Jean A. Petershack

Ibuprofen-Induced Patent Ductus Arteriosus Closure: Physiologic, Histologic, and Biochemical Effects on the Premature Lung

OBJECTIVE. The goal was to study the pulmonary, biochemical, and morphologic effects of a persistent patent ductus arteriosus in a preterm baboon model of bronchopulmonary dysplasia. METHODS. Preterm baboons (treated prenatally with glucocorticoids) were delivered at 125 days of gestation (term: 185 days), given surfactant, and ventilated for 14 days. Twenty-four hours after birth, newborns were randomly assigned to receive either ibuprofen (to close the patent ductus arteriosus; n = 8) or no drug (control; n = 13). RESULTS. After treatment was started, the ibuprofen group had significantly lower pulmonary/systemic flow ratio, higher systemic blood pressure, and lower left ventricular end diastolic diameter, compared with the control group. There were no differences in cardiac performance indices between the groups. Ventilation index and dynamic compliance were significantly improved with ibuprofen. The improved pulmonary mechanics in ibuprofen-treated newborns were not attributable to changes in levels of surfactant protein B, C, or D, saturated phoshatidylcholine, or surfactant inhibitory proteins. There were no differences in tracheal concentrations of cytokines commonly associated with the development of bronchopulmonary dysplasia. The groups had similar messenger RNA expression of genes that regulate inflammation and remodeling in the lung. Lungs from ibuprofen-treated newborns were significantly drier (lower wet/dry ratio) and expressed 2.5 times more epithelial sodium channel protein than did control lungs. By 14 days after delivery, control newborns had morphologic features of arrested alveolar development (decreased alveolar surface area and complexity), compared with age-matched fetuses. In contrast, there was no evidence of alveolar arrest in the ibuprofen-treated newborns. CONCLUSIONS. Ibuprofen-induced patent ductus arteriosus closure improved pulmonary mechanics, decreased total lung water, increased epithelial sodium channel expression, and decreased the detrimental effects of preterm birth on alveolarization.

Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Mediate cAMP Induction of α-Epithelial Na+ Channels (α-ENaC)

A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (α, β, and γ). The α‐subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG‐C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of α‐ENaC mRNA and protein expression and amiloride‐sensitive short‐circuit current (ISC). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H‐89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP‐induced α‐ENaC protein formation and ISC. Exposure of SMG‐C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H‐89. Dominant‐negative CREB decreased DbcAMP‐induced α‐ENaC expression. Suppression of the extracellular signal‐regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen‐activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP‐induced α‐ENaC protein levels in SMG‐C6 cells. DbcAMP‐induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP‐induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H‐89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of α‐ENaC observed after 24 h of treatment in SMG‐C6 epithelial cells. J. Cell. Physiol. 215: 101–110, 2008.

Ontogeny and regulation of cardiac angiotensin types 1 and 2 receptors during fetal life in sheep.

Previous studies have shown that the expression of cardiac angiotensin II(ANG II) type 1 (AT1) and type 2 (AT2) receptors are developmentally regulated, although factors modulating these receptors have not been well investigated. The present study was designed 1) to characterize the ontogeny of cardiac AT1 and AT2 gene expression during the last third trimester of gestation in fetal sheep and newborn lambs, 2) to determine the influence of ANG II on modulating cardiac AT1 and AT2 gene expression during fetal life, and 3) to investigate the role of AT1 receptor activity on the regulation of AT1 and AT2 mRNA levels during fetal cardiac development. Using sheep AT1 and AT2 cDNA probes, we demonstrated that cardiac AT1 gene expression is relatively unchanged during fetal (90-135 d of gestation, term 145 d) and newborn life. In contrast, cardiac AT2 mRNA expression was high during fetal development and decreased rapidly after birth. Continuous i.v. infusion of ANG II (9.5 nM/h) for 24 h, which raised ANG II levels from 84± 9 to 210 ± 21 pg/mL had no effect on the expression of cardiac AT1 or AT2 mRNA, but increased adrenal and decreased liver AT1 mRNA levels. Administration of the AT1 receptor antagonist losartan (1.2 mg kg-1 h-1) significantly decreased arterial blood pressure in fetuses at 110- and 135-d, but not 95-d gestation. Except for increased AT1 receptor gene expression in the right atrium at 95-and 135-d gestation, and left ventricle at 110-d gestation, cardiac AT1 and AT2 mRNA levels were unaltered by AT1 receptor blockade. In summary, this study demonstrates that cardiac AT2 but not AT1 receptor gene expression is regulated by the transition from fetal to newborn life. Neither ANG II nor blockade of AT1 receptors significantly alter the expression of AT1 or AT2 mRNA in the fetal heart. Endogenous ANG II also appears to significantly contribute to the maintenance of blood pressure homeostasis during the final third of gestation in fetal lambs.

Recruiting and Retaining Community-Based Preceptors: A Multicenter Qualitative Action Study of Pediatric Preceptors

Purpose The recruitment and retention of community preceptors to teach medical students is difficult. The authors sought to characterize the underlying motivational factors for becoming a preceptor and to identify strategies for recruiting and retaining community-based pediatric preceptors. Method This multicenter qualitative action study included semistructured interviews with community-based pediatric preceptors affiliated with 12 institutions from August to December 2015. Only active preceptors were included, and participating institutions were diverse with respect to geographic location and class size. Interviews were conducted over the telephone and transcribed verbatim. Six investigators used deidentified transcripts to develop a codebook. Through a constant comparative method, codes were revised as data were analyzed and disagreements were resolved through discussion. All investigators organized the themes into dimensions. Results Fifty-one preceptors were interviewed. Forty-one themes coalesced into four dimensions: (1) least liked aspects of teaching, (2) preparation to teach, (3) inspiration to teach, and (4) ways to improve recruitment and retention. Time constraints and patient care demands were the most commonly cited deterrents to teaching. Successful preceptors balanced their clinical demands with their desire to teach using creative scheduling. External rewards (e.g., recognition, continuing medical education credit) served as incentives. Internal motivation inspired participants to share their enthusiasm for pediatrics and to develop longitudinal relationships with their learners. Conclusions Changes in health care delivery have imposed more time constraints on community-based preceptors. However, this study identified underlying factors motivating physicians to volunteer as preceptors. Strategies to recruit new and retain current preceptors must be collaborative.

Conducting Quantitative Medical Education Research: From Design to Dissemination

Rigorous medical education research is critical to effectively develop and evaluate the training we provide our learners. Yet many clinical medical educators lack the training and skills needed to conduct high-quality medical education research. We offer guidance on conducting sound quantitative medical education research. Our aim is to equip readers with the key skills and strategies necessary to conduct successful research projects, highlighting new concepts and controversies in the field. We utilize Glassicks criteria for scholarship as a framework to discuss strategies to ensure that the research question of interest is worthy of further study and how to use existing literature and conceptual frameworks to strengthen a research study. Through discussions of the strengths and limitations of commonly used study designs, we expose the reader to particular nuances of these decisions in medical education research and discuss outcomes generally focused on, as well as strategies for determining the significance of consequent findings. We conclude with information on critiquing research findings and preparing results for dissemination to a broad audience. Practical planning worksheets and comprehensive tables illustrating key concepts are provided in order to guide researchers through each step of the process. Medical education research provides wonderful opportunities to improve how we teach our learners, to satisfy our own intellectual curiosity, and ultimately to enhance the care provided to patients.

Role of Glucocorticoids in the Maturation of Renal Cortical Na+, K+-ATPase During Fetal Life in Sheep 295

In sheep and humans, circulating glucocorticoid levels increase at the time of birth coincident with an increased ability of the kidney to reabsorb sodium. Proximal tubule Na+/H+ exchanger activity and mRNA abundance as well as basolateral Na,K-ATPase activity and abundance increase at this time. We have previously shown that cortisol induces a maturation of proximal tubule apical membrane Na+/H+ exchanger activity and mRNA levels in near-term fetal sheep. The regulation of Na,K-ATPase at birth is poorly understood. Therefore, we have studied the effects of this cortisol treatment on third trimester fetal sheep renal cortical Na,K-ATPase activity, mRNA levels and protein abundance. Nine pairs of twin fetal sheep were chronically instrumented at 127 days gestation (term is 145 days). After 72 hr recovery, one twin was given a continuous intraperitoneal infusion of cortisol at 3 mg/hr for 48 hr, the second twin received nothing. At the end of the infusion, both twins were sacrificed and their renal cortices were excised and frozen in liquid nitrogen. Na,K-ATPase activity (μmol PO4/mg protein/30 min) increased from 0.60±0.09 in controls to 1.22±0.11 (p< 0.005) in treated fetuses. Treated animals had levels of Na,K-ATPase activity equal to those of untreated 140 days gestation fetal sheep. Northern blots were probed for the α1 and β1 subunits of Na,K-ATPase; α1 mRNA abundance increased by 60±11% (p< 0.005), β1 abundance increased by 56±17% (p<0.005) with cortisol treatment. Immunoblots were probed with sheep-specific antisera to the α1 and β1 subunits; subunit protein abundances increased by 24±4% (p< 0.005) and 34±4% (p< 0.005) respectively with cortisol treatment. In summary, Na,K-ATPase activity increased to a greater degree than did subunit mRNA or protein levels. We conclude that cortisol may increase Na,K-ATPase activity in fetal sheep kidney by both transcriptional and post-transcriptional mechanisms. A substantial part of this cortisol effect may result from an activation of preexisting Na,K-ATPase.

Effects of Losartan on Angiotension Receptors During Fetal Cardiac Development in Sheep. † 137

We have previously shown in sheep that AT2 receptor mRNA expression is elevated early during fetal life and decreases rapidly near term, while AT1 receptor expression increases during the last trimester of gestation(Pediatr Res 38:896-904, 1995). The present study was designed to determine if a rise in AT1 receptor activity during fetal cardiac development regulated the decrease in AT2 receptor expression in near term fetuses.

Influence of AT1 Receptor Activity on AT1 and AT2 Receptor Expression During Fetal Life. |[dagger]| 284

The present study was designed to test the hypothesis that an increase in AT1 receptor activity participates in the decline in AT2 mRNA expression associated with fetal maturation (Pediatr Res 38:896904, 1995). To test this hypothesis, we studied eleven pairs of chronically instrumented twin fetal sheep at 95d gestation and seven pairs at 110d gestation (term is 145d). One twin received intravenous infusion of losartan (L) (10mg/kg bolus followed by 20 μg/kg/min for 48 hr) while the other served as a salinetreated control (C). In the 95d animals, no change in heart rate, mean arterial blood pressure (MABP), plasma renin activity (PRA), or aldosterone levels were observed during L infusion. Furthermore, L did not alter AT1 and AT2 mRNA expression in 95d fetuses; AT1 and AT2 mRNA levels were consistent in C and L animals kidney cortex, kidney medulla, liver, and adrenal. In contrast, in 110d fetuses, L decreased MABP from 40.1±1mmHg to 33±1mmHg(p<0.001) while PRA increased from 4.8±1.5 to 9.8±3 ngAI/ml/hr (p<0.05). In addition, L decreased 110d kidney medulla AT1 by 32±12% (p<0.05) and reduced kidney medulla AT2 by 32±14%(p<0.05) mRNA expression. Likewise, adrenal AT2 fell by 14±11%. To determine if these changes in mRNA expression during L were secondary to changes in MABP, three additional sets of 110d twin fetuses were studied; one twin received L while the other twin received L plus phenylephrine to keep MABP from falling. When compared to L fetuses, keeping MABP normal prevented changes in AT1 and AT2 mRNA levels.

TISSUE-SPECIFIC AND DIFFERENTIAL REGULATION OF AT1 AND AT2 GENE EXPRESSION FOLLOWING ADMINISTRATION OF AN AT1 ANTAGONIST DURING FETAL LIFE. |[dagger]| 2186

Fetal kidney AT2 mRNA levels are elevated early in gestation and decrease with fetal maturation, whereas AT1 mRNA expression increases rapidly before birth. The present study was designed to determine if the developmental rise in AT1 expression contributes to the decrease in AT2 expression with advancing gestational age. To test this hypothesis, eight pairs of chronically-instrumented twin fetal sheep (111±2 d gestation, term 145 d) were studied; one of the fetuses received an intravenous infusion of the AT1 antagonist losartan (L) (20μg/kg/min for 48 h) while the other served as a saline-treated control (C)(0.9% NaCl at 0.1 ml/h). Mean arterial blood pressure did not change in C, but decreased from 40±1 to 33±2 mmHg in L-treated fetuses(p<0.01). Heart rate did not change in C or L fetuses. Plasma renin activity increased significantly (p<0.01) in L-treated fetuses from 4.3±1.3 to 10.7±3.8 ng AI/ml/h. L produced significant(p<0.05) decreases in kidney medulla AT1 (-32.4±12.6%) and AT2 mRNA levels (-7.4±2.9%) but did not affect AT1 or AT2 expression in the kidney cortex. In the adrenal, L did not alter AT1 but increased AT2 expression by 246±130% (p<0.05). L did not alter AT1 or AT2 expression in fetal liver. The present results demonstrate that inhibition of AT1 receptor activity produces tissue-specific and differential regulation of both AT1 and AT2 genes during fetal life, and that the decrease in kidney AT2 expression during fetal development is not dependent on a rise in AT1 receptor activity.