Cytoprotective-selective activated protein C attenuates Pseudomonas aeruginosa-induced lung injury in mice.

Inhibition of the small GTPase RhoA attenuates the development of pulmonary edema and restores positive alveolar fluid clearance in a murine model of Pseudomonas aeruginosa pneumonia. Activated protein C (aPC) blocks the development of an unfavorably low ratio of small GTPase Rac1/RhoA activity in lung endothelium through endothelial protein C receptor (EPCR)/protease-activated receptor-1 (PAR-1)-dependent signaling mechanisms that include transactivating the sphingosine-1-phosphate (S1P) pathway. However, whether aPC's cytoprotective effects can attenuate the development of pulmonary edema and death associated with P. aeruginosa pneumonia in mice remains unknown. Thus, we determined whether the normalization of a depressed ratio of activated Rac1/RhoA by aPC would attenuate the P. aeruginosa-mediated increase in protein permeability across lung endothelial and alveolar epithelial barriers. Pretreatment with aPC significantly reduced P. aeruginosa-induced increases in paracellular permeability across pulmonary endothelial cell and alveolar epithelial monolayers via an inhibition of RhoA activation and a promotion of Rac1 activation that required the EPCR-PAR-1 and S1P pathways. Furthermore, pretreatment with aPC attenuated the development of pulmonary edema in a murine model of P. aeruginosa pneumonia. Finally, a cytoprotective-selective aPC mutant, aPC-5A, which lacks most of aPC's anticoagulant activity, reproduced the protective effect of wild-type aPC by attenuating the development of pulmonary edema and decreasing mortality in a murine model of P. aeruginosa pneumonia. Taken together, these results demonstrate a critical role for the cytoprotective activities of aPC in attenuating P. aeruginosa-induced lung vascular permeability and mortality, suggesting that cytoprotective-selective aPC-5A with diminished bleeding risks could attenuate the lung damage caused by P. aeruginosa in critically ill patients.

Transforming growth factor beta1 inhibits cystic fibrosis transmembrane conductance regulator-dependent cAMP-stimulated alveolar epithelial fluid transport via a phosphatidylinositol 3-kinase-dependent mechanism.

Exogenous or endogenous beta(2)-adrenergic receptor agonists enhance alveolar epithelial fluid transport via a cAMP-dependent mechanism that protects the lungs from alveolar flooding in acute lung injury. However, impaired alveolar fluid clearance is present in most of the patients with acute lung injury and is associated with increased mortality, although the mechanisms responsible for this inhibition of the alveolar epithelial fluid transport are not completely understood. Here, we found that transforming growth factor beta1 (TGF-beta1), a critical mediator of acute lung injury, inhibits beta(2)-adrenergic receptor agonist-stimulated vectorial fluid and Cl(-) transport across primary rat and human alveolar epithelial type II cell monolayers. This inhibition is due to a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis mediated by a phosphatidylinositol 3-kinase (PI3K)-dependent heterologous desensitization and down-regulation of the beta(2)-adrenergic receptors. Consistent with these in vitro results, inhibition of the PI3K pathway or pretreatment with soluble chimeric TGF-beta type II receptor restored beta(2)-adrenergic receptor agonist-stimulated alveolar epithelial fluid transport in an in vivo model of acute lung injury induced by hemorrhagic shock in rats. The results demonstrate a novel role for TGF-beta1 in impairing the beta- adrenergic agonist-stimulated alveolar fluid clearance in acute lung injury, an effect that could be corrected by using PI3K inhibitors that are safe to use in humans.

PAI-1 is an essential component of the pulmonary host response during Pseudomonas aeruginosa pneumonia in mice.

RATIONALE: Elevated plasma and bronchoalveolar lavage fluid plasminogen activator inhibitor 1 (PAI-1) levels are associated with adverse clinical outcome in patients with pneumonia caused by Pseudomonas aeruginosa. However, whether PAI-1 plays a pathogenic role in the breakdown of the alveolar-capillary barrier caused by P aeruginosa is unknown. OBJECTIVES: The role of PAI-1 in pulmonary host defence and survival during P aeruginosa pneumonia in mice was tested. The in vitro mechanisms by which P aeruginosa causes PAI-1 gene and protein expression in lung endothelial and epithelial cells were also examined. METHODS AND RESULTS: PAI-1 null and wild-type mice that were pretreated with the PAI-1 inhibitor Tiplaxtinin had a significantly lower increase in lung vascular permeability than wild-type littermates after the airspace instillation of 1×10(7) colony-forming units (CFU) of P aeruginosa bacteria. Furthermore, P aeruginosa in vitro induced the expression of the PAI-1 gene and protein in a TLR4/p38/RhoA/NF-κB (Toll-like receptor 4/p38/RhoA/nuclear factor-κB) manner in lung endothelial and alveolar epithelial cells. However, in vivo disruption of PAI-1 signalling was associated with higher mortality at 24 h (p<0.03) and higher bacterial burden in the lungs secondary to decreased neutrophil migration into the distal airspace in response to P aeruginosa. CONCLUSIONS: The results indicate that PAI-1 is a critical mediator that controls the development of the early lung inflammation that is required for the activation of the later innate immune response necessary for the eradication of P aeruginosa from the distal airspaces of the lung.

Serotonin decreases alveolar epithelial fluid transport via a direct inhibition of the epithelial sodium channel.

Hypoxia and epithelial stretch that are commonly observed in patients with acute lung injury have been shown to promote the release of serotonin (5-hydroxytryptamine, 5-HT) in vitro. However, whether 5-HT contributes to the decrease of alveolar epithelial fluid transport, which is a hallmark of lung injury, is unknown. Thus, we investigated the effect of 5-HT on ion and fluid transport across the alveolar epithelium. 5-HT caused a dose-dependent inhibition of the amiloride-sensitive current across primary rat and human alveolar epithelial type II cell monolayers, but did not affect Na(+)/K(+) ATPase function. Furthermore, we found that the 5-HT induced inhibition of ion transport across the lung epithelium was receptor independent, as it was not prevented by the blockade of 5-HT2R (5-HT receptor 2), 5-HT3R (5-HT receptor 3), or by pretreatment with an intracellular calcium-chelating agent, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester). In addition, the stimulation of 5-HT1R (5-HT receptor 1), 5-HT2R (5-HT receptor 2), 5-HT4R (5-HT receptor 4), and 5-HT7R (5-HT receptor 7) failed to reproduce the 5-HT effect on amiloride-sensitive sodium transport. We ascertained that 5-HT directly inhibited the function of rat alphabetagamma epithelial sodium channel (ENaC), as determined by heterologous expression of rat ENaC in Xenopus oocytes that do not express endogenous ENaC nor 5-HT receptors (5-HTR). Exposure of mice to hypoxia for 1 hour induced a 30% increase of 5-HT secretion into the distal airways of mice. Finally, the intratracheal instillation of 5-HT inhibited the amiloride-sensitive fraction of alveolar fluid clearance in mice. Together, these results indicate that 5-HT inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport via a direct interaction with ENaC, and thus can be an endogenous inhibitor of this ion channel.

Activation of the stress protein response inhibits the STAT1 signalling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70.

BACKGROUND AND AIM: Alveolar fluid clearance is impaired by inducible nitric oxide synthase (iNOS)/nitric oxide (NO)-dependent mechanisms in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The activation of the stress protein response (SPR) in alveolar macrophages on iNOS-dependent NO production in response to interferon gamma (IFNgamma), a major cytokine present in the airspace of patients with ALI, was investigated. METHODS: The SPR was activated in murine and primary human alveolar macrophages prior to analysis of signal transducer and activator of transcription factor 1 (STAT1) activation, iNOS mRNA and protein synthesis, and NO production. RESULTS: SPR activation resulted in inhibition of IFNgamma-mediated NO production (p=0.001) with >95% detergent insolubilisation of the STAT1 protein. Its subsequent proteasomal degradation was partially reversed with pretreatment of cells with the chemical chaperone glycerol. This early effect of the SPR was caused by the complete disruption of heat shock protein 90 (Hsp90)-STAT1 binding, as shown by immunoprecipitation. Recovery of STAT1 activation and recovery of iNOS synthesis occurred within 12 h after SPR activation (p=0.02). NO production (as compared with non-SPR controls) did not occur until 48 h later (p=0.02). SPR-induced Hsp70 (Hsp70i) expression caused a late inhibition of NO production (p=0.02). Inhibiting >50% Hsp70i expression recovered NO production to control levels whereas overexpressing Hsp70i in the absence of the SPR inhibited NO production (p=0.02). CONCLUSION: Early inhibition of STAT1 following its dissociation from Hsp90, and later inhibition of iNOS activity by Hsp70i, represent novel mechanisms by which SPR activation modulates the IFNgamma signalling in alveolar macrophages. These results highlight a potential clinical application for Hsp90 inhibitors in modulating NO signalling during the early phase of acute lung injury.

Critical role of the small GTPase RhoA in the development of pulmonary edema induced by Pseudomonas aeruginosa in mice.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia in critically ill patients. We have reported previously that P. aeruginosa exotoxins S and T mediate in vitro the increase in protein permeability across lung endothelial cell monolayers via a RhoA-dependent mechanism. However, whether inhibition of RhoA would significantly attenuate P. aeruginosa-mediated lung injury in mice is unknown. METHODS: P. aeruginosa-induced paracellular protein permeability was measured across bovine lung endothelial and rat alveolar epithelial type II cell monolayers with I-albumin. Some cell monolayers were pretreated with RhoA inhibitor CGX0287 1 h before P. aeruginosa exposure. At 4 h after exposure, lung endothelial and epithelial permeability, bacterial counts, bronchoalveolar lavage fluid levels of keratinocyte-derived chemokine, myeloperoxidase activity, and alveolar fluid clearance were measured. Some mice were treated intraperitoneally with CGX0287 1 h before or after airspace instillation of P. aeruginosa. RESULTS: RhoA inhibition attenuated in vitro P. aeruginosa-mediated increase in lung endothelial and epithelial permeability to protein and in vivo the development of pulmonary edema and inhibition of alveolar fluid clearance associated with P. aeruginosa pneumonia. Furthermore, RhoA inhibition decreased the systemic dissemination of P. aeruginosa and neutrophil activity in the lung tissue observed after airspace instillation of these bacteria. CONCLUSIONS: The small GTPase RhoA plays a critical role in mediating lung injury associated with P. aeruginosa pneumonia in mice. Thus, transient blockade of RhoA could attenuate lung damage caused by P. aeruginosa in critically ill patients.

Interleukin-1beta causes acute lung injury via alphavbeta5 and alphavbeta6 integrin-dependent mechanisms.

Interleukin (IL)-1beta has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1beta-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1beta production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta attenuated the IL-1beta-induced ALI. The results of in vitro studies indicated that IL-1beta caused the activation of transforming growth factor-beta via RhoA/alphavbeta6 integrin-dependent mechanisms and the inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta signaling completely blocked the IL-1beta-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1beta increased protein permeability across lung endothelial cell monolayers via RhoA- and alphavbeta5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the alphavbeta5 and alphavbeta6 integrins had an additive protective effect against IL-1beta-induced ALI. In summary, these results demonstrate a critical role for the alphavbeta5/beta6 integrins in mediating the IL-1beta-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.

Role of small GTPases and alphavbeta5 integrin in Pseudomonas aeruginosa-induced increase in lung endothelial permeability.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia associated with airspace flooding with protein-rich edema in critically ill patients. The type III secretion system is a major virulence factor and contributes to dissemination of P. aeruginosa. However, it is still unknown which particular bacterial toxin and which cellular pathways are responsible for the increase in lung endothelial permeability induced by P. aeruginosa. Thus, the first objective of this study was to determine the mechanisms by which this species causes an increase in lung endothelial permeability. The results showed that ExoS and ExoT, two of the four known P. aeruginosa type III cytotoxins, were primarily responsible for bacterium-induced increases in protein permeability across the lung endothelium via an inhibition of Rac1 and an activation of the RhoA signaling pathway. In addition, inhibition of the alphavbeta5 integrin, a central regulator of lung vascular permeability, prevented these P. aeruginosa-mediated increases in albumin flux due to endothelial permeability. Finally, prior activation of the stress protein response or adenoviral gene transfer of the inducible heat shock protein Hsp72 also inhibited the damaging effects of P. aeruginosa on the barrier function of lung endothelium. Taken together, these results demonstrate the critical role of the RhoA/alphavbeta5 integrin pathway in mediating P. aeruginosa-induced lung vascular permeability. In addition, activation of the stress protein response with pharmacologic inhibitors of Hsp90 may protect lungs against P. aeruginosa-induced permeability changes.

Early release of high mobility group box nuclear protein 1 after severe trauma in humans: role of injury severity and tissue hypoperfusion.

INTRODUCTION: High mobility group box nuclear protein 1 (HMGB1) is a DNA nuclear binding protein that has recently been shown to be an early trigger of sterile inflammation in animal models of trauma-hemorrhage via the activation of the Toll-like-receptor 4 (TLR4) and the receptor for the advanced glycation endproducts (RAGE). However, whether HMGB1 is released early after trauma hemorrhage in humans and is associated with the development of an inflammatory response and coagulopathy is not known and therefore constitutes the aim of the present study. METHODS: One hundred sixty eight patients were studied as part of a prospective cohort study of severe trauma patients admitted to a single Level 1 Trauma center. Blood was drawn within 10 minutes of arrival to the emergency room before the administration of any fluid resuscitation. HMGB1, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, von Willebrand Factor (vWF), angiopoietin-2 (Ang-2), Prothrombin time (PT), prothrombin fragments 1+2 (PF1+2), soluble thrombomodulin (sTM), protein C (PC), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and D-Dimers were measured using standard techniques. Base deficit was used as a measure of tissue hypoperfusion. Measurements were compared to outcome measures obtained from the electronic medical record and trauma registry. RESULTS: Plasma levels of HMGB1 were increased within 30 minutes after severe trauma in humans and correlated with the severity of injury, tissue hypoperfusion, early posttraumatic coagulopathy and hyperfibrinolysis as well with a systemic inflammatory response and activation of complement. Non-survivors had significantly higher plasma levels of HMGB1 than survivors. Finally, patients who later developed organ injury, (acute lung injury and acute renal failure) had also significantly higher plasma levels of HMGB1 early after trauma. CONCLUSIONS: The results of this study demonstrate for the first time that HMGB1 is released into the bloodstream early after severe trauma in humans. The release of HMGB1 requires severe injury and tissue hypoperfusion, and is associated with posttraumatic coagulation abnormalities, activation of complement and severe systemic inflammatory response.

Sensitization of mesothelioma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by heat stress via the inhibition of the 3-phosphoinositide-dependent kinase 1/Akt pathway.

Heat stress may enhance the effect of apoptosis-inducing agents in resistant tumor cells. One such agent is the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which has attracted intense interest for its ability to induce apoptosis in tumors without affecting nonmalignant cells. We therefore tested whether heat stress potentiates TRAIL-induced apoptosis in mesothelioma cells, its cell type being resistant to TRAIL alone. We found that heat stress enhanced the apoptosis caused by TRAIL but not by chemotherapy. To explain this potentiation, we found that heat stress decreased Akt phosphorylation via the dissociation of heat shock protein 90 (Hsp90) from its client protein 3-phosphoinositide-dependent kinase 1 (PDK-1), a major Akt kinase. The role of Hsp90 and the Akt pathway was confirmed by showing that inhibitors of Hsp90 and the phosphatidyilinositol-3 kinase/Akt pathway reproduced the effect of heat stress on TRAIL-induced apoptosis and that the effect of inhibiting Hsp90 on TRAIL-induced apoptosis could be overcome by activating the Akt pathway with a constitutively active construct of the Akt kinase PDK-1. The effect of heat stress involved multiple steps of the apoptotic machinery. Heat stress potentiated the death receptor pathway, as shown by an increase in TRAIL-induced caspase 8 cleavage. Nonetheless, knockdown of Bid, the main intermediary molecule from the death receptor pathway to the mitochondria, inhibited the effect of heat stress, showing that mitochondrial amplification was required for potentiation by heat stress. In summary, these results support the novel concept that heat stress inhibits the Akt pathway by dissociating PDK-1 from its chaperone Hsp90, leading to potentiation of TRAIL-induced apoptosis in resistant malignant cells.

Activation of the heat shock response attenuates the interleukin 1ß-mediated inhibition of the amiloride-sensitive alveolar epithelial ion transport.

Acute lung injury (ALI) is a clinical syndrome characterized by hypoxia, which is caused by the breakdown of the alveolar capillary barrier. Interleukin 1ß (IL-1ß), a cytokine released within the airspace in ALI, downregulates the α subunit of the epithelial sodium channel (αENaC) transcription and protein expression via p38 MAP kinase-dependent signaling. Although induction of the heat shock response can restore alveolar fluid clearance compromised by IL-1ß following the onset of severe hemorrhagic shock in rats, the mechanisms are not fully understood. In this study, we report that the induction of the heat shock response prevents IL-1ß-dependent inhibition of αENaC mRNA expression and subsequent channel function. Heat shock results in IRAK1 detergent insolubility and a disruption of Hsp90 binding to IRAK1. Likewise, TAK1, another client protein of Hsp90 and signaling component of the IL-1ß pathway, is also detergent insoluble after heat shock. Twenty-four hours after heat shock, both IRAK1 and TAK1 are again detergent soluble, which correlates with the IL-1ß-dependent p38 activation. Remarkably, IL-1ß-dependent p38 activation 24 h after heat shock did not result in an inhibition of αENaC mRNA expression and channel function. Further analysis demonstrates prolonged preservation of αENaC expression by the activation of the heat shock response that involves inducible Hsp70. Inhibition of Hsp70 at 24 h after heat shock results in p38-dependent IL-1ß inhibition of αENaC mRNA expression, whereas overexpression of Hsp70 attenuates the p38-dependent IL-1ß inhibition of αENaC mRNA expression. These studies demonstrate new mechanisms by which the induction of the heat shock response protects the barrier function of the alveolar epithelium in ALI.

Extracellular heat shock protein 72 is a marker of the stress protein response in acute lung injury.

Previous studies have shown that heat shock protein 72 (Hsp72) is found in the extracellular space (eHsp72) and that eHsp72 has potent immunomodulatory effects. However, whether eHsp72 is present in the distal air spaces and whether eHsp72 could modulate removal of alveolar edema is unknown. The first objective was to determine whether Hsp72 is released within air spaces and whether Hsp72 levels in pulmonary edema fluid would correlate with the capacity of the alveolar epithelium to remove alveolar edema fluid in patients with ALI/ARDS. Patients with hydrostatic edema served as controls. The second objective was to determine whether activation of the stress protein response (SPR) caused the release of Hsp72 into the extracellular space in vivo and in vitro and to determine whether SPR activation and/or eHsp72 itself would prevent the IL-1beta-mediated inhibition of the vectorial fluid transport across alveolar type II cells. We found that eHsp72 was present in plasma and pulmonary edema fluid of ALI patients and that eHsp72 was significantly higher in pulmonary edema fluid from patients with preserved alveolar epithelial fluid clearance. Furthermore, SPR activation in vivo in mice and in vitro in lung endothelial, epithelial, and macrophage cells caused intracellular expression and extracellular release of Hsp72. Finally, SPR activation, but not eHsp72 itself, prevented the decrease in alveolar epithelial ion transport induced by exposure to IL-1beta. Thus SPR may protect the alveolar epithelium against oxidative stress associated with experimental ALI, and eHsp72 may serve as a marker of SPR activation in the distal air spaces of patients with ALI.

Serum levels of Hsp60 correlate with the development of acute lung injury after trauma.

BACKGROUND: Previous studies have shown that heat shock protein 60 (Hsp60) is a danger signal for the immune system and appears to be a key endogenous inflammatory mediator that activates the toll-like receptors and causes the release of proinflammatory cytokines and nitric oxide by immune competent cells, but no data are available for trauma patients. The purpose of this study was to determine whether Hsp60 could be detected in the serum of patients early after severe trauma and whether its serum level might correlate with the development of acute lung injury (ALI) in trauma patients. METHODS: Clinical data were collected prospectively during a 12-month period for trauma patients who were ventilated mechanically for more than 24 h and who met the following inclusion criteria: Injury Severity Score > or =16, age >18 years. Physiological data for quantitative assessment of organ dysfunction were collected for each patient. Hsp60 levels were measured in the serum of trauma patients. RESULTS: Sixty-four patients with severe trauma were enrolled in the study. Eighteen patients developed ALI (28%). Trauma patients who later developed ALI had significantly higher serum values of Hsp60 than those who did not (4.21 +/- 2.24 ng/mL versus 0.73 +/- 0.26 ng/mL, P < 0.05, mean +/- SE). Furthermore, immature but not mature recombinant Hsp60 induced in vitro the release of nitric oxide (NO) from RAW 264.7 murine macrophages. CONCLUSION: Serum levels of Hsp60 detected within 30 min after trauma correlate with the development of ALI. Immature but mature Hsp60 causes in vitro the release of NO by macrophages, suggesting that the extracellular release of the immature Hsp60 associated with traumatic cell necrosis could be involved in the release of NO by immune competent cells, leading to an activation of the inflammatory response within the lung or other organs.

Mammalian osmolytes and S-nitrosoglutathione promote Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function.

In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed Delta F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of Delta F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the Delta F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that Delta F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact Delta F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted Delta F508 CFTR maturation and function. S-Nitrosoglutathione-corrected Delta F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting Delta F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize Delta F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to explain why Delta F508 homozygous patients do not present with kidney dysfunction.

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.