Fibroblast-derived factors stimulate epithelial sodium transport in fetal alveolar cells

Preterm birth is a leading cause of neonatal morbidity and mortality, primarily due to pulmonary immaturity, which also predisposes infants to chronic lung disease later in life. A major consequence of lung immaturity is impaired alveolar fluid clearance (AFC). AFC depends on vectorial sodium transport across the alveolar epithelium via the epithelial sodium channel (ENaC), which generates the osmotic gradient required for fluid absorption at birth. Although glucocorticoids enhance AFC, their use is limited by adverse effects, highlighting the need for alternatives. Epithelial–mesenchymal interactions may provide such a potential alternative by modulating epithelial differentiation and sodium transport. Supporting this notion, we identified a previously unrecognized ability of fetal lung fibroblasts to stimulate epithelial sodium transport. The present study aimed to identify the underlying fibroblast-derived factor responsible for this effect. Conditioned medium from fetal lung fibroblasts (FCM) was fractionated using reversed phase (RPC), hydrophilic interaction (HILIC), size exclusion (SEC) and ion exchange (IEC) chromatography, combined with extraction and filtration procedures, followed by mass spectrometry analysis. The effects of FCM fractions on ENaC expression and activity were evaluated in primary fetal distal lung epithelial cells after 24 h using real-time qPCR, Ussing chamber measurements and a novel membrane-potential-based assay. FCM significantly enhanced ENaC activity and increased β-ENaC mRNA expression by ~20% after 24 h. Membrane-potential assay provided a robust, high-throughput approach for assessing ENaC activity, consistent with qPCR results. The ENaC-stimulatory activity was resistant to proteinase K, drying, and heating up to 96°C, indicating a structurally stable, non-proteinaceous factor. RPC failed to retain the active component, while HILIC showed weak retention, suggesting a highly polar factor. Liquid–liquid extractions with MTBE, DCM, iPrOH, and TCM confirmed exclusive activity in the aqueous phase, further supporting its polar nature. Ultrafiltration and SEC localized the activity to a