Lactate produced by alveolar type II cells suppresses inflammatory alveolar macrophages in acute lung injury.

Alveolar inflammation is a hallmark of acute lung injury (ALI), and its clinical correlate is acute respiratory distress syndrome-and it is as a result of interactions between alveolar type II cells (ATII) and alveolar macrophages (AM). In the setting of acute injury, the microenvironment of the intra-alveolar space is determined in part by metabolites and cytokines and is known to shape the AM phenotype. In response to ALI, increased glycolysis is observed in AT II cells, mediated by the transcription factor hypoxia-inducible factor (HIF) 1α, which has been shown to decrease inflammation. We hypothesized that in acute lung injury, lactate, the end product of glycolysis, produced by ATII cells shifts AMs toward an anti-inflammatory phenotype, thus mitigating ALI. We found that local intratracheal delivery of lactate improved ALI in two different mouse models. Lactate shifted cytokine expression of murine AMs toward increased IL-10, while decreasing IL-1 and IL-6 expression. Mice with ATII-specific deletion of Hif1a and mice treated with an inhibitor of lactate dehydrogenase displayed exacerbated ALI and increased inflammation with decreased levels of lactate in the bronchoalveolar lavage fluid; however, all those parameters improved with intratracheal lactate. When exposed to LPS (to recapitulate an inflammatory stimulus as it occurs in ALI), human primary AMs co-cultured with alveolar epithelial cells had reduced inflammatory responses. Taken together, these studies reveal an innate protective pathway, in which lactate produced by ATII cells shifts AMs toward an anti-inflammatory phenotype and dampens excessive inflammation in ALI.

Release of extracellular superoxide dismutase into alveolar fluid protects against acute lung injury and inflammation in Staphylococcus aureus pneumonia.

Acute respiratory distress syndrome (ARDS) remains a significant cause of morbidity and mortality in critically ill patients. Oxidative stress and inflammation play a crucial role in the pathogenesis of ARDS. Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and is an important enzymatic defense against superoxide. Human single-nucleotide polymorphism in matrix binding region of EC-SOD leads to the substitution of arginine to glycine at position 213 (R213G) and results in release of EC-SOD into alveolar fluid, without affecting enzyme activity. We hypothesized that R213G EC-SOD variant protects against lung injury and inflammation via the blockade of neutrophil recruitment in infectious model of methicillin-resistant S. aureus (MRSA) pneumonia. After inoculation with MRSA, wild-type (WT) mice had impaired integrity of alveolar-capillary barrier and increased levels of IL-1ß, IL-6, and TNF-α in the broncho-alveolar lavage fluid (BALF), while infected mice expressing R213G EC-SOD variant maintained the integrity of alveolar-capillary interface and had attenuated levels of proinflammatory cytokines. MRSA-infected mice expressing R213G EC-SOD variant also had attenuated neutrophil numbers in BALF and decreased expression of neutrophil chemoattractant CXCL1 by the alveolar epithelial ATII cells, compared with the infected WT group. The decreased neutrophil numbers in R213G mice were not due to increased rate of apoptosis. Mice expressing R213G variant had a differential effect on neutrophil functionality-the generation of neutrophil extracellular traps (NETs) but not myeloperoxidase (MPO) levels were attenuated in comparison with WT controls. Despite having the same bacterial load in the lung as WT controls, mice expressing R213G EC-SOD variant were protected from extrapulmonary dissemination of bacteria.

Intense light-elicited alveolar type 2-specific circadian PER2 protects from bacterial lung injury via BPIFB1.

Circadian amplitude enhancement has the potential to be organ protective but has not been studied in acute lung injury (ALI). Consistent light and dark cycles are crucial for the amplitude regulation of the circadian rhythm protein Period2 (PER2). Housing mice under intense instead of ambient light for 1 wk (light: dark cycle:14h:10h), we demonstrated a robust increase of pulmonary PER2 trough and peak levels, which is consistent with circadian amplitude enhancement. A search for the affected lung cell type suggested alveolar type 2 (ATII) cells as strong candidates for light induction of PER2. A head-to-head comparison of mice with cell-type-specific deletion of Per2 in ATII, endothelial, or myeloid cells uncovered a dramatic phenotype in mice with an ATII-specific deletion of Per2. During Pseudomonas aeruginosa-induced ALI, mice with Per2 deletion in ATII cells showed 0% survival, whereas 85% of control mice survived. Subsequent studies demonstrated that intense light therapy dampened lung inflammation or improved the alveolar barrier function during P. aeruginosa-induced ALI, which was abolished in mice with an ATII-specific deletion of Per2. A genome-wide mRNA array uncovered bactericidal/permeability-increasing fold-containing family B member 1 (BPIFB1) as a downstream target of intense light-elicited ATII-PER2 mediated lung protection. Using the flavonoid and PER2 amplitude enhancer nobiletin, we recapitulated the lung-protective and anti-inflammatory effects of light and BPIFB1, respectively. Together, our studies demonstrate that light-elicited amplitude enhancement of ATII-specific PER2 is a critical control point of inflammatory pathways during bacterial ALI.

Targeting alveolar-specific succinate dehydrogenase A attenuates pulmonary inflammation during acute lung injury.

Acute lung injury (ALI) is an inflammatory lung disease, which manifests itself in patients as acute respiratory distress syndrome (ARDS). Previous studies have implicated alveolar-epithelial succinate in ALI protection. Therefore, we hypothesized that targeting alveolar succinate dehydrogenase SDH A would result in elevated succinate levels and concomitant lung protection. Wild-type (WT) mice or transgenic mice with targeted alveolar-epithelial Sdha or hypoxia-inducible transcription factor Hif1a deletion were exposed to ALI induced by mechanical ventilation. Succinate metabolism was assessed in alveolar-epithelial via mass spectrometry as well as redox measurements and evaluation of lung injury. In WT mice, ALI induced by mechanical ventilation decreased SDHA activity and increased succinate in alveolar-epithelial. In vitro, cell-permeable succinate decreased epithelial inflammation during stretch injury. Mice with inducible alveolar-epithelial Sdha deletion (Sdhaloxp/loxp SPC-CreER mice) revealed reduced lung inflammation, improved alveolar barrier function, and attenuated histologic injury. Consistent with a functional role of succinate to stabilize HIF, Sdhaloxp/loxp SPC-CreER experienced enhanced Hif1a levels during hypoxia or ALI. Conversely, Hif1aloxp/loxp SPC-CreER showed increased inflammation with ALI induced by mechanical ventilation. Finally, wild-type mice treated with intra-tracheal dimethlysuccinate were protected during ALI. These data suggest that targeting alveolar-epithelial SDHA dampens ALI via succinate-mediated stabilization of HIF1A. Translational extensions of our studies implicate succinate treatment in attenuating alveolar inflammation in patients suffering from ARDS.

Endothelial cell PHD2-HIF1α-PFKFB3 contributes to right ventricle vascular adaptation in pulmonary hypertension.

Humans and animals with pulmonary hypertension (PH) show right ventricular (RV) capillary growth, which positively correlates with overall RV hypertrophy. However, molecular drivers of RV vascular augmentation in PH are unknown. Prolyl hydroxylase (PHD2) is a regulator of hypoxia-inducible factors (HIFs), which transcriptionally activates several proangiogenic genes, including the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We hypothesized that a signaling axis of PHD2-HIF1α-PFKFB3 contributes to adaptive coupling between the RV vasculature and tissue volume to maintain appropriate vascular density in PH. We used design-based stereology to analyze endothelial cell (EC) proliferation and the absolute length of the vascular network in the RV free wall, relative to the tissue volume in mice challenged with hypoxic PH. We observed increased RV EC proliferation starting after 6 h of hypoxia challenge. Using parabiotic mice, we found no evidence for a contribution of circulating EC precursors to the RV vascular network. Mice with transgenic deletion or pharmacological inhibition of PHD2, HIF1α, or PFKFB3 all had evidence of impaired RV vascular adaptation following hypoxia PH challenge. PHD2-HIF1α-PFKFB3 contributes to structural coupling between the RV vascular length and tissue volume in hypoxic mice, consistent with homeostatic mechanisms that maintain appropriate vascular density. Activating this pathway could help augment the RV vasculature and preserve RV substrate delivery in PH, as an approach to promote RV function.

MicroRNA dysregulation in lung injury: the role of the miR-26a/EphA2 axis in regulation of endothelial permeability.

MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression in many diseases, although the contribution of miRNAs to the pathophysiology of lung injury remains obscure. We hypothesized that dysregulation of miRNA expression drives the changes in key genes implicated in the development of lung injury. To test our hypothesis, we utilized a model of lung injury induced early after administration of intratracheal bleomycin (0.1 U). Wild-type mice were treated with bleomycin or PBS, and lungs were collected at 4 or 7 days. A profile of lung miRNA was determined by miRNA array and confirmed by quantitative PCR and flow cytometry. Lung miR-26a was significantly decreased 7 days after bleomycin injury, and, on the basis of enrichment of predicted gene targets, it was identified as a putative regulator of cell adhesion, including the gene targets EphA2, KDR, and ROCK1, important in altered barrier function. Lung EphA2 mRNA, and protein increased in the bleomycin-injured lung. We further explored the miR-26a/EphA2 axis in vitro using human lung microvascular endothelial cells (HMVEC-L). Cells were transfected with miR-26a mimic and inhibitor, and expression of gene targets and permeability was measured. miR-26a regulated expression of EphA2 but not KDR or ROCK1. Additionally, miR-26a inhibition increased HMVEC-L permeability, and the disrupted barrier integrity due to miR-26a was blocked by EphA2 knockdown, shown by VE-cadherin staining. Our data suggest that miR-26a is an important epigenetic regulator of EphA2 expression in the pulmonary endothelium. As such, miR-26a may represent a novel therapeutic target in lung injury by mitigating EphA2-mediated changes in permeability.

Restoration of Megalin-Mediated Clearance of Alveolar Protein as a Novel Therapeutic Approach for Acute Lung Injury.

Acute respiratory distress syndrome constitutes a significant disease burden with regard to both morbidity and mortality. Current therapies are mostly supportive and do not address the underlying pathophysiologic mechanisms. Removal of protein-rich alveolar edema-a clinical hallmark of acute respiratory distress syndrome-is critical for survival. Here, we describe a transforming growth factor (TGF)-ß-triggered mechanism, in which megalin, the primary mediator of alveolar protein transport, is negatively regulated by glycogen synthase kinase (GSK) 3ß, with protein phosphatase 1 and nuclear inhibitor of protein phosphatase 1 being involved in the signaling cascade. Inhibition of GSK3ß rescued transepithelial protein clearance in primary alveolar epithelial cells after TGF-ß treatment. Moreover, in a bleomycin-based model of acute lung injury, megalin+/- animals (the megalin-/- variant is lethal due to postnatal respiratory failure) showed a marked increase in intra-alveolar protein and more severe lung injury compared with wild-type littermates. In contrast, wild-type mice treated with the clinically relevant GSK3ß inhibitors, tideglusib and valproate, exhibited significantly decreased alveolar protein concentrations, which was associated with improved lung function and histopathology. Together, we discovered that the TGF-ß-GSK3ß-megalin axis is centrally involved in disturbances of alveolar protein clearance in acute lung injury and provide preclinical evidence for therapeutic efficacy of GSK3ß inhibition.

TGF-ß inhibits alveolar protein transport by promoting shedding, regulated intramembrane proteolysis, and transcriptional downregulation of megalin.

Disruption of the alveolar-capillary barrier is a hallmark of acute respiratory distress syndrome (ARDS) that leads to the accumulation of protein-rich edema in the alveolar space, often resulting in comparable protein concentrations in alveolar edema and plasma and causing deleterious remodeling. Patients who survive ARDS have approximately three times lower protein concentrations in the alveolar edema than nonsurvivors; thus the ability to remove excess protein from the alveolar space may be critical for a positive outcome. We have recently shown that clearance of albumin from the alveolar space is mediated by megalin, a 600-kDa transmembrane endocytic receptor and member of the low-density lipoprotein receptor superfamily. In the currents study, we investigate the molecular mechanisms by which transforming growth factor-ß (TGF-ß), a key molecule of ARDS pathogenesis, drives downregulation of megalin expression and function. TGF-ß treatment led to shedding and regulated intramembrane proteolysis of megalin at the cell surface and to a subsequent increase in intracellular megalin COOH-terminal fragment abundance resulting in transcriptional downregulation of megalin. Activity of classical protein kinase C enzymes and γ-secretase was required for the TGF-ß-induced megalin downregulation. Furthermore, TGF-ß-induced shedding of megalin was mediated by matrix metalloproteinases (MMPs)-2, -9, and -14. Silencing of either of these MMPs stabilized megalin at the cell surface after TGF-ß treatment and restored normal albumin transport. Moreover, a direct interaction of megalin with MMP-2 and -14 was demonstrated, suggesting that these MMPs may function as novel sheddases of megalin. Further understanding of these mechanisms may lead to novel therapeutic approaches for the treatment of ARDS.

Acute respiratory distress syndrome following cardiovascular surgery: current concepts and novel therapeutic approaches.

PURPOSE OF REVIEW: This review gives an update on current treatment options and novel concepts on the prevention and treatment of the acute respiratory distress syndrome (ARDS) in cardiovascular surgery patients. RECENT FINDINGS: The only proven beneficial therapeutic options in ARDS are those that help to prevent further ventilator-induced lung injury, such as prone position, use of lung-protective ventilation strategies, and extracorporeal membrane oxygenation. In the future also new approaches like mesenchymal cell therapy, activation of hypoxia-elicited transcription factors or targeting of purinergic signaling may be successful outside the experimental setting. Owing to the so far limited treatment options, it is of great importance to determine patients at risk for developing ARDS already perioperatively. In this context, serum biomarkers and lung injury prediction scores could be useful. SUMMARY: Preventing ARDS as a severe complication in the cardiovascular surgery setting may help to reduce morbidity and mortality. As cardiovascular surgery patients are of greater risk to develop ARDS, preventive interventions should be implemented early on. Especially, use of low tidal volumes, avoiding of fluid overload and restrictive blood transfusion regimes may help to prevent ARDS.

Hypercapnia impairs lung neutrophil function and increases mortality in murine pseudomonas pneumonia.

Hypercapnia, an elevation of the level of carbon dioxide (CO2) in blood and tissues, is a marker of poor prognosis in chronic obstructive pulmonary disease and other pulmonary disorders. We previously reported that hypercapnia inhibits the expression of TNF and IL-6 and phagocytosis in macrophages in vitro. In the present study, we determined the effects of normoxic hypercapnia (10% CO2, 21% O2, and 69% N2) on outcomes of Pseudomonas aeruginosa pneumonia in BALB/c mice and on pulmonary neutrophil function. We found that the mortality of P. aeruginosa pneumonia was increased in 10% CO2-exposed compared with air-exposed mice. Hypercapnia increased pneumonia mortality similarly in mice with acute and chronic respiratory acidosis, indicating an effect unrelated to the degree of acidosis. Exposure to 10% CO2 increased the burden of P. aeruginosa in the lungs, spleen, and liver, but did not alter lung injury attributable to pneumonia. Hypercapnia did not reduce pulmonary neutrophil recruitment during infection, but alveolar neutrophils from 10% CO2-exposed mice phagocytosed fewer bacteria and produced less H2O2 than neutrophils from air-exposed mice. Secretion of IL-6 and TNF in the lungs of 10% CO2-exposed mice was decreased 7 hours, but not 15 hours, after the onset of pneumonia, indicating that hypercapnia inhibited the early cytokine response to infection. The increase in pneumonia mortality caused by elevated CO2 was reversible when hypercapnic mice were returned to breathing air before or immediately after infection. These results suggest that hypercapnia may increase the susceptibility to and/or worsen the outcome of lung infections in patients with severe lung disease.

Efficient gene delivery to primary alveolar epithelial cells by nucleofection.

Primary alveolar epithelial cells play a pivotal role in lung research, particularly when focusing on gas exchange, barrier function, and transepithelial transport processes. However, efficient transfection of primary alveolar epithelial cells continues to be a major challenge. In the present study, we applied nucleofection, a novel method of gene and oligonucleotide delivery to the nucleus of cells by electroporation, to achieve highly efficient transfection of primary alveolar epithelial type II (ATII) cells. To quantify the amount of ATII cells effectively transfected, we applied a plasmid expressing GFP and assessed the amount of GFP-expressing cells by flow cytometry. Analysis of the nucleofected ATII cells revealed a concentration-dependent transfection efficiency of up to 50% when using 3-8 µg plasmid DNA without affecting cell viability. Nucleofection of cultured A549 and H441 cells yielded similar transfection rates. Importantly, nucleofection of ATII cells did not interfere with the integrity of ATII monolayers even with use of relatively high concentrations of plasmid DNA. In subsequent studies, we also efficiently delivered small interfering RNAs to ATII cells by nucleofection, thereby silencing Akt and the multiligand receptor megalin, which has been recently shown to play a key role in removal of excess protein from the alveolar space, and effectively inhibited megalin-driven uptake and transcellular transport of albumin in ATII cells. Thus we report successful transfection of primary rat alveolar epithelial cells with both plasmids and oligonucleotides via nucleofection with high viability and consistently good transfection rates without impairing key physiological properties of the cells.

CNP-miR146a Decreases Inflammation in Murine Acute Infectious Lung Injury.

Acute respiratory distress syndrome (ARDS) has approximately 40% in-hospital mortality, and treatment is limited to supportive care. Pneumonia is the underlying etiology in many cases with unrestrained inflammation central to the pathophysiology. We have previously shown that CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA(miR)-146a, reduces bleomycin- and endotoxin-induced acute lung injury (ALI) by decreasing inflammation. We therefore hypothesized that CNP-miR146a would decrease inflammation in murine infectious ALI. Mice were injured with intratracheal (IT) MRSA or saline followed by treatment with IT CNP-miR146a or saline control. Twenty-four hours post-infection, bronchoalveolar lavage fluid (BALF) and whole lungs were analyzed for various markers of inflammation. Compared to controls, MRSA infection significantly increased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1ß; p < 0.05), BALF proinflammatory cytokines (IL-6, IL-8, TNFα, IL-1ß; p < 0.01), and inflammatory cell infiltrate (p = 0.03). CNP-miR146a treatment significantly decreased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1ß; p < 0.05), bronchoalveolar proinflammatory protein leak (IL-6, IL-8, TNFα; p < 0.05), and inflammatory infiltrate (p = 0.01). CNP-miR146a decreases inflammation and improves alveolar-capillary barrier integrity in the MRSA-infected lung and has significant promise as a potential therapeutic for ARDS.

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.

Elevated CO(2) levels cause mitochondrial dysfunction and impair cell proliferation.

Elevated CO(2) concentrations (hypercapnia) occur in patients with severe lung diseases. Here, we provide evidence that high CO(2) levels decrease O(2) consumption and ATP production and impair cell proliferation independently of acidosis and hypoxia in fibroblasts (N12) and alveolar epithelial cells (A549). Cells exposed to elevated CO(2) died in galactose medium as well as when glucose-6-phosphate isomerase was knocked down, suggesting mitochondrial dysfunction. High CO(2) levels led to increased levels of microRNA-183 (miR-183), which in turn decreased expression of IDH2 (isocitrate dehydrogenase 2). The high CO(2)-induced decrease in cell proliferation was rescued by α-ketoglutarate and overexpression of IDH2, whereas proliferation decreased in normocapnic cells transfected with siRNA for IDH2. Also, overexpression of miR-183 decreased IDH2 (mRNA and protein) as well as cell proliferation under normocapnic conditions, whereas inhibition of miR-183 rescued the normal proliferation phenotype in cells exposed to elevated levels of CO(2). Accordingly, we provide evidence that high CO(2) induces miR-183, which down-regulates IDH2, thus impairing mitochondrial function and cell proliferation. These results are of relevance to patients with hypercapnia such as those with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchopulmonary dysplasia, and muscular dystrophies.

Plasma microRNA and metabolic changes associated with pediatric acute respiratory distress syndrome: a prospective cohort study.

Acute respiratory distress syndrome is a heterogeneous pathophysiological process responsible for significant morbidity and mortality in pediatric intensive care patients. Diagnosis is defined by clinical characteristics that identify the syndrome after development. Subphenotyping patients at risk of progression to ARDS could provide the opportunity for therapeutic intervention. microRNAs, non-coding RNAs stable in circulation, are a promising biomarker candidate. We conducted a single-center prospective cohort study to evaluate random forest classification of microarray-quantified circulating microRNAs in critically ill pediatric patients. We additionally selected a sub-cohort for parallel metabolomics profiling as a pilot study for concurrent use of miRNAs and metabolites as circulating biomarkers. In 35 patients (n = 21 acute respiratory distress, n = 14 control) 15 microRNAs were differentially expressed. Unsupervised random forest classification accurately grouped ARDS and control patients with an area under the curve of 0.762, which was improved to 0.839 when subset to only patients with bacterial infection. Nine metabolites were differentially abundant between acute respiratory distress and control patients (n = 4, both groups) and abundance was highly correlated with miRNA expression. Random forest classification of microRNAs differentiated critically ill pediatric patients who developed acute respiratory distress relative to those who do not. The differential expression of microRNAs and metabolites provides a strong foundation for further work to validate their use as a prognostic biomarker.

HIF1A-dependent induction of alveolar epithelial PFKFB3 dampens acute lung injury.

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.

Ubiquitination participates in the lysosomal degradation of Na,K-ATPase in steady-state conditions.

The alveolar epithelial cell (AEC) Na,K-ATPase contributes to vectorial Na(+) transport and plays an important role in keeping the lungs free of edema. We determined, by cell surface labeling with biotin and immunofluorescence, that approximately 30% of total Na,K-ATPase is at the plasma membrane of AEC in steady-state conditions. The half-life of the plasma membrane Na,K-ATPase was about 4 hours, and the incorporation of new Na,K-ATPase to the plasma membrane was Brefeldin A sensitive. Both protein kinase C (PKC) inhibition with bisindolylmaleimide (10 microM) and infection with an adenovirus expressing dominant-negative PKCzeta prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPase alpha1-subunit lacking the PKC phosphorylation sites, the plasma membrane Na,K-ATPase had a moderate increase in half-life. We also found that the Na,K-ATPase was ubiquitinated in steady-state conditions and that proteasomal inhibitors prevented its degradation. Interestingly, mutation of the four lysines described to be necessary for ubiquitination and endocytosis of the Na,K-ATPase in injurious conditions did not have an effect on its half-life in steady-state conditions. Lysosomal inhibitors prevented Na,K-ATPase degradation, and co-localization of Na,K-ATPase and lysosomes was found after labeling and chasing the plasma membrane Na,K-ATPase for 4 hours. Accordingly, we provide evidence suggesting that phosphorylation and ubiquitination are necessary for the steady-state degradation of the plasma membrane Na,K-ATPase in the lysosomes in alveolar epithelial cells.