TGF-ß directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury.

TGF-ß is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-ß pathway is described, which rapidly promoted internalization of the αßγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-ß applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which--together with patch-clamp and flow cytometry studies--identified ENaC as the target of TGF-ß. TGF-ß rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of ßENaC, the subunit responsible for cell-surface stability of the αßγENaC complex. ENaC internalization was dependent on oxidation of ßENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove ßENaC internalization, which was inhibited by a TGF-ß neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-ß signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-ß-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.

Megalin mediates transepithelial albumin clearance from the alveolar space of intact rabbit lungs.

The alveolo-capillary barrier is effectively impermeable to large solutes such as proteins. A hallmark of acute lung injury/acute respiratory distress syndrome is the accumulation of protein-rich oedema fluid in the distal airspaces. Excess protein must be cleared from the alveolar space for recovery; however, the mechanisms of protein clearance remain incompletely understood. In intact rabbit lungs 29.8 ± 2.2% of the radio-labelled alveolar albumin was transported to the vascular compartment at 37°C within 120 min, as assessed by real-time measurement of 125I-albumin clearance from the alveolar space. At 4°C or 22°C significantly lower albumin clearance (3.7 ± 0.4 or 16.2 ± 1.1%, respectively) was observed. Deposition of a 1000-fold molar excess of unlabelled albumin into the alveolar space or inhibition of cytoskeletal rearrangement or clathrin-dependent endocytosis largely inhibited the transport of 125I-albumin to the vasculature, while administration of unlabelled albumin to the vascular space had no effect on albumin clearance. Furthermore, albumin uptake capacity was measured as about 0.37 mg ml−1 in cultured rat lung epithelial monolayers, further highlighting the (patho)physiological relevance of active alveolar epithelial protein transport. Moreover, gene silencing and pharmacological inhibition of the multi-ligand receptor megalin resulted in significantly decreased albumin binding and uptake in monolayers of primary alveolar type II and type I-like and cultured lung epithelial cells. Our data indicate that clearance of albumin from the distal air spaces is facilitated by an active, high-capacity, megalin-mediated transport process across the alveolar epithelium. Further understanding of this mechanism is of clinical importance, since an inability to clear excess protein from the alveolar space is associated with poor outcome in patients with acute lung injury/acute respiratory distress syndrome.

The lectin-like domain of tumor necrosis factor-alpha improves alveolar fluid balance in injured isolated rabbit lungs.

OBJECTIVE: Identification of mechanisms that preserve optimal alveolar fluid balance during pulmonary edema is of great clinical importance. This study was performed to determine whether the lectin-like domain of tumor necrosis factor-alpha (designated TIP) can improve fluid balance in experimental lung injury by affecting alveolocapillary permeability and/or fluid clearance. DESIGN: Prospective, randomized laboratory investigation. SETTING: University-affiliated laboratory. SUBJECTS: Adult male rabbits. INTERVENTIONS: TIP, a scrambled peptide (scrTIP), dibutyryl cyclic adenosine monophosphate (db-cAMP), or saline was applied to isolated, ventilated, and buffer-perfused rabbit lungs by ultrasonic nebulization, after which hydrostatic edema or endo/exotoxin-induced lung injury was induced and edema formation was assessed. In studies evaluating the resolution of alveolar edema, 2.5 mL of excess fluid was deposited into the alveolar space of isolated lungs by nebulization in the absence or presence of TIP, scrTIP, amiloride, or ouabain or combinations thereof. MEASUREMENTS AND MAIN RESULTS: Microvascular permeability was largely increased during hydrostatic edema and endo/exotoxin-induced lung injury in saline-treated lungs, or lungs that received scrTIP, as assessed by capillary filtration coefficient (K(f,c)) and fluorescein isothiocyanate-labeled albumin flux across the alveolocapillary barrier. In contrast, TIP- or db-cAMP-treated lungs exhibited significantly lower vascular permeability upon hydrostatic challenge. Similarly, extravascular fluid accumulation, as assessed by fluid retention, wet weight to dry weight ratio, and epithelial lining fluid volume measurements, was largely inhibited by TIP or db-cAMP pretreatment. Furthermore, TIP increased sodium-potassium adenosine triphosphatase (Na,K-ATPase) activity 1.6-fold by promoting Na,K-ATPase exocytosis to the alveolar epithelial cell surface and increased amiloride-sensitive sodium uptake, resulting in a 2.2-fold increase in active Na+ transport, and hence improved clearance of excess fluid from the alveolar space. CONCLUSIONS: Aerosolized TIP improved alveolar fluid balance by both reducing vascular permeability and enhancing the absorption of excess alveolar fluid in experimental lung injury. These data may suggest a role for TIP as a potential therapeutic agent in pulmonary edema.