ENaC‐a mediates the protective effect of the TNF‐derived TIP peptide in pneumolysin‐induced capillary barrier dysfunction

Background Streptococcus pneumoniae is the major etiologic agent of bacterial pneumonia. Autolysis and antibiotic‐mediated lysis of pneumococci releases the pore‐forming toxin, pneumolysin (PLY), which is the primary cause of acute lung injury. PLY unleashes the perfect storm, by inhibiting alveolar liquid clearance (ALC) and by potently compromising alveolar‐capillary barrier function, which together cause potentially lethal permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. Apart from being the entry step in Na + reabsorption across Na + ‐transporting epithelia, the epithelial sodium channel (ENaC) was recently suggested to affect stiffness in the large vasculature, but its role in barrier function in the microvasculature has not been investigated so far. In this study, we therefore investigated the consequences of specific ENaC activation or depletion on capillary barrier function, in the presence or absence of PLY. Methods Expression of α, β and γ subunits of ENaC and protein phosphorylation status in HL‐MVEC were assessed in Western blotting. Barrier function in untreated and PLY‐treated human lung microvascular endothelial cell (HL‐MVEC) monolayers was monitored using electrical cell‐substrate impedance sensing (ECIS). In order to investigate the role of ENaC in barrier function in HL‐MVEC, we either activated the channel, using the TNF‐derived TIP peptide, which directly binds to the α subunit, or we depleted the crucial ENaC‐α subunit with specific siRNA. Results HL‐MVEC express all three subunits of ENaC. TIP peptide‐mediated ENaC activation significantly strengthened barrier function in PLY‐treated HL‐MVEC. By contrast, ENaC‐α depletion significantly increased sensitivity to PLY‐induced hyperpermeability and blunted the protective effect of the TIP peptide. PLY significantly increased phosphorylation of the actin‐binding protein filamin A, which was shown to directly bind to ENaC‐α and which promotes the interaction between ENaC with MARCKS, an important mechanism for activation of the channel's open probability. TIP peptide‐mediated activation of ENaC blunted phosphorylation of both calmodulin dependent Kinase II (CaMKII) and its substrate filamin A. Conclusions Taken together, our results demonstrate a previously unrecognized role for ENaC, and more specifically for ENaC‐α, in strengthening barrier function of human lung capillaries. As such, these data suggest that strategies aiming to activate ENaC have the potential to not only improve ALC in the alveolar epithelium, but also barrier function in the capillary endothelium during pneumonia.