Molecular mechanisms of Na,K-ATPase dysregulation driving alveolar epithelial barrier failure in severe COVID-19.

A significant number of patients with coronavirus disease 2019 (COVID-19) develop acute respiratory distress syndrome (ARDS) that is associated with a poor outcome. The molecular mechanisms driving failure of the alveolar barrier upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain incompletely understood. The Na,K-ATPase is an adhesion molecule and a plasma membrane transporter that is critically required for proper alveolar epithelial function by both promoting barrier integrity and resolution of excess alveolar fluid, thus enabling appropriate gas exchange. However, numerous SARS-CoV-2-mediated and COVID-19-related signals directly or indirectly impair the function of the Na,K-ATPase, thereby potentially contributing to disease progression. In this Perspective, we highlight some of the putative mechanisms of SARS-CoV-2-driven dysfunction of the Na,K-ATPase, focusing on expression, maturation, and trafficking of the transporter. A therapeutic mean to selectively inhibit the maladaptive signals that impair the Na,K-ATPase upon SARS-CoV-2 infection might be effective in reestablishing the alveolar epithelial barrier and promoting alveolar fluid clearance and thus advantageous in patients with COVID-19-associated ARDS.

INTRODUCTION

Coronavirus disease 2019 (COVID-19) is a potentially life-threatening condition that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Up to date, three types of coronaviruses have been identified that directly damage lungs: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 (1–3). Although the majority of COVID-19 cases, particularly those in young and before the infection healthy subjects, are mild or even asymptomatic, approximately 2% of the infected patients and 15% of those who require hospitalization develop acute respiratory distress syndrome (ARDS) (4–6). Given that as of March 2021 over 115 million SARS-CoV-2 infections have been confirmed worldwide, the number of patients with ARDS secondary to COVID-19 is enormously high. One of the consequences of COVID-19-associated ARDS is a prolonged stay at the intensive care unit (ICU), often due to major lung damage and slow resolution of injury (3, 7). During the past few months, extensive research has been focused on the potential mechanisms that may drive lung injury or block its resolution upon SARS-CoV-2 infection.

ARDS is characterized by an acute disruption of the alveolar-capillary barrier that leads to flooding of the alveolar space and thus, to marked impairment of gas exchange (8). It is well established that, if unresolved, pulmonary edema is associated with a poor outcome in this patient population (8, 9). In order to clear pulmonary edema effectively, it is essential that 1) a tight alveolar epithelial barrier is reestablished and that 2) a sodium gradient across the alveolar epithelium is created, which then drives reabsorption of water from the alveolar space. This Na+ gradient is primarily achieved by the activity of the sodium-potassium adenosine triphosphatase (Na,K-ATPase). The Na,K-ATPase is a heterodimeric transporter from the family of the P-type ATPases and is widely expressed in various tissues and organs. As a plasma membrane (PM) transporter, the Na,K-ATPase is localized at the basolateral side of polarized cells, where the primary function of the enzyme is an active exchange of sodium and potassium ions in an ATP-dependent manner, thus establishing sodium and potassium gradients (10). The transporter consists of a catalytic α-, a regulatory β-, and an axillary γ-subunit (11). To date, four α-subunits, three β-subunits, and seven FXYD subunits have been characterized, of which the Na,K-ATPase α1:β1 subunit combination is the most abundant one. The Na,K-ATPase isoforms vary in a cell- and tissue-specific manner, thus affecting the function of the enzyme (11). Importantly, the Na,K-ATPase is the only transporter that discards sodium from alveolar epithelial cells (12, 13). Subsequently, Na+ enters the cells through apically situated epithelial Na+ channels with various Na+ selectivity, including the highly selective ENaC channels (14, 15).

In addition to driving vectorial sodium transport in the lungs, the Na,K-ATPase acts as a cell adhesion and tight junction molecule in epithelial cells by forming adherence junctions between the extracellular domains of Na,K-ATPase-β molecules (16, 17) and regulates permeability of the tight junctions via phosphorylation of occludin (18). Thus, proper function of the Na,K-ATPase reduces formation of pulmonary edema by (re)establishing the alveolar-epithelial barrier and also drives resolution of the excess alveolar fluid. In contrast, decreased activity or reduced PM abundance of Na,K-ATPase leads to inability of the alveolar epithelium to reabsorb alveolar fluid and thus may result in progression of acute lung injury (13, 19).

Furthermore, the Na,K-ATPase has been found to act as signaling molecule by regulating intracellular Ca2+ concentrations (20), responding to oxidative stress (21, 22), controlling the actin cytoskeleton, cellular volume and motility, and interacting with cellular receptors, such as EGFR (22, 23), as well as participating in various signaling pathways including control of programmed cellular death by autosis (21, 24). Apart from the pulmonary alveolar epithelium, several other cell types of various tissues and organs utilize the Na+ gradient generated by the Na,K-ATPase to maintain their functions. For instance, the Na,K-ATPase plays an important role in heart muscle metabolism, endothelial and vascular function, regulating neurotransmitter reuptake in neurons, reabsorption of amino acids and glucose in kidneys, regulation of electrolyte balance, and maintaining blood pH and pressure (11, 25–27). It is now well established that patients with a SARS-CoV-2 infection and certain comorbidities, such as chronic lung disease, hypertension, diabetes, obesity, and chronic kidney disease have worse outcomes (28). Of note, it has been reported that cardiac muscle hypertrophy, chronic heart failure (27), diabetes (29), obesity (30), and acute and chronic renal failure (25, 31) are associated with aberrant Na,K-ATPase expression and function.

Therefore, it is plausible that Na,K-ATPase dysfunction secondary to SARS-CoV-2 infection contributes to alveolar epithelial barrier failure leading to further aggravation and persistence of lung injury. Deregulation of the Na,K-ATPase may also lead to extrapulmonary manifestations of COVID-19. On the following pages, we highlight direct and indirect mechanisms mediated by SARS-CoV-2 that may impair the Na,K-ATPase and thus cause disease progression.

DIRECT EFFECTS OF SARS-COV-2 INFECTION ON EXPRESSION, MATURATION, AND TRAFFICKING OF Na,K-ATPASE IN INFECTED CELLS

In general, viruses exploit for their own replication the transcriptional and translational machinery, the endoplasmic reticulum (ER), the Golgi apparatus, and endosomal and trafficking pathways of the infected host cells (32, 33). The Na,K-ATPase, similarly to other PM proteins, undergoes transcription, translation, and posttranslational modifications in the ER and Golgi before trafficking to the PM (34–36). A disruption of any of these critical maturation steps of the Na,K-ATPase may result in decreased PM abundance and thus activity of the enzyme. Of note, SARS-CoV-2 is not the only respiratory virus that negatively impacts the Na,K-ATPase in the lungs. For example, it has been previously shown that influenza virus infection impairs Na,K-ATPase activity and alveolar fluid clearance (AFC) in part by downregulating expression of the enzyme and also by targeting the Na,K-ATPase to the apical surface of the polarized alveolar epithelium, thus leading to dysfunction of the transporter (37–39).

Transcriptional and Translational Machinery

SARS-CoV-2 encodes 27 proteins, which participate in virus replication and packaging. These include four structural proteins: nucleocapsid (N), envelope (E), membrane (M), and glycosylated spike (S) proteins. Additionally, there are 16 nonstructural proteins (NSP1–NSP16) and seven accessory proteins (ORF3a–ORF8) (3, 40). Accumulating evidence suggests that coronaviruses, including SARS-CoV-2 affect the transcriptional and translational processing in infected cells. Recent reports establish that SARS-CoV-2 NSPs directly interfere with the cellular mRNA and miRNA machinery. For example, it has been shown that NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs, thus suppressing global mRNA splicing (41). Additionally, in SARS-CoV infected cells, NSP1 has been found to suppress host gene expression through RNA degradation (42). Moreover, recent evidence suggests that SARS-CoV-2 may also affect the mRNA by depletion of host-specific miRNAs (43). Transcriptional regulation of the Na,K-ATPase is complex, subunit-specific, and involves various transcription factors, as well as epigenetic modifications (44). For example, the Na,K-ATPase α1-subunit contains two mineralocorticoid/glucocorticoid response element (MRE/GRE)-binding sites, the potential bindings regions for activator protein-1 (AP-1), AP-2, AP-3, and SP1. In addition to MRE/GRE and SP1-binding sites, the Na,K-ATPase β1-subunit contains prostaglandin-, hypoxia-, thyroid-, calcium-, and serum-response elements (44). Of note, it has been previously demonstrated that both SARS-CoV and SARS-CoV-2 alter the activity of AP-1 (45, 46) and SP1 transcriptional pathways (47). Moreover, two recent publications demonstrated that infection of various lung epithelial cells with SARS-CoV-2 leads to decreased expression of the α1-, α2-, α3-, β1-, and β3-subunits of the Na,K-ATPase at the transcriptional level (48, 49). Similar results were obtained in another study, which documented reduced gene expression of the Na,K-ATPase β1-subunit upon infection of human lung epithelial cells with SARS-CoV-2 (50). In addition, differential gene expression analysis of postmortem lung tissue samples from patients with COVID-19 revealed significant downregulation of gene expression of the Na,K-ATPase α1-subunit (48). Furthermore, an analysis of the proteome of a SARS-CoV-2-infected epithelial cell line showed a significant alteration of the expression of Na,K-ATPase α1-, β1-, and β3-subunits at the protein level (51). In terms of translation, SARS-CoV-2 NSP1 has been found to directly bind to the ribosomal 18S and 40S molecules resulting in global shutdown of mRNA translational in the infected cells (41, 52). These mechanisms may also play a central role in downregulation of Na,K-ATPase expression in infected respiratory cells.

Protein Maturation and Trafficking

Protein folding in the ER is regulated by the coordinated action of specific ER-resident chaperons, such as calnexin, calreticulin, and binding immunoglobulin protein (BiP, also known as GRP78) (53). It has been shown that nascent unfolded or partially folded Na,K-ATPase subunits are bound to calnexin and BiP (54), and only assembled Na,K-ATPase α:β complexes can leave the ER and proceed for the subsequent maturation in the Golgi (36). In addition, the Na,K-ATPase subunits undergo N-glycosylation in the ER (35), which are of critical importance for proper delivery of the transporter the PM (55). Of note, a decreased interaction of the Na,K-ATPase subunits with BiP or mutations in the α:β interacting regions result in a reduced PM abundance of the transporter (34, 36, 54). It has been demonstrated that coronaviruses substantially utilize secretory and protein control pathways in the ER and Golgi of the host cells during their replication (56). It has been shown that the spike protein of SARS-CoV-2 and particularly its receptor-binding domain are extensively glycosylated and that the N- and O-glycans of the S protein play a central role in viral entry into cells (57). Notably, the S proteins of the SARS-CoV and MERS-CoV viruses utilize both calnexin and BiP (58, 59) for virus replication and have N-glycosylation sites, suggesting N-glycan viral processing (60). Thus, SARS-CoV-2 may also inhibit maturation of the Na,K-ATPase in the ER and Golgi and impair trafficking of the enzyme to the PM by affecting calnexin, BiP, and glycosyltransferases in infected respiratory cells.

Alterations in protein folding in the ER result in the accumulation of misfolded/unfolded proteins, ER stress, and thus activation of the unfolded protein response (UPR) (61). Typically, changes of the ER Ca2+ concentrations and alterations of redox homeostasis, as well as ATP depletion lead to ER stress and activate UPR (62). Depending on the activation and coordinated action of the ER receptors, inositol-requiring enzyme 1 (IRE1), protein kinase RNA-activated (PKR)-like ER kinase (PERK), and activating transcription factor-6 (ATF6), UPR may be adaptive or maladaptive (63). Indeed, it has been shown that cells infected with SARS-CoV express elevated levels of the chaperon proteins GRP78 and GRP94 and show increased phosphorylation levels of protein kinase-R (PKR), PERK, and eukaryotic initiation factor 2α (eIF2α), suggesting ER stress and accumulation of the unfolded/misfolded proteins in the ER (64–66). In addition, it has been shown that perturbations in the ER oxidative homeostasis or decreased ATP levels negatively impact Na,K-ATPase PM abundance and function (67). Recent data suggest that coronaviruses require high levels of ATP during replication (68) and have a negative impact on mitochondrial function of infected cells (69, 70). Thus, based on these findings, decreased cellular ATP levels and changes in the ER redox status upon SARS-CoV-2 infection may lead to ER stress and misfolding of the Na,K-ATPase resulting in reduced PM expression of the enzyme in infected lung epithelial cells. Of note, in addition to the above-mentioned inhibitory effects of SARS-CoV-2 NSP proteins on mRNA transcription and ribosomal translation, the activation of eIF2α suggests a translational shutdown and thus inhibition of the canonical 5′-cap-dependent translation initiation. This mechanism may additionally block translation of the Na,K-ATPase subunits. Whether the Na,K-ATPase can be translated upon such stress conditions, for example, by using internal ribosome entry sites, which allow translation independently from eIF2α, remains to be investigated.

Cell-Surface Abundance

After delivery of the Na,K-ATPase to the PM, various intracellular messengers, and kinases in response to diverse extra- and intracellular stimuli regulate the abundance of the transporter. In the alveolar epithelium, elevated Ca2+ levels (71, 72), and phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) (73), AMP-activated protein kinase (AMPK) (39, 72, 74), c-Jun N-terminal kinase (JNK) (75, 76), protein kinase C (PKC) (72, 77), and protein kinase A (PKA) (78) have been shown to promote endocytosis of the Na,K-ATPase from the PM, thus reducing abundance and activity of the enzyme. Recent evidence suggests that an infection with coronaviruses activates ERK1/2, JNK, and PKC, which have been found to be involved in virus replication and downregulation of cellular host defense (79–81). Of note, it has been shown that the SARS-CoV N-protein induces phosphorylation of JNK (82). Moreover, SARS-CoV 3b-protein stimulates the expression of AP-1 by activating JNK and ERK (46). Furthermore, it has been shown that the SARS-CoV-2 spike protein has a number of phosphorylation sites that induce cAMP and PKC signaling pathways (83). Therefore, activation of these signals by SARS-CoV-2 may additionally reduce the PM abundance and thus function of the Na,K-ATPase in infected respiratory cells, thus impairing tightness of the epithelium and AFC. Of note, two proteins of SARS-CoV-2, NSP8 and NSP9, have also been found to interfere with trafficking of various proteins to the cell membrane (41). If this is relevant for proper targeting of the Na,K-ATPase to the PM will need to be investigated in further research.

INDIRECT EFFECTS OF SARS-COV-2 INFECTION ON THE Na,K-ATPASE

Gas Exchange Disturbances

Hallmarks of ARDS secondary to critical COVID-19 include a severe impairment of gas exchange (hypoxia and occasionally hypercapnia), in part due to massive pulmonary inflammatory responses and coagulopathy (84, 85). Of note, it is very well documented that hypoxia rapidly downregulates the function of the Na,K-ATPase by promoting endocytosis of the transporter from the PM by a Ca2+-/AMPK-/PKC-ζ-mediated pathway (71, 77). Similarly, cell surface abundance of ENaC is also markedly reduced upon hypoxia, thus significantly impairing the Na,K-ATPase/ENaC-driven AFC (86–88). Furthermore, sustained hypoxia markedly inhibits expression of Na,K-ATPase α- and β-subunits at the level of transcription (44) and drives Na,K-ATPase-regulated autosis (24), which may contribute to the epithelial cell death observed in lungs during SARS-CoV-2 infection.

Although carbon dioxide (CO2) diffuses faster than O2 across the alveolar-capillary barrier and thus patients with acute respiratory failure are primarily hypoxic, mechanical ventilation of patients with ARDS secondary to COVID-19 with relatively low tidal volumes, to limit further ventilator-induced lung injury, often leads to hypercapnia, an elevation in CO2 levels (89, 90). Additionally, deleterious lung remodeling in ventilated patients with COVID-19-ARDS, ranging from organizing pneumonia to irreversible lung fibrosis, may further exacerbate hypercapnia (91, 92). Importantly, elevated levels of CO2 have been shown to decrease Na,K-ATPase PM abundance by both impairing maturation of the enzyme in the ER (67) and driving endocytosis of the Na,K-ATPase, and ENaC from the PM by activation of ERK1/2, AMPK, JNK, and PKA (72, 73, 75, 76, 78, 93, 94). Therefore, gas exchange disturbances secondary to SARS-CoV-2 infection probably impair expression and function of the Na,K-ATPase, leading to diminished alveolar-capillary barrier function.

Inflammation and Coagulopathy

Other typical characteristics of severe COVID-19 include an uncontrolled immune response and an activation of coagulation pathways. The levels of various inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α), are generally strongly elevated in patients with severe and critical COVID-19 (7, 95, 96). Of note, activation of these inflammatory cytokines in this group of patients negatively correlates with the levels of immunoglobulin G and is associated with worse outcomes (97). Importantly, it is also well described that proinflammatory cytokines such as TNF-α (98, 99) and IL-1β (100) downregulate expression and activity of the Na,K-ATPase. Notably, pharmacological inhibition of Na,K-ATPase activity in macrophages activates the NF-κB signaling pathway and thus increases the production of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and monocyte chemoattractant protein-1 (101). This viscous cycle of paracrine effects may substantially contribute to alveolar epithelial barrier dysfunction and impaired lung fluid balance. Interestingly, it is now increasingly evident that dexamethasone is beneficial in patients with severe and critical COVID-19 (102), which is thought to be mediated by attenuation of the injurious inflammatory response to SARS-CoV-2. Importantly, glucocorticoids are also well known to upregulate the activity of the Na,K-ATPase (44, 103) that may contribute to the advantageous effects of corticosteroid therapy. Indeed, several studies have demonstrated that dexamethasone stimulates expression and activity of the Na,K-ATPase via transcriptional, and in part, translational or posttranslational mechanisms (87, 104, 105).

An activation of coagulation pathways is frequently observed in patients with COVID-19, resulting, among others, in an elevation of thrombin levels and increased thromboembolic complications (106). Notably, it has been shown that thrombin via the action of reactive oxygen species and translocation of PKC-ζ from intracellular stores to the PM, promotes retrieval of the Na,K-ATPase from the cell surface thus limiting the function of the transporter and impairing AFC (107). Therefore, the systemic inflammatory response and increased coagulation activity may also lead to impaired Na,K-ATPase function in patients with COVID-19 patients, which might further aggravate the disease.

Collectively, increasing evidence suggests that various molecular mechanisms that are key in the regulation of transcription, translation, maturation, and trafficking of the Na,K-ATPase are negatively regulated by SARS-CoV-2 (Fig. 1). As the Na,K-ATPase plays a pivotal role in maintaining cell volume and specifically in (re)establishing the alveolar epithelial barrier and promoting an optimal lung fluid balance, further research on these mechanisms is warranted, particularly as many of these pathways are potentially targetable. A tailored therapeutic modality selectively inhibiting the maladaptive signals that dysregulate the Na,K-ATPase upon SARS-CoV-2 infection might be of value.

Figure 1.

Schematic representation of mechanisms that may impair Na,K-ATPase function in COVID-19. SARS-CoV-2 may directly inhibit transcription, translation, maturation, and trafficking of the Na,K-ATPase, thus reducing the abundance and activity of the transporter at the plasma membrane. Sequels of COVID-19, such as gas exchange disturbances, uncontrolled inflammation, and activation of coagulation may additionally inhibit function of the Na,K-ATPase and lead to impaired alveolar epithelial barrier function and thus progressive respiratory failure. ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PKC, protein kinase C; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.

GRANTS

This work was supported by grants from the Federal Ministry of Education and Research [German Center for Lung Research (DZL/ALI 1.5 and 3.4)], the Hessen State Ministry of Higher Education, Research and the Arts [Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE)]), the von Behring Röntgen Foundation (Project 66-LV07), the German Research Foundation (DFG/KFO309, P5) (to I.V.), and an MD/PhD start-up grant (DFG/KFO309, MD/PhD) (to V.K.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

V.K. prepared figures; V.K. and I.V. drafted manuscript; I.V. edited and revised manuscript; V.K. and I.V. approved final version of manuscript.