Post-Transcriptional Modulation of aENaC mRNA in Alveolar Epithelial Cells: Involvement of its 3' Untranslated Region.

BACKGROUND/AIMS: The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays a major role in lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that αENaC mRNA expression is downregulated in part via posttranscriptional regulation of mRNA stability. In the present work, the role of the αENaC 3' untranslated region (3'UTR) in the regulation of mRNA stability was studied further. METHODS: Quantitative reverse transcription PCR (qRT-PCR) was performed to investigate the expression of αENaC in alveolar epithelial cells. The role of the αENaC 3'UTR was evaluated through sequential deletions. RNA affinity chromatography and mass spectrometry were achieved to investigate the nature of the proteins that could bind this sequence. The function of these proteins was assessed through knockdown and overexpression in vitro. RESULTS: First, we found that αENaC mRNA half-life was much shorter than expected when using a transcriptionally controlled plasmid expression system compared to Actinomycin D treatment. Sequential deletions of the αENaC 3'UTR revealed that the αENaC 3'UTR plays an important role in the modulation of αENaC mRNA stability, and that there is a complex stabilizing and destabilizing interplay between different regions of the 3'UTR that modulate this process. Finally, we identified RNA-binding proteins that interact with the αENaC 3'UTR and showed that Dhx36 and Tial1 are involved in the decrease in αENaC mRNA stability via the proximal region of its 3'UTR. CONCLUSION: Taken together, these findings indicate that the αENaC 3'UTR plays an important role in modulating transcript levels, and Dhx36 and Tial1 seem to be involved in posttranscriptional regulation of αENaC expression in alveolar epithelial cells.

Loss of barrier integrity in alveolar epithelial cells downregulates ENaC expression and activity via Ca2+ and TRPV4 activation.

The epithelial Na channel (ENaC) plays an essential role in lung physiology by modulating the amount of liquid lining the respiratory epithelium. Here, we tested the effect of breaking alveolar epithelial cell barrier integrity on ENaC expression and function. We found that either mechanical wounding by scratching the monolayer or disruption of tight junction with EDTA induced a ~ 50% decrease of α,ß and γENaC mRNA expression and an 80% reduction of ENaC short-circuit current (Isc) at 6 h. Scratching the cell monolayer generated a Ca2+ wave that spread from the margin of the scratch to distant cells. Pretreatment with BAPTA-AM, an intracellular Ca2+ chelator, abolished the effect of mechanical wounding and EDTA on αENaC mRNA expression, suggesting that [Ca2+]i is important for this modulation. We tested the hypothesis that a mechanosensitive channel such as TRPV4, a cationic channel known to increase [Ca2+]i, could mediate this effect. Activation of the channel with the TRPV4 specific agonist GSK-1016790A (GSK) decreased αENAC mRNA expression and almost completely abolished ENaC Isc. Pretreatment of alveolar epithelial cells with HC-067047 (HC0), a specific TRPV4 antagonist, reduced the extent of αENAC mRNA downregulation by mechanical wounding and EDTA. Altogether, our results suggest that mechanical stress induced by wounding or TRPV4-mediated loss of tight junction increases [Ca2+]i and elicits a Ca2+ wave that affects ENaC expression and function away from the site of injury. These data are important to better understand how Ca2+ signaling affects lung liquid clearance in injured lungs.

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 µM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.

Cycloheximide and lipopolysaccharide downregulate αENaC mRNA via different mechanisms in alveolar epithelial cells.

Active Na(+) transport mediated by epithelial Na(+) channel (ENaC) is vital for fetal lung fluid reabsorption at birth and pulmonary edema resolution. Previously, we demonstrated that αENaC expression and activity are downregulated in alveolar epithelial cells by cycloheximide (Chx) and Pseudomonas aeruginosa. The regulatory mechanisms of αENaC mRNA expression by Chx and lipopolysaccharide (LPS) from P. aeruginosa were further studied in the present work. Both agents decreased αENaC mRNA expression to 50% of control values after 4 h. Chx repressed αENaC expression in a dose-dependent manner independently of protein synthesis. Although extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways were activated by the two treatments, their mechanisms of ENaC mRNA modulation were different. First, activation of the signaling pathways was sustained by Chx but only transiently by LPS. Second, ERK1/2 or p38 MAPK inhibition attenuated the effects of Chx on αENaC mRNA, whereas suppression of both signaling pathways was necessary to alleviate the outcome of LPS on αENaC mRNA. The molecular mechanisms involved in the decrease of αENaC expression were investigated in both conditions. LPS, but not Chx, significantly reduced αENaC promoter activity via the ERK1/2 and p38 MAPK pathways. These results suggest that LPS attenuates αENaC mRNA expression via diminution of transcription, whereas Chx could trigger some posttranscriptional mechanisms. Although LPS and Chx downregulate αENaC mRNA expression similarly and with similar signaling pathways, the mechanisms modulating ENaC expression are different depending on the nature of the cellular stress.

Hypotonic shock modulates Na(+) current via a Cl(-) and Ca(2+)/calmodulin dependent mechanism in alveolar epithelial cells.

Alveolar epithelial cells are involved in Na(+) absorption via the epithelial Na(+) channel (ENaC), an important process for maintaining an appropriate volume of liquid lining the respiratory epithelium and for lung oedema clearance. Here, we investigated how a 20% hypotonic shock modulates the ionic current in these cells. Polarized alveolar epithelial cells isolated from rat lungs were cultured on permeant filters and their electrophysiological properties recorded. A 20% bilateral hypotonic shock induced an immediate, but transient 52% rise in total transepithelial current and a 67% increase in the amiloride-sensitive current mediated by ENaC. Amiloride pre-treatment decreased the current rise after hypotonic shock, showing that ENaC current is involved in this response. Since Cl(-) transport is modulated by hypotonic shock, its contribution to the basal and hypotonic-induced transepithelial current was also assessed. Apical NPPB, a broad Cl(-) channel inhibitor and basolateral DIOA a potassium chloride co-transporter (KCC) inhibitor reduced the total and ENaC currents, showing that transcellular Cl(-) transport plays a major role in that process. During hypotonic shock, a basolateral Cl(-) influx, partly inhibited by NPPB is essential for the hypotonic-induced current rise. Hypotonic shock promoted apical ATP secretion and increased intracellular Ca(2+). While apyrase, an ATP scavenger, did not inhibit the hypotonic shock current response, W7 a calmodulin antagonist completely prevented the hypotonic current rise. These results indicate that a basolateral Cl(-) influx as well as Ca(2+)/calmodulin, but not ATP, are involved in the acute transepithelial current rise elicited by hypotonic shock.

Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKCα-ß1 in alveolar epithelial cells.

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⁺ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca²âº chelation and protein kinase C (PKCα-ß1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca²âº-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca²âº/PKCα-ß1 pathway independent of ATP signaling.

K+ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells.

Active Na+ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and KATP K+ channel activities exerts sustained control in Na+ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the alpha-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K+ channels, and 2) to determine the physiological impact of K+ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and KATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24 h) increased alpha-ENaC expression, similarly to KATP activation by pinacidil. Conversely, pharmacological KvLQT1 and KATP inhibition or silencing with siRNAs down-regulated alpha-ENaC expression. Furthermore, K+ channel blockers significantly decreased alpha-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and KATP activation dose-dependently enhanced alpha-ENaC promoter activity. Finally, we noted a physiological impact of changes in K+ channel functions on ERK activity, alpha-, beta-, gamma-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K+ channels regulate alpha-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.

Modulation of epithelial sodium channel activity by lipopolysaccharide in alveolar type II cells: involvement of purinergic signaling.

Pseudomonas aeruginosa is a gram-negative bacterium that causes chronic infection in cystic fibrosis patients. We reported recently that P. aeruginosa modulates epithelial Na(+) channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested whether LPS from P. aeruginosa alters ENaC expression and activity in alveolar epithelial cells. We found that LPS induces a approximately 60% decrease of ENaC apical current without significant changes in intracellular ENaC or surface protein expression. Because a growing body of evidence reports a key role for extracellular nucleotides in regulation of ion channels, we evaluated the possibility that modulation of ENaC activity by LPS involves extracellular ATP signaling. We found that alveolar epithelial cells release ATP upon LPS stimulation and that pretreatment with suramin, a P2Y(2) purinergic receptor antagonist, inhibited the effect of LPS on ENaC. Furthermore, ET-18-OCH3, a PLC inhibitor, and Go-6976, a PKC inhibitor, were able to partially prevent ENaC inhibition by LPS, suggesting that the actions of LPS on ENaC current were mediated, in part, by the PKC and PLC pathways. Together, these findings demonstrate an important role of extracellular ATP signaling in the response of epithelial cells to LPS.

[Na+ Transport in the lungs: differential impact of ENaC in the airways and alveoli]. / Transport du Na+ dans les poumons : impact différentiel du canal ENaC dans les voies aériennes et les alvéoles.

Na+ transport by airway epithelial cells, in conjunction with Cl- secretion is crucial for maintaining an adequate level of airway surface liquid (ASL) for an effective mucociliary clearance by the ciliated airway epithelial cells. It is also an important mechanism for lung liquid absorption at birth and oedema absorption during an acute respiratory distress syndrome (ARDS). The epithelial Na+ channel (ENaC) is the channel mostly involved in this process. The consequences of an imbalance in ENaC activity in the airways and in the distal lung are different. Experimental over expression of ENaC in the airways leads to a decrease in mucociliary clearance and inflammation similar to cystic fibrosis and chronic bronchitis. However, bacterial and viral pathogens, as well as pro-inflammatory cytokines present during lung infection downregulate ENaC expression and activity in airway and alveolar epithelial cells. ENaC downregulation by pathogens or inflammatory products could participate in the modulation of the severity of ARDS. Pharmacological strategies that modulate ENaC expression or activity could be important in the treatment of different lung diseases since it is actively involved in the lung innate defence mechanisms.

Pharmacologic modulation of alveolar liquid clearance in transplanted lungs by phentolamine and FK506.

BACKGROUND: The lung's capacity to clear alveolar fluid can determine the severity of the edema seen after transplantation. We recently observed that alveolar liquid clearance was decreased in transplanted lungs. This study evaluates the ability of phentolamine and FK506 to modulate the severity of lung injury and the decline in alveolar liquid clearance after transplantation. METHODS: A canine orthotopic single-lung transplantation model was used. The lungs were preserved with a low-potassium-dextran solution (50 mL/kg) and transplanted after 3 hours of cold ischemia. The experimental protocol included a control group, a phentolamine group, in which donor lungs were infused with phentolamine (2 mg/kg), and a FK506 group, in which the animals received FK506 (25 mg/kg per hour) intravenously during reperfusion. After 4 hours of reperfusion, alveolar liquid clearance, wet-to-dry ratios, lung epithelial Na(+) channel expression, and the response to beta-adrenergic stimulation were measured. RESULTS: The increase in wet-to-dry ratios of transplanted lungs was less pronounced in the phentolamine and FK506 groups. The FK506 treatment led to improvement of alveolar liquid clearance. Neither phentolamine nor FK506 influenced lung epithelial Na(+) channel expression in transplanted lungs or preserved alveolar cell ability to respond to beta-adrenergic stimulation. CONCLUSIONS: Phentolamine or FK506 treatment during reperfusion improves alveolar liquid clearance and decreases the severity of lung injury.

The regulation of amiloride-sensitive epithelial sodium channels by tumor necrosis factor-alpha in injured lungs and alveolar type II cells.

Alveolar liquid clearance, which mainly depends on sodium transport in alveolar epithelial cells, is an important mechanism by which excess water in the alveoli is reabsorbed during the resolution of pulmonary edema. In this study, we examined the regulation of epithelial sodium channel (ENaC), the main contributor to sodium transport, during acute lung injury and the direct impact of tumor necrosis factor-alpha (TNF-alpha), one of the important cytokines in acute lung injury, on the ENaC regulation. During the development of pulmonary edema, the increases in the number of neutrophils and the levels of TNF-alpha in blood and bronchoalveolar lavage were seen. In parallel, the mRNA expression of the alpha-, beta- and gamma-ENaC subunits in the whole lung tissue was inhibited to 72.0, 47.8 and 53.9%, respectively. The direct exposure of rat alveolar type II cells to TNF-alpha inhibited the mRNA expression of alpha- and gamma-ENaC to 64.0 and 78.0%, but not that of the beta-ENaC. TNF-alpha also inhibited the ENaC function as indicated by the reduction of amiloride-sensitive current (control 4.4, TNF-alpha 1.9 microA/cm(2)). These data suggest that TNF-alpha may affect the pathophysiology of acute lung injury and pulmonary edema through the inhibition of alveolar liquid clearance and sodium transport.

Omega-3 polyunsaturated fatty acids improve host response in chronic Pseudomonas aeruginosa lung infection in mice.

Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 wk with specifically designed diets with high contents in either omega-3 (omega-3) or omega-6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally, and the animals were studied for 7 days. On the 4th day, the mice fed with the omega-3 diet had a higher lean body mass gain and a lower omega-6:omega-3 ratio of fatty acids extracted from the lung tissue compared with the other groups (P < 0.05). The omega-3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the omega-3 group on the 4th day. The effect on DAFC was independent of alpha-epithelial Na(+) channels (alpha-ENaC), beta-ENaC, and alpha(1)-Na-K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in omega-3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the omega-6/omega-3 PUFA balance may represent an interesting target in the prevention and/or control of P. aeruginosa infection in patients.

Dexamethasone inhibits the action of TNF on ENaC expression and activity.

We have reported that TNF, a proinflammatory cytokine present in several lung pathologies, decreases the expression and activity of the epithelial Na(+) channel (ENaC) by approximately 70% in alveolar epithelial cells. Because dexamethasone has been shown to upregulate ENaC mRNA expression and is well known to downregulate proinflammatory genes, we tested if it could alleviate the effect of TNF on ENaC expression and activity. In cotreatment with TNF, we found that dexamethasone reversed the inhibitory effect of TNF and upregulated alpha, beta, and gammaENaC mRNA expression. When the cells were pretreated for 24 h with TNF before cotreatment, dexamethasone was still able to increase alphaENaC mRNA expression to 1.8-fold above control values. However, in these conditions, beta and gammaENaC mRNA expression was reduced to 47% and 14%, respectively. The potential role of TNF and dexamethasone on alphaENaC promoter activity was tested in A549 alveolar epithelial cells. TNF decreased luciferase (Luc) expression by approximately 25% in these cells, indicating that the strong diminution of alphaENaC mRNA must be related to posttranscriptional events. Dexamethasone raised Luc expression by fivefold in the cells and augmented promoter activity by 2.77-fold in cotreatment with TNF. In addition to its effect on alphaENaC gene expression, dexamethasone was able to maintain amiloride-sensitive current as well as the liquid clearance abilities of TNF-treated cells within the normal range. All these results suggest that dexamethasone alleviates the downregulation of ENaC expression and activity in TNF-treated alveolar epithelial cells.

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells.

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and Gö6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.

Evidence of a functional CFTR Cl(-) channel in adult alveolar epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the fetal lung, but during lung development it gradually disappears in cells of future alveolar spaces. Recent studies have implicated the CFTR in fluid transport by the adult alveolar epithelium, but its presence has not been demonstrated directly. This study re-evaluated CFTR expression and activity in the adult pulmonary epithelium by using freshly isolated rat alveolar type II (ATII) cells. CFTR mRNA was detected by semiquantitative polymerase chain reaction on the day of cell isolation but was rapidly reduced by 60% after 24 h of cell culture. This was paralleled by a similar decrease of surfactant protein A expression and alkaline phosphatase staining, markers of the ATII cell phenotype. CFTR expression increased significantly on day 4 in cells grown on filters at the air-liquid interface compared with cells submerged or grown on plastic. Significantly higher CFTR expression was detected in distal lung tissue compared with the trachea. The CFTR was also found at the protein level in Western blot experiments employing lysates of freshly isolated alveolar cells. Whole cell patch-clamp experiments revealed cAMP-stimulated, 5-nitro-2-(3-phenylpropylamino)-benzoate-sensitive Cl(-) conductance with a linear current-voltage relationship. In cell-attached membrane patches with 100 microM amiloride in pipette solution, forskolin stimulated channels of approximately 4 pS conductance. Our results indicate that 50-250 of functional CFTR Cl(-) channels occur in adult alveolar cells and could contribute to alveolar liquid homeostasis.

Downregulation of ENaC activity and expression by TNF-alpha in alveolar epithelial cells.

Sodium absorption by an amiloride-sensitive channel is the main driving force of lung liquid clearance at birth and lung edema clearance in adulthood. In this study, we tested whether tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine involved in several lung pathologies, could modulate sodium absorption in cultured alveolar epithelial cells. We found that TNF-alpha decreased the expression of the alpha-, beta-, and gamma-subunits of epithelial sodium channel (ENaC) mRNA to 36, 43, and 16% of the controls after 24-h treatment and reduced to 50% the amount of alpha-ENaC protein in these cells. There was no impact, however, on alpha(1) and beta(1) Na(+)-K(+)-ATPase mRNA expression. Amiloride-sensitive current and ouabain-sensitive Rb(+) uptake were reduced, respectively, to 28 and 39% of the controls. A strong correlation was found at different TNF-alpha concentrations between the decrease of amiloride-sensitive current and alpha-ENaC mRNA expression. All these data show that TNF-alpha, a proinflammatory cytokine present during lung infection, has a profound influence on the capacity of alveolar epithelial cells to transport sodium.

Transforming growth factor-beta1 decreases expression of the epithelial sodium channel alphaENaC and alveolar epithelial vectorial sodium and fluid transport via an ERK1/2-dependent mechanism.

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells. The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-beta1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-beta1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity.

Alveolar liquid clearance and sodium channel expression are decreased in transplanted canine lungs.

To determine the impact of transplantation-associated injury on the clearance mechanisms of pulmonary edema, we created a canine single lung transplant model. After 3 hours of preservation and 4 hours of reperfusion, right native lungs and left transplanted lungs were used to measure alveolar liquid clearance (ALC) in ex vivo liquid-filled lung preparations. We also examined the role of the pulmonary circulation in edema clearance in in vivo liquid-filled lungs between 4 and 8 hours of reperfusion. To study molecular modifications in ALC, we also measured expression levels of the epithelial sodium channel (ENaC) and sodium-potassium-adenosine triphosphatase (ATPase). We found that ALC was significantly lower in transplanted than in right native lungs ex vivo (p < 0.05) and that transplanted lungs did not respond to the beta-adrenergic agonist terbutaline. Our in vivo study confirmed the ex vivo results. Molecular analyses revealed that ENaC messenger RNA but not sodium-potassium-ATPase was significantly decreased in transplanted lungs (p < 0.01). Furthermore, there was a significant decrease in ENaC protein expression. Therefore, we conclude that the current investigation indicates that the lung injury caused by lung preservation and transplantation significantly reduces the edema clearance ability of transplanted lungs.