FXYD1 negatively regulates Na+/K+-ATPase activity in lung alveolar epithelial cells
Introduction
Acute respiratory distress syndrome (ARDS) is a rapidly evolving clinical syndrome resulting in acute respiratory failure. Damage to the alveolo-capillary barrier increases permeability, and blunts lung fluid reabsorption, resulting in accumulation and persistence of protein-rich edema fluid in the alveolar airspaces, the hallmark of ARDS. The protein-rich edema fluid causes surfactant dysfunction, perturbs the oncotic gradients in the lung, and increased lining fluid volume inhibits effective gas exchange (Matthay and Zemans, 2011, Ware, 2006). It is accepted that edema clearance is a key factor in patient outcome (Matthay, 2002, Matthay, 2014, Sznajder, 2001), indeed, alveolar edema is considered a Na+ transport defect (Wilson, 2004). Thus, understanding molecular mechanisms of alveolar fluid clearance is an important line of research into the pathobiology of this devastating syndrome (Berthiaume and Matthay, 2007, Brune et al., 2015, Downs and Helms, 2013, Herold et al., 2013).
The major force driving water movement in the lung is Na+ transport from the alveolar airspaces across the epithelium via the apically-located epithelial sodium channel (ENaC) and the basolaterally-located Na+/K+-transporting adenosine 5′-triphosphatase (Na+/K+-ATPase), into the interstitium (Dobbs and Johnson, 2007, Kemp and Kim, 2004, Mairbäurl, 2006, Mutlu and Sznajder, 2005). There is much interest in understanding normal regulation and pathological deregulation of these ion transporters, for example, by growth factors, cytokines, proteases, and regulatory subunits (Olver et al., 2004), as potential pathomechanisms leading to the formation and persistence of alveolar edema in ARDS patients.
Members of the transforming growth factor (TGF)-β family have been credited with pathogenic roles in disturbances to alveolo-capillary barrier failure, since elevated levels of TGF-β have been noted, and correlated with prognosis, in bronchoalveolar lavage fluids of patients with ARDS (Budinger et al., 2005, Fahy et al., 2003, Peters et al., 2014). These observations are important, since TGF-β is a mediator of experimental acute lung injury, ostensibly by reducing transepithelial electrical resistance and increasing permeability of the epithelial barrier (Pittet et al., 2001, Willis et al., 2003). TGF-β is also known to dysregulate ENaC and Na+/K+-ATPase function (Morty et al., 2007, Vadász et al., 2007). For example, TGF-β downregulates expression of the α1‑ENaC subunit (Frank et al., 2003), and TGF-β drives ENaC endocytosis (Peters et al., 2014).
Many ion transport studies have addressed roles for subunits of the heteromeric Na+/K+-ATPase, which consists of the transporting (α), membrane anchoring (β) and accessory regulatory (γ) subunits (Jorgensen et al., 2003), in ion transport. The β-subunit has received much attention as β-subunit expression is downregulated in experimental acute lung injury (Akbarshahi et al., 2014, Wesselkamper et al., 2005). Furthermore, gene transfer of the ATP1B1 gene (encoding the β1-subunit) improves Na+ transport, alveolar fluid clearance, and pulmonary edema in experimental cell, organ, and animal models (Adir et al., 2003, Adir et al., 2008, Azzam et al., 2002, Factor et al., 2000, Factor et al., 1998a, Factor et al., 1998b, Stern et al., 2000), indicating that alterations to Na+/K+-ATPase subunit stoichiometry has consequences for ion transport in the lung.
To date, no studies have examined possible roles for the Na+/K+-ATPase γ-subunit in alveolar ion transport. The γ-subunit includes seven members of the FXYD domain-containing ion transport regulator family, encoded by seven different genes, designated FXYD1-FYXD7 in humans (Garty and Karlish, 2006). The FXYD proteins, which are not well-studied in lung pathology, are expressed in a tissue-dependent manner and function as stabilizers and modulators of Na+/K+-ATPase activity (Geering, 2006, Geering, 2008).
Given the pathogenic roles for TGF-β and blunted ion channel and pump function in ARDS, it was hypothesized here that (i) the expression of FXYD genes is changed in ARDS patients, (ii) TGF-β can modulate the expression of FXYD genes, and (iii) FXYD subunits regulate Na+/K+-ATPase activity in alveolar epithelial cells.
Section snippets
Patient material
Investigations using human material were approved by the Ethik-Kommission (Institutional Review Board) of the Justus Liebig University School of Medicine in Giessen (approval number 29/01). All patients met clinical American–European Consensus Conference criteria for ARDS and died in the early phase, with a mean duration of mechanical ventilation of 92 h. The clinical characteristics of these patients are illustrated in Table 1. Control lung specimens were obtained at autopsy from four patients
The expression of FXYD genes in the lung is altered in ARDS
The expression of the FXYD genes in apparently healthy human donor lungs or lungs from ARDS patients was assessed by real-time RT-PCR (Fig. 2). All seven FXYD genes were expressed in human lungs. The expression of three FXYD genes, namely, FXYD1, FXYD3 and FXYD5 was upregulated in the lungs of ARDS patients. The authors do acknowledge that the patient groups represent an extremely small sample size, and that this is an important limitation of the current study. However, given that lung tissue
Discussion
In the present manuscript, it has been demonstrated that (i) FXYD1, FXYD3 and FXYD5 gene expression was upregulated in the lungs of ARDS patients, (ii) TGF-β, a critical mediator of ARDS, can modulate FXYD gene expression in vitro, and (iii) overexpression of FXYD1 and FXYD3 modulate transepithelial ion transport in human lung epithelial cells.
This study is the first study that has profiled the expression of all FXYD family members in the lungs of patients with ARDS. FXYD proteins are emerging
Competing interests
The authors have no conflict of interest.
Acknowledgements
The authors thank Sonja Engelhardt for excellent technical assistance. This work was supported by the Max Planck Society; the German Center for Lung Research (DZL); the von Behring-Röntgen Foundation (grant 51-0031); the University Medical Center Giessen and Marburg (grant 62580935); the German Research Foundation through Excellence Cluster 147 “Cardio-Pulmonary System” and grants Mo-1789/1 (to R.E.M.), Wy-119/1-3 (to M.W.), and Ma-1503/28-1 (to H.M.); the Federal Ministry of Higher Education,
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