NKCC-1 and ENaC are down-regulated in nitrofen-induced hypoplastic lungs with congenital diaphragmatic hernia

Abstract

Congenital diaphragmatic hernia (CDH) is accompanied by pulmonary hypoplasia and pulmonary hypertension. Fetal lung growth is dependent on the secretion of lung liquid, which normally is absorbed at partus. The ion channel NKCC-1 is involved in this secretory process, but has recently also been reported to be implicated in absorption. CDH patients show a disturbed transition from secretion to absorption. alpha- and beta-ENaC are essential for lung liquid absorption. Common for all transcellular ion transport is the need for Na/K-ATPase as a primary driving force. The aim of the study was first to map the normal pulmonary expression of the above proteins during late gestation and secondly to see if the expression was affected in a CDH rat model. Pregnant Sprague-Dawley rat dams were given nitrofen on gestational day 9.5 to induce CDH. The fetuses were removed on gestational days E18 and E21. In addition, newborn rats were harvested postpartum on day P2. The fetuses were put into one of two groups: hypoplastic lungs without CDH (N-CDH) and hypoplastic lungs with CDH (N+CDH). The pulmonary expression of NKCC-1, alpha-/beta-ENaC and Na/K-ATPase was then analyzed using Western blot. We found that the protein levels of NKCC-1 on gestational days E18 and E21 were significantly lower among fetuses with N+CDH as well as N-CDH compared to controls. The expression of beta-ENaC was also significantly down-regulated in both the groups on E18 and E21. The protein levels of alpha-ENaC and Na/K-ATPase were not found to be significantly decreased, but both showed a tendency towards down-regulation. The marked down-regulation of NKCC-1 in fetal hypoplastic lungs with CDH indicates a possibly decreased lung liquid production. This may be one of the mechanisms behind the disturbed pulmonary development in CDH. We also show that beta-ENaC is down-regulated. Down-regulation of beta-ENaC may result in abnormal lung liquid absorption, which could be one of the mechanisms behind the respiratory distress seen in CDH patients postpartum.