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Synthetic glucocorticoids are commonly given to pregnant women when premature delivery threatens. Antenatal administration of clinically relevant doses of betamethasone to pregnant sheep causes sex-specific compromises of renal function and increases in blood pressure in adult offspring. However, it is unclear whether such effects are present in immature lambs. Therefore, the aims of the present study were to determine whether antenatal betamethasone at 80-81 days of gestation increases blood pressure and adversely impacts renal function in adolescent ewes and rams. Prenatal steroid exposure increased blood pressure significantly in the young male (84 +/- 2 vs. 74 +/- 3 mmHg) and female sheep (88 +/- 5 vs. 79 +/- 4), but it did not alter basal glomerular filtration rate, renal blood flow (RBF), or sodium excretion in either sex. However, antenatal betamethasone exposure blocked increases in RBF (P = 0.001), and enhanced excretion of an acute Na load (P < 0.05) in response to systemic infusions of angiotensin (ANG)-(1-7) at 10 pmol.kg(-1).min(-1) in males. In females, the natriuretic response to combined ANG-(1-7), and Na load was significantly altered by prenatal betamethasone exposure. These findings indicate that blood pressure is increased in immature animals in response to antenatal steroid exposure and that sex-specific effects on renal function also exist. These changes may reflect greater risk for further loss of renal function with age.

Prenatal glucocorticoids have long-term effects on the kidney and blood pressure that may be mediated by the renin-angiotensin system (RAS). We studied the effects of antenatal betamethasone administration on renin in fetal and adult female sheep. Pregnant sheep received 2 doses of betamethasone or vehicle, at 80 and 81 days of gestation (dGA). Fetuses were delivered within 24 hours following treatment, at 135 dGA, or allowed to continue to term. Plasma and kidney samples were collected from fetal and 1-year-old sheep. Plasma and renal renin and renin messenger RNA (mRNA) were measured. Significant decreases in plasma and renal renin and renin mRNA were apparent in female betamethasone fetuses at 80 dGA (P < .05). At 135 dGA, renal renin concentrations were significantly increased in betamethasone fetuses. At 1 year, renin levels were similar in the 2 groups. These findings suggest that prenatal betamethasone has an immediate effect on expression and secretion of renin. The downregulation of renin at 80 dGA may affect nephron development.

This study examined whether renin expression and secretion and plasma angiotensin II (Ang II) levels were altered in adult sheep exposed to antenatal betamethasone. Pregnant sheep received injections of 0.17 mg/kg betamethasone or vehicle, at 80 and 81 days of gestation, and offspring were studied at 6 and 18 months of age. At 6 months, plasma prorenin concentrations were significantly lower in betamethasone animals (4.63 +/- 0.64 vs 7.09 +/- 0.83 ng angiotensin I/mL/h, P < .01). The percentage of plasma active renin was significantly higher in the betamethasone group (31.93 +/- 4.09% vs 18.57 +/- 2.79%, P < .01). Plasma and renocortical renin levels were similar in both groups at 18 months, but plasma renin activity was lower than at 6 months. Ang II levels were suppressed by betamethasone. The data indicate that prenatal exposure to betamethasone alters processing and secretion of renin in offspring at 6 months, but that this difference is not apparent at 18 months.

In late gestation fetal sheep, the pituitary becomes increasingly responsive to stimulation by arginine vasopressin (AVP). This change appears to be one important factor mediating the plasma cortisol surge, a critical developmental event. It is not known precisely why pituitary corticotropes become more responsive at this time. In this study we examined the possibility that changes in second messenger generation [inositol trisphosphate (IP(3))] are responsible. Two studies were undertaken. The first was an ontogeny study, where pituitaries were isolated from 100-, 120-, and 140-d gestational age (dGA) fetal sheep. Cells were cultured, stimulated with AVP, and the formation of IP(3) assessed. The amount of IP(3) generated increased with gestational age (percent increases from unstimulated controls were 4.6, 11.5, and 21.5 for 100, 120, and 140 dGA, respectively), with significant differences between the 140-dGA group and both earlier groups apparent. The second study examined the impact of 120-dGA hypothalamo-pituitary disconnection (HPD), which prevents corticotrope maturation, on responsiveness of pituitary cells isolated from 140-dGA fetuses. Cells were stimulated with AVP, and the formation of IP(3) and secretion of ACTH were assessed. Significantly less IP(3) was formed, and ACTH secreted in cells from HPD compared with control fetuses (IP(3) and ACTH levels were 50% and 35% lower, respectively). Results from the HPD study demonstrate that the ontogenic changes in IP(3) after AVP require an intact hypothalamic-pituitary-adrenal axis. These findings suggest that heightened second messenger generation may be a key reason for increased ACTH secretory responsiveness to AVP in the late gestation sheep fetus.

Fetal renin-angiotensin system (RAS) activity is developmentally regulated, increasing in late gestation toward term. At the same time, fetal hemodynamic parameters change, with blood pressure increasing and heart rate decreasing. During this period, fetal plasma thyroid hormone concentrations also increase significantly. In this study we utilized the technique of thyroidectomy (TX), which removes thyroid hormone from the circulation, to investigate the importance of thyroid hormone on the developmental changes in the RAS (in plasma, kidney, heart, and lung) and hemodynamic regulation in fetal sheep. TX was performed at 120 days of gestational age (dGA), and control fetuses were sham operated. Immediately before necropsy ( approximately 137 dGA), fetuses were infused with isoproterenol and the hemodynamic responses were noted. TX significantly decreased plasma thyroid hormone concentrations and renal renin mRNA and renal active renin levels but did not change fetal plasma active renin levels. TX decreased both angiotensin II receptor subtype 1 (AT1) mRNA and protein levels in kidney and lung but not in the left ventricle. TX also was associated with increased ANG II receptor subtype 2 (AT2) mRNA and protein at the 44-kDa band in kidney, whereas AT2 protein was decreased at the 78-kDa level in kidney and lung tissue only. TX fetuses had significantly lower basal mean arterial blood pressures (MAP) and heart rates than controls. Isoproterenol infusion decreased MAP in TX fetuses. These findings support the hypothesis that thyroid hormone is important in modulating maturation of RAS and cardiovascular function in the late-gestation fetal sheep.

Epidemiologic studies have yielded controversial information regarding an association between antenatal steroid administration and elevations in arterial blood pressure (BP). The aim of the study was to determine whether antenatal administration of a clinically relevant dose of steroids at a time when fetal nephrogenesis is at its highest results in abnormal kidney development and adult hypertension. Pregnant sheep were treated with either vehicle or betamethasone. Maternal injections were given 24 h apart at 80 d of gestational age (dGA; 0.55 of gestation). Animals were studied either as fetuses or as immature adults. Fetuses were delivered by cesarean section at 135 dGA. Adults were studied at 6 mo of age. Betamethasone administration did not induce premature labor or intrauterine growth restriction. In the betamethasone-exposed group, we found at 135 dGA a 25.5% decrease in the number of glomeruli with no differences in fetal kidney weight. In adults, mean, systolic, and diastolic arterial BPs were significantly higher, whereas there were no significant differences in heart rate over the same study period. The major finding of this study is that a single course of antenatal steroids alters renal development and is associated with elevations in arterial BP in lambs at 6 mo of age. We conclude that antenatal glucocorticoid administration under the National Institutes of Health consensus guidelines may alter human fetal renal development.

The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106-108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121-123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE(2) receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.

Responsiveness of the hypothalamo-pituitary-adrenal axis is decreased during pregnancy. Therefore, the objective of the present study was to determine if responsiveness at the level of individual corticotrophs to corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) is decreased during pregnancy in sheep. Anterior pituitaries (APs) were collected from pregnant and nonpregnant ewes. Half of the APs were dispersed, and cells were placed on immobilon and treated with vehicle, CRH (10 nM), or AVP (100 nM) for 2 h. Cells were then fixed and incubated with ACTH or pro-opiomelanocortin (POMC) antibodies. The percentage of cells staining positive for immunoreactive (ir) ACTH or POMC, the percentage of cells secreting irACTH or POMC, and the area of irACTH or POMC secretion were measured. RNA was extracted from the other half of the APs to quantify CRH type 1 (CRH-R1) and vasopressin type 1b (V1b) receptor mRNA by ribonuclease protection assay. CRH treatment increased the percentage of corticotrophs with relatively large areas of irACTH and POMC secretion in nonpregnant, but not in pregnant, ewes. AVP treatment significantly increased the percentage of irACTH- and POMC-secreting cells in nonpregnant, but not in pregnant, ewes. V1b receptor mRNA, but not CRH-R1 receptor mRNA, was significantly decreased during pregnancy. These results suggest that corticotroph responsiveness to CRH and AVP is decreased during pregnancy in sheep. Therefore, reduced corticotroph responsiveness may contribute to stress hyporesponsivity during pregnancy.

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. Angiotensinconverting 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. [ABSTRACT FROM AUTHOR]

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 ETA or ETB 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 ETA receptor via the cyclic ADPR/ryanodine pathway. [ABSTRACT FROM AUTHOR]

This article presents information from a study that tested whether or not alterations in angiotensin-converting enzyme and neprilysin in serum, urine, and proximile tubules are associated with the increase in mean arterial blood pressure.

Cycloxygenase 2 (COX-2) is important for development of the fetal kidney. Precisely how renal COX-2 expression is regulated in fetal life is unclear. The hypothesis that thyroid hormone positively regulates COX-2 and PGE2 levels in the late gestation fetal kidney cortex was tested. Sham, thyroidectomized (TX), and TX + thyroid hormone replacement (R) fetal sheep were studied. TX was performed at 120 days gestational age (dGA). TX + R fetuses were continuously infused with thyroxine from 3 days after surgery until study completion. Fetal kidney cortex was obtained at 137 dGA for measurement of renal cydooxygenase type-2 (COX-2) protein and PGE2 metabolites. Renocortical COX-2 and PGE2 levels were significantly lower in TX compared with sham and TX + R fetuses. There were no differences between sham and TX + R fetuses. These findings demonstrate that thyroid hormone positively regulates renal COX-2 and PGE2 expression in the late gestation fetal sheep kidney. [ABSTRACT FROM AUTHOR]

Despite the evidence that angiotensin-converting enzyme (ACE)2 is a component of the renin-angiotensin 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 [1251]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 NH2 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 ± 17 fmol·mg-1·min-1) and ACE2 FANG II to ANG-(1-7): 253 ± 11 fmol·mg-1·min-1, but lower neprilysin activity [ANG II to ANG-(1-4): 119 ± 24 fmol·mg-1·min-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). [ABSTRACT FROM AUTHOR]

The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of ~118 and 138 days of gestational age (dGA) were infused with ACTH-(1-24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness. [ABSTRACT FROM AUTHOR]

Objective: This study was designed to determine the role of adenosine in the hypoxic release of corticotropin in fetal sheep.Study Design: The adenosine receptor antagonist 8-phenyltheophylline or the vehicle was infused intra-arterially to chronically catheterized fetal sheep (>0.8 term) during an hour of fetal hypoxemia (Pa O 2 congruent with 14 mm Hg). Control studies were also performed in which 8-phenyltheophylline or the vehicle was administered to normoxic fetuses.Results: 8-Phenyltheophylline abolished hypoxia-induced bradycardia and hypertension and produced a nearly 5-fold greater rise in fetal plasma concentrations of corticotropin and approximately a 3-fold greater increase in plasma cortisol levels. During normoxia 8-phenyltheophylline increased plasma cortisol concentrations by 2-fold without altering corticotropin levels, mean arterial blood pressure, or heart rate.Conclusion: Adenosine blunts fetal corticotropin release during hypoxia, which in turn reduces cortisol secretion. At lower corticotropin concentrations, adenosine also appears to dampen the cortisol response through direct effects on the adrenals. [ABSTRACT FROM AUTHOR]

Presents a study which seeks to determine whether concentrations of plasma cortisol increase at the same time and to the same extent in twin sheep fetuses as they approach term. Importance of gestational ages of fetuses to the study; Question whether activation of adrenal steroidogenesis occurs synchronously in twins; Conclusion of the study.

Studies the effect of exogenous cortisol on plasma immunoreactive adrenocorticotropic hormone (ACTH) in sheep fetuses. Increased plasma cortisol concentrations during saline infusions; Suppression of bioactive ACTH secretion by cortisol selectivity; ACTH response to cortisol inhibition between 126 and 140 days.

Presents information on a study on the pattern of plasma active renin concentration (ARC), prorenin concentration (PRC), renal renin concentration and the renin mRNA levels in ovine fetuses subjected to reduced renal perfusion pressure. Goals of the study; Relationship between ARC and PRC; What the data gathered suggest.

Highlights: [•] BMX decreases Mas receptor expression in brain medulla at 0.5- and 1.8-years [•] Decreased Mas partially explains previous reports of decreased autonomic function [•] Aogen processing is augmented at 0.5-years and reduced at 1.8-years in BMX sheep [•] Peptides ratios are shifted in favor of the Ang II in BMX animals at 0.5-years [•] Ang-(1-7) levels show a positive correlation with the Mas receptor at 0.5-years [Copyright &y& Elsevier]