Nancy K. Valego

Gender differences in the effects of antenatal betamethasone exposure on renal function in adult sheep

Exposure to clinically relevant doses of glucocorticoids during fetal life increases blood pressure in adult male and female sheep. The purpose of this study was to evaluate the effects of prenatal exposure to betamethasone at 80-81 days of gestation on renal function in ewes and rams at 1.5 yr of age. In prenatal betamethasone-exposed males, compared with the vehicle-exposed animals, basal glomerular filtration rate (GFR) (1.93 +/- 0.08 vs. 2.27 +/- 0.10 ml.min(-1).kg body wt(-1)) and the ability to excrete an acute Na+ load (37.1 +/- 4.4 vs. 53.7 +/- 9.7%) were reduced. (P < 0.03 and P = 0.03, respectively). In contrast, prenatal betamethasone exposure had no effect on basal GFR, Na+ excretion, or the percentage of the Na+ load excreted during the experiment in females. Systemic infusions of ANG-(1-7) at 9 ng.min(-1).kg(-1) for 2 h had minimal effects on basal GFR, renal plasma flow, and Na+ excretion in males but increased Na+ excretion in females. However, the percentage of Na+ load excreted during ANG-(1-7) infusion did not change in prenatal betamethasone-exposed females (113.1 +/- 14.2 vs. 98.1 +/- 12.2%) compared with the significant increase in vehicle females (139.2 +/- 22.3 vs. 92.2 +/- 7.5%) (P = 0.01). The data indicate that antenatal betamethasone exposure produces gender-specific alternations in renal function and thus suggest that different mechanisms underlie the antenatal steroid-induced elevations in blood pressure in male and female offspring.

Prenatal betamethasone exposure alters renal function in immature sheep: sex differences in effects.

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.

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.

Cortisol infusion depresses the ratio of bioactive to immunoreactive ACTH in adrenalectomized sheep fetuses

We examined the effects of exogenous cortisol on plasma immunoreactive adrenocorticotropic hormone (iACTH), bioactive ACTH (bACTH), and ACTH-(1-39) in nine adrenalectomized fetuses at 126-130 and 136-140 days of gestation. Fetuses received 4 h of cortisol (2 μg ⋅ kg-1 ⋅ min-1) or saline infusions on consecutive days. Blood was obtained before and at intervals during infusions. Arterial blood gases and hematocrits were normal and did not change with age. Plasma cortisol did not change during saline infusions but increased significantly (range 30-70 ng/ml) during cortisol infusions. Basal plasma iACTH, bACTH, ACTH-(1-39), and bACTH-to-iACTH and ACTH-(1-39)-to-iACTH ratios were significantly higher in the older fetuses. Cortisol infusions decreased plasma iACTH, bACTH, and ACTH-(1-39) in both groups, and the suppression as a percent of the baseline was similar. The bACTH-to-iACTH ratio declined to the same level at 126-130 (0.201 ± 0.040 to 0.051 ± 0.002) and 136-140 (0.389 ± 0.088 to 0.046 ± 0.002) days of gestation. These data suggest that physiological concentrations of cortisol selectively inhibit bACTH secretion, and the ACTH response to cortisol inhibition is not different between 126 and 140 days of gestation in adrenalectomized sheep fetuses.

Active renin, prorenin, and renin gene expression after reduced renal perfusion pressure in term ovine fetuses

We studied the pattern of plasma active renin concentration (ARC), prorenin concentration (PRC), renal renin concentration, and the renin mRNA levels in ovine fetuses subjected for 24 h to reduced renal perfusion pressure (RPP). The results obtained in five animals (133.8 +/- 1.4 days of gestation) in which RPP was reduced by 10 mmHg were compared with those in seven control fetuses (130.3 +/- 0. 8 days of gestation) without pressure reduction. Plasma samples were obtained before and at intervals of 24 h after initiating reduced RPP. The plasma ARC increased within 60 min of reduced RPP, reaching a maximum (13.0 +/- 4.7 vs. 115.7 +/- 23.8, P < 0.01) at 3 h. The ARC then declined toward control values. In contrast, plasma PRC did not increase consistently until 4 h into reduced RPP, with maximal levels at 24 h (8.2 +/- 2.4 vs. 87.7 +/- 21.9, P = 0.016). Within the kidney PRC, but not ARC, increased significantly, by 2.5-fold. Reduced RPP also increased renal renin mRNA levels (P = 0.004). We conclude that a chronic reduction in RPP in the near-term ovine fetus increases renal PRC and is associated with increased plasma prorenin levels. The data suggest that the conversion of prorenin to active renin is an important regulation point of the renin ANG system during development.We studied the pattern of plasma active renin concentration (ARC), prorenin concentration (PRC), renal renin concentration, and the renin mRNA levels in ovine fetuses subjected for 24 h to reduced renal perfusion pressure (RPP). The results obtained in five animals (133.8 ± 1.4 days of gestation) in which RPP was reduced by 10 mmHg were compared with those in seven control fetuses (130.3 ± 0.8 days of gestation) without pressure reduction. Plasma samples were obtained before and at intervals of 24 h after initiating reduced RPP. The plasma ARC increased within 60 min of reduced RPP, reaching a maximum (13.0 ± 4.7 vs. 115.7 ± 23.8, P < 0.01) at 3 h. The ARC then declined toward control values. In contrast, plasma PRC did not increase consistently until 4 h into reduced RPP, with maximal levels at 24 h (8.2 ± 2.4 vs. 87.7 ± 21.9, P = 0.016). Within the kidney PRC, but not ARC, increased significantly, by 2.5-fold. Reduced RPP also increased renal renin mRNA levels ( P = 0.004). We conclude that a chronic reduction in RPP in the near-term ovine fetus increases renal PRC and is associated with increased plasma prorenin levels. The data suggest that the conversion of prorenin to active renin is an important regulation point of the renin ANG system during development.

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 ACTH1-24, 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.

Developmental Aspects of Ovine Adrenal Adrenocorticotropic Hormone Receptor Expression

Objective: Maturation of adrenocortical function is important because a prepartum increase in fetal plasma glucocorticoids is required for survival after birth. Adrenal maturation may include alterations in the regulation of adrenocorticotropic hormone (ACTH) receptor expression. Therefore, we quantitated ACTH receptor expression in the ovine adrenal cortex during development and after manipulations to better understand the regulation of the adrenal receptor in vivo. Methods: For the ontogeny study, adrenals were obtained from fetuses at different stages of development, and the cortical tissue was stored at -80C until total RNA was extracted. The ACTH binding studies were done on adrenal membranes obtained from fetuses at two different ages using I125 (Phe-2, Nle-4) ACTH as the ligand. Plasma ACTH was measured by two-site immunoradiometric assay, and cortisol was measured by radioimmunoassay. ACTH receptor mRNA was quantitated by ribonuclease protection assay. The data were analyzed by analyses of variance. Results: ACTH receptor mRNa level progressively increased in fetal life; relative changes in receptor mRNA and binding were similar (3.0-fold and 2.4-fold, respectively). Physiologic increases in fetal plasma cortisol decreased adrenal ACTH receptor mRNA concentration, and there was a strong (r2 = 0.76, P < .002) linear relationship between fetal plasma ACTH concentration and receptor mRNA levels. Receptor mRNA stability increased in development, and message half-life was greater in adulthood than in fetal life. Conclusion: The data suggest that developmental increases in receptor expression are part of the maturation process in the fetal adrenal and that plasma ACTH concentration plays a major role in regulating ACTH receptor mRNA levels in vivo.

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.

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 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 cyclooxygenase 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.

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-Ala7-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-Ala7-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.