Na+ reabsorption in airway and alveolar epithelia is critically important at the time of birth and continues to play a significant role after birth and throughout life to regulate the ionic composition, and volume of pulmonary secretions. Immature or dysregulated Na+ and fluid reabsorption in the airway and alveoli may contribute to the pathophysiology of respiratory distress syndrome (RDS) of the newborn and of lung disorders that are characterized by excess airway liquid such as that seen in pulmonary edema or following a toxic, infectious or inflammatory lung injury. In this research plan we propose to study two important regulators of perinatal Na+ transport in the lung, cyclic AMP and glucocorticoids, focusing on the role of sgk1, a kinase that appears to be a point of convergence for many signaling pathways that stimulate Na+ transport. Our hypothesis is that cyclic AMP and GC, acting through sgk1 stimulate Na+ transport in distal lung epithelia and in fetal lung and that regulation of sgk1 expression occurs primarily at the level of sgk1 gene transcription. We will first investigate if cyclic AMP-and GC-regulated Na+ transport in human fetal lung and in fetal distal lung epithelial cells occurs, in part, through stimulation of sgk1. Na+ transport will be measured by short-circuit current in a distal lung epithelial cell line and amiloride-sensitive changes in lumen volume measured in human fetal lung. The role of sgk1 will be investigated by expression of dominant negative sgk1 or by antisense oligonucleotides. We will also test the hypothesis that adenoviral transfer of sgk1 can enhance cyclic AMP- and GC-mediated Na+ transport in distal lung epithelia and in fetal lung. Second, we will determine if elevation of cyclic AMP stimulates sgk1 expression in fetal lung and in distal lung epithelia and identify the pathways of regulation. The proposed experiments will measure sgk1 mRNA and protein; determine the mechanism of regulation in response to cyclic AMP stimulation and map pathways of regulation. Finally, we will identify the cyclic AMP-stimulated enhancer elements of the sgk1 gene in distal lung epithelia by transient transfection, gel mobility shift assays and by chromatin immunoprecipitation assays. Understanding the mechanisms of regulation of Na+ transport by hormone mediated events and second messenger systems offers the potential for modulating Na+ transport in respiratory epithelia in a variety of pathophysiological conditions including RDS.
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