Hypercapnia decreases Na,K-ATPase plasma membrane abundance by impairing endoplasmic reticulum maturation of its beta-subunit in alveolar epithelial cells
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.
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