Epithelial Sodium Channel-α Mediates the Protective Effect of the TNF-Derived TIP Peptide in Pneumolysin-Induced Endothelial Barrier Dysfunction

Abstract

BackgroundStreptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar–capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The α subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function.MethodsThe presence of α, β, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α.ResultsHL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance.ConclusionOur results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

The TNF-derived TIP peptide activates the epithelial sodium channel and ameliorates experimental nephrotoxic serum nephritis

“…The TIP peptide specifically binds to the a subunit of ENaC, but not to the b and g subunits. 20,22 Because depletion of ENaC-a abrogated the protective effects of the TIP peptide in barrier function in toxin-treated human lung microvascular endothelial cell 25 as well as in TNF-induced p38 activation and PGE 2 generation in glomerular endothelial cells (this study), we hypothesize that ENaC-a is the specific receptor for the peptide, although the existence of alternative receptors cannot be fully excluded.…”

“…We demonstrated that the TIP peptide binds to the a subunit of the epithelial sodium channel (ENaC), 20,22 which can be expressed in both epithelial and endothelial cells. 25,26 Support for this experimental direction is provided by the finding that inhaled TIP peptide (a.k.a. AP301 and solnatide) was recently found to be safe in a phase 1 clinical trial in volunteers 27 and displayed promising activities on lung function in 2 phase 2a clinical trials in patients with acute lung injury 28 and after lung transplantation.…”

Upregulation of CBP by PLY can cause permeability of blood‐brain barrier to increase meningitis

“…However, researchers pointed to that TNF‐α‐mediated host responses can resist to pneumococcal infection . In addition, other research showed that PLY can impair endothelial barrier function through decreasing the activity of endothelial sodium channel (ENaC) . Previously demonstrated by other studies, PLY can recognize TLR4, which can also confer the resistance to the bacteria during the infection, and then it can trigger the releasing of TNF‐α .…”

Impact of Bacterial Toxins in the Lungs

“…LLO causes a dysfunction in the ENaC channel in airway epithelial cells, especially by targeting the crucial α subunit [21,315]. ENaC-α is not only crucial for promoting Na + reabsorption across Na + -transporting epithelia but also for its role in strengthening capillary barrier function in lung microvascular endothelial cells [315,316]. Thus, targeting ENaC-α expression impairs structural and functional properties of ENaC and weakens the capillary barrier function [9,316].…”

“…ENaC-α is not only crucial for promoting Na + reabsorption across Na + -transporting epithelia but also for its role in strengthening capillary barrier function in lung microvascular endothelial cells [315,316]. Thus, targeting ENaC-α expression impairs structural and functional properties of ENaC and weakens the capillary barrier function [9,316]. LLO decreases the expression of ENaC-α at least partially through disabling positive regulators of ENaC including serum and glucocorticoid-dependent kinase (Sgk-1) and protein kinase B (Akt-1).…”