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.

Antenatal use of bosentan and/or sildenafil attenuates pulmonary features in rats with congenital diaphragmatic hernia

“…[29] In groups 3, 4 and 5 which received sildenafil and/ or bosentan, it was possible to attenuate pulmonary morphological, architectural and structural changes; especially those related to the abnormal development of the pulmonary vascular wall, as the characteristic of PPHN in patients with CDH and are the main cause of death of such patients. [30][31][32] Thus, group 3 which received sildenafi l, a specific phosphodiesterase 5 inhibitor, showed a dilated pulmonary vasculature and antiproliferative effects on cells of the pulmonary artery. [33] There was a significant reduction in arterial wall thickness in microns, which helped to mitigate the reduction of the vessel's lumen, expressed as the arterial-wall-lumen ratio.…”

Low Pulmonary Expression of Epithelial Na⁺ Channel and Na⁺, K⁺-ATPase in Newborn Infants with Congenital Diaphragmatic Hernia

“…Expression of the ␣ 1 -subunit in rat and mouse lung increases from low levels throughout gestation and peaks just before or at birth [43,44] . No significant changes have been demonstrated in Na + , K + -ATPase ␣ 1 expression in nitrofen-induced CDH in rats, even though a tendency towards downregulation has been reported by our group before [24] . The decrease that we observe in CDH patients is pronounced.…”

“…This association was first showed in 2006 by Folkesson et al [22] , who reported a markedly impaired postnatal lung fluid transport and decreased protein levels of ENaC in nitrofen-induced CDH in rats. A recent study from our group has demonstrated low protein levels of ␤ -ENaC and Na + -K + -2Cl -cotransporter (NKCC-1) in hypoplastic CDH rat lungs [24] .…”

“…In 2006, Folkesson et al [22] reported that in nitrofen-induced CDH in rats, the hypoplastic lungs showed an impaired capacity for lung fluid absorption and a decreased expression of ␣ -and ␤ -ENaC, resulting in fluid-filled lungs and respiratory distress at birth. Our group has also reported perinatal downregulation of pulmonary ion channels in CDH in rats and the expression of AQP5 was recently found to be decreased in another animal study [23,24] . To our knowledge, no data exist on the expression of lung ion and water channels in human newborn infants with CDH and pulmonary hypoplasia.…”

“…After electrophoresis (8 or 12% gel, 20 g protein/lane), detection of immunosignals was performed using primary antibodies as described previously [24] . The antibody signal was normalized for total protein amount calculated from the amido black staining in each lane.…”