G. Angela Massmann

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.

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.

Corticotropin-Releasing Hormone Type I Receptor Messenger Ribonucleic Acid and Protein Levels in the Ovine Fetal Pituitary: Ontogeny and Effect of Chronic Cortisol Administration

In sheep, the ACTH secretory response to CRH in vivo or in vitro changes as a function of development, with peak responses occurring several weeks before term (145 days of gestation). CRH-stimulated ACTH secretion is mediated via the G protein-coupled CRH type I (CRH R1) receptor. We used a quantitative ribonuclease protection assay and Western immunoblotting to determine messenger RNA (mRNA) and protein levels of the CRH R1 receptor in immature and mature fetuses and adults. In addition, we precociously elevated fetal plasma cortisol levels to determine whether the fetal CRH R1 receptor is sensitive to increases in plasma cortisol. CRH R1 receptor mRNA levels decreased markedly throughout gestation and into the transition to adult life (immature fetus, 1.24 ± 0.17; mature fetus, 0.75 ± 0.13; adult, 0.18 ± 0.093 pg/μg total anterior pituitary RNA). Also, continuous cortisol infusion in immature fetuses significantly decreased CRH R1 mRNA levels by 41%. Similar decreases were noted in protein levels. Thus,...

Developmental and regional expression patterns of Type I Nitric Oxide Synthase mRNA and protein in fetal sheep brain during the last third of gestation.

Type I NOS (nNOS) catalytic activity represents the activity of full-size protein and truncated protein variants originated from many different spliced mRNA variants. Splice mRNA variants are thought to be important in determining the differential organ and subcellular expression of Type I NOS. The present study was directed to increase our understanding of the developmental regulation of Type I NOS in fetal brain. In four discrete areas of the fetal brain, we measured steady-state mRNA levels and catalytic activity and protein mass in the soluble and particulate fractions. Under general anesthesia, we collected sensory-motor cortex, striatum, hippocampus and cerebellum from sheep fetuses at 105, 115, 125 and 135 days gestation (32 fetuses). NOS protein in the soluble and particulate fractions was characterized using Western blot (molecular weight) and arginine to citrulline conversion (enzymatic activity). At the mRNA level, steady state levels were determined using probes directed against exon 2 and exon 21/22 by ribonuclease protection assay (RPA). Our data show that NOS catalytic activity is regulated in a region, subcellular and temporal manner. NOS activity was higher in the soluble fraction in all brain regions and significantly higher levels were found in the soluble fraction of striatum and particulate fraction of hippocampus (P<0.05 by ANOVA). Western blot analysis revealed three distinct molecular weight bands for Type I NOS (155, 144 and 136 kDa). The bands were present in all brain regions and in both cellular compartments with the 155 kDa band being the most abundant molecular form. Truncated protein variants accounted for 25% and 15% of total Type I NOS protein in the soluble fraction and particulate fraction respectively. RPA analysis showed a differential regulation of mRNA variants with exon 2 frame deletions in striatum and hippocampus. A coordinated increase with advancing gestational age of catalytic activity, the full-length protein, the protein variants and steady state mRNA levels was observed in cortex and striatum as demonstrated by higher levels at 125 and 135 days gestation (P<0.05 by ANOVA). NOS enzymatic activity was Ca(2+) and calmodulin dependent. However, in the particulate fraction 20% of the NOS activity was resistant to calmodulin inhibition. In summary, fetal brain Type I NOS mRNA variants are differentially regulated according to brain regions. Our data suggests that exon 2 deleted mRNA variants have low translation efficiency as indicated by the lack of parallel expression of truncated Type I NOS protein variants.

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.

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.