Alveolar fluid clearance driven by active epithelial Na + and secondary Cl − absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na + channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl − secretion and alveolar Cl − influx were quantified by radionuclide tracing and alveolar Cl − imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl − secretion and alveolar Cl − influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na + -K + -Cl − cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR −/− mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl − secretion that were again CFTR-, NKCC-, and Na + -K + -ATPase-dependent. Our findings show a reversal of transepithelial Cl − and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl − and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.epithelial Cl − transport | pulmonary edema T raditionally, the formation of cardiogenic pulmonary edema has been attributed to passive fluid filtration across an intact alveolocapillary barrier along an increased hydrostatic pressure gradient. However, recent studies show that cardiogenic edema is critically regulated by active signaling processes. Activation of mechanosensitive endothelial ion channels increases lung vascular permeability (1), whereas alveolar epithelial cells lose their physiological ability to clear the distal airspaces from excess fluid by their capacity to actively transport ions across the epithelial barrier (2-4).In the intact lung, the predominant force driving alveolar fluid clearance is an active transepithelial Na + transport from the alveolar into the interstitial space. A major portion of the apical Na + entry is mediated by the amiloride-inhibitable epithelial Na + channel (ENaC), with basolateral Na + extrusion through the Na + -K + -ATPase (5). Cl − and water are considered to follow paracellularly for electroneutrality and osmotic balance. In cardiogenic lung edema, the physiological protection against alveolar flooding provided by an intact alveolar fluid clearance is largely attenuated (3, 4). Previously, ...

Platelet-activating factor (PAF) is a mediator of pulmonary oedema in acute lung injury that increases vascular permeability within minutes, partly through activation of acid sphingomyelinase (ASM). Since caveolae are rich in sphingomyelin and caveolin-1, which block endothelial nitric oxide (NO) synthase (eNOS) by direct binding, we examined the relationship between ASM, caveolin-1 and eNOS activity in the regulation of vascular permeability by PAF.In caveolar fractions from pulmonary vascular endothelial cells (isolated from perfused rat lungs) the abundance of caveolin-1 and eNOS increased rapidly after PAF perfusion. PAF treatment decreased endothelial NO (eNO) formation as assessed by in situ fluorescence microscopy. Restoration of eNO levels with PAPA-NONOate ((Z)-1-[N-(3-ammoniopropyl)-N-(npropyl)amino]diazen-1-ium-1,2-diolate) mitigated the PAF-induced oedema.PAF treatment increased the ASM activity in caveolar fractions and perfusion with ASM decreased eNO production. Pharmacological inhibition of the ASM pathway with imipramine, D609 or dexamethasone blocked the PAF-induced increase of caveolin-1 and eNOS in caveolae, and the decrease in eNO production and oedema formation.We conclude that PAF causes ASM-dependent enrichment of caveolin-1 in caveolae of endothelial cells, leading to decreased eNO production which contributes to pulmonary oedema formation. These findings suggest rapid reduction in eNO production as a novel mechanism in the regulation of vascular permeability.

Hydrostatic lung edema evolves from increased fluid filtration and inhibition of epithelial Na+ channels (ENaC) that facilitate alveolar fluid clearance. Recently, we identified alveolar fluid secretion (AFS) as new key component in edema formation. Here, we tested whether Cl− secretion via cystic fibrosis transmemberane conductance regulator (CFTR) and Na+‐K+‐Cl− cotransporter 1 (NKCC1) may mediate AFS at elevated left atrial pressure (PLA) and may be induced by inhibition of ENaC.In isolated lungs, we quantified AFS by a double indicator dilution technique, and transepithelial Cl− flux by radionuclide tracing and alveolar Cl− imaging. PLA elevation induced lung edema and AFS that coincided with transepithelial Cl− secretion and alveolar Cl− influx. These effects were blocked by inhibitors of CFTR, NKCC or Na+‐K+‐ATPase, and CFTR−/− mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride at physiologicalPLA induced AFS and Cl− secretion that were again CFTR‐, NKCC‐ and Na+‐K+‐ ATPase dependent.We conclude that transepithelial Cl− and fluid flux reverse from absorptive to secretory mode at hydrostatic stress as a result of ENaC inhibition, and are mediated by NKCC and CFTR.