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A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31−CD45−tdTomatoSPC−T1α+ for ATI and EpCAM+CD31−CD45−tdTomatoSPC+T1α− for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.

Protein transcription, translation, and folding occur continuously in every living cell and are essential for physiological functions. About one-third of all proteins of the cellular proteome interacts with the endoplasmic reticulum (ER). The ER is a large, dynamic cellular organelle that orchestrates synthesis, folding, and structural maturation of proteins, regulation of lipid metabolism and additionally functions as a calcium store. Recent evidence suggests that both acute and chronic hypercapnia (elevated levels of CO2) impair ER function by different mechanisms, leading to adaptive and maladaptive regulation of protein folding and maturation. In order to cope with ER stress, cells activate unfolded protein response (UPR) pathways. Initially, during the adaptive phase of ER stress, the UPR mainly functions to restore ER protein-folding homeostasis by decreasing protein synthesis and translation and by activation of ER-associated degradation (ERAD) and autophagy. However, if the initial UPR attempts for alleviating ER stress fail, a maladaptive response is triggered. In this review, we discuss the distinct mechanisms by which elevated CO2 levels affect these molecular pathways in the setting of acute and chronic pulmonary diseases associated with hypercapnia.

Several acute and chronic lung diseases are associated with alveolar hypoventilation leading to accumulation of CO2 (hypercapnia). The β-subunit of the Na,K-ATPase plays a pivotal role in maintaining epithelial integrity by functioning as a cell adhesion molecule and regulating cell surface stability of the catalytic α-subunit of the transporter, thereby, maintaining optimal alveolar fluid balance. Here, we identified the E3 ubiquitin ligase for the Na,K-ATPase β-subunit, which promoted polyubiquitination, subsequent endocytosis and proteasomal degradation of the protein upon exposure of alveolar epithelial cells to elevated CO2 levels, thus impairing alveolar integrity. Ubiquitination of the Na,K-ATPase β-subunit required lysine 5 and 7 and mutating these residues (but not other lysines) prevented trafficking of Na,K-ATPase from the plasma membrane and stabilized the protein upon hypercapnia. Furthermore, ubiquitination of the Na,K-ATPase β-subunit was dependent on prior phosphorylation at serine 11 by protein kinase C (PKC)-ζ. Using a protein microarray, we identified the tumor necrosis factor receptor-associated factor 2 (TRAF2) as the E3 ligase driving ubiquitination of the Na,K-ATPase β-subunit upon hypercapnia. Of note, prevention of Na,K-ATPase β-subunit ubiquitination was necessary and sufficient to restore the formation of cell-cell junctions under hypercapnic conditions. These results suggest that a hypercapnic environment in the lung may lead to persistent epithelial dysfunction in affected patients. As such, the identification of the E3 ligase for the Na,K-ATPase may provide a novel therapeutic target, to be employed in patients with acute or chronic hypercapnic respiratory failure, aiming to restore alveolar epithelial integrity.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO2 levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO2 concentrations caused a significant retention of NKA-&beta, in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-&alpha, /&beta, complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-&beta, subunits were only partially assembled with NKA &alpha, subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO2 levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-&beta, oxidation. Treatment with &alpha, ketoglutaric acid (&alpha, KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO2 concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO2 levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.

The main hallmarks of acute respiratory distress syndrome (ARDS) are impaired gas exchange and alveolar edema, which are often associated with elevated levels of CO2 (hypercapnia) due to the disruption of the alveolar-capillary barrier and in part as a consequence of lung-protective mechanical ventilation by using low tidal volumes. Both decreased alveolar fluid clearance (AFC) and hypercapnia have been shown to be associated with worse outcomes in patients with ARDS. The resolution of alveolar edema directly correlates with AFC, which is driven by a vectorial Na+ transport, mediated by the coordinated action of the apically-localized epithelial Na+ channel (ENaC) on the apical and the Na,K-ATPase (NKA) on the basolateral side. The endoplasmic reticulum (ER) is the main organelle that is involved in the proper maturation of glycoproteins. NKA is a heterodimeric glycoprotein that in order to be delivered to the plasma membrane must be assembled in the ER. A disturbance in the ER maturation may result in a decreased plasma membrane abundance of the transporter and an impaired alveolar fluid clearance. Here, we provide evidence that hypercapnia (pCO2=120 mmHg; pHe=7.4) decreases the NKA plasma membrane abundance by affecting the ER folding of the beta-subunit of the enzyme in alveolar epithelial cells. We found that the short-term exposure of cells to elevated CO2 levels (up to 1 hour) results in depletion of the ER Ca2+ stores by a leakage through 1,4,5-triphosphate receptors (IP3R). The rapid activation of serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1alpha (IRE1alpha) triggers ER-associated degradation (ERAD) of the NKA beta-subunit, which subsequently decreases the cell surface expression of the transporter. The inhibition of Ca2+ release through IP3R receptors stabilizes the levels of the ER-resident NKA-beta and increases the plasma membrane abundance of the enzyme. In contrast, long-term hypercapnia (up to 72 hours) promotes significant retention of the NKA beta-subunit in the ER. This is followed by increased protein oxidation in the ER and the disruption of the Na,K-ATPase alpha/beta-complex formation. Furthermore, disturbances in ER homeostasis activate the adaptive unfolded protein response (UPR) by increasing the phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) by protein kinase R-like endoplasmic reticulum kinase (PERK). Moreover, we demonstrate that administration of alpha-ketoglutaric acid to hypercapnia-exposed cells prevents ER protein oxidation and restores plasma membrane abundance of the Na,K-ATPase. Taken together, short- and long-term exposure to elevated CO2 levels result in misfolding of the Na,K-ATPase beta-subunit in the ER and decrease the plasma membrane expression of the Na,K-ATPase alpha/beta-complex, which impairs alveolar fluid clearance. Understanding the mechanisms of hypercapnic respiratory failure may provide new approaches in the treatment of patients with ARDS. Die Hauptmerkmale des akuten Atemnotsyndroms (ARDS) umfassen ein Lungenödem und einen gestörten Gasaustausch, die häufig mit einem erhöhten CO2-Gehalt (Hyperkapnie), aufgrund einer Störung der alveolo-kapillären Barriere, und einer lungenschützenden mechanischen Beatmung, mithilfe eines niedrigen Tidalvolumen, verbunden sind. Es zeigte sich, dass das Zusammenspiel von Hyperkapnie und einer verminderten Clearance der Alveolarflüssigkeit (AFC) zu einem schlechteren Krankheitsverlauf bei ARDS-Patienten führt. Das Ausschwämmen des Lungenödems korreliert direkt mit der AFC, welche durch einen zielgerichteten vektoriellen Na+-Transport, einerseits durch den apikal lokalisierten epithelialen Na+-Kanals (ENaC) und anderseits durch die basolateral lokalisierten Na,K-ATPase (NKA) bestimmt wird. Das endoplasmatische Retikulum (ER) ist die Hauptorganelle, die an der ordnungsgemäßen Reifung von Glykoproteinen beteiligt ist. NKA ist ein heterodimeres Glykoprotein, dessen Untereinheiten im ER aufgebaut und danach von der Plasmamembran freigegeben wird. Eine Störung im Reifungsprozess des ER kann zu einer verringerten Transporterfreisetzung führen, was eine beeinträchtigte Alveolarflüssigkeitsclearance nach sich ziehen könnte. Unsere vorliegenden Daten belegen, dass die Hyperkapnie (pCO2=120 mmHg; pHe=7.4) den Zusammenbau einer Enzym-beta-Untereinheit im ER von Alveolarepithelzellen beeinflusst, was die NKA-Freisetzung an den Plasmamembranen verringert. Es zeigte sich, dass die kurzfristige Erhöhung des CO2-Spiegels (bis zu 1 Stunde) zu einer Erschöpfung des Ca2+-Speichers führte, was wiederum in einem Verlust der IP3R-Rezeptoren und der raschen Aktivierung von IRE1alpha im ER resultierte. Anschließend löste dies den ER-assoziierten Abbau (ERAD) der NKA-beta-Untereinheit aus und verringerte die Transporterexpression an der Zellöberflache. Eine Hemmung der Ca2+-Freisetzung durch die IP3R-Rezeptoren stabilisierte die Anzahl von ER-residenten NKA-beta und erhöhte die Emission des Enzyms an der Plasmamembran. Dagegen führte eine langfristige Hyperkapnie (bis zu 72 Stunden) signifikant zu einer verminderten Freisetzung der NKA-beta-Untereinheit im ER, gefolgt von einer erhöhten Oxidation des ER-Proteins und einer Bildungsstörung des Transporter-alpha:beta-Komplexes. Darüber hinaus kam es zu einer adaptiv entwickelten Proteinreaktion durch eine aktivierte Störung der ER-Homöostase, indem die Phosphorylierung durch PERK von eIF2alpha erhöht wurde. Zusätzlich konnten wir zeigen, dass durch die Zugabe von alpha-Ketoglutarsäure zu den Hyperkanie-exponierten Zellen die Proteinoxidation im ER und die Freisetzung der Na, K-ATPase wiederhergestellt werden konnte. Zusammenfassend führt eine kurz- und langfristige Exposition mit erhöhten CO2-Spiegeln zu einer Fehlbildung der beta-Untereinheit im ER und einer verminderten Expression der Na, K-ATPase an der Plasmamembran, was zu einer verschlechterten Clearance der Alveolarflüssigkeit führen kann. Diese neu erworbenen Kenntnisse über die Mechanismen der durch Hyperkapnie induzierten respiratorischen Insuffizienz, könnten neue Ansätze in der Pathophysiologie und in der Behandlung von Patienten mit ARDS liefern.