**Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the** β**-Subunit of the Enzyme in Alveolar Epithelial Cells**

**Vitalii Kryvenko 1,2, Miriam Wessendorf 1, Rory E. Morty 1,2,3, Susanne Herold 1,2, Werner Seeger 1,2,3, Olga Vagin 4,5, Laura A. Dada 6, Jacob I. Sznajder <sup>6</sup> and István Vadász 1,2,\***


Received: 3 February 2020; Accepted: 17 February 2020; Published: 21 February 2020

**Abstract:** 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-β 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-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-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-β oxidation. Treatment with α-ketoglutaric acid (α-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.

**Keywords:** carbon dioxide; hypercapnia; Na,K-ATPase; endoplasmic reticulum; sodium transport; protein oxidation; alveolar epithelium

#### **1. Introduction**

Na,K-ATPase (NKA) is a heterodimeric enzyme and a member of the P-type ATPase family. NKA is located at the basolateral plasma membrane (PM) of polarized cells, where the primary function of the enzyme is to extrude three sodium ions while taking up two potassium ions per pump cycle in an ATP-dependent manner [1,2]. A functional NKA requires a catalytic α-subunit and a regulatory β-subunit [2]. Additionally, a γ-subunit has also been identified, which represents a family

of single-span transmembrane proteins containing the FXYD motif that is not an integral part of the transporter but rather regulates the activity and membrane abundance of the enzyme [3,4]. In the alveolar epithelium of the lung, the activity of NKA creates an Na<sup>+</sup> gradient that drives reabsorption of fluid from the alveolar space, which keeps the alveoli relatively "dry," which is essential for an effective gas exchange. The catalytic α-subunit of the transporter contains the binding sites for Na+, K+, and ATP [2]. The NKA β-subunit, which is a type II membrane glycoprotein, has a pivotal role in delivery and appropriate insertion of the NKA-α subunit in the PM [1]. Remarkably, mice deficient in the NKA-β subunit in alveolar epithelial cells have reduced alveolar fluid clearance, which results in aggravation of acute lung injury (ALI) and further underlies the pivotal role of NKA-β in the overall transporter function [5]. Additionally, numerous reports have shown that the function of NKA-β is not limited to regulation of NKA-α, but is centrally involved in establishing epithelial cell polarity, formation of adherens junctions, and regulation of paracellular permeability, which are key for maintaining a functional epithelial barrier [6–10].

Carbon dioxide (CO2) is a byproduct of mitochondrial respiration and cellular metabolism. Excess of CO2, in mammals, is eliminated by the lungs under physiological conditions [11,12]. Thus, any condition that leads to alveolar hypoventilation or impairs diffusion of CO2 across the alveolar-capillary barrier results in retention of CO2 in the blood, which is termed hypercapnia. During ALI and in patients with acute respiratory distress syndrome (ARDS), disruption of the alveolar-capillary barrier, and, thus, accumulation of edema fluid in the interstitial and alveolar spaces, may result in hypercapnia. Moreover, hypercapnia is often further potentiated or even directly caused by protective ventilation strategies with low tidal volumes to limit further lung damage [12,13]. Remarkably, hypercapnia was found to decrease alveolar fluid clearance and resolution of alveolar edema by decreasing the PM abundance of the Na,K-ATPase [14–16]. Since ALI is often associated with hypercapnia and is characterized by alveolar edema and disruption of epithelial junctions [17], further understanding of the mechanisms impairing the NKA function and of the potential rescue mechanisms might be of critical importance promoting the resolution of epithelial injury, alveolar repair processes, and edema resolution.

About one-third of all cellular proteins interact with the endoplasmic reticulum (ER) during folding and maturation processes [18]. Of note, in the ER, the NKA-β undergoes various post-translational modifications, including glycosylation and assembly with NKA-α, before leaving the ER and, subsequently, being transferred to the PM [19,20]. Whether hypercapnia affects ER protein folding of NKA has not been previously investigated. In the current study, we explored how elevated CO2 levels influence the ER environment and expression, PM abundance, and function of the NKA-β subunit. Understanding the molecular mechanisms underlying the effects of hypercapnia on the folding and maturation of the NKA-β in the alveolar epithelium might provide new therapeutic strategies for the treatment of patients with ARDS and hypercapnia.

### **2. Results**

#### *2.1. Hypercapnia Increases the Endoplasmic Reticulum Fraction of the Na,K-ATPase* β*-Subunit*

The NKA-β subunit is a glycoprotein that undergoes posttranslational maturation processing in the ER and Golgi prior to delivery to the plasma membrane. After the initial step of ER folding, the addition of an oligosaccharide core results in the formation of a specific high mannose N-glycan type NKA-β, which resides exclusively in the ER [19]. To investigate whether hypercapnia affects cellular levels of NKA-β, we exposed alveolar epithelial cells (AEC) for up to 72 h to physiological or increased levels of CO2 and analyzed the protein expression pattern of NKA-β and NKA-α (Figure 1A).

We observed a transient and time-dependent increase in the abundance of ER-resident NKA-β, which reached a maximum at 12 h and lasted for at least 24 h upon hypercapnic exposure. Our subsequent experiments were performed after a 12-h hypercapnia exposure, where the highest ER-resident NKA-β abundance was observed. To demonstrate whether this effect was directly driven

by CO2 or the elevated CO2-associated acidosis, AEC were treated with increasing CO2 concentrations (up to 120 mmHg) with normal (pH = 7.4) or acidic (pH = 7.2) extracellular pH (Figure 1B). Of note, we observed that the acidic environment per se did not affect the levels of the ER-resident NKA-β. In addition, we did not find significant differences in the total protein levels of NKA-α and NKA-β subunits upon hypercapnic exposure for up to 12 h (Figure 1C,D).

**Figure 1.** Hypercapnia increases Na,K-ATPase (NKA)-β abundance in the endoplasmic reticulum (ER). (**A**) A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for the different time-points up to 72 h. Total cellular level of NKA-β was measured by immunoblotting. Representative immunoblots of NKA-β are shown. Bars represent ER-resident NKA-β/β-actin ratio. Values are expressed as mean ± SD (*n* = 3, \*\*\* *p* < 0.001). (**B**) A549 cells were treated with 40, 60, 80, and 120 mmHg of CO2 with an extracellular pH = 7.4 or to 40 mmHg CO2 with a pH = 7.2 for 12 h. NKA-β levels were measured by immunoblotting. Representative immunoblots of NKA-β are shown. Bars represent total NKA-β/β-actin ratio. Values are expressed as mean ± SD (*n* = 3, \* *p* < 0.05, \*\*\* *p* < 0.001). (**C**) A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for different time-points. Total cellular levels of NKA-α were measured by immunoblotting. Representative immunoblots of NKA-α are shown. Bars represent total NKA-α/β-actin ratio. Values are expressed as mean ± SD (*n* = 3). (**D**) A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) for different time-points. Total cellular levels of NKA-β were measured by immunoblotting. Representative immunoblots of NKA-β are shown. Bars represent total NKA-β/β-actin ratio. Values are expressed as mean ± SD (*n* = 3).
