Respiratory Physiology & Neurobiology 216 (2015) 52–62

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Regulation of epithelial sodium channel expression by oestradiol and progestogen in alveolar epithelial cells Ling Luo a , Jia Deng b , Dao-xin Wang a,∗ , Jing He a , Wang Deng a a b

Department of Respiratory Medicine, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China First Department of Internal Medicine, Traditional Chinese Medical Hospital of Jiangbei District, Chongqing, China

a r t i c l e

i n f o

Article history: Received 12 December 2014 Received in revised form 15 May 2015 Accepted 1 June 2015 Available online 4 June 2015 Keywords: Oestradiol Progestogen Epithelial Na+ channel 11␤-Hydroxysteroid dehydrogenase type 2 acute lung injury Acute respiratory distress syndrome

a b s t r a c t Oestrogen (E) and progestogen (P) exert regulatory effects on the epithelial Na+ channel (ENaC) in the kidneys and the colon. However, the effects of E and P on the ENaC and on alveolar fluid clearance (AFC) remain unclear, and the mechanisms of action of these hormones are unknown. In this study, we showed that E and/or P administration increased AFC by more than 25% and increased the expression of the ˛ and  subunits of ENaC by approximately 35% in rats subjected to oleic acid-induced acute lung injury (ALI). A similar effect was observed in the dexamethasone-treated group. Furthermore, E and/or P treatment inhibited 11␤-hydroxysteroid dehydrogenase (HSD) type 2 (11␤-HSD2) activity, increased corticosterone expression and decreased the serum adrenocorticotrophic hormone (ACTH) levels. These effects were similar to those observed following treatment with carbenoxolone (CBX), a nonspecific HSD inhibitor. Further investigation showed that CBX further significantly increased AFC and ˛-ENaC expression after treatment with a low dose of E and/or P. In vitro, E or P alone inhibited 11␤-HSD2 activity in a dose-dependent manner and increased ˛-ENaC expression by at least 50%, and E combined with P increased ˛-ENaC expression by more than 80%. Thus, E and P may augment the expression of ˛-ENaC, enhance AFC, attenuate pulmonary oedema by inhibiting 11␤-HSD2 activity, and increase the active glucocorticoid levels in vivo and in vitro. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Acute respiratory distress syndrome (ARDS) manifests as pulmonary oedema, respiratory distress and consequent severe hypoxemia. Despite multimodal treatments such as assuming a prone position, applying positive end-expiratory pressure, performing restrictive volume therapy and pulmonary surfactant replacement, and administering glucocorticoids and nitric oxide

Abbreviations: ACTH, adrenocorticotropic hormone; AFC, alveolar fluid clearance; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; 11␤-HSD, 11␤-hydroxysteroid dehydrogenase; 11␤-HSD1, 11␤-hydroxysteroid dehydrogenase type 1; 11␤-HSD2, 11␤-hydroxysteroid dehydrogenase type 2; CBX, carbenoxolone; E, oestrogen; ENa, Cepithelial Na+ channel; GR, glucocorticoid receptor; HSD, hydroxysteroid dehydrogenase; IL, interleukin; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; P, progestogen; RDS, respiratory distress syndrome; RT-PCR, reverse transcriptionpolymerase chain reaction; SGK1, serum glucocorticoid-regulated kinase 1; W/D, wet-to-dry weight ratio. ∗ Corresponding author at: Department of Respiratory Medicine, Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, Chongqing 400010, China. Tel.: +86 23 6369 3094. E-mail address: [email protected] (D.-x. Wang). http://dx.doi.org/10.1016/j.resp.2015.06.001 1569-9048/© 2015 Elsevier B.V. All rights reserved.

(NO), the mortality rate of ARDS remains at approximately 30–50% (Rubenfeld and Herridge, 2007; Mutlu and Budinger, 2006). Recent research has demonstrated that alveolar fluid clearance (AFC) is critical for the effective treatment of acute lung injury (ALI)/ARDS (Berthiaume and Matthay, 2007; Morty et al., 2007; Mutlu and Budinger, 2006) and that a reduced net rate of AFC has been associated with higher mortality in clinical settings. Preliminary studies have revealed that AFC occurs via both passive and active modes of transport; the active mode is the primary mechanism for the transport of water and Na+ . In the alveolar epithelium, the active transport system is composed of the epithelial Na+ channel (ENaC), the Na+ –K+ -adenosine triphosphatase and aquaporin. ENaC displays a high selectivity for Na+ ions, and ENaC primarily functions in intracellular Na+ uptake. ENaC is responsible for most oedema fluid absorption and is composed of three homologous subunits (˛, ˇ and ) (Althaus et al., 2011; Canessa et al., 1994), each of which plays a key role in AFC. Female sex steroids may also be involved in the regulation of AFC; women with ALI have higher rates of AFC and of survival than males, and maximal AFC rates were more commonly observed in females (Bastarache et al., 2011; Ware and Matthay, 2001). Two studies reported that genetic susceptibility to ARDS varies depend-

L. Luo et al. / Respiratory Physiology & Neurobiology 216 (2015) 52–62

ing on gender (Gong et al., 2004; Sheu et al., 2009). Moreover, preterm infants suffering from respiratory distress syndrome (RDS) showed decreased oestrogen (E) and progestogen (P) concentrations in plasma (Parker et al., 1987). After receiving E substitution, preterm infants with RDS had higher survival rates and less respiratory distress (Hallman and Haataja, 2003; Helve et al., 2004). Trotter et al. found that the pharmacological deprivation of E and P during pregnancy inhibited the formation of alveoli and decreased AFC in newborn piglets (Trotter et al., 2006). However, the mechanisms underlying these effects are not understood. The sex-specific regulation of ENaC was observed in female rat kidneys (Kienitz et al., 2009). Female gonadal steroids differentially modulate the mRNA expression of ENaC subunits in rat kidneys. The abundance of ˛-, ˇ- and -ENaC mRNA was significantly higher in female rat kidneys than in male rat kidneys. These differences were abolished in ovariectomised rats. The treatment of ovariectomised rats with E increased ˛-ENaC mRNA abundance in the kidney, whereas treatment with P increased -ENaC mRNA expression (Gambling et al., 2004). A low concentration both E and P stimulates ENaC activity, whereas a high concentration of E almost completely inhibits the stimulation of ENaC activity in renal collecting tubule cells (Chang et al., 2007). Other studies showed that E decreases the plasma aldosterone levels and the expression of renal sodium transporters. Upon the elimination of aldosterone, E does not affect the expression of sodium transporters, which indicates that the effect of E on renal sodium transporters is at least partly influenced by aldosterone (Heo et al., 2012). Moreover, the ENaC levels are increased in rat foetal distal lung epithelial cells in response to exposure to E alone or to both E and P (Laube et al., 2011). The lungs of sexually mature female rats have higher levels of ˛- and -ENaC mRNA than those of males because the female rats have higher E and P levels (Sweezey et al., 1998). These findings indicate that female hormones may regulate ENaC activity and expression in the alveolar epithelium, representing a pathway potentially involved in AFC and ALI/ARDS. However, the underlying mechanism warrants further study. Glucocorticoids have been proposed to promote the absorption of fluid from alveolar spaces. The intracellular levels and the biological activity of endogenous glucocorticoids are controlled by 11␤-hydroxysteroid dehydrogenase (11␤-HSD) prior to their binding to glucocorticoid receptors (GRs). Active glucocorticoids include cortisol in humans and corticosterone in rats. There are two types of 11␤-HSD: 11␤-HSD type 1 (11␤-HSD1) and 11␤-HSD type 2 (11␤-HSD2). The 11␤-HSD1 protein appears in hepatic tissues, adipose tissues, the brain and the placenta and acts as a reductase to convert inactive corticosterone to active cortisol, thus increasing the glucocorticoid levels in local tissues. It possesses both oxidase and reductase activities to interconvert inactive 11-oxo metabolites to their active forms in vivo; this process requires nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. 11␤-HSD2 serves as an oxidase to convert active cortisol to inactive cortisone using nicotinamide adenine dinucleotide (NAD) as a cofactor. This enzyme has been detected in lung epithelial cells of the ciliated airway epithelium and the alveolar epithelium, where fluid absorption may occur in humans (Suzuki et al., 1998) and rats (Suzuki et al., 2001). Markedly increased expression of 11␤-HSD2 was found in the lungs upon autopsy in patients with ARDS (Suzuki et al., 2003). The function of 11␤-HSD2 is inhibited by carbenoxolone (CBX), which is an agent derived from liquorice root that acts as a nonspecific hydroxysteroid dehydrogenase (HSD) inhibitor. Treatment with CBX significantly augments ENaC mRNA expression and AFC in rats due to the increased activity of endogenous bioactive glucocorticoids on lung cells via the reduction of local steroid breakdown (Suzuki et al., 2004). Both E and P are inhibitors of 11␤-HSD2 activity in term human placenta. P also reduces 11␤-HSD2 the mRNA levels in vitro (Sun et al., 1998). Based on the available evidence,

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we hypothesised that treatment with E and P would enhance the effects of endogenous glucocorticoids on lung cells by inhibiting steroid breakdown. We examined the effects of this treatment on ENaC mRNA and protein expression and on AFC in rats and in A549 cells. 2. Methods 2.1. Materials All protocols involving rats were approved by the Institutional Review Board of Chongqing Medical University. All SD rats (130 ± 20 g, Animal Centre of Chongqing Medical University) received humane care. The A549 human alveolar epithelial cell line, foetal bovine serum and 1640 medium were purchased from the Institute of Life Science of Chongqing Medical University (Chongqing, China). Polyclonal antibodies against ˛-ENaC, ˇ-ENaC and -ENaC were purchased from Abcam (Cambridge, London, England). The secondary antibody (IgG) was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). The RNA extraction kit was purchased from BioTeke Corporation (Beijing, China). [3 H]-corticosterone was purchased from New England Nuclear (Boston, MA, USA). NAD+ was purchased from Sigma (St. Louis, MO, USA). 2.2. Animals and preparation A total of 35 immature female rats were maintained in accordance with the experimental guidelines of Chongqing Medical University. The rats were randomly divided into 7 groups of 5 animals each. The control group was intravenously injected with 8 mL kg−1 saline; the others were intravenously injected with 0.1 mL kg−1 oleic acid to establish the ALI model. The rats were subcutaneously injected with 0.1 mL saline or the same volume of saline containing 1 ␮g E (0.01 mg/mL), 1 mg P (10 mg/mL), a combination of both hormones; 1 mg CBX (10 mg/mL), or a mixture of E, P and CBX at 12 and 36 h prior to oleic acid injection. All rats were euthanised at 6 h after oleic acid injection. Blood samples were collected from the heart via direct puncture. The lungs were removed immediately after the rats were euthanised (Fig. 1A). In another experiment, according to the methods mentioned above, a total of 15 immature female rats were randomly divided into the control group, the ALI group, and the dexamethasone group (5 animals each). After the ALI model was established, the rats were subcutaneously injected with 0.1 mL saline or the same volume containing 20 ␮g dexamethasone (0.2 mg/mL). As a supplemental experiment, a total of 30 immature female rats were randomly divided into 6 groups of 5 animals each. After the ALI model was established, the rats were subcutaneously injected with 0.06 mL saline or the same volume containing 0.6 mg CBX (10 mg/mL) separately or together with 0.06 ␮g E (0.01 mg/mL) and/or 0.6 mg P (10 mg/mL). 2.3. Measurement of AFC AFC was evaluated as previously described (Sakuma et al., 2004). Briefly, the isolated left lung was placed in a humidified incubator at 37 ◦ C, and a 5% albumin solution containing Evans blue (0.15 mg/mL), an alveolar volume marker, was instilled into the airway of the right lung. This instillation was followed by the instillation of 2 mL of oxygen to ensure that the solution infiltrated all of the alveolar spaces. The lungs were then inflated to an airway pressure of 7 cm H2 O using 100% oxygen for 1 h after instillation. The concentrations of Evans blue-labelled albumin in the initial and final solutions were measured using a spectrophotometer (Beckman Coulter, Los Angeles, CA, USA) at 620 nm.

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Fig. 1. Flow chart illustrating the animal and cell-culture experiments. In vitro study (A). The intervention groups included the E, P, E + P, CBX and E + P + CX groups. In another experiment, the intervention drug was dexamethasone. Finally, in the low dose experiments, the intervention groups included CBX, E + CBX, P + CBX and E + P + CBX. The flow chart of cell culture experiments is illustrated as follows (B,C). C: control group; ALI: ALI model group; E: oestrogen group; P: progestogen group; E + P: oestrogen and progestogen group; CBX: carbenoxolone group; E + P + CBX: oestrogen, progestogen and carbenoxolone group; E + CBX: oestrogen and carbenoxolone group; P + CBX: progestogen and carbenoxolone group.

AFC was calculated as follows: AFC(%) =

 Vi − Vf  Vi

(Vi × EBi) × 100%; Vf = EBf

In these equations, Vi and Vf represent the initial and final volumes of alveolar fluid, respectively, and EBi and EBf represent the initial and final concentrations of the Evans blue-labelled 5% albumin solution, respectively. 2.4. Lung wet-to-dry weight ratio and lung coefficient measurement We calculated the lung wet-to-dry weight ratio (W/D) and the lung coefficient to quantify the extent of pulmonary oedema and to assess the severity of ALI/ARDS. After the rats were euthanized, the body weight and the lung wet weight of the lungs were determined, and the lung coefficient  was calculated  as follows: × 100%. Lungcoefficient = wetlungweight bodyweight Portions of the right lungs were harvested and weighed using an automated electric balance to obtain the wet weight. These samples were then placed in an oven at 80 ◦ C for 48 h and weighed again to obtain the dry weight. The lung W/D was then calculated. 2.5. Lung histology evaluation The right lower lung lobes were harvested and fixed in formalin followed by embedding in paraffin and staining with haematoxylin and eosin for microscopic observation. 2.6. Measurement of the corticosterone and ACTH levels Blood samples were collected and centrifuged. The levels of corticosterone were determined via gas chromatography-mass spectrometry (Audige et al., 2002), and the plasma ACTH levels were analysed using enzyme-linked immunosorbent assay kits. 2.7. 11ˇ-HSD2 activity in lung homogenates Lung tissues were processed in homogenisation buffer on ice using a rotor homogeniser. The lung homogenates (100 ␮g) were then incubated in 2 nM [3 H]-corticosterone in the presence of 0.5 mM NAD+ at 37 ◦ C for 4 h. The steroids were extracted into

an ethanol solution containing unlabelled corticosterone and 11hydroxycorticosterone and then separated on plastic silica gel plates using chloroform/ethanol (92:8) as a solvent. The areas corresponding to cold carriers were visualised under ultraviolet light, cut out, placed in scintillation vials, and counted. The results are expressed as the steroid conversion rate (percentage of corticosterone that was converted to 11-hydroxycorticosterone).

2.8. Treatment of A549 cells A549 cells are widely used as a model of alveolar epithelial cells. The cells were cultured in 1640 medium containing 10% calf serum in a humidified incubator containing 5% CO2 at 37 ◦ C. The cells were treated with 0.25% trypsin for subculturing. For protein and mRNA expression analysis, the cells were seeded at a density of 2 × 106 cells per insert. The medium was replaced daily with serum-free complete medium. The cells were then treated with E (0.001, 0.01, 0.1, or 1 ␮M) and/or P (0.001, 0.01, 0.1, or 1 ␮M; Fig. 1B), E (1 ␮M) together with P (1 ␮M) in the presence or absence of CBX (1 ␮M), or 1 ␮M CBX alone at days zero, one, two and four. The control cells were not treated with E, P or CBX (Fig. 1C). The assessments were performed on the fifth day.

2.9. 11ˇ-HSD2 activity in A549 cells To measure the conversion activity, a mixture containing [3 H]cortisol and cortisone (40 ␮g each) was added to the collected medium to identify the steroids via thin-layer chromatography (TLC). We extracted the steroids from the medium using ethyl acetate, dried the extract, reconstituted it in 100 ␮L of ethyl acetate, and applied it to the TLC plate. Cortisol and cortisone were separated using a solvent system containing chloroform/ethanol (95:5 v:v). The steroids were visualized under ultraviolet light, scraped off the plate, and extracted with ethyl acetate. The solvent was dried, and scintillation fluid was added. The radioactivity was then measured in a liquid scintillation counter. 11␤-HSD2 activity was expressed as the percentage of [3 H]-cortisone formed from [3 H]-cortisol in cultured cells. Each treatment was performed in triplicate for each cell preparation.

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2.10. Analysis of ENaC mRNA expression via RT-PCR Total RNA was extracted from the lung tissue homogenate or A549 cells using an RNA extraction kit according to the manufacturer’s instructions. The quality and the quantity of the RNA were estimated via agarose gel electrophoresis and ultraviolet spectrophotometry. The primer sequences for ˛-ENaC, ˇ-ENaC, -ENaC and ␤-actin were as follows: ˛-ENaC (330 bp), 5 -TTCTGGGCGGTGCTGTGGCT-3  (forward) and 3 -GCGTCTGCTCCGTGATGCGG-5 (reverse); (319 bp), 5 -TGCAGGCCCAATGCCGAGGT-3 (forˇ-ENaC ward) and 3 -GGGCTCTGTGCCCTGGCTCT-5 (reverse); -ENaC (278 bp), 5 -CACGCCAGCCGTGACCCTTC-3 (forward) and (reverse); and ␤-actin 3 -CTCGGGACACCACGATGCGG-5 (290 bp), 5 -CATGAAGATCCTCACCGAGC-3 (forward) and 5 GTGGACATCCGCAAAGACCT-3 (reverse). A reaction system was established according to the instructions provided with the onestep SYBR RT-PCR kit. Amplification was then performed using a PCR amplification analyser. The reverse transcription reaction was incubated at 42 ◦ C for 1 h. The PCR cycling conditions were as follows: 2 min at 94 ◦ C followed by 35 cycles of 30 s at 94 ◦ C, 30 s at 58 ◦ C, and 4 min at 72 ◦ C (˛-ENaC); 32 cycles of 30 s at 94 ◦ C, 30 s at 58 ◦ C, and 3 min at 72 ◦ C (ˇ-ENaC); 30 cycles of 30 s at 94 ◦ C, 30 s at 59 ◦ C, and 2.5 min at 72 ◦ C (-ENaC); or 35 cycles of 30 s at 94 ◦ C, 30 s at 57 ◦ C, and 3 s at 72 ◦ C (␤-actin). Polymerization was performed at 72 ◦ C for 60 s. Each PCR product was mixed with 5x loading buffer and electrophoresed on a 1.2% agarose gel containing ethidium bromide; the result was visualized using a Gel Imaging System (Bio-Rad, Hercules, CA, USA). Each reaction was repeated at least 3 times. The ratio of ˛-, ˇ- or -ENaC to ␤-actin was calculated to determine the relative mRNA expression of ˛-, ˇ- and -ENaC, respectively. 2.11. Measurement of ENaC protein expression Western blot for the ˛-, ˇ- and -ENaC subunits was performed as described previously (Thome et al., 2003). Protein samples were separated via 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After 1 h in a blocking solution containing 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20, the membranes were incubated in antibodies against ˛-, ˇ- or -ENaC (1:1000) or ␤-actin (1:2000) overnight at 4 ◦ C, followed by incubation in horseradish peroxidase-conjugated secondary antibodies (1:5000) (Santa Cruz Biotechnology) at room temperature for 1 h. Horseradish peroxidase activity was detected using the enhanced chemiluminescence method. Protein bands were visualised using an ultraviolet photometry gel imaging system (Upland, CA, USA). Each reaction was repeated at least 3 times. The ratio of ˛-, ˇ- or -ENaC to ␤-actin was calculated to determine the relative protein expression of ˛-, ˇ- and -ENaC, respectively. 2.12. Statistics All data were analysed via one-way analysis of variance using SPSS 17.0 software (SPSS Inc., USA) followed by the least significant difference test for multiple comparisons. The data are presented as the means ± standard deviations. A P-value < 0.05 was considered to be statistically significant. 3. Results 3.1. E and P attenuate pulmonary oedema Compared with the control group, the W/D (Fig. 2A1 ) and the lung coefficient (Fig. 2B1 ) were significantly increased in the other

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groups. E and/or P, especially the combination of E and P in the presence or absence of CBX, significantly reduced the oleic acid-induced increase in the W/D and the lung coefficient (P < 0.05, Fig. 2A1 and B1 ). CBX did not further reduce the W/D or the lung coefficient in the presence of both E and P. 3.2. E and P augment AFC AFC decreased by 60% in the ALI model group (P < 0.05, Fig. 2C1–3 ) compared with the control group. The rats treated with E and/or P demonstrated a significantly higher AFC than untreated rats subjected to ALI (P < 0.05). There were 26% and 29% increases in AFC in the rats treated with E and P, respectively, compared with the untreated rats subjected to ALI. The level of AFC in the group treated with both E and P was nearly twofold that in the ALI group (Fig. 2C1 ). 3.3. E and P attenuate lung injury Evident pulmonary oedema, bleeding and inflammatory cell infiltration were observed in the ALI group. However, E and/or P, especially the combination of E and P, markedly decreased the extent of pulmonary oedema, bleeding and inflammatory cell infiltration. CBX did not further attenuate the oleic acid-induced pulmonary oedema, bleeding or inflammation when administered with E and P (Fig. 3). 3.4. Expression of corticosterone and ACTH in plasma There were significant 8%, 9% and 18% increases in the levels of corticosterone in the rats treated with E, P or both E and P, respectively, compared with the untreated rats subjected to ALI (P < 0.05). Decreases in the expression of ACTH of approximately 50% were observed in the rats treated with E or P compared with those in the ALI group. A decrease of greater than 70% was observed in the rats treated with E together with P compared with the untreated rats subjected to ALI (P < 0.05). No significant differences were found in the levels of corticosterone or ACTH between the groups treated with E and P with or without CBX administration (Fig. 4A and B). 3.5. 11ˇ-HSD2 activity in lung homogenates The rats treated with E, P, or both E and P showed significant decreases (16%, 19% and 43%, respectively) in 11␤-HSD2 activity compared with the untreated rats subjected to ALI (P < 0.05) (Fig. 4C). 3.6. E and P up-regulate ENaC mRNA expression in vivo The expression of ˛-ENaC mRNA decreased by 37% in the ALI model group (P < 0.05, Fig. 5A) compared with the control group. The ˛-ENaC mRNA level increased by 30%, 31% and 43% following treatment with E, P or both E and P, respectively, compared to the control treatment (P < 0.05, Fig. 5A). There were no statistically significant changes in ˇ- or -ENaC mRNA expression between the groups, with the exception of the control group (Fig. 5C). 3.7. E and P up-regulate ENaC protein expression in vivo E and P significantly increased the oleic acid-induced decrease in the ˛-ENaC and -ENaC protein levels, especially in rats treated with E and P (P < 0.05, Fig. 6A, C and D). The ˛-ENaC protein level was increased by approximately 40% and the -ENaC protein level was increased by approximately 30% in the rats treated with E and P compared with the untreated rats subjected to ALI. CBX did not further promote the effect of the combination of E and P on the protein expression of ˛- and -ENaC. There were no statistically significant

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Fig. 2. Changes in the lung coefficient, the lung wet-to-dry weight ratio (W/D) and alveolar fluid clearance (AFC). The W/D (A1–3 ) and the lung coefficient (B1–3 ) were increased and AFC (C1–3 ) was significantly decreased in the ALI model group compared with the control group. Oestrogen and/or progestogen, especially the combination of oestrogen and progestogen, significantly reduced the oleic acid-induced increase in the W/D and the lung coefficient (A1 ,B1 ). AFC in the group treated with both oestrogen and progestogen was nearly twofold that in the ALI group (C1 ). Carbenoxolone together with oestrogen and progestogen did not further decrease the W/D or the lung coefficient or further increase the AFC compared with oestrogen and progestogen (A1 ,B1 ,C1 ). Dexamethasone significantly reduced the W/D and the lung coefficient and enhanced AFC by approximately 15%, 40% and 40%, respectively, compared with ALI alone (A2 ,B2 ,C2 ). Treatment with a low dose of carbenoxolone separately or together with a low dose of oestrogen and/or progestogen further decreased the W/D and the lung coefficient and increased AFC by approximately 5–13%, 20–35% and 20–50%, respectively, compared with ALI alone. Compared with CBX alone, CBX combined with oestrogen or progestogen decreased the W/D and the lung coefficient and increased AFC by approximately 6%, 11% and 10%, respectively. In the group treated with CBX combined with oestrogen and progestogen, the changes were more significant: approximately 9%, 20% and 28%, respectively (A3 ,B3 ,C3 ). Control: control group; ALI: ALI model group; E: oestrogen group; P: progestogen group; E + P: oestrogen and progestogen group; CBX: carbenoxolone group; E + P + CBX: oestrogen, progestogen and carbenoxolone group (A1 ,B1 ,C1 ). Control: control group; ALI: ALI model group; Dexamethasone, dexamethasone group (A2 ,B2 ,C2 ). Control: control group; ALI: ALI model group; CBX: low dose of carbenoxolone group; E + CBX: low doses of oestrogen and carbenoxolone group; P + CBX: low doses of progestogen and carbenoxolone group; E + P + CBX: low doses of oestrogen, progestogen and carbenoxolone group (A3 ,B3 ,C3 ). *Compared with the ALI group, P < 0.05; cˆ ompared with the E group, P > 0.05; a compared with the CBX group, P > 0.05; b compared with the E + CBX group, P > 0.05.

differences in the ˇ-ENaC protein level between the groups (Fig. 6B and D).

3.8. Dexamethasone induced changes in pulmonary oedema, AFC and ENaC expression in lung tissue. Dexamethasone significantly reduced the W/D and the lung coefficient and enhanced AFC by approximately 15%, 40%, and 40%, respectively, compared with ALI alone (Fig. 2A2 –C2 ). Dexamethasone induced an approximately 30% decrease in the ˛-ENaC mRNA and protein levels in ALI lung tissue (Fig. 7). In contrast, no apparent changes in ˇ- or -ENaC mRNA or protein expression were observed (not shown).

3.9. Low doses of CBX, E, and P induce changes in pulmonary oedema, AFC and ENaC expression in lung tissue. Further investigation revealed that after treatment with a low dose of CBX separately or together with a low dose of oestrogen and/or progestogen, CBX further decreased the W/D by approximately 5–13% and the lung coefficient by approximately 20–35%, increased the AFC by approximately 20–50%, and increased ˛-ENaC mRNA and protein expression by approximately 13–30% compared with ALI alone (Fig. 2A3 –C3 , Fig. 8). Compared with CBX alone, CBX combined with E or P further increased AFC by approximately 10%, increased ˛-ENaC mRNA and protein expression by approximately 5%. CBX combined with E and P produced a more significant increase of AFC and ˛-ENaC nearly 28%, 15%, respectively (Fig. 8).

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Fig. 3. Pathological changes: administration of oestrogen (E) and/or progestogen (P), especially both E and P, attenuated oleic acid-induced pulmonary oedema, bleeding and inflammation. A: control group; B: ALI model group; C: oestrogen group; the changes observed in the progestogen group and the CBX group were similar to those observed in the oestrogen group; D: oestrogen + progestogen group; the changes observed in the oestrogen + progestogen + CBX group were similar to those observed in the oestrogen + progestogen group.

Fig. 4. Changes in the serum corticosterone and adrenocorticotropic hormone (ACTH) levels and in 11␤-hydroxysteroid dehydrogenase type 2 (11␤-HSD2) activity in the lung. After treatment with oestrogen and/or progestogen, the levels of corticosterone were slightly increased (A), whereas the expression of ACTH was clearly decreased (B) and 11␤-HSD2 activity was significantly decreased (C). The effects were especially evident in the group treated with both oestrogen and progestogen (A, B and C). The addition of CBX did not further increase the corticosterone levels or decrease the ACTH level or 11␤-HSD2 activity compared to treatment with both E and P. Control: control group; ALI: ALI model group; E: oestrogen group; P: progestogen group; E + P: oestrogen and progestogen group; CBX: carbenoxolone group; E + P + CBX: oestrogen, progestogen and carbenoxolone group. Compared with the ALI group, P < 0.05; cˆ ompared with the E group, P < 0.05.

However, no apparent differences in ˇ- or -ENaC subunit expression were observed.

by 78% (Fig. 9A and B). No substantial changes in the expression of the ˇ- or -ENaC subunit were observed (data not shown).

3.10. Effect of E and P on ENaC mRNA expression in vitro

3.12. Effect of E and P on 11ˇ-HSD2 activity in vitro

Analysis of the mRNA levels in A549 cells revealed that the ion transporter subunits showed a differential expression pattern. ˛ENaC mRNA expression was elevated by 55%, 57% and 84%in the groups treated with E, P, or both E and P, respectively, compared with the control group (Fig. 9A). No substantial changes in the mRNA expression of ˇ- or -ENaC were observed (data not shown).

Both E and P inhibited 11␤-HSD2 activity in A549 cells in a dosedependent manner (Fig. 10A and B). These inhibitory effects were apparent at a concentration of 0.001 ␮M and were significantly augmented at 0.1 ␮M. At 1 ␮M, the effects were most notable after treatment with E or P; the cortisol-to-cortisone conversion percentages in the A549 cells in the E and P groups were 57.43 ± 1.61% and 43.86 ± 2.80% of the control value, respectively (Fig. 10C, P < 0.05). This inhibition was further enhanced by treatment with both E and P, resulting in a cortisol-to-cortisone conversion percentage of 39.30 ± 1.69% of the control value (Fig. 10C).

3.11. Effect of E and P on the ENaC protein levels in vitro A549 cells obtained from the experiments described above were used for Western blot. The signals corresponding to the ˇ- and -ENaC subunits were considerably weaker than the signal corresponding to the ˛-ENaC subunit; however, no substantial changes in expression were observed. The expression of the ˛-ENaC subunit was increased by more than 50% in response to E or P. The combination of E and P increased the expression of the ˛-ENaC subunit

4. Discussion A substantial body of clinical research indicates that controlling pulmonary oedema at an early stage in patients with ARDS promotes AFC, protects against respiratory failure and reduces

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Fig. 5. Changes in the ˛-, ˇ- and -epithelial sodium channel (ENaC) subunit mRNA levels in lung tissue. Oestrogen and/or progestogen decreased the ˛-ENaC mRNA levels in lung tissue subjected to ALI (A). In contrast, no apparent increases in the ˇ- or -ENaC mRNA levels were observed (B,C). Control: control group; ALI: ALI model group; E: oestrogen group; P: progestogen group; E + P: oestrogen and progestogen group; CBX: carbenoxolone group; E + P + CBX: oestrogen, progestogen and carbenoxolone group; M: marker; Lane 1: control; Lane 2: E; Lane 3: P; Lane 4: E + P; Lane 5: CBX; Lane 6: E + P + CBX. *Compared with the ALI group, P < 0.05; cˆ ompared with the E group, P < 0.05.

Fig. 6. Changes in the ˛-, ˇ- and -epithelial Na+ channel (ENaC) subunit protein levels in lung tissue. Oestrogen and/or progestogen, especially the combination of oestrogen and progestogen, promoted the oleic acid-induced decrease in the ˛-ENaC and -ENaC protein levels. The ˛-ENaC protein levels were increased by approximately 40% (A), and the -ENaC protein levels were increased by approximately 30% (C). Control: control group; E: oestrogen group; P: progestogen group; E + P: oestrogen and progestogen group; CBX: carbenoxolone group; E + P + CBX: oestrogen, progestogen and carbenoxolone group; Lane 1: control; Lane 2: E; Lane 3: P; Lane 4: E + P; Lane 5: CBX; Lane 6: E + P + CBX. *Compared with the ALI group, P < 0.05; cˆ ompared with the E group, P < 0.05.

mortality. In the present study, we demonstrated that E and P play a therapeutic role in oleic acid-induced ALI and pulmonary oedema. Previous studies have demonstrated that AFC is associated with ENaC. Enhancing the amiloride-sensitive Na+ current and the membrane density of Na+ channels would promote the function of ENaC

(Wu et al., 2011) and thus remove excess pulmonary oedema fluid from the alveolar space. All three (˛, ˇ and ) subunits of ENaC are necessary for removing pulmonary oedema fluid. Matalon et al. found that ˛-ENaC knockout mice died within 48 h after birth due to an inability to clear alveolar oedema fluid (Matalon et al.,

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Fig. 7. Dexamethasone induced changes in the ˛-epithelial Na+ channel (ENaC) mRNA and protein levels in lung tissue. Dexamethasone decreased ˛-ENaC mRNA and protein expression by approximately 30% in lung tissue following ALI. In contrast, no apparent change in ˇ- or -ENaC mRNA or protein was observed (not shown). Control: control group; ALI: ALI model group; Dexamethasone, dexamethasone group. Compared with the ALI group, P < 0.05.

Fig. 8. Low doses of carbenoxolone, oestrogen and progestogen induced changes in the ˛-epithelial Na+ channel (ENaC) mRNA and protein levels in lung tissue. Treatment with a low dose of carbenoxolone separately or together with a low dose of oestrogen and/or progestogen further increased ˛-ENaC mRNA and protein expression by approximately 13–30% as compared with ALI alone. Compared with CBX alone, CBX combined with oestrogen or progestogen further decreased the ˛-ENaC mRNA and protein levels by approximately 5%. In the group treated with CBX combined with oestrogen and progestogen group, these changes were more significant, nearly 15%. In contrast, no apparent change in ˇ- or -ENaC mRNA or protein expression was observed between the groups (not shown). Control: control group; ALI: ALI model group; CBX: low dose of carbenoxolone group; E + CBX: low doses of oestrogen and carbenoxolone group; P + CBX: low doses of progestogen and carbenoxolone group; E + P + CBX: low doses of oestrogen, progestogen and carbenoxolone group. Compared with the ALI group, P < 0.05; a compared with the CBX group, P > 0.05; b compared with the E + CBX group, P < 0.05.

2002). Hummler et al. observed that knockout mice that did not express the ˛-ENaC subunit died after birth due to defective neonatal fluid clearance and fluid accumulation in the lungs (Hummler et al., 1996). Consistent with this result, lung-specific knockdown of ˛-ENaC using siRNA decreased baseline fluid clearance in rats

(Li and Folkesson, 2006). Genetic variations in ˛-ENaC can also influence AFC (Foxx-Lupo et al., 2011). Adequate ˇ-ENaC expression in alveolar epithelial cells appears to be required for AFC in mice (Randrianarison et al., 2008). The ˇ- and -ENaC subunits are essential for maximal net fluid absorption by distal lung epithe-

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Fig. 9. Changes in the ˛-epithelial Na+ channel (ENaC) mRNA and protein levels in A549 cells. The ˛-ENaC subunit expression level was increased by more than 50% in response to E or P and by more than 70% in response to the combination of oestrogen and progestogen. Control: control group; E: oestrogen group; P: progestogen group; E + P: oestrogen and progestogen group; CBX: carbenoxolone group; E + P + CBX: oestrogen, progestogen and carbenoxolone group; M: marker; Lane 1: control; Lane 2: E; Lane 3: P; Lane 4: E + P; Lane 5: CBX; Lane 6: E + P + CBX. *Compared with the control group, P < 0.05; cˆ ompared with the oestrogen group, P < 0.05.

Fig. 10. Oestrogen and progestogen inhibited 11␤-hydroxysteroid dehydrogenase type 2 (11␤-HSD2) activity in A549 cells. Oestrogen (A) and progestogen (B) inhibited 11␤-HSD2 activity in A549 cells in a dose-dependent manner. This inhibitory effect was observed at a concentration of 0.001 ␮M, and significant changes were observed at 0.1 ␮M. At 1 ␮M, the inhibitory effect was most evident in the group treated with oestrogen and progestogen (A,B). At 1 ␮M, the percentage of cortisol that was converted to cortisone in A549 cells in the oestrogen, progestogen and combined oestrogen and progestogen groups was 57.43 ± 1.61%, 43.86 ± 2.80%, and 39.30 ± 1.69% of the control value, respectively (C). Control: control group; E: oestrogen group; P: progestogen group; E + P: oestrogen and progestogen group; CBX: carbenoxolone group; E + P + CBX: oestrogen, progestogen and carbenoxolone group. *Compared with the control group, P < 0.05.

lia (Elias et al., 2007). However, the ˛-, ˇ- and -ENaC subunits showed distinct changes in the renal connecting tubule (Jacquillet et al., 2013; Zheng et al., 2011), the colon (Bertog et al., 2008; Zeissig et al., 2006), Reissner’s membrane epithelium (Kim et al., 2009), and vascular smooth muscle cells (Jernigan et al., 2009), among other areas including the lung. Therefore, we performed further in vitro and in vivo experiments in our lab and discovered the following. (1) E and P significantly enhanced the oleic acid-induced decrease in AFC and in ˛-ENaC protein and mRNA expression in vivo. After lung injury induced by oleic acid, the ˛-, ˇ- and -ENaC mRNA levels decreased by approximately 40% in the ALI model group, the ENaC protein levels decreased by approximately 30%, and AFC decreased by 60% compared with the control treatment. However, after treatment with E and/or P, AFC was significantly increased and the ˛-ENaC mRNA and protein levels were increased. Further anal-

ysis showed that the change in AFC was more pronounced than the changes in the ˛-ENaC levels; this finding showed that although the change in AFC is primarily associated with the changes in the ˛-ENaC levels, the alteration of the ˛-ENaC levels is not the only factor that is involved in the change in AFC. (2) The expression level of the ˛-ENaC subunit was enhanced in response to E and P in vitro. Although human A549 adenocarcinoma cells are not completely accurate representations of human alveolar epithelial cells, we chose to conduct our experiments using human A549 cells for several reasons. First, primary alveolar epithelial cells die easily and are difficult to culture. Moreover, some cells that are stated to be primary human alveolar epithelial cells have actually been infected by a virus; as a consequence, the structure and the function of these cells are altered, and these cells are no longer true primary cells. Second, the primary focus of our study was to eval-

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uate the expression levels of ENaC, and ENaC is not involved in the proliferation or the differentiation of A549 cells. Comparisons were readily drawn between groups because in vitro experiments were conducted using A549 cells in all groups. Finally, A549 cells are easily cultured at a low cost. (3) There were no substantial changes in ˇ-ENaC mRNA or protein expression in vivo or in vitro. (4) The protein expression of -ENaC displayed a 30% increase in response to E and/or P in vivo, and no substantial changes were observed in vitro. These results showed that the female sex steroids E and P regulate the expression of ENaC in vivo and in vitro. The reason for these distinct changes remains unclear; however, based on the data from the present study, these changes may be related to variability in species, in the extent of sexual maturity, in the ages of the experimental animals, or in the intracellular environment (Boyd and Naray-Fejes-Toth, 2007; Chang et al., 2007; Gambling et al., 2004; Greenlee et al., 2013; Heo et al., 2012; Kienitz et al., 2009; Laube et al., 2011; Yang et al., 2011). The mechanism by which these hormones interact within alveolar epithelial cells to affect ENaC expression remains unknown. Glucocorticoids are widely known to be effective antiinflammatory therapeutic medicines. Additionally, accumulating evidence indicates that glucocorticoids may promote the absorption of fluid from the alveolar space by stimulating ENaC, and this process is indirectly mediated by an increase in serum- and glucocorticoid-regulated kinase (SGK1) expression (Itani et al., 2002; Menniti et al., 2010; Renauld et al., 2010; Vasquez et al., 2008). However, other studies have demonstrated that SGK1 is not required for the regulation of colonic ENaC activity (McTavish et al., 2009; Rexhepaj et al., 2006). Glucocorticoids, which are steroid hormones, have been shown to substantially modify the biophysical properties of ENaC (Lazrak et al., 2000). The activity and the metabolism of these hormones are modulated by 11␤-HSD before they bind to GRs. In our study, we found that exogenous dexamethasone significantly enhanced AFC by approximately 40% and further decreased ˛-ENaC subunit expression by approximately 30% compared with ALI alone. The female sex steroids E and P can act either individually or in combination to increase the serum corticosterone levels and to decrease the conversion of corticosterone to 11-hydroxycorticosterone in lung tissue homogenates. We concluded that lung epithelial cells use more corticosterone in the presence of E and P because these factors reduce the local breakdown of endogenous bioactive glucocorticoids by inhibiting 11␤-HSD2 in the lung. Furthermore, our study demonstrated that E and P inhibited 11␤-HSD2 in a dose-dependent manner in A549 cells. We also found that E and P function either independently or in combination to significantly decrease the serum ACTH concentration. Therefore, the existence of a negative feedback system in the hypothalamic-pituitary-adrenal axis may explain the results obtained from the blood examinations. We conclude that the reduction of 11␤-HSD enzymatic activity induced by female sex steroids suppresses the ACTH concentration in the circulation. CBX is a compound that is derived from liquorice root and that is used for the treatment of digestive tract ulcers, especially stomach ulcers. CBX is structurally similar to natural and synthetic glucocorticoids. However, its affinity for GRs is very low, and CBX is a competitive inhibitor of 11␤-HSD (Naray-Fejes-Toth and FejesToth, 1997; Soro et al., 1997). After further experimentation, we found that E and/or P not only increased the expression of the ˛ENaC subunit but also enhanced AFC, and stimulated 11␤-HSD2 activity; these effects were similar to those of CBX. However, the administration of CBX together with E and P exerted no additional inhibitory effect on AFC, ENaC expression or 11␤-HSD2 activity in either rats or cultured A549 cells. We surmised that this observation may have occurred because the combination effect of E and P exerted maximal effects and, therefore, the addition of CBX did not (and could not) further increase these effects. Thus, we conducted

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further experiments using reduced doses of CBX, E and P and found CBX enhanced the effects of E and P. The expression of the ˛-, ˇ- and -ENaC subunits showed distinct changes after treatment with E and P. In vitro, E and P increased only the expression of the ˛-ENaC subunit and had no effect on ˇ- or -ENaC subunit expression. Alternatively, in vivo, these hormones increased the expression of the -ENaC subunit in addition to the ˛-ENaC subunit. Previous studies have shown that E and P influence the expression of the ˇ-ENaC subunit; however, we did not observe the same findings. This disparity may be related to the species studied, the extent of sexual maturity of the animals studied, the ages of the experimental animals, or the intracellular environment (Boyd and Naray-Fejes-Toth, 2007; Chang et al., 2007; Gambling et al., 2004; Greenlee et al., 2013; Heo et al., 2012; Kienitz et al., 2009; Laube et al., 2011; Yang et al., 2011). Further investigation is needed to explore these possibilities. It has been shown that exogenous dexamethasone may promote the absorption of fluid from alveolar spaces by stimulating ENaC, as demonstrated in our study. Dexamethasone also augments GR expression in the cell membrane (Dagenais et al., 2006; Jiang et al., 2014; Kim et al., 2014, 2009; Mustafa et al., 2004; Nakamura et al., 2002; Xinmin et al., 2006). However, it is unclear whether this increase in GR expression is accompanied by an increase in the endogenous glucocorticoid levels after treatment with exogenous E and P. Thus, further research in this area is necessary. In summary, we conclude that the female steroid hormones E and P may augment ˛-ENaC subunit expression, enhance AFC, and attenuate pulmonary oedema by inhibiting 11␤-HSD2 activity and increasing the active glucocorticoid levels in vivo and in vitro. This new information may open new avenues in the search for a cure for ALI/ARDS. Author contributions Conception and design: Ling Luo and Dao-xin Wang. Financial support: Dao-xin Wang. Collection and assembly of data: Ling Luo and Jia Deng. Data analysis and interpretation: Ling Luo, Jia Deng, Jing He, Wang Deng, and Dao-xin Wang. Manuscript writing: Ling Luo, Dao-xin Wang, and Jia Deng. Acknowledgement This study was supported by grants from the National Natural Science Foundation of China (Grant No. 30971301). I am grateful to all of the members of our laboratory for their invaluable advice and help. References Althaus, M., Clauss, W.G., Fronius, M., 2011. Amiloride-sensitive sodium channels and pulmonary edema. Pulmon. Med. 2011, 830320. Audige, A., Dick, B., Frey, B.M., Frey, F.J., Corman, B., Vogt, B., 2002. Glucocorticoids and 11 beta-hydroxysteroid dehydrogenase type 2 gene expression in the aging kidney. Eur. J. Clin. Invest. 32, 411–420. Bastarache, J.A., Ong, T., Matthay, M.A., Ware, L.B., 2011. Alveolar fluid clearance is faster in women with acute lung injury compared to men. J. Crit. Care 26, 249–256. Berthiaume, Y., Matthay, M.A., 2007. Alveolar edema fluid clearance and acute lung injury. Respir. Physiol. Neurobiol. 159, 350–359. Bertog, M., Cuffe, J.E., Pradervand, S., Hummler, E., Hartner, A., Porst, M., Hilgers, K.F., Rossier, B.C., Korbmacher, C., 2008. Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for liddle’s syndrome. J. Physiol. 586, 459–475. Boyd, C., Naray-Fejes-Toth, A., 2007. Steroid-mediated regulation of the epithelial sodium channel subunits in mammary epithelial cells. Endocrinology 148, 3958–3967. Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.D., Rossier, B.C., 1994. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367, 463–467.

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