the endoplasmic reticulum, nucleus, and mitochondria (Mito). Indeed, angiotensin (ANG) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively, in previous studies. We undertook a biochemical analysis of the Mito RAS from male and female sheep kidney cortex. Mito were isolated by differential centrifugation followed by a discontinuous Percoll gradient and were coenriched in Mito membrane markers VDAC and ATP synthase, but not ß-actin or cathepsin B. Two distinct renin antibodies identified a 37-kDa protein band in Mito; angiotensinogen (Aogen) conversion was abolished by the inhibitor aliskiren. Mito Aogen was detected by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the ANG I N terminus. ANG peptides were quantified by three direct RIAs; mitochondrial ANG II and ANG-(1-7) contents were higher compared with ANG I (23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; P < 0.01, n = 3). 125I-ANG I metabolism primarily revealed the formation of 125I-ANG-(1-7) in Mito that reflects the endopeptidases neprilysin and thimet oligopeptidase. Last, immunoblot studies utilizing the ANG-(1-7)/Mas receptor antibody revealed the protein in isolated Mito from sheep renal cortex. Collectively, the current data demonstrate that Mito actively metabolize the RAS precursor protein Aogen, suggesting that ANG-(1-7) may be generated within Mito to establish an intramitochondrial RAS tone and contribute to renal mitochondrial function.

Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner.

We have shown a sex-specific effect of fetal programming on Na(+) excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na(+) uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and female sheep, we measured Na(+) uptake under basal conditions and after stimulation with ANG II. To gain insight into the mechanisms involved, we also measured nitric oxide (NO) levels, ANG II receptor mRNA levels, and expression of Na(+)/H(+) exchanger 3. Basal Na(+) uptake increased more in cells from Beta-exposed male sheep than in cells from vehicle-exposed male sheep (400% vs. 300%, P < 0.00001). ANG II-stimulated Na(+) uptake was also greater in cells from Beta-exposed males. Beta exposure did not increase Na(+) uptake by RPTCs from female sheep. NO production was suppressed more by ANG II in RPTCs from Beta-exposed males than in RPTCs from either vehicle-exposed male or female sheep. Our data suggest that one site of the sex-specific effect of Beta-induced fetal programming in the kidney is the RPTC and that the enhanced Na(+) uptake induced by antenatal Beta in male RPTCs may be related to the suppression of NO in these cells.

An angiotensin-(1-7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme.

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. J Neurochem 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 fmol·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 mercury-containing 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(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-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.

Early Pregnancy Maternal Lipid Profiles and the Risk of Gestational Diabetes Mellitus Stratified for Body Mass Index.

OBJECTIVE: To determine associations between lipid profiles in early pregnancy stratified by body mass index (BMI) and risk of developing gestational diabetes mellitus (GDM). STUDY DESIGN: A total of 2488 healthy pregnant women were enrolled prospectively. Fasting plasma lipid profiles were measured at mean 11 weeks of gestation including triglycerides (TGs), total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cholesterol (CHO). We assessed early pregnancy maternal lipid concentrations in different tertiles in association with the risk of GDM stratified for BMI. Multivariable logistic regression analyses were used to estimate the relative risk of GDM by calculating odds ratios and 95% confidence intervals (CIs). RESULTS: In univariate analyses, pregnant women with GDM had significantly increased serum TG, CHO, LDL concentrations, LDL/HDL ratio, and decreased LDL concentrations, compared to control groups, each P < .01, respectively. After adjustment for confounders, there was a 1.8-fold increase in risk for GDM in the lean group (95% CI: 1.2-2.7) and 2.7-fold increase in the obese group (95% CI: 1.1-6.6), respectively, if TG ≥ 1.58 mmol/L. About a 50% decrease in the risk of GDM was observed in lean women with HDL ≥ 2.22 mmol/L (95% CI: 0.3-0.9). No significant correlations of other lipid profiles with the risk of developing GDM were observed. CONCLUSION: Early pregnancy dyslipidemia is associated with the risk of developing GDM. Lean or obese women with higher TG concentrations are at an increased risk for developing GDM while lean women with high HDL are protected.

Sex-specific effect of antenatal betamethasone exposure on renal oxidative stress induced by angiotensins in adult sheep.

Prenatal glucocorticoid administration in clinically relevant doses reduces nephron number and renal function in adulthood and is associated with hypertension. Nephron loss in early life may predispose the kidney to other insults later but whether sex influences increases in renal susceptibility is unclear. Therefore, we determined, in male and female adult sheep, whether antenatal glucocorticoid (betamethasone) exposure increased 8-isoprostane (marker of oxidative stress) and protein excretion after acute nephron reduction and intrarenal infusions of angiotensin peptides. We also examined whether renal proximal tubule cells (PTCs) could contribute to alterations in 8-isoprostane excretion in a sex-specific fashion. In vivo, ANG II significantly increased 8-isoprostane excretion by 49% and protein excretion by 44% in male betamethasone- but not in female betamethasone- or vehicle-treated sheep. ANG-(1-7) decreased 8-isoprostane excretion but did not affect protein excretion in either group. In vitro, ANG II stimulated 8-isoprostane release from PTCs of male but not female betamethasone-treated sheep. Male betamethasone-exposed sheep had increased p47 phox abundance in the renal cortex while superoxide dismutase (SOD) activity was increased only in females. We conclude that antenatal glucocorticoid exposure enhances the susceptibility of the kidney to oxidative stress induced by ANG II in a sex-specific fashion and the renal proximal tubule is one target of the sex-specific effects of antenatal steroids. ANG-(1-7) may mitigate the impact of prenatal glucocorticoids on the kidney. P47 phox activation may be responsible for the increased oxidative stress and proteinuria in males. The protection from renal oxidative stress in females is associated with increased SOD activity.

Evidence for an angiotensin-(1-7) neuropeptidase expressed in the brain medulla and CSF of sheep.

Angiotensin-(1-7) [Ang-(1-7)] is an alternative product of the brain renin-angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang-(1-7) to the inactive metabolite product Ang-(1-4) in CSF of adult sheep. This study purified the peptidase 1445-fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o-phenanthroline and EDTA, as well as the mercury compound p-chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin-converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV-390 was a potent inhibitor of Ang-(1-7) hydrolysis (Ki = 0.8 nM). Kinetic studies using (125) I-labeled Ang-(1-7), Ang II, and Ang I revealed comparable apparent Km values (2.6, 2.8, and 4.3 µM, respectively), but a higher apparent Vmax for Ang-(1-7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang-(1-7) to Ang-(1-4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin-13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang-(1-7) within the brain. Angiotensin-(1-7) actions are mediated by the AT7 /Mas receptor and include reduced blood pressure, decreased oxidative stress, enhanced baroreflex sensitivity, and increased nitric oxide (NO). Ang-(1-7) is directly formed from Ang I by neprilysin (NEP). We identify a new pathway for Ang-(1-7) metabolism in the brain distinct from angiotensin-converting enzyme-dependent hydrolysis. The Ang-(1-7) endopeptidase (A7-EP) degrades the peptide to Ang-(1-4) and may influence central Ang-(1-7) tone.

Enhanced activity of an angiotensin-(1-7) neuropeptidase in glucocorticoid-induced fetal programming.

We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1-7) [Ang-(1-7)] to Ang-(1-5) and Ang-(1-4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1-7) to Ang-(1-4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1-7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1-7) (5µM) and Ang II (3µM), but lower values for Ala(1)-Ang-(1-7) and Ang-(2-7) of 1.8 and 2.0µM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32µM) than Ang-(1-7) while neurotensin was a poor competitor. Mean arterial pressure (78±1 vs. 94±2mmHg, N=4-5, P<0.01) and Ang-(1-7) peptidase activity (14.2±1 vs 32±1.5fmol/min/ml CSF, N=5, P<0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1-7) content in CSF. Lower Ang-(1-7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1-7) peptidase may contribute to lower CSF Ang-(1-7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming.

Antenatal betamethasone exposure is associated with lower ANG-(1-7) and increased ACE in the CSF of adult sheep.

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.

Antenatal glucocorticoid exposure enhances the inhibition of adrenal steroidogenesis by leptin in a sex-specific fashion.

Antenatal treatment with glucocorticoids (GC) poses long-lasting effects on endocrine and cardiovascular function. Given that leptin attenuates adrenal function and the reported sex differences in plasma leptin concentration, we hypothesized that antenatal GC will affect leptin levels and leptin modulation of adrenal function in a sex-specific manner. Pregnant sheep were randomly given betamethasone or vehicle at 80 days of gestational age, and offspring were allowed to deliver at term. Adrenocortical cells (ADC) were studied from male and female animals at 1.5 yr of age. Plasma leptin was increased 66% in male and 41% in female GC-treated animals (P < 0.05), but adrenal leptin mRNA was increased only in GC-treated males (P < 0.05). Whereas mRNA expression of adrenal leptin receptor isoforms showed sex (Ob-Ra and Ob-Rb) and treatment-dependent (Ob-Rb) differences, protein expression remained unchanged. GC-treated females showed greater plasma cortisol and greater ACTH-stimulated cortisol production (P < 0.05) in ADC. Leptin exerted a greater inhibitory effect on basal and stimulated cortisol by ADC from GC-treated males (P < 0.05), with no differences in females. Similarly, greater inhibitory effects on basal and ACTH-stimulated StAR and ACTH-R mRNA expression by leptin were observed in cells from GC males (P < 0.05), with no changes in females. Persistent effects of antenatal GC on leptin levels and leptin modulation of adrenal function are expressed in a sex-specific manner; males are more sensitive than females to the inhibitory influences of leptin on adrenal function, and this effect appears to be mediated by a greater inhibition of StAR and ACTH-R expression in adrenals of adult GC-treated males.

Fetal betamethasone exposure attenuates angiotensin-(1-7)-Mas receptor expression in the dorsal medulla of adult sheep.

Glucocorticoids including betamethasone (BM) are routinely administered to women entering into early preterm labor to facilitate fetal lung development and decrease infant mortality; however, fetal steroid exposure may lead to deleterious long term consequences. In a sheep model of fetal programming, BM-exposed (BMX) offspring exhibit elevated mean arterial pressure (MAP) and decreased baroreflex sensitivity (BRS) for control of heart rate by 0.5-years of age associated with changes in the circulating and renal renin-angiotensin systems (RAS). In the brain solitary tract nucleus, angiotensin (Ang) II actions through the AT1 receptor oppose the beneficial actions of Ang-(1-7) at the Mas receptor for BRS regulation. Therefore, we examined Ang peptides, angiotensinogen (Aogen), and receptor expression in this brain region of exposed and control offspring of 0.5- and 1.8-years of age. Mas protein expression was significantly lower (>40%) in the dorsal medulla of BMX animals at both ages; however, AT1 receptor expression was not changed. BMX offspring exhibited a higher ratio of Ang II to Ang-(1-7) (2.30±0.36 versus 0.99±0.28; p<0.01) and Ang II to Ang I at 0.5-years. Although total Aogen was unchanged, Ang I-intact Aogen was lower in 0.5-year BMX animals (0.78±0.06 vs. 1.94±0.41; p<0.05) suggesting a greater degree of enzymatic processing of the precursor protein in exposed animals. We conclude that in utero BM exposure promotes an imbalance in the central RAS pathways of Ang II and Ang-(1-7) that may contribute to the elevated MAP and lower BRS in this model.

Antenatal betamethasone has a sex-dependent effect on the in vivo response to endothelin in adult sheep.

Antenatal steroid administration is associated with multiple cardiometabolic alterations, including hypertension; however, the mechanisms underlying this phenomenon are unclear. The aim of the present study was to ascertain, in vivo, the contribution of the endothelin system to the development of hypertension in the adult offspring and the signaling pathway involved. Pregnant sheep were treated with two doses of betamethasone (n = 23) or vehicle (n = 22) at 80 days (~0.55) gestation and allowed to deliver at term. Adult sheep were chronically instrumented under general anesthesia to place vascular catheters and a femoral artery flow probe. Blood pressure and flow were recorded continuously, and femoral artery vascular resistance was calculated before and during administration of endothelin 1 (ET-1). Selective blockers (dantrolene, BQ123, niacinamide) or saline were administered simultaneously. Betamethasone-exposed animals exhibited a significant elevation in mean blood pressure (female: 98 ± 1.8 vs. 92 ± 2.1; males: 97 ± 3.4 vs. 90 ± 2.3; mmHg; P < 0.05). ET-1 elicited a significant increase in blood pressure (F = 56.4; P < 0.001) and in vascular resistance (F = 44.3; P < 0.001) in all groups. A betamethasone effect in the vascular resistance response to ET-1 (F = 25.7; P < 0.001) was present in females only, and the effect was partially blunted by niacinamide (F = 6.6; P < 0.01). Combined administration of niacinamide and BQ123, as well as of dantrolene abolished the betamethasone effect on vascular resistance. No significant differences in mRNA expression of ET(A) or ET(B) in endothelial or smooth muscle cells of resistance-size arteries were observed. We conclude that the betamethasone effect on vascular resistance is mediated by an enhanced response to ET-1 through ET(A) receptor via the cyclic ADPR/ryanodine pathway.

Antenatal betamethasone exposure alters renal responses to angiotensin-(1-7) in uninephrectomized adult male sheep.

Antenatal corticosteroid exposure reduces renal function and alters the intrarenal renin-angiotensin system to favor angiotensin activation of angiotensin type 1 receptor (AT1R) mediated responses in ovine offspring. This study aimed to assess whether antenatal steroid exposure would affect renal responses to the direct intrarenal infusion of angiotensin-(1-7) in rams and the angiotensin receptors involved in mediating responses to the peptide. Adult, uninephrectomized rams exposed to either betamethasone or vehicle before birth received intrarenal angiotensin-(1-7) infusions (1 ng/kg/min) alone or in combination with antagonists to angiotensin receptors for 3 h. Basal sodium excretion (UNa) was significantly lower and mean arterial pressure was significantly higher in betamethasone- compared to the vehicle-treated sheep. Angiotensin-(1-7) decreased UNa more in betamethasone- than in vehicle-treated sheep. Candesartan reversed the response to angiotensin-(1-7) but D-Ala(7)-angiotensin-(1-7) did not. Angiotensin-(1-7) infusion decreased effective renal plasma flow in both groups to a similar extent and the response was reversed by candesartan, but was not blocked by D-Ala(7)-angiotensin-(1-7). Glomerular filtration rate increased significantly in both groups after 3 h infusion of angiotensin-(1-7) plus candesartan. These results suggest that antenatal exposure to a clinically relevant dose of betamethasone impairs renal function in rams. Moreover, angiotensin-(1-7) appears capable of activating the AT1R in uninephrectomized rams.

Leptin alters adrenal responsiveness by decreasing expression of ACTH-R, StAR, and P450c21 in hypoxemic fetal sheep.

The late gestation increase in adrenal responsiveness to adrenocorticotropin (ACTH) is dependent upon the upregulation of the ACTH receptor (ACTH-R), steroidogenic acute regulatory protein (StAR), and steroidogenic enzymes in the fetal adrenal. Long-term hypoxia decreases the expression of these and adrenal responsiveness to ACTH in vivo. Leptin, an adipocyte-derived hormone which attenuates the peripartum increase in fetal plasma cortisol is elevated in hypoxic fetuses. Therefore, we hypothesized that increases in plasma leptin will inhibit the expression of the ACTH-R, StAR, and steroidogenic enzymes and attenuate adrenal responsiveness in hypoxic fetuses. Spontaneously hypoxemic fetal sheep (132 days of gestation, PO(2) ≈ 15 mm Hg) were infused with recombinant human leptin (n = 8) or saline (n = 7) for 96 hours. An ACTH challenge was performed at 72 hours of infusion to assess adrenal responsiveness. Plasma cortisol and ACTH were measured daily and adrenals were collected after 96 hours infusion for messenger RNA (mRNA) and protein expression measurement. Plasma cortisol concentrations were lower in leptin- compared with saline-infused fetuses (14.8 ± 3.2 vs 42.3 ± 9.6 ng/mL, P < .05), as was the cortisol:ACTH ratio (0.9 ± 0.074 vs 46 ± 1.49, P < .05). Increases in cortisol concentrations were blunted in the leptin-treated group after ACTH(1-24) challenge (F = 12.2, P < .0001). Adrenal ACTH-R, StAR, and P450c21 expression levels were reduced in leptin-treated fetuses (P < .05), whereas the expression of Ob-Ra and Ob-Rb leptin receptor isoforms remained unchanged. Our results indicate that leptin blunts adrenal responsiveness in the late gestation hypoxemic fetus, and this effect appears mediated by decreased adrenal ACTH-R, StAR, and P450c21 expression.

Angiotensin-(1-7) deficiency and baroreflex impairment precede the antenatal Betamethasone exposure-induced elevation in blood pressure.

Betamethasone is administered to accelerate lung development and improve survival of premature infants but may be associated with hypertension later in life. In a sheep model of fetal programming resulting from exposure at day 80 of gestation to Betamethasone (Beta-exposed), adult sheep at 6 to 9 months or 1.8 years of age have elevated mean arterial pressure (MAP) and attenuated spontaneous baroreflex sensitivity (sBRS) for control of heart rate compared to age-matched controls associated with imbalances in angiotensin (Ang) II vs Ang-(1-7) tone. At 6 weeks of age, evoked BRS is already low in the Beta-exposed animals. In this study, we assessed the potential contribution of the renin-angiotensin system to the impaired sBRS. Female lambs (6 weeks old) with Beta exposure in utero had similar MAP to control lambs (78±2 vs 77±2 mm Hg, n=4-5 per group), but lower sBRS (8±1 vs 16±3 ms/mm Hg; P<0.05) and impaired heart rate variability. Peripheral AT1 receptor blockade using candesartan lowered MAP in both groups (≈10 mm Hg) and improved sBRS and heart rate variability in Beta-exposed lambs to a level similar to control. AT7 receptor blockade by infusion of D-ala Ang-(1-7) (700 ng/kg/min for 45 minutes) reduced sBRS 46%±10% in Beta-exposed vs in control lambs (P<0.15) and increased MAP in both groups (≈6±2 mm Hg). Our data reveal that Beta exposure impairs sBRS and heart rate variability at a time point preceding the elevation in MAP via mechanisms involving an imbalance in the Ang II/Ang-(1-7) ratio consistent with a progressive loss in Ang-(1-7) function.

Exaggerated sympathetic mediated responses to behavioral or pharmacological challenges following antenatal betamethasone exposure.

Glucocorticoid administration to women at risk for preterm delivery is standard practice to enhance neonatal survival. However, antenatal betamethasone exposure (ß-exposure) increases mean arterial pressure (MAP) in adult sheep (1.8 yr old) and results in impaired baroreflex sensitivity (BRS) for control of heart rate (HR). In the current studies we tested the hypothesis that enhanced sympathetic nervous system and hypothalamo-pituitary-adrenal (HPA) axis-mediated responses are evident at an early age in ß-exposed sheep. Pregnant ewes were administered betamethasone (0.17 mg/kg twice over 24 h) or vehicle (Veh-control) on the 80th day of gestation, and offspring were delivered at full term. Female ß-exposed and control offspring instrumented at age 42 ± 3 days for conscious continuous recording of MAP and HR had similar resting values at baseline. However, BRS was ~45% lower in ß-exposed offspring. ß-Exposed lambs allowed to suckle for 10 min had a greater elevation in MAP than Veh-control lambs (19 ± 1 vs 12 ± 2 mmHg; n = 4-5, P < 0.05). MAP was reduced by 20% from baseline via sodium nitroprusside infusion (SNP) over 10 min, which triggered a rebound increase in MAP only in ß-exposed lambs. HR increased with the reduction in MAP during SNP infusion in Veh-control lambs, whereas there was no change in HR with the reduction in MAP in ß-exposed lambs. Combined vasopressin-CRF injection caused greater increases in MAP in the ß-exposed lambs. Cortisol and ACTH responses were higher in response to SNP hypotension in the ß-exposed lambs. The data reveal enhanced sympathetic and HPA axis responses associated with impaired BRS preceding differences in resting MAP in preweanling female lambs exposed in utero to glucocorticoids. The consequences of these alterations at an early age include eventual development of higher blood pressure in this ovine model of fetal programming.