RESUMO
BACKGROUND: We showed that intrauterine growth restriction (IUGR) increases distal airspace wall thickness at birth (postnatal age 0; P0) in rat pups (saccular stage of lung development). However, that report did not assess whether the saccular phenotype persisted postnatally or occurred in males or females, nor did the report identify a potential molecular pathway for the saccular phenotype at P0. We hypothesized that IUGR persistently delays alveolar formation and disrupts retinoic acid receptor (RAR) mRNA and protein levels in the lung of rat pups in a postnatal age- and sex-specific manner. METHODS: IUGR was induced in pregnant rats by bilateral uterine artery ligation. Alveolar formation and expression of RARα, -ß, and -γ were quantified at P0, P6 (alveolar stage), and P21 (postalveolarization). RESULTS: IUGR increased distal airspace wall thickness in female pups at P0 only. IUGR did not affect male pups at any age. IUGR transiently increased lung RAR-ß protein abundance, which inhibits alveolar formation, at P0 in female pups. Serum retinol concentration was normal at all ages. CONCLUSION: IUGR alone is not sufficient to persistently delay postnatal alveolar formation or disrupt expression of RARs. We speculate that for IUGR to delay alveolar formation postnatally, a second insult is necessary.
Assuntos
Retardo do Crescimento Fetal , Pulmão/metabolismo , Alvéolos Pulmonares/embriologia , Receptores do Ácido Retinoico/metabolismo , Animais , Feminino , Pulmão/embriologia , Gravidez , RatosRESUMO
Pediatric traumatic brain injury (TBI) is a major cause of acquired cognitive dysfunction in children. Hippocampal Brain Derived Neurotrophic Factor (BDNF) is important for normal cognition. Little is known about the effects of TBI on BDNF levels in the developing hippocampus. We used controlled cortical impact (CCI) in the 17 day old rat pup to test the hypothesis that CCI would first increase rat hippocampal BDNF mRNA/protein levels relative to SHAM and Naïve rats by post injury day (PID) 2 and then decrease BDNF mRNA/protein by PID14. Relative to SHAM, CCI did not change BDNF mRNA/protein levels in the injured hippocampus in the first 2 days after injury but did decrease BDNF protein at PID14. Surprisingly, BDNF mRNA decreased at PID 1, 3, 7 and 14, and BDNF protein decreased at PID 2, in SHAM and CCI hippocampi relative to Naïve. In conclusion, TBI decreased BDNF protein in the injured rat pup hippocampus 14 days after injury. BDNF mRNA levels decreased in both CCI and SHAM hippocampi relative to Naïve, suggesting that certain aspects of the experimental paradigm (such as craniotomy, anesthesia, and/or maternal separation) may decrease the expression of BDNF in the developing hippocampus. While BDNF is important for normal cognition, no inferences can be made regarding the cognitive impact of any of these factors. Such findings, however, suggest that meticulous attention to the experimental paradigm, and possible inclusion of a Naïve group, is warranted in studies of BDNF expression in the developing brain after TBI.
Assuntos
Lesões Encefálicas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Encéfalo/crescimento & desenvolvimento , Animais , Lesões Encefálicas/genética , Fator Neurotrófico Derivado do Encéfalo/genética , Hipocampo/lesões , Hipocampo/metabolismo , Masculino , Proteínas do Tecido Nervoso/biossíntese , RNA/biossíntese , RNA/isolamento & purificação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Técnicas EstereotáxicasRESUMO
Intrauterine growth restricted (IUGR) infants have increased susceptibility to infection associated with higher risk of illness and death. Dual specificity phosphatase 1 (DUSP1), which is transcribed in the thymus, increases in quantity as T cells mature and differentiate into CD4+ cells. Little is known about how IUGR affects DUSP1 levels and T-cell subpopulations over time. We hypothesized that IUGR would decrease cell count, CD4+ and CD8+ subpopulations of T lymphocytes, and DUSP1 levels in IUGR rat thymus and spleen. Bilateral uterine artery ligation produced IUGR rats. Thymus and spleen were harvested at P0 and P21. Flow cytometry was used to compare CD4+ and CD8+ lymphocyte populations. Real-time RT-PCR and Western blotting were used to determine DUSP1 quantity. IUGR significantly decreased total cell count in P0 and P21 IUGR male and female thymus. IUGR significantly increased CD4+ cells in IUGR P0 males and females, significantly decreased CD4+ cells in P21 female thymus, and significantly altered DUSP1 levels in the IUGR female thymus at P0 and P21, although it is not yet known whether the change in DUSP1 levels is due to a change in the level per cell or to a change in cellular composition of the thymus.
Assuntos
Diferenciação Celular/imunologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Retardo do Crescimento Fetal/enzimologia , Retardo do Crescimento Fetal/imunologia , Linfócitos T/imunologia , Timo/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Western Blotting , Relação CD4-CD8 , Contagem de Células , Primers do DNA/genética , Feminino , Citometria de Fluxo , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2 , 11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Epigênese Genética , Retardo do Crescimento Fetal/enzimologia , Fatores de Transcrição/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , 11-beta-Hidroxiesteroide Desidrogenases/genética , Animais , Regulação para Baixo , Éxons , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Rim/metabolismo , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Artéria UterinaRESUMO
A quantitative real-time PCR (qRT-PCR) assay was developed to measure cytokine transcription profiles and viral load during sub-clinical and clinical infection with murine cytomegalovirus (MCMV). Primers/fluorogenic probes specific for mouse cytokines and for the immediate early gene 1 (IE1) of MCMV were used to quantitate cytokine responses and viral load in various organs of MCMV infected mice. Increased mRNA levels of TNF-alpha, INF-gamma and IL-10 were detected in the spleens, lungs and livers of clinically infected mice at 5 days post-infection. Transcription of these cytokines was 2-5-fold lower (p=0.07 for each cytokine) in the spleens and 10-100-fold lower in the lungs (p=0.03 for INFgamma, not significant for IL-10 and TNFalpha) and livers (p<0.05 for each cytokine) of sub-clinically infected mice. Clinical MCMV infection induced high levels of IL-6 in the lungs and spleens of infected animals, while no significant transcription of IL-6 was detected in any organ during sub-clinical infection (p<0.05). The timing of peak amounts of INF-gamma, IL-10 and IL-6 observed in the spleens of clinically infected mice correlated with high viral loads in these organs. Cytokine expression rose in the salivary glands later, at day 15, corresponding to the increase in salivary gland viral load. The qRT-PCR demonstrates that infection with MCMV induces an organ-specific cytokine response characterized by the production of TNF-alpha, INF-gamma, IL-6 and IL-10 which correlates with severity of the disease (sub-clinical versus clinical) and with viral load. In summary, qRT-PCR is a sensitive and accurate method to study MCMV infection and host responses to the virus.
Assuntos
Citocinas/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Reação em Cadeia da Polimerase/métodos , Transcrição Gênica , Carga Viral , Animais , Citocinas/análise , Feminino , Fígado/imunologia , Fígado/virologia , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise , RNA Viral/análise , Glândulas Salivares/imunologia , Glândulas Salivares/virologia , Sensibilidade e Especificidade , Baço/imunologia , Baço/virologiaRESUMO
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.
Assuntos
Modelos Animais de Doenças , Retardo do Crescimento Fetal/metabolismo , Hipertensão/complicações , Glomérulos Renais/patologia , Rim/metabolismo , Circulação Placentária , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Corticosterona/sangue , Creatinina/urina , Primers do DNA , Feminino , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testosterona/sangue , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Clinical and animal studies indicate that intrauterine growth restriction (IUGR) following uteroplacental insufficiency (UPI) reduces nephron number and predisposes toward renal insufficiency early in life and increased risk of adult-onset hypertension. In this study, we hypothesized that the inducible enzyme cyclooxygenase-2 (COX-2), a pivotal protein in nephrogenesis, constitutes a mechanism through which UPI and subsequent glucocorticoid overexposure can decrease nephron number. We further hypothesized that UPI downregulates the key enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts corticosterone to inert 11-dehydrocorticosterone, thereby protecting both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) from the actions of corticosterone. Following bilateral uterine ligation on the pregnant rat, UPI significantly decreased renal COX-2, 11beta-HSD2, and GR mRNA and protein levels, but upregulated expression of MR at birth. At day 21 of life, 11beta-HSD2, GR, and also MR mRNA and protein levels were downregulated. UPI did not affect blood pressures (BP) at day 21 of life but significantly increased systolic BP in both genders at day 140. We conclude that 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.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Retardo do Crescimento Fetal , Hipertensão , Rim/crescimento & desenvolvimento , Circulação Placentária/fisiologia , Insuficiência Placentária/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Envelhecimento , Animais , Ciclo-Oxigenase 2/genética , Regulação para Baixo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rim/patologia , Gravidez , RNA Mensageiro/metabolismo , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Caracteres SexuaisRESUMO
Uteroplacental insufficiency (UPI) leads to intrauterine growth restriction (IUGR), which predisposes infants toward renal insufficiency early in life and increases the risk of kidney-related adult morbidities, such as hypertension. This compromised in utero environment has been demonstrated to impair nephrogenesis, as evidenced by a reduced nephron endowment in humans and in rats rendered IUGR by UPI. Concordantly, we have observed that IUGR rats have increased kidney p53 protein levels associated with increased apoptosis. Several factors can regulate p53 gene expression and activity, including posttranslational modifications and protein-protein interactions in the cell. Among these, two important mechanisms are 1) phosphorylation of the amino terminal serine 15 [phospho-p53 (Ser15)], which increases p53 stability and apoptotic activity, and 2) the murine double-minute (MDM2) functional circuit that limits further p53-induced apoptosis by promoting proteosomal degradation of p53. We hypothesize that UPI induces an increase in phospho-p53 (Ser15) in association with an absent MDM2 response, predisposing the kidney to increased apoptosis. To test our hypothesis, we induced IUGR through bilateral uterine artery ligation of the pregnant rat. UPI significantly increased phospho-p53 (Ser15), as well as ataxia teleangiectasia-mutated kinase/A-T-related kinase and dsDNA-activated protein kinase kinase levels, which induce phosphorylation of p53. In contrast, UPI induced no increase in kidney MDM2 mRNA and protein levels in IUGR pups. We conclude that among multiple mechanisms that affect nephrogenesis, UPI induces an increase in p53 phosphorylation without a corresponding increase in MDM2 expression, and we speculate that this response may contribute to the increased apoptosis previously described in the IUGR kidney.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Rim/metabolismo , Circulação Placentária , Insuficiência Placentária/fisiopatologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Feminino , Fosforilação , Gravidez , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Uteroplacental insufficiency (UPI) induces persistent changes in hepatic gene expression secondary to altered chromatin dynamics in the intrauterine growth- restricted (IUGR) rat liver. The glucocorticoid receptor (GR) is a transcription factor that when activated can induce changes in chromatin structure. To begin the process of identifying pathways by which IUGR affects chromatin structure, we hypothesized that UPI in the rat induces a significant increase in endogenous glucocorticoids (corticosterone) and increases GR expression and activation. To prove our hypothesis, we induced IUGR through bilateral uterine artery ligation of the pregnant rat. At day 1, UPI significantly increased corticosterone levels and was associated with increased total GR mRNA and protein levels in the liver, as well as increased hepatic phosphorylation of GR serine 211. Moreover, cyclin-dependent kinase 2 (CDK2) cyclinA/CDK2 protein levels, which selectively phosphorylate GR serine 211, were also significantly increased. To assess activity of the GR, we measured protein levels of the transcription factor p53 whose levels are downregulated, at least in part, by active GR. In this study, UPI decreased p53 protein and its downstream target Bax mRNA levels. We conclude that UPI in rats affects GR expression and activity in the liver. We speculate that these alterations early in life may contribute to the changes in chromatin structure and gene expression previously described in the IUGR liver.