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.

A specific CRH antagonist attenuates ACTH-stimulated cortisol secretion in ovine adrenocortical cells.

Corticotropin releasing hormone (CRH) has been detected in the adrenal gland of many species and may be involved in regulation of glucocorticoid secretion. In cultured human fetal adrenal definitive/transitional zone cells, CRH upregulates the adrenocorticotropic hormone (ACTH) receptor and steroidogenic enzymes and is blocked by the selective CRH type 1 receptor (CRH(1)) antagonist, antalarmin. Based on these findings and evidence that antalarmin infusion into sheep suppressed prepartum increases in cortisol, we hypothesized that antalarmin would influence adrenal cortisol secretion. Antalarmin strongly attenuated ACTH and forskolin (FSK)-stimulated cortisol and cyclic adenosine monophosphate (cAMP) release from cultured ovine adrenocortical cells but did not prevent ACTH binding to cells or ACTH-induced proliferation in adult cells. Corticotropin releasing hormone was minimally effective as a secretagogue but increased the cortisol response to subsequent ACTH. These results suggest that antalarmin attenuates ACTH-induced cortisol secretion from cultured ovine adrenal cortical cells at a site distal to the ACTH receptor. Although CRH may modulate the secretory response to ACTH, it is probably not a direct cortisol secretagogue in the sheep.

Fetal and postnatal renin secretion in female sheep exposed to prenatal betamethasone.

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.

Plasma and renal renin concentrations in adult sheep after prenatal betamethasone exposure.

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.

Thyroid hormone regulates renocortical COX-2 and PGE2 expression in the late gestation fetal sheep.

Cyclooxygenase 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 PGE(2) 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 cyclooxygenase type-2 (COX-2) protein and PGE(2) metabolites. Renocortical COX-2 and PGE(2) 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 PGE(2) expression in the late gestation fetal sheep kidney.

Thyroid hormone replacement normalizes renal renin and angiotensin receptor expression in thyroidectomized fetal sheep.

Previous studies have suggested that thyroid hormone influences maturation of the renin-angiotensin system (RAS) and cardiovascular function in the late-gestation fetal sheep. To further examine the importance of thyroid hormone in this regard, we used the technique of thyroidectomy (TX) to remove endogenous thyroid hormone from the circulation and then replaced it with physiological amounts of exogenous thyroxine. We hypothesized that the previously observed changes in RAS activity and cardiovascular function associated with TX would be normalized. TX was performed at 120 days of gestational age (dGA), and control fetuses were sham operated. After 3 days of recovery, TX fetuses were continuously intravenously infused with thyroxine until delivery by cesarean section close to term (around 138 dGA). Immediately before necropsy, fetuses were infused with isoproterenol, and the hemodynamic responses were noted. Thyroid hormone replacement normalized not only plasma triiodothyronine (T3) and thyroxine (T4) levels but also the TX-induced decreases in renal renin mRNA and renal renin content. Renal ANG II subtype receptor expression levels were also normalized for both mRNA and protein. Decreased basal heat rate and systolic blood pressure associated with TX returned to normal following replacement; however, changes in mean blood pressure and isoproterenol-induced changes in mean blood pressure were not altered. These findings demonstrate that replacement of thyroid hormone in hypothyroid sheep fetuses can restore renal ANG II receptor and renin expression and secretion to normal.

Combined thyroidectomy and renal denervation suppress renin expression and secretion in fetal sheep.

BACKGROUND AND OBJECTIVES: Activity of the fetal renin-angiotensin system (RAS) is developmentally regulated, increasing in late gestation toward term. Thyroid hormone and the renal nerves are both important modulators of renal RAS maturation; however, ablation of either influence alone does not totally block the aforementioned developmental late gestation increase in RAS in fetal sheep. In the current study, we used the technique of thyroidectomy combined with bilateral renal denervation (TX+D), which removes thyroid hormone from the circulation and abolishes effects of renal nerve activity, to determine if simultaneous removal of their effects on the kidney would markedly alter renin expression and secretion in late gestation. METHODS: TX+D was performed at 120 days of gestation age (dGA). Control fetuses were sham-operated. Immediately before necropsy (approximately 138 dGA), fetuses were infused with isoproterenol to examine plasma active and prorenin changes in response to beta-adrenergic stimulation. RESULTS: TX+D decreased plasma thyroid hormone concentrations, renal renin mRNA, renal active and prorenin levels, and plasma active and prorenin concentrations. Isoproterenol-induced increases in plasma active renin were also reduced in TX+D fetuses. TX+D did not alter renal angiotensin (Ang) II subtype receptor (AT2) expression close to term. CONCLUSION: These findings suggest that TX+D synergize in the suppression of fetal renin expression.

Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep.

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 approximately 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.

Ontogeny and effects of thyroid hormone on beta1-adrenergic receptor mRNA expression in ovine fetal kidney cortex.

OBJECTIVE: Previous studies indicate that thyroidectomy (TX) decreases renin gene expression in ovine fetal renal cortex in late gestation. Fetal ovine renin-containing renocortical cells become increasingly responsive to beta-adrenergic stimulation as gestation proceeds. Increases in plasma thyroid hormone concentrations parallel this change, suggesting that there is a positive developmental relationship between the two. To examine this hypothesis, we determined the ontogeny of beta1-adrenergic receptor (beta1R) mRNA expression, and the effect of thyroid hormone on in vivo and in vitro expression in fetal sheep. METHODS: Renocortical tissue was obtained from naive, TX, and sham-operated fetuses to determine beta1R mRNA levels. Renin-containing renocortical cells from TX or sham fetuses were treated with isoproterenol (Iso) or forskolin (FSK) for analysis of cellular cyclic adenosine monophosphate (cAMP) levels. Renocortical cells from naive fetuses were treated with triiodothyronine (T3) to assess cellular beta1R mRNA levels. Fetal plasma thyroxine (T4) level was determined. RESULTS: Renocortical beta1R mRNA expression increased significantly between 100 and 140 days' gestational age (dGA), while TX attenuated this increase (P <.01). Renocortical cellular cAMP levels were higher in sham compared to TX fetuses following incubation with Iso or FSK (P <.05). Cells incubated with T3 exhibited significantly increased beta1R mRNA expression (P <.05). CONCLUSION: The data suggest that thyroid hormone may be involved in modulating ovine fetal renocortical beta1R gene expression during development. We speculate that the increased beta1R mRNA expression in renal cortical cells as development progresses may mediate the increases in renin gene response to beta-adrenergic stimulation in late gestation.

Developmental changes in adrenocorticotrophin (ACTH)-induced expression of ACTH receptor and steroid acute regulatory protein mRNA in ovine fetal adrenal cells.

OBJECTIVES: Adrenocorticotrophin (ACTH) plays an important role in mediating the increase in cortisol output in the late gestation sheep fetus. At the adrenal itself, heightened expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) appear to be important parallel changes. This study examined how ACTH affects ACTH-R and StAR mRNA expression, and cortisol production in adrenocortical cells isolated from fetuses of varying gestational age (dGA). We hypothesized that the ability of ACTH to stimulate its receptor and StAR mRNA expression would be greater close to term than earlier in development. METHODS: Adrenals were obtained from fetuses (100-105, 120, or 135-139 dGA), and the cortical cells were dispersed. After 3 days of culture, cells were stimulated with ACTH(1-24), and the cells and medium were collected at different time points (0, 3, 6, 9, 12, and 24 hours) for measurement of cortisol and ACTH-R and StAR mRNA. RESULTS: Cortisol secretion was increased after ACTH treatment in all three age cohorts. Cells from the 135-139 dGA group secreted the most cortisol, followed by the 100-105 and then the 120 dGA groups (P <.05). ACTH-R mRNA levels before and after ACTH were higher in the late compared to both earlier groups. StAR mRNA levels before and after ACTH were higher in the 100-105 and 135 than in the 120 dGA group. The time to peak ACTH-R mRNA response was age-dependent, with the 100-105 dGA cells taking longer to attain maximum levels. Maximal StAR mRNA levels were not age-related. CONCLUSION: The data suggest that ACTH-R and StAR are indeed key mediators of fetal adrenocortical responsiveness, and that ACTH is able to up-regulate responsiveness, and hence cortisol production, by increasing their expression.

Thyroid hormone modulates renin and ANG II receptor expression in fetal sheep.

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.

Hypothalamic-pituitary disconnection in fetal sheep blocks the peripartum increases in adrenal responsiveness and adrenal ACTH receptor expression.

Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestation, and catheters were placed for blood sampling. At approximately 138 days of gestation, fetuses were killed, and adrenals were removed for cell culture and analyses of ACTH-R mRNA and protein. After 48 h, adrenocortical cells were stimulated with ACTH for 2 h, and the medium was collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analyses. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2 +/- 1.2 vs. 47.3 +/- 11.1 ng.ml(-1).2 h(-1)) after the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, whereas overnight incubation with ACTH or FSK increased levels by approximately twofold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.

The effect of hypothalamo-pituitary disconnection on the renin-angiotensin system in the late-gestation fetal sheep.

The activity of the renin-angiotensin system (RAS) increases significantly in the late-gestation fetal sheep. Fetal cortisol is also increased during this time, and it is thought that the increase in cortisol may modulate the RAS changes. Previous studies have examined the effects of cortisol infusion on RAS activity, but the effects of blocking the peripartum increase in cortisol concentrations on the developmental changes in the RAS are not known. Therefore, we utilized the technique of hypothalamic-pituitary disconnection (HPD), which prevents the cortisol surge from occurring, to investigate the importance of the late-gestation increase in cortisol on the ontogenic changes in RAS activity. HPD of fetal sheep was performed at 120 days of gestational age (dGA), and fetuses were delivered between 135 and 139 dGA. Control fetuses were sham operated. HPD blocked the late-gestation cortisol increase but did not alter renal renin mRNA, renal renin or prorenin protein content, nor plasma renin levels compared with sham operated. However, HPD fetuses had increased ANG II receptor subtype 1 (AT1) mRNA and protein expression in the kidney and lungs. ANG II receptor subtype 2 (AT2) expression was not altered in these tissues at either mRNA or protein level. HPD did not change AT1 or AT2 mRNA in the left ventricle but did result in decreased protein levels for both receptors. These studies demonstrate that blockade of the naturally occurring increase in fetal cortisol concentration in late gestation is associated with tissue-specific alterations in expression of AT1 and AT2 receptors. These changes may impact on fetal tissue maturation and hence have consequences in postnatal life.

The role of hypothalamic input on corticotroph maturation in fetal sheep.

Corticotropin-releasing hormone receptor type 1 (CRH-R1) expression and vasopressin type 1b (V1b) receptor protein decrease in late-gestation fetal sheep. Because hypothalamo-pituitary disconnection (HPD) has been demonstrated to prevent the morphological maturation of corticotrophs, we hypothesized that hypothalamic input is necessary for the maturational changes in CRH-R1 and V1b receptor levels. We measured CRH-R1 and V1b receptor expression in the anterior pituitaries of fetuses at 140 days gestational age (dGA) that underwent HPD or sham surgery at 120 dGA. CRH-R1 mRNA decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. However, CRH-R1 protein levels were elevated in HPD fetuses compared with sham and were not different from 120 dGA values. V1b protein levels decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. We conclude that hypothalamic input to the pituitary is necessary for the decrease in CRH-R1 receptor protein levels in late-gestation fetal sheep. However, hypothalamic input is not necessary for the decrease in V1b receptor expression seen in late gestation.

Inhibition of cyclooxygenase-2: effects on renin secretion and expression in fetal lambs.

The importance of prostaglandins in the regulation of the renin-angiotensin system during development is not known. These experiments were conducted to examine the effects of prostaglandin synthesis inhibitors on basal and isoproterenol-induced plasma renin concentration and renin gene expression in the late-gestation fetal lamb. Eighteen lamb fetuses ranging in gestational age from 129 to 138 days underwent surgical insertion of femoral arterial and venous catheters under general endotracheal anesthesia. After a period of recovery, animals underwent an infusion of isoproterenol after administration of a saline bolus (control experiments); 24-48 h later a second study was performed after administration of NS-398, a cyclooxygenase (COX)-2 inhibitor, or saline for a second control study. Administration of COX-2 inhibitor significantly reduced baseline plasma renin levels and attenuated responses in fetal renin secretion to isoproterenol infusions. Renal cortical cells from animals receiving COX-2 inhibitor had significantly lower levels of renin mRNA compared with animals receiving only saline. Renal cortical cells in culture from animals receiving only saline exhibited increased levels of renin mRNA when treated with isoproterenol, forskolin, or IBMX. Only forskolin increased renin mRNA levels in renal cortical cells in culture from animals receiving COX-2 inhibitor. We conclude that prostaglandins play a stimulatory role in the regulation of the renin-angiotensin system and are necessary for beta-adrenergic stimulation of renin secretion and gene expression in the late-gestation fetal lamb.