Prenatal administration of all-trans retinoic acid upregulates leptin signaling in hypoplastic rat lungs with experimental congenital diaphragmatic hernia

Pulmonary hypoplasia (PH), characterized by alveolar immaturity, is one of the leading causes of respiratory insufficiency in newborns with congenital diaphragmatic hernia (CDH). Leptin (Lep) and its receptor (Lep-R) play an important role in fetal lung growth by stimulating alveolar differentiation and maturation. Lep and Lep-R are strongly expressed by alveolar cells during the saccular stage of fetal lung development. Lep-deficient mice exhibit decreased alveolarization with reduced pulmonary surfactant phospholipid synthesis, similar to human and nitrofen-induced PH. Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced PH. Recent studies have demonstrated that Lep and Lep-R expression in developing lungs is regulated by ATRA. We hypothesized that prenatal treatment with ATRA increases pulmonary Lep and Lep-R expression in the nitrofen model of CDH-associated PH.

Time-mated rats received either 100 mg nitrofen or vehicle via oral-gastric lavage on embryonic day 9.5 (E9.5). Control and nitrofen-exposed dams were randomly assigned to either intraperitoneal ATRA (5 mg/kg/d) or placebo administration on E18.5, E19.5 and E20.5. Fetal lungs were harvested on E21.5, and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo and Nitrofen+ATRA. Alveolarization was assessed using stereo- and morphometric analysis techniques. Surfactant phospholipid synthesis was analyzed by labeling for surfactant protein B (SP-B). Pulmonary gene expression levels of Lep and Lep-R were determined using quantitative real-time polymerase chain reaction. Immunohistochemical staining for Lep and Lep-R was performed to evaluate alveolar protein expression and localization.

In vivo administration of ATRA resulted in significantly increased lung-to-body weight ratio with enhanced radial alveolar count and decreased mean linear intercept compared to placebo treatment. Immunofluorescence analysis demonstrated markedly increased pulmonary SP-B expression in Nitrofen+ATRA compared to Nitrofen+Placebo. Relative mRNA expression of Lep and Lep-R was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo. Lep and Lep-R immunoreactivity was markedly increased in interstitial and alveolar epithelial cells of Nitrofen+ATRA compared to Nitrofen+Placebo.

Increased Lep and Lep-R expression after prenatal administration of ATRA in nitrofen-induced PH suggests that ATRA may have therapeutic potential in attenuating CDH-associated PH by stimulating alveolarization and de novo surfactant production.