Inactivation of Channel-Activating Protease 1 in mouse distal lung impairs ENaC-mediated lung fluid clearance

Abstract

Transepithelial sodium (Na + ) transport via amiloride-sensitive epithelial Na + channels (ENaC) expressed in the apical membrane of alveolar epithelial cells provides the driving force for removal of fluid from the alveolar space. In vitro , the membrane-anchored channel-activating serine protease 1 (CAP1) strongly activates ENaC channels at the cell surface by increasing their open probability through proteolytic cleavage of γ-ENaC subunit, but the physiological relevance of this process in vivo is unknown. We investigated whether inactivation of CAP1 gene in mouse distal lung epithelium would affect in vivo transepithelial alveolar sodium transport and alveolar fluid clearance. Mice lacking CAP1 in the alveolar epithelium were generated by CreLoxP-recombination inducible in utero by doxycycline (Tet-ON system). Specificity and efficiency of Cre-recombination as well as expression of ENaC subunits were examined by PCR, real-time RT-PCR and Western-blotting in freshly isolated alveolar epithelial cells. Alveolar fluid clearance was measured in vivo in an in situ lung model under basal and β 2 -agonist-stimulated conditions using radiolabeled albumin as a tracer of alveolar fluid volume. Cre-recombination was restricted to alveolar epithelial cells by PCR and led to a 95% decrease in CAP1 mRNA expression by quantitative RT-PCR, without affecting ENaC mRNA and protein levels. CAP1-deficient mice had normal growth and survival rate, and normal lung histology. Basal alveolar fluid clearance was reduced by 30% in CAP1-deficient mice as compared with control littermates (p<0.01), due to a 50%-decrease in amiloride-sensitive clearance reflecting ENaC activity (p<0.001). β 2 -agonists failed to stimulate alveolar fluid clearance in CAP1-deficient mice. Basal alveolar fluid clearance and stimulation by β 2 -agonists were fully restored by addition in the alveolar instillate of human neutrophil elastase, a serine protease that reproduces the effect of CAP1 on ENaC. Conditional inactivation of CAP1 in mouse alveolar epithelium impairs ENaC-mediated alveolar fluid clearance, and abolishes the response to β 2 -agonists. These data provide in vivo evidence that epithelium-derived CAP1 is a potent activator of ENaC in mouse distal lung. Funded by Inserm, SPLF, Fondation du Legs Poix and Swiss National Foundation