Sex-dependent effects of antenatal glucocorticoids on insulin sensitivity in adult sheep: role of the adipose tissue renin angiotensin system.

Exposure to glucocorticoids in utero is associated with changes in organ function and structure in the adult. The aims of this study were to characterize the effects of antenatal exposure to glucocorticoids on glucose handling and the role of adipose tissue. Pregnant sheep received betamethasone (Beta, 0.17 mg/kg) or vehicle 24 h apart at 80 days of gestation and allowed to deliver at term. At 9 mo, male and female offspring were fed at either 100% of nutritional allowance (lean) or ad libitum for 3 mo (obese). At 1 yr, they were chronically instrumented under general anesthesia. Glucose tolerance was evaluated using a bolus of glucose (0.25 g/kg). Adipose tissue was harvested after death to determine mRNA expression levels of angiotensinogen (AGT), angiotensin-converting enzyme (ACE) 1, ACE2, and peroxisome proliferator-activated receptor γ (PPAR-γ). Data are expressed as means ± SE and analyzed by ANOVA. Sex, obesity, and Beta exposure had significant effects on glucose tolerance and mRNA expression. Beta impaired glucose tolerance in lean females but not males. Superimposed obesity worsened the impairment in females and unmasked the defect in males. Beta increased ACE1 mRNA in females and males and AGT in females only (P < 0.05 by three-way ANOVA). Obesity increased AGT in females but had no effect on ACE1 in either males or females. PPAR-γ mRNA exhibited a significant sex (F = 42.8; P < 0.01) and obesity (F = 6.9; P < 0.05) effect and was significantly higher in males (P < 0.01 by three-way ANOVA). We conclude that adipose tissue may play an important role in the sexually dimorphic response to antenatal glucocorticoids.

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.

Differential effects of clinical doses of antenatal betamethasone on nephron endowment and glomerular filtration rate in adult sheep.

Antenatal steroid administration is associated with alterations in fetal kidney development and hypertension. However, a causal relationship between nephron deficit and hypertension has not been established. In this study, we measured nephron number, renal function, and blood pressure in sheep exposed antenataly to betamethasone. Pregnant sheep were given 2 betamethasone doses (0.17 mg/kg) or vehicle at 80 and 81 days gestational age and allowed to deliver at term. Data were obtained from a fetal cohort and 2 adult cohorts and were analyzed by analysis of variance (ANOVA) and/or 2 sample t test. Antenatal betamethasone induced a 26% reduction in the number of nephrons in both males and females in the absence of intrauterine growth restriction and/or prematurity. Adult males presented a reduction in glomerular filtration rate (GFR; 132 +/- 12.7 vs 114 +/- 7.0 mL/min, P < .05). Betamethasone administration was also associated with an increase in arterial blood pressure of similar magnitude in male (mean arterial pressure [MAP] in mm Hg; 98 +/- 2.7 vs 105 +/- 2.4) and female (96 +/- 1.9 vs 105 +/- 2.4) adult sheep and the increase in blood pressure preceded the decrease in GFR in the males. Furthermore, we found no significant association between the magnitude of the decrease in nephron number and the magnitude of the increase in arterial blood pressure. Our data thus support the conclusion that exposure to glucocorticoids at a time of rapid kidney growth is associated with an elevation in blood pressure that does not appear related solely to the reduction in nephron number.

Mild chronic hypoxia modifies the fetal sheep neural and cardiovascular responses to repeated umbilical cord occlusion.

We have shown that 5 days of mild hypoxia has significant effects on fetal ECoG activity, heart rate and blood pressure. We now studied if mild prolonged hypoxemia had an adverse effect on the fetal cardiovascular and neural responses to repeated cord occlusion and on the magnitude of neuronal damage. Fetal and maternal catheters were placed at 120 days' gestation and animals allocated at random to receive intratracheal maternal administration of nitrogen (n=8) or compressed air in controls (n=7). Five days after surgery, nitrogen infusion was adjusted to reduce fetal brachial artery pO(2) by 25%. After 5 days of chronic hypoxemia, the umbilical cord was completely occluded for 5 min every 30 min for a total of four occlusions. Data are presented as mean+/-SEM and were analyzed by two-way ANOVA or two-sample t-test. Nitrogen infusion decreased fetal pO(2) by 26% (20.5+/-1.7 vs. 14.3+/-0.8 mm Hg) without changing fetal pCO(2) or pH. Pre-existing hypoxia fetuses had a greater terminal fall in heart rate in occlusions II, III and IV, and also had a more severe terminal hypotension in the final occlusion. Pre-existing hypoxia was associated with a greater fall in spectral edge frequency during occlusions from 14.4+/-0.9 Hz to 6.9+/-0.4 Hz vs. 13.6+/-1.64 Hz to 10.6+/-0.77 Hz in controls, p<0.05. In addition, during the three-day post-occlusion period, the contribution of theta and alpha band frequencies to total ECoG activity was significantly lower in the pre-existing hypoxia fetuses (p<0.05). These effects were associated with increased neuronal loss in the striatum (p<0.05). In summary, the cardiovascular and neural response indicates a detrimental effect of pre-existing mild hypoxia on fetal outcome following repeated umbilical cord occlusions.

Prolonged mild hypoxia alters fetal sheep electrocorticogram activity.

OBJECTIVE: To assess the effects of prolonged mild hypoxemia on fetal brain electrocorticogram (ECoG) in late gestation. STUDY DESIGN: Fetal and maternal catheters were placed under general anesthesia and animals allocated at random to receive intratracheal maternal administration of either nitrogen (n = 8) or compressed air (n = 8). Five days after surgery (125 days' gestational age), nitrogen infusion was adjusted to reduce fetal brachial artery PO2 by 25%. The targeted decrease in fetal oxygenation was maintained for 5 days while fetal ECoG activity and fetal and maternal cardiovascular variables were continuously recorded. Data are presented as mean +/- SEM and were analyzed by two-way analysis of variance (ANOVA) or two-sample t test. RESULTS: Nitrogen infusion decreased fetal Po2 by 26% (20.5 +/- 1.7 versus 14.3 +/- 0.8) without changing fetal PCO2 or pH. Mild fetal hypoxemia was associated with fetal tachycardia and increased fetal blood pressure (P < .05). Fetal ECoG in hypoxic fetuses showed a significant decrease in the time spent in high voltage (HV) (P < .05) and an increase in the time spent in low voltage (LV) and in the number of low voltage events (P < .05). Also, a significant decrease in the proportion of 1-4 Hz and an increase in the proportion of 13-20 Hz frequencies was observed in LV events without a significant change in the frequency profile of HV events (P < .05). CONCLUSION: Prolonged mild hypoxemia significantly altered fetal homeostasis as reflected by the sustained tachycardia and increased blood pressure. Fetal ECoG activity was affected significantly in a qualitatively and quantitative manner by mild prolonged hypoxemia.

Acute and long-term effects of clinical doses of antenatal glucocorticoids in the developing fetal sheep kidney.

OBJECTIVES: The controversy regarding potential long-term side effects of antenatal steroid administration for accelerating fetal lung maturation is still unresolved despite more than 30 years of experience. Studies in animals have demonstrated that administration of glucocorticoids during pregnancy alters renal expression of several key regulatory molecules at different developmental stages followed in most cases with the development of hypertension in the adult. We studied the effects of betamethasone on the expression of (1) NA,K-ATPAse pump; (2) the Na/H exchanger 3 (NAHE3); (3) angiotensin receptor (AT1 and AT2); and (4) the type 1 dopamine receptor (D1R). METHODS: Pregnant sheep were treated with either 0.17 mg/kg betamethasone or vehicle 24 hours apart at 80 and 81 days' gestation. Fetal kidneys were harvested at 81 and 135 days' gestation. Protein and mRNA levels were measured in kidney cortex. RESULTS: Betamethasone had acute and long-term effects on fetal kidney cortex gene expression. Acutely, mRNA abundance for AT2 was significantly lower and that of NHE3 significantly higher than in controls (0.4 +/- 0.02 vs 0.7 +/- 0.05; 1.2 +/- 0.16 vs 0.4 +/- 0.04; P < .05). At 135 days' gestation, AT2 receptor abundance remained lower than control (0.2 +/- 0.02 vs 0.4 +/- 0.02; P < .05), whereas D1R expression was higher (0.8 +/- 0.17 vs 0.5 +/- 0.06; P < .05). No changes in Na,K-ATPase of AT1 receptor at either of the two time points studied were observed. Antenatal steroid administration was not associated with premature labor or a reduction in either body weight or kidney weight. CONCLUSION: Our findings strongly suggest that antenatal glucocorticoid administration according to National Institutes of Health (NIH) consensus guidelines may alter human fetal renal development. Further studies are needed to establish a direct relationship between alterations in fetal renal gene expression and the development of hypertension in adulthood.

Alterations in fetal kidney development and elevations in arterial blood pressure in young adult sheep after clinical doses of antenatal glucocorticoids.

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.

Prolonged mild fetal hypoxia up-regulates type I nitric oxide synthase expression in discrete areas of the late-gestation fetal sheep brain.

OBJECTIVE: Our purpose was to study the effects of prolonged mild hypoxemia on type I nitric oxide synthase (NOS) messenger RNA, protein, and enzymatic activity in the fetal sheep brain. STUDY DESIGN: Pregnant sheep were randomly allocated to receive maternal nitrogen (n = 8) or compressed air (controls, n = 5) to reduce fetal brachial artery PO(2) by 25% for 5 days. Type I NOS mRNA (determined by ribonuclease protection assay) protein (determined by Western blot) and enzymatic activity (determined by citrulline assay) were measured in the hippocampus, striatum, cerebellum, and frontal cortex. Data are presented as mean +/- SEM and were compared by means of one-way analysis of variance or two-sample t test. RESULTS: The reduction in maternal inspired oxygen concentration decreased fetal PO(2) by 26% and oxygen content by 25% without an associated change in PCO(2) or pH. Fetal hypoxemia increased type I NOS mRNA by threefold in the striatum and by 2-fold in the frontal cortex and cerebellum, but it did not change mRNA expression in the hippocampus (P <.05). Type I NOS protein and catalytic activity increased only in the striatum (P <.05). CONCLUSION: Prolonged mild hypoxemia has a differential effect on type I NOS mRNA in fetal sheep brain areas. Type I NOS protein and catalytic activity significantly increased only in the striatum. Our data suggest that fetal type I NOS gene expression is regulated at transcriptional, post-transcriptional, and translational levels.