Role of ion and water channels for lung growth in congenital diaphragmatic hernia

Early lung development is based on a continuous cross-talk of growth and transcription factors between the developing lung bud and the surrounding parenchyma. Throughout fetal life, pulmonary growth and differentiation depend on Cl- driven lung liquid secretion. The prospective fetal airways are thus filled with lung liquid and the fetus is provided with oxygen by the mother through the placenta. Cl- secretion involves the basolateral Na+-K+-2Cl- co-transporter 1 (NKCC-1), coupled to an apical Cl- exit pathway. A decrease in lung liquid renders pulmonary hypoplasia, while an in utero applied tracheal occlusion (TO), trapping lung liquid within the airways, results in pulmonary hyperplasia. In order to eliminate residual lung fluid and to make way for an effective gas exchange, the lungs switch prenatally to active fluid absorption by apical Na+ transport through the amiloride sensitive epithelial sodium channel (ENaC-alpha/-beta/-gamma). The basolaterally located Na+, K+- ATPase acts as the primary driving force. Four water channels, aquaporin (AQP) 1, 3, 4 and 5 are also expressed in the lung and may contribute to fluid absorption.

Pulmonary hypoplasia is most frequently associated with congenital diaphragmatic hernia (CDH), a birth defect present in 1 in 2000-3000 live newborns; approximately 8% of all major congenital anomalies. About 30 newborns per year are diagnosed with CDH in Sweden. Survival rates differ significantly, but averages on 50%. The disease includes herniation of abdominal viscera into the thoracic cavity through a diaphragmatic defect. The pathophysiology of CDH is constituted by pulmonary hypoplasia (PH) and an increase in lung vessel resistance, resulting in pulmonary artery hypertension (PAH). This leads to respiratory distress at birth. The work in this thesis aimed to explore the role of lung liquid metabolism for the development and function of hypoplastic lungs in CDH. The work also includes a study on a possible new drug candidate for the treatment of PAH in CDH.

In study I, we investigated the expression of NKCC-1, ENaC and Na+, K+-ATPase during different developmental stages in a rat model of CDH. NKCC-1 together with beta-ENaC were significantly down-regulated in fetuses with CDH and PH in mid- and late gestation. This indicates that lung liquid metabolism might be affected in CDH and the ability for lung liquid production and absorption may be decreased.

In study II, we examined the role of the chloride channels ClC-2, -3 and -5 in the development of hypoplastic lungs and TO-induced hyperplastic lungs. ClC-2 and -3 were both downregulated just before birth in PH lungs, while lung hyperplasia created by TO up-regulated the expression of ClC-2. NKCC-1 showed a tendency towards up-regulation in hyperplastic lungs. ClC-5 were not affected in any group. ClC-2 is therefore an interesting potential target in the development of novel, non-invasive therapies for CDH treatment.

In study III, we performed a global gene expression analysis on the nitrofen-induced animal model of CDH. Significantly decreased expression was found for several growth factors and growth factor receptors involved in lung development, transcription factors, water and ion channels including ENaC and AQP1 and -4, genes involved in pulmonary angiogenesis and extracellular matrix formation. These data supports our hypothesis that CDH with PH is connected to a reduced ability for lung liquid clearance at birth and that PH is not mainly a consequence of mechanical compression, but primarily an intrinsic pulmonary disease. In study IV, we conducted a clinical study examining the pulmonary expression of ion and water transporters in newborn CDH patients. beta-ENaC, gamma-ENaC and Na+, K+-ATPase alpha1 collected at 18 h postnatally were significantly lower in CDH infants compared to control infants and remained unchanged on the second day of life. These results support earlier reports from animal studies and may result in an abnormal lung fluid absorption contributing to the respiratory distress in newborn CDH patients.

Study V was inspired by successful results from our group after administration of the platelet derived growth factor (PDGF) inhibitor, imatinib, to a CDH patient with intractable PAH. The pulmonary artery pressure decreased and the patient s condition gradually improved. In the present study performed in rats, we showed that the typical medial wall hyperplasia of pulmonary arteries seen in CDH, leading to the increased pulmonary vascular resistance, could be counteracted and almost normalized after antenatal imatinib administration. Imatinib treated CDH fetuses also showed a significant reduction in the number of fully muscularized pulmonary arteries. The increase in smooth muscle cell proliferation which was found in the nitrofen treated rats compared to controls was also significantly reduced by imatinib treatment.

We concluded that imatinib is a very interesting candidate as a novel therapeutic approach for counteracting PAH in CDH before birth.

List of scientific papers

I. Ringman A, Zelenina M, Eklöf AC, Aperia A, Frenckner B (2008). "NKCC-1 and ENaC are down-regulated in nitrofen-induced hypoplastic lungs with congenital diaphragmatic hernia." Pediatr Surg Int 24(9): 993-1000.
https://doi.org/10.1007/s00383-008-2209-6

II. Ringman Uggla A, Zelenina M, Eklöf AC, Aperia A, Frenckner B (2009). "Expression of chloride channels in trachea-occluded hyperplastic lungs and nitrofen-induced hypoplastic lungs in rats." Pediatr Surg Int 25(9): 799-806.
https://doi.org/10.1007/s00383-009-2423-x

III. Mesas Burgos C, Ringman Uggla A, Fagerström-Billai F, Eklöf AC, Frenckner B, Nord M (2010). "Gene expression analysis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia". [Accepted]
https://doi.org/10.1016/j.jpedsurg.2009.09.023

IV. Ringman Uggla A, von Schewelov K, Zelenina M, Aperia A, Frenckner B (2010). "Low pulmonary expression of ENaC and Na+, K+-ATPase in newborn infants with congenital diaphragmatic hernia." [Submitted]

V. Ringman Uggla A, Chang Y, Tran Lundmark K, Eklöf AC, Tran K, Frenckner B (2010). "Antenatal imatinib treatment reverses pulmonary vascular remodeling in a rat model of congenital diaphragmatic hernia." [Manuscript]