β‐ENaC associated with cilia indicates the distribution of absorptive epithelial cells in the lumen of small airways

Airway surface liquid (ASL) is crucial for optimal clearance of mucus, pathogens and debris from the pulmonary bronchial tree and is managed by a balance between secretory and absorptive activities along the luminal airway surfaces. Immunohistochemically, we recently demonstrated that in small airways, about half of the lining epithelium consists of cells with a secretory phenotype that are confined to the contraluminal regions (pleats) of the plicated luminal epithelium (1). However, the distribution/location of absorptive airway epithelial cells has not been demonstrated. The epithelial sodium channel (ENaC) is responsible for Na(+) absorption across the apical membranes of many salt absorbing epithelia including airways. Herein, we hypothesized that localization of β‐ENaC subunits would define the distribution of absorptive cells in airway epithelia. We obtained small pieces of peripheral porcine lung tissue of approximately 0.5 cm 3 and pieces of trachea of approximately 0.5 cm 2 that were dissected and immediately snap‐frozen at −80°C. Serial tissue sections (7 μm thick) containing cross‐sections of small airways, with a diameter between 0.5 and 1 mm, were used for immunohistochemistry. We used antibodies directed to b‐subunit of ENaC (courtesy C. Fuller), as well antibodies directed to β‐Tubulin and the tight junction protein, ZO1. Western blot analysis demonstrated ENaC expression in cell extracts derived from isolated small airways and trachea. Confocal microscopy of immunostained sections of airway tissue defined the distribution of β‐ENaC subunits in the apical regions of a sub‐set of cells in the small airways and trachea. In small airways, strong staining for β‐subunit was generally restricted to ciliated cells in the region around the tips of folds in the luminal epithelium well separated from the sub‐sets of secretory cells in base of the epithelial pleats where staining was generally absent or faint, but strong for the secretory marker, NKCC1, seen previously (1). In trachea, β‐ENaC subunit staining was mainly observed at the surface of the stratified ciliated epithelium, and absent in regions where submucosal glands were present. Importantly, we observed that β‐subunit of ENaC appeared to co‐localize with the distribution β‐tubulin, a biomarker for cilia. These findings suggest that small airway epithelium contains a subset of epithelial cells with absorptive phenotype, recognized by the high expression of β‐subunit of ENaC. Support or Funding Information Supported by the Cystic Fibrosis Foundation‐MCCC, CFRI and the Nancy Olmsted Trust for Pediatric Respiratory Medicine