Respiratory Physiology & Neurobiology 220 (2016) 54–61

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FXYD1 negatively regulates Na+ /K+ -ATPase activity in lung alveolar epithelial cells Łukasz A. Wujak a,b,c , Anna Blume a , Emel Balo˘glu d,e , Małgorzata Wygrecka a,c , Jegor Wygowski b , Susanne Herold a , Konstantin Mayer a , István Vadász a , Petra Besuch f , Heimo Mairbäurl c , Werner Seeger a,b , Rory E. Morty a,b,∗ a Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany b Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany c Department of Biochemistry, University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany d Department of Sports Medicine, Medical Clinic VII, University Hospital Heidelberg, University of Heidelberg, Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL), Heidelberg, Germany e Department of Medical Pharmacology, Acibadem University, I˙ stanbul, Turkey f Department of Pathology, Klinikum Frankfurt (Oder) GmbH, Frankfurt (Oder), Germany

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Article history: Received 8 May 2015 Received in revised form 17 September 2015 Accepted 20 September 2015 Available online 26 September 2015 Keywords: ARDS TGF-␤ Na+ /K+ -ATPase FXYD Ion transport Edema

a b s t r a c t Acute respiratory distress syndrome (ARDS) is clinical syndrome characterized by decreased lung fluid reabsorption, causing alveolar edema. Defective alveolar ion transport undertaken in part by the Na+ /K+ ATPase underlies this compromised fluid balance, although the molecular mechanisms at play are not understood. We describe here increased expression of FXYD1, FXYD3 and FXYD5, three regulatory subunits of the Na+ /K+ -ATPase, in the lungs of ARDS patients. Transforming growth factor (TGF)-␤, a pathogenic mediator of ARDS, drove increased FXYD1 expression in A549 human lung alveolar epithelial cells, suggesting that pathogenic TGF-␤ signaling altered Na+ /K+ -ATPase activity in affected lungs. Lentivirus-mediated delivery of FXYD1 and FXYD3 allowed for overexpression of both regulatory subunits in polarized H441 cell monolayers on an air/liquid interface. FXYD1 but not FXYD3 overexpression inhibited amphotericin B-sensitive equivalent short-circuit current in Ussing chamber studies. Thus, we speculate that FXYD1 overexpression in ARDS patient lungs may limit Na+ /K+ -ATPase activity, and contribute to edema persistence. © 2015 Elsevier B.V. All rights reserved.

1. 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

∗ Corresponding author at: Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Parkstrasse 1, D-61231 Bad Nauheim, Germany. Fax: +49 6032 705 360. E-mail address: [email protected] (R.E. Morty). http://dx.doi.org/10.1016/j.resp.2015.09.008 1569-9048/© 2015 Elsevier B.V. All rights reserved.

is a key factor in patient outcome (Matthay, 2002, 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

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Table 1 The clinical characteristics of acute respiratory distress syndrome patients. Patient

Age

Sex

Background

Modified APACHE II

PaO2 /FiO2 (mmHg)

1 2 3 4 5

40 51 48 59 67

Female Female Female Female Male

Pneumonia Trauma Pancreatitis Trauma Sepsis

11 6 20 16 20

83.0 181.5 127.2 137.1 109.0

Abbreviations: APACHE, acute physiology and chronic health evaluation; FiO2 , fraction of inspired oxygen; PaO2 , partial pressure of oxygen in arterial blood.

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)-␤ family have been credited with pathogenic roles in disturbances to alveolocapillary barrier failure, since elevated levels of TGF-␤ 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-␤ 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␤ is also known to dysregulate ENaC and Na+ /K+ -ATPase function (Morty et al., 2007; Vadász et al., 2007). For example, TGF-␤ downregulates expression of the ␣1 -ENaC subunit (Frank et al., 2003), and TGF-␤ 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 (␣), membrane anchoring (␤) and accessory regulatory (␥) subunits (Jorgensen et al., 2003), in ion transport. The ␤-subunit has received much attention as ␤-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 ␤1 -subunit) improves Na+ transport, alveolar fluid clearance, and pulmonary edema in experimental cell, organ, and animal models (Adir et al., 2003, 2008; Azzam et al., 2002; Factor et al., 2000, 1998a,b; 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 ␥-subunit in alveolar ion transport. The ␥-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, 2008). Given the pathogenic roles for TGF-␤ 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-␤ can modulate the expression of FXYD genes, and (iii) FXYD subunits regulate Na+ /K+ -ATPase activity in alveolar epithelial cells.

specimens were obtained at autopsy from four patients who died of myocardial infarction, with no evidence of pulmonary disease. 2.2. RNA isolation, reverse transcription and real-time RT-PCR Gene expression analysis in human lung tissues and cultured cells was undertaken as described previously (Madurga et al., 2014; Schwartze et al., 2014; Witsch et al., 2014; Wujak et al., 2015). Briefly, total RNA was isolated from lung tissue by TRIzol® /chloroform fractionation. Total RNA was isolated from cultured cells using a NucleoSpin® RNA II kit (Macherey-Nagel, Düren Germany). RNA was reverse-transcribed using MuLV reverse transcriptase and random hexamer oligodeoxyribonucleotides (Applied Biosystems, Waltham, USA). Gene expression analysis was performed by quantitative real-time (RT)-polymerase chain reaction (PCR) using a Platinum® SYBR® Green qPCR SuperMix UDG kit (Invitrogen, Waltham, U.S.A.) and a StepOnePlusTM Real-Time PCR System (Applied Biosystems, Waltham, U.S.A.), and the intronspanning primers listed in Table 2. Gene expression levels are represented as Ct values, using expression of the HPRT1 gene as a reference. 2.3. Cell culture and TGF-ˇ stimulation The A549 human lung adenocarcinoma epithelial cell-line, a model of distal lung epithelial cells (including type I and type II cells), was purchased form the American type Culture Collection (CCL-18; distributed by LGC Standards, Wesel, Germany) and was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (vol./vol.) fetal calf serum (FCS), on plastic tissue-culture plates in liquid culture. The H441 human bronchiolar epithelial cell-line, a model of Clara cells, was purchased from the American Type Culture Collection Collection (HTB-174; distributed by LGC Standards, Wesel, Germany), and was cultured in RPMI 1640/l-glutamine medium (PAA, Cölbe, Germany) supplemented with 10% (vol./vol.) FCS, 1 mM sodium pyruvate Table 2 Primers employed for gene expression analysis. Gene

Sequences

FXYD1

F: 5 -TCGCCGGGATCCTCTTCATCC-3 R: 5 -CCCTCCTCTTCATCGGGTTCCCC-3 F: 5 -AATGGGGGCCTGATCTTC-3 R: 5 -CTTCTTATTGCCCCCACAGC-3 F: 5 -AGCGCTCTGACATGCAGAAGGTG-3 R: 5 -TCTTCTAGGTCATTGGCGTCCAGG-3 F: 5 -GCGGACTGATCTGCGGAGGG-3 R: 5 -GCTGCTTCTGGCTGCTCTTGC-3 F: 5 -AGCAACTGGAAGGAACGGAT-3 R: 5 -GGGTCTGTCTGGACGTCTGT-3 F: 5 -CCCCAGAAAGCAGAGAACTG-3 R: 5 -GGCCGGTTTTCTTAAGCATC-3 F: 5 -TGTGGGCATGACTCTGGCAACC-3 R: 5 -TGGAGTCCGCCTTCCTGCAC-3 F: 5 -AAGGACCCCACGAAGTGTTG-3 R: 5 -GGCTTTGTATTTTGCTTTTCCA-3

FXYD2

2. Material and methods 2.1. Patient material

FXYD3 FXYD4 FXYD5

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

FXYD6 FXYD7 HPRT

Abbreviations: F, forward; R, reverse.

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and 1% (vol./vol.) insulin–transferrin–sodium selenite supplement (ITS; Sigma, Taufkirchen, Germany), and an air/liquid interface (Althaus et al., 2009, 2011). The HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (vol./vol.) FCS. Antibiotics were not supplemented in any of the tissue culture media. All three cell-lines were cultured at 37 ◦ C in humidified atmosphere containing 5% (vol./vol.) CO2 . For the TGF␤ stimulation experiments, A549 cells were treated with 0, 2, 6 or 10 ng/ml TGF-␤1 (R&D Systems, Minneapolis, U.S.A.) for 24 or 48 h, as indicated; or with TGF-␤ vehicle [4 mM HCl containing 0.1% (mass/vol.) bovine serum albumin] alone.

A Viability (% of 0 mg/ml Polybrene)

56

5000; - serum 7500; - serum 10000; - serum

250 200

5000; + serum 7500; + serum 10000; + serum

150 100

50 Polybrene:

2

4

6

8

10

12

14 (mg/ml)

2.4. Optimization of lentiviral transduction conditions

2.5. Generation of FXYD1 and FXYD3-encoding lentiviral particles The FXYD1- or FXYD3-encoding lentiviral particles were constructed using the Gateway® cloning technology (Invitrogen, Waltham, U.S.A.) according to the manufacturer’s instructions. Briefly, the FXYD1 and FXYD3 coding sequences were amplified by RT-PCR with following primer pairs: 5 -ATG GCG TCT CTT GGC CAC ATC-3 and 5 -CTA CCG CCT GCG GGT GGA CA-3 for FXYD1, and 5 -ATG CAG AAG GTG ACC CTG GGC-3 and 5 -TCA GCT TTG GGC

Viability (% of 0 mg/ml Polybrene)

B

5000; - serum 7500; - serum 10000; - serum

150 125

5000; + serum 7500; + serum 10000; + serum

100 75

50 Polybrene:

2

4

6

8

10

12

14 (mg/ml)

C promoter/DMEM only ratio

The use of S2-level genetically-modified viruses was approved for the promoter screening (approval number IV44-53r.30.03.MPP07.13.16) and FXYD1 and FXYD3 overexpression (approval number IV44-53r.30.03.MPP07.13.14) by the Regierungspräsidium Gießen, which houses the responsible government authority. To optimize lentiviral transduction conditions, H441 cell viability was first assessed under a spectrum of transduction conditions. The H441 cells were seeded at a density of 5000–10,000 cells/0.32 cm2 transwell insert, in medium containing sodium pyruvate and ITS with or without FCS, and treated with 0–14 mg/ml polybrene® (Sigma, St. Louis, U.S.A.) for 6 h or 24 h. Cells viability under these conditions was assessed by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, screening was undertaken to identify the optimal promoter for FXYD1 and FXYD3 overexpression in H441 cells. This was performed with a SMARTchoice shRNA Promoter Selection Plate (Dharmacon, Lafayette, USA) according to the manufacturer’s instructions. Briefly, 7500 cells/0.32 cm2 transwell insert were transduced for 24 h using 8 mg/ml Polybrene® in the presence of FCS with different SMARTvector 2.0 Empty Vector Lentiviral Particles encoding Turbo green fluorescent protein (TurboGFP) under the control of following promoters: human cytomegalovirus immediate early promoter (hCMV), mouse cytomegalovirus immediate early promoter (mCMV), human elongation factor 1␣ promoter (hEF1␣), mouse elongation factor 1␣ promoter (mEF1␣), chicken ␤-actin hybrid promoter (CAG), mouse phosphoglycerate kinase promoter (PGK) or human ubiquitin C promoter (UBC); all at multiplicity of infection (MOI) of 2.5–80. Afterwards, transduction medium was removed and cells were cultured in FCS containing medium for 48 h, followed by assessment of promoter activity by fluorescence analysis of cells in a microplate fluorimeter. The preliminary methodological optimization data addressed polybrene concentration, cell-seeding density, the use of serum, and the duration of the transduction protocol (Fig. 1A and B). These data led the investigators to select a cell density of 7500 cells/0.32 cm2 transwell insert, under serum-containing conditions and 8 mg/ml polybrene. Under these conditions, cell viability was optimal. Subsequent studies on promoter screening (Fig. 1C) revealed that only the mouse and human CMV promoters resulted in detectable and robust gene expression in H441 cells after lentiviral-mediated delivery.

1.6

hCMV mCMV hEF1alpha mEF1alpha CAG PGH UBC

1.4 1.2 1.0

0.8 MOI:

2.5

5

10

20

40

80

Fig. 1. The human and mouse CMV promoters drive gene expression in H441 cells. Viral delivery and expression of the TurboGFP reporter gene was optimized first by demonstrating H441 cell viability over a range of H441 cell densities (5000–10,000 cells/0.32 cm2 filter), polybrene concentrations (2–14 mg/ml), in the presence or absence of serum. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. H441 cell viability was assessed 48 h after a (A) 6-h or (B) 24-h treatment with polybrene. (C) The efficiency at which seven different promoters drove TurboGFO expression after lentiviral delivery in H441 cells seeded at a density of 7500 cells/0.32 cm2 transwell insert, was assessed over a range of multiplicity of infection (MOI; 2.5–80), using 8 mg/ml polybrene in the presence of serum. After a 24-h viral transduction period, cells were incubated for an additional 48 h in cell-culture medium. Green fluorescent protein fluorescence was assessed in cells, as a measure of promoter activity, by spectrofluorimetry. Data are presented as the ratio between GFP fluorescence in virus-treated cells, normalized for GFP fluorescence in untreated cells. Error bars have been omitted to avoid cluttering the graph. Abbreviations: hCMV, human cytomegalovirus immediate early promoter; mCMV, mouse cytomegalovirus immediate early promoter; hEF1␣, human elongation factor 1␣ promoter; mEF1␣, mouse elongation factor 1␣ promoter; CAG, chicken ␤-actin hybrid promoter; PGK, mouse phosphoglycerate kinase promoter; UBC, human ubiquitin C promoter.

TGA GCC TGG-3 for FXYD3. The resulting amplicons were then ligated into a pCRTM 8/GW/TOPO® Gateway® entry vector (Invitrogen, Waltham, U.S.A.). Next, FXYD1- and FXYD3-coding sequences were transferred using LR ClonaseTM (Invitrogen, Waltham, U.S.A.) from pCRTM 8/GW/TOPO® Gateway® entry vector to the pLenti7.3/V5DESTTM destination vector (Invitrogen, Waltham, U.S.A.), under control of the human cytomegalovirus immediate early promoter. To produce lentiviral particles, HEK293T cells were co-transfected with either the FXYD1- or the FXYD3-encoding pLenti7.3/V5-

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Fig. 2. The expression of FXYD1 and FXYD3 is upregulated in lungs of patients with acute respiratory distress syndrome. The expression of genes encoding the seven members of the FXYD family in humans, FXYD1-FXYD7, was assessed in lung tissue from five patients with ARDS and four apparently healthy lung donors, by real-time reversetranscription-polymerase chain reaction, using the HPRT gene as reference. Data are expressed as mean Ct ± S.E.M. Significant differences between groups were assessed by Student’s t-test.

DESTTM vector, together with the ViraPowerTM Packaging Vectors Mix (Invitrogen, Waltham, U.S.A.) for 24 h, using TurboFect transfection reagent (Thermo Scientific, Waltham, U.S.A.). After 48 h, medium containing lentiviral particles was collected, centrifuged and stored at −80 ◦ C.

2.6. Overexpression of FXYD1 and FXYD3 in H441 cells To transduce H441 cells with FXYD1- or FXYD3-encoding lentiviral particles, 500,000 H441 cells were seed onto the upper surface of 12-mm Corning® Transwell® polycarbonate membrane cell cul-

Fig. 3. The expression of FXYD genes is regulated by TGF-␤ in A549 cells. The expression of FXYD1-FXYD7 was assessed in A549 cells treated either with TGF-␤ vehicle [4 mM HCl containing 0.1% (mass/vol.) bovine serum albumin] alone (the “0 ng/ml TGF-␤” group), or with TGF-␤ (2–10 ng/ml for 24 h or 48 h) by real-time reverse-transcriptionpolymerase chain reaction, using the HPRT gene as reference. Data are expressed as mean Ct ± S.E.M. (n = 3, per group). Significant differences between groups were assessed by one-way ANOVA with a Bonferroni post hoc test.

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ture inserts (Corning, Corning, U.S.A.) in medium supplemented with FCS, 1 ␮M dexamethasone (Sigma, St. louis, U.S.A.), Na+ pyruvate and ITS. After 48 h, medium was aspirated and cells were transduced for 24 h with 150 ␮l of FXYD1- or FXYD3-expressing lentivirus particles, or empty lentivirus particles (as control), and 8 mg/ml polybrene® . The lentiviral particle concentration was not assessed; rather, unadulterated HEK293T cell supernatants were used as a source of lentiviral particles without further lentiviral particle purification. Afterwards, transduction medium was removed and cells were cultured under air/liquid conditions in medium containing FCS, 1 ␮M dexamethasone, 1 mM sodium pyruvate and 1% (vol./vol.) ITS for seven days, with media exchange every two days, to allow monolayers to develop sufficient transepithelial electrical resistance. 2.7. Ussing chamber studies Ussing chamber studies were undertaken essentially as described previously (Balo˘glu et al., 2009, 2011). Briefly, after 7 d under air/liquid conditions in the presence of dexamethasone, the monolayers developed a transepithelial resistance of approximately 300 /cm2 , as previously optimized (Althaus et al., 2009, 2011), and were used for Ussing chamber studies from Day 7. Confluent monolayers on membrane supports were mounted in Ussing chambers and bathed from both sides with physiological saline containing 141 mM NaCl, 5.4 mM KCl, 0.78 mM NaH2 PO4 , 1.8 mM CaCl2 , 8 mM MgCl2 , 15 mM HEPES, and 5 mM glucose (37 ◦ C, pH 7.4, HEPES-NaOH). Monolayers were maintained under open-circuit conditions, spontaneous transepithelial potential (VT ) was monitored until stable. The transepithelial potential (VT ) was monitored under current clamp conditions, and equivalent short circuit current (Ieq ) was calculated from the transepithelial electrical resistance using a TECC-2 apparatus (EPS, Leuven, Belgium). All experiments were performed at 37 ◦ C. To estimate the activity of the basolaterally localized Na+ /K+ -ATPase, 10 ␮M amiloride was applied to the apical bath to block epithelial sodium channels, and 5 ␮M amphotericin B was added to the apical bath to permeabilize the apical membrane. The current was completely blocked with 100 ␮M ouabain.

meaningful, and suggests that subsequent studies with larger sample sizes are indicated. 3.2. TGF-ˇ regulates FXYD gene expression in A549 cells Since it is known that TGF-␤ is a mediator of ARDS (Budinger et al., 2005; Fahy et al., 2003; Peters et al., 2014), and that TGF-␤ can deregulate the expression and activity of key ion transporting molecules in the lung epithelium (Akbarshahi et al., 2014; Frank et al., 2003; Peters et al., 2014; Wesselkamper et al., 2005), the impact of TGF-␤ on FXYD gene expression in A549 cells was assessed (Fig. 3). With the exception of FXYD7, the expression of all other FXYD genes in A549 cells was influenced by TGF-␤, after both 24-h and 48-h stimulations. Judging by the magnitude of the Ct values, the most dramatic effects were seen on the expression of FXYD1 and FXYD3. An increased expression of FXYD1 was driven by TGF-␤, where a Ct ≈ 4 was attained, translating to a 16-fold increase in mRNA expression, when A549 cells were stimulated with TGF-␤ for at 2–10 ng/ml for 24 h, or 6–10 ng/ml for 48 h. In contrast, TGF-␤ repressed the expression of FXYD3, where a Ct ≈ 1 was attained after 24 h (in the TGF-␤ range 6–10 ng/ml), translating to a 2-fold down-regulation; while after 48 h (in the TGF-␤ range 2–10 ng/ml), stimulation with TGF-␤ yielded a Ct ≈ 2.5, translating to a 5.5-fold down-regulation of FXYD3 expression. Concerning other FXYD genes, TGF-␤ also downregulated FXYD2 and FXYD4 expression, while upregulating FXYD5 and FXYD6 expression in A549 cells. Since FXYD1, FXYD3, and FXYD5 gene expression was both upregulated in lungs from ARDS patients (Fig. 2), further study on the contribution of FXYD1, FXYD3, and FXYD5 to Na+ /K+ -ATPase function was desirable. An examination of the effects of the modulation of expression levels of FXYD5 on Na+ /K+ -ATPase activity in the context of cystic fibrosis has already been undertaken (Miller and Davis, 2008), where FXYD5 was identified as a positive regulator of Na+ /K+ -ATPase activity. For this reason, FXYD5 was not considered further in this study, and FXYD1 and FXYD3 were selected for subsequent functional studies in polarized lung epithelial cells. 3.3. H441 cells over-express FXYD1 and FXYD3 genes after lentivirus delivery with CMV promoters

2.8. Statistics Values represent mean ± S.E.M. Data sets were screened for significant outliers by Grubbs’ test. All data sets were screened for normal distribution by the Anderson–Darling test. All data presented are normally distributed. Statistical comparisons between two samples were made with an unpaired Student’s t-test. For more than two samples, a one-way ANOVA followed by a Bonferroni posthoc test was employed. P values