Mariana Baserga

Fetal growth restriction alters transcription factor binding and epigenetic mechanisms of renal 11β-hydroxysteroid dehydrogenase type 2 in a sex-specific manner

Intrauterine growth restriction (IUGR) increases the risk of serious adult morbidities such as hypertension. In an IUGR rat model of hypertension, we reported a persistent decrease in kidney 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA and protein levels from birth through postnatal (P) day 21. This enzyme deficiency can lead to hypertension by limiting renal glucocorticoid deactivation. In the present study, we hypothesized that IUGR affects renal 11beta-HSD2 epigenetic determinants of chromatin structure and alters key transcription factor binding to the 11beta-HSD2 promoter in association with persistent downregulation of its mRNA expression. To test this hypothesis, we performed bilateral uterine artery ligation on embryonic day 19.5 pregnant rats and harvested kidneys at day 0 (P0) and P21. Key transcription factors that can affect 11beta-HSD2 expression include transcriptional enhancers specificity protein 1 (SP1) and NF-kappaB p65 and transcriptional repressors early growth response factor (Egr-1) and NF-kappaB p50. Our most important findings were as follows: 1) IUGR significantly decreased SP1 and NF-kappaB (p65) binding to the 11beta-HSD2 promoter in males, while it increased Egr-1 binding in females and NF-kappaB (p50) binding in males; 2) IUGR increased CpG methylation status, as well as modified the pattern of methylation in several CpG sites of 11beta-HSD2 promoter at P0 also in a sex-specific manner; and 3) IUGR decreased trimethylation of H3K36 in exon 5 of 11beta-HSD2 at P0 and P21 in both genders. We conclude that IUGR is associated with altered transcriptional repressor/activator binding in connection with increased methylation in the 11beta-HSD2 promoter region in a sex-specific manner, possibly leading to decreased transcriptional activity. Furthermore, IUGR decreased trimethylation of H3K36 of the 11beta-HSD2 gene in both genders, which is associated with decreased transcriptional elongation. We speculate that alterations in transcription factor binding and chromatin structure play a role in in utero reprogramming.

Uteroplacental insufficiency affects kidney VEGF expression in a model of IUGR with compensatory glomerular hypertrophy and hypertension

Low nephron endowment secondary to intrauterine growth restriction (IUGR) results in compensatory hypertrophy of the remaining glomeruli, which in turn is associated with hypertension. However, gender differences exist in the response of the kidney to injury, and IUGR female offspring seems protected from an unfavorable outcome. We previously reported differences in gender-specific gene expression in the IUGR kidney as well as increased circulating corticosterone levels following uteroplacental insufficiency (UPI). Vascular endothelial growth factor (VEGF), which is critical for renal development, is an important candidate in the IUGR kidney since its expression can be regulated by sex-steroids and glucocorticoids. We hypothesize that IUGR leads to altered kidney VEGF expression in a gender-specific manner. Following uterine ligation in the pregnant rat, UPI decreases renal VEGF levels in male and female IUGR animals at birth and through postnatal day 21. However, by day 120 of life, IUGR females have increased kidney VEGF expression, not present in the IUGR males. In addition, IUGR males exhibit increased serum testosterone levels as well as proteinuria. These findings are intriguing in light of the difference in glomerular hypertrophy observed: IUGR males show increased glomerular area when compared to IUGR females. In this model characterized by decreased nephron number and adult onset hypertension, UPI decreases renal VEGF expression during nephrogenesis. Our most intriguing finding is the increased renal VEGF levels in adult IUGR females, associated with a more benign phenotype. We suggest that the mechanisms underlying renal disease in response to IUGR are most likely regulated in a gender specific manner.

Darbepoetin administration to neonates undergoing cooling for encephalopathy: a safety and pharmacokinetic trial

Background:Despite therapeutic hypothermia, neonates with encephalopathy (NE) have high rates of death or disability. Darbepoetin alfa (Darbe) has comparable biological activity to erythropoietin, but has extended circulating half-life (t1/2). Our aim was to determine Darbe safety and pharmacokinetics as adjunctive therapy to hypothermia.Study design:Thirty infants (n = 10/arm) ≥36 wk gestation undergoing therapeutic hypothermia for NE were randomized to receive placebo, Darbe low dose (2 μg/kg), or high dose (10 μg/kg) given intravenously within 12 h of birth (first dose/hypothermia condition) and at 7 d (second dose/normothermia condition). Adverse events were documented for 1 mo. Serum samples were obtained to characterize Darbe pharmacokinetics.Results:Adverse events (hypotension, altered liver and renal function, seizures, and death) were similar to placebo and historical controls. Following the first Darbe dose at 2 and 10 μg/kg, t1/2 was 24 and 32 h, and the area under the curve (AUCinf) was 26,555 and 180,886 h*mU/ml*, respectively. In addition, clearance was not significantly different between the doses (0.05 and 0.04 l/h). At 7 d, t1/2 was 26 and 35 h, and AUCinf was 10,790 and 56,233 h*mU/ml*, respectively (*P < 0.01).Conclusion:Darbe combined with hypothermia has similar safety profile to placebo with pharmacokinetics sufficient for weekly administration.

Organ-Specific Defects in Insulin-Like Growth Factor and Insulin Receptor Signaling in Late Gestational Asymmetric Intrauterine Growth Restriction in Cited1 Mutant Mice

Late gestational placental insufficiency resulting in asymmetric intrauterine organ growth restriction (IUGR) is associated with an increased incidence of diabetes, cardiovascular and renal disease in adults. The molecular mechanisms mediating these defects are poorly understood. To explore this, we investigated the mechanisms leading to IUGR in Cited1 knockout mice, a genetic model of late gestational placental insufficiency. We show that loss of placental Cited1 leads to asymmetric IUGR with decreased liver, lung, and kidney sizes and preservation of fetal brain weight. IGF and insulin signaling regulate embryonic organ growth. IGF-I and IGF-II protein and mRNA expression are reduced in livers, lungs, and kidneys of embryonic d 18.5 embryos with IUGR. Decreased IGF-I is associated with reduced activating phosphorylation of the type 1 IGF receptor (pIGF-IR) in the kidney, whereas reduced IGF-II is associated with decreased phosphorylation of the insulin receptor (pIR) in the lung. In contrast, decreased pIR is associated with reduced IGF-I but not IGF-II in the liver. However, pancreatic β-cell mass and serum insulin levels are also decreased in mice with IUGR, suggesting that hepatic IR signaling may be regulated by alterations in fetal insulin production. These findings contrast with observations in IUGR fetal brains in which there is no change in IGF-IR/IR phosphorylation, and IGF-I and IGF-II expression is actually increased. In conclusion, IUGR disrupts normal fetal IGF and insulin production and is associated with organ-specific defects in IGF-IR and IR signaling that may regulate asymmetric IUGR in late gestational placental insufficiency.

UTEROPLACENTAL INSUFFICIENCY AFFECTS RENAL EXPRESSION OF ESTROGEN RECEPTORS IN INTRAUTERINE GROWTH-RESTRICTED RATS.: 65

Background Uteroplacental insufficiency results in intrauterine growth restriction (IUGR). IUGR newborns are at an increased risk for later development of morbidities, including hypertension, at an incidence that varies between genders. Although we have previously demonstrated differences in gender-specific gene expression in the IUGR rat kidney, it is unknown whether IUGR affects kidney expression of either the androgen or estrogen receptors. Objective We hypothesized that IUGR would alter renal mRNA levels of the androgen receptor (AR), estrogen receptor 1 (ER1), and estrogen receptor 2 (ER2) in a gender-specific manner in day 0 (d0) and day 21 (d21) IUGR rat pups. Methods Bilateral uterine artery ligation was performed on day 19 pregnant Sprague-Dawley rats to render them IUGR, and pups were then harvested on day 21 (term). Levels of AR, ER1, and ER2 mRNA were quantified using real-time RT-PCR from whole kidneys on days 0 and 21. Results mRNA levels expressed as % of control ⊆ SEM. IUGR significantly increased renal ER1 mRNA levels in d21 males to 177 ⊆ 16.* In contrast, ER2 mRNA levels were significantly decreased to 57 ⊆ 6** in day 21 female kidneys. Identical changes in the ER1 and ER2 mRNA levels were seen in day 0 males and females but did not achieve statistical significance. *p Conclusions IUGR increases ER1 receptor expression in IUGR male rats while decreasing ER2 receptor expression in IUGR female rats. Interestingly, the differences in gene expression widens from day 0 to day 21 in the IUGR rat kidney. We speculate that such differences contribute to the gender-specific characteristics of the IUGR rat kidney.

276 UTEROPLACENTAL INSUFFICIENCY ALTERS KIDNEY CYCLOOXYGENASE 2 EXPRESSION AND CAUSES ADULT-ONSET HYPERTENSION.

Background IUGR predisposes the fetus towards increased corticosterone levels, decreased nephron number, and adult-onset hypertension. Rats rendered IUGR by uteroplacental insufficiency (UPI) also have increased corticosterone levels and suffer reduced nephron number. A possible mechanism through which steroids decrease nephron number is regulation of the inducible enzyme cyclooxygenase 2 (COX-2), a pivotal protein in nephrogenesis; however, the effect of UPI upon COX-2 expression is unknown. Furthermore, the association of reduced nephron number and adult-onset hypertension has not been assessed in our animal model. Hypothesis We hypothesized that IUGR and subsequent glucocorticoid overexposure of the fetus lead to decreased kidney COX-2 expression and protein levels at birth and predispose towards adult-onset hypertension. Methods Bilateral uterine artery ligation was performed on day e19 pregnant Sprague-Dawley rats and pups were harvested at term (e21.5). Levels of COX-2 mRNA, and protein were quantified using real-time RT-PCR and Western blotting from whole kidneys on d0 and d21 of life (juvenile rat). Systolic (SBP), diastolic (DBP), and mean blood pressure (MBP) were measured on d21 and d120 of life by tail cuff method. Results On d0, IUGR was associated with decreased kidney COX-2 mRNA levels (79 ± 7* % of control) and protein levels (69 ± 3%** of control). Kidney COX-2 mRNA and protein levels were unaffected in d21 IUGR juvenile rats. Though blood pressure was not significantly different on d21 of life among groups, SBP was significantly higher in both males and females IUGR adults on d120 of life when compared to control animals (176 ± 7 vs 159 ± 4*** and 149 ± 9 vs 128 ± 7**, respectively). MBP and DBP were also significantly higher in IUGR female rats but not affected in IUGR males on d120 (*p < .05, **p < .01, ***p < .001). Conclusions In vitro studies demonstrate that steroid exposure decreases COX-2 expression in renal cells. In vivo studies demonstrate that COX-2 inhibition impairs nephrogenesis. In our animal model, UPI decreases fetal COX-2 expression during a period of active nephrogenesis in the IUGR rat, which is also characterized by decreased nephron number and adult-onset hypertension. We speculate that decreased IUGR kidney COX-2 levels reduce nephron number and lead to adult-onset hypertension. Supported by the University of Utah Department of Pediatrics CHRC.

467 UTEROPLACENTAL INSUFFICIENCY ALTERS HEPATIC GLUCOCORTICOID RECEPTOR AND 11 β HYDROXYSTEROID DEHYDROGENASE TYPE I LEVELS IN FETAL AND JUVENILE IUGR RATS

Background IUGR predisposes towards long-term morbidities such as insulin resistance. Corticosterone regulates hepatic gluconeogenesis. The glucocorticoid receptor (GR) and its phosphorylation (S211) status, and 11 beta hydroxysteroid dehydrogenase (11β-HSD 1) are key components of the steroid metabolism. Hypothesis We hypothesized that IUGR and subsequent paracrine glucocorticoid overexposure of the fetus would lead to short and long-term alterations in the steroid metabolism key components. Methods Bilateral uterine artery ligation was performed on d19 pregnant Sprague-Dawley rats and pups were harvested at term. Plasma corticosterone levels (CORT) were measured at d0 and d21 by ELISA. Hepatic GR and 11 β-HSD 1 mRNA levels were quantified using real time-PCR. Protein levels of GR, 11 β-HSD 1 and serine 211 (S211) were measured by Western blot. Results On d0, CORT and mRNA GR increased in IUGR pups, but 11 β-HSD 1mRNA decreased. On d21, CORT remained elevated in IUGR rats, with decreased mRNA GR and 11 β-HSD 1in IUGR females. (Table) On d0, there was increased hepatic GR (S211) phosphorylation in IUGR rats (162±10 % of control values, p≤0.01). However, we found decreased S211 levels in d21 IUGR males (40±3 % of control values, p≤0.001), with preservation of GR phosphorylation in IUGR females (104±%of control values, NS). Conclusion Uteroplacental insufficiency is associated with prenatal and postnatal glucocorticoid overexposure that most likely affects steroid metabolism in a time and gender-specific manner. We speculate that increased glucocorticoid levels at day 0 and day 21 may contribute to the molecular mechanisms underlying insulin resistance in adulthood.