Transgenic SPAK knockout mice
The mouse mutants for SPAK were provided by Dr Sung-Sen Yang (National Defense Medical Center, Taipei, Taiwan). SPAK+/– littermates were generated as described previously [23, 24]. SPAK+/– littermates were intercrossed to generate SPAK–/– (SPKA-KO) and wild-type (WT) mice. Approval for the project protocol was obtained from the Institutional Animal Care and Use Committee of the National Defense Medical Center. The mice were bred and maintained in pathogen-free animal facilities at the Laboratory Animal Center of the National Defense Medical Center (Taipei, Taiwan).
Animal model of hyperoxic acute lung injury
The study was performed with 10 to 12-week-old male mice. The mice were randomly allotted into four groups comprising two control groups and two hyperoxic groups with and without SPAK knockout (n = 6 per group; totally 24 mice were used). We achieved random allocation by tossing a coin. In the control group, mice were kept in identical chambers and exposed to room air only (n = 6 per cage). In the hyperoxic group, mice were exposed to >99% oxygen in an airtight chamber with a ventilation flow of 5 L/min for 64 hours (n = 6 per cage). The CO2 concentration was kept at <0.1%, and the temperature was controlled between 25 and 26°C. In each cage, all mice were provided access to food and water ad libitum to minimise potential confounders such as the order of measurements and animal location.
At the end of the animal experiment, the mice were anaesthetized through intraperitoneal injection of Zoletil (Virbac, Carros, France; 35 mg/kg body weight) and Rompun (Bayer, Leverkusen, Germany; 10 mg/kg body weight). We performed a tracheostomy and a median sternotomy under general anesthesia. Then, the mice were euthanized by cardiac puncture before regaining consciousness. After euthanasia, bronchoalveolar lavage ﬂuid (BALF) was obtained by lavaging the left lung twice with 0.5 mL of saline from the tracheostomy. The blood samples and right lung tissues were collected for further evaluation.
The numbers of polymorphonuclear neutrophils and lung injury score in the lung tissue were analyzed. In brief, the lung tissues were fixed, sectioned, and stained with eosin and hematoxylin. Morphological examinations were performed using light microscopy. A minimum of 10 randomly selected fields were examined for neutrophil infiltration in the airspace or vessel wall. The thickening of the alveolar wall was also observed. The lung damage was scored as follows using a four-point scale: none (0), mild (1), moderate (2), or severe (3). The scoring was performed by two pathologists who were blinded to the experimental conditions. The two resulting scores were summed to represent the lung injury score.
Bronchoalveolar lavage ﬂuid protein
The BALF was centrifuged at 200g for 10 minutes to remove all cells and cellular debris. The protein concentrations were determined using a PierceTM BCA protein assay kit (Thermo Fisher Scientiﬁc).
Transmission electron microscopy
Ultrastructural characterization of the cytological alterations was performed by a following a procedure that is detailed in previous publication. Briefly, lung tissue blocks (maximal 1 mm3) were immediately dissected after euthanasia, kept overnight at 4°C in fixative (4% paraformaldehyde and 2.5% glutaraldehyde in 1xPBS; pH 7.4), and postfixed in 1% OsO4 in the same buffer. After dehydration in graded ethanol the blocks were finally embedded in Spurr’s resin (Spurr Low Viscosity Embedding Kit; EMS ®). Semithin sections (0.5μm thick) were cut with a glass knife on a Leica EM UC7 ultramicrotome and stained with toluidine blue. For TEM, ultrathin sections were cut on a Leica® Ultracut UC7 Ultramicrotome with a diamond knife. The sections were stained with uranyl acetate and lead citrate and examined with a FEI Tecnai G2 F20 S-TWIN Electron Microscope at 120 kV.
MLE-12 cells and exposure to hyperoxia
MLE-12 cells, the type II mouse-lung epithelial cell, were purchased from ATCC (Manassas, VA). Cells were cultured in a 50:50 mixed medium of DMEM and Ham’s F-12 supplemented with 4% FBS, insulin (5 μg/mL), transferrin (10 μg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), β-estradiol (10 nM), HEPES (10 nM), and L-glutamine (2 mM). In transgenic studies, MLE-12 cells were cultured on 6-well plates. At 60–75% confluence, transient transfection was carried out using SPAK siRNA (50 nM) (Dharmacon RNA Technologies) as the SPAK-knockdown (SPAK-KD) or siCONTROL Non-Targeting siRNA (50 nM) as the negative control. In the hyperoxic group, cells were placed in an incubator filled with 95% O2 and 5% CO2 at 37°C for 48 hours. In the control group, cells were kept in 21% O2 and 5% CO2 at 37°C for 48 hours.
Transepithelial electric resistance measurements
Electric cell-substrate impedance sensing (ECIS) measurements were performed using 8W1E+ electrode arrays on an ECIS Zθ instrument (Applied Biophysics, Troy, NY). The measurements were performed as described previously [25, 26]. A baseline was established using culture medium (400 μL·well-1). The resistance was recorded in units of Ω at a frequency of 500 Hz. At 48 hours after transfection, the cells were sub-cultured on an ECIS array. Exposure to hyperoxia or normoxia began when the electrode was covered with a monolayer of cells. The ECIS allows for a sensitive determination of the amount of current passing between cells and the resistance of the barrier (Rb) (in units of Ω cm2). Rb is a robust reporter of barrier function .
Transwell monolayer permeability assay
To measure the paracellular permeability, MLE-12 cells were grown as a monolayer in 6.5-mm-diameter transwell filter inserts with a pore size of 3.0 μm (Corning Life Sciences, Lowell, MA). After 48 hours of exposure to hyperoxia, the medium of the upper chamber was replaced with medium containing albumin-fluorescein isothiocyanate (4 kDa, 2 mg/ml). Four hours later, 100-μL samples from the lower chambers were collected and analyzed for fluorescein isothiocyanate intensity using a fluorometric plate reader with an excitation of 494 nm and emission at 520 nm.
Immunoﬂuorescence staining was performed using a published procedure . We treated lung sections with primary rabbit polyclonal antibody, claudin-18 (diluted 1:200, Proteintech, IL, USA), and phosphorylated-SPAK (p-SPAK) (diluted 1:100, OriGene, MD, USA) for immunoﬂuorescent labeling. The secondary antibody was goat anti-mouse IgG-FITC (diluted 1:200, Santa Cruz Biotechnology, USA) and Rhodamine (TRITC) AffiniPure Goat Anti-Rabbit IgG (diluted 1:200, Jackson ImmunoResearch Inc. PA, USA). The slides were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, Inc. CA, USA) and DAPI. Images were obtained using a DeltaVision system (Applied Precision) comprising a wide-ﬁeld inverted microscope (model IX-71; Olympus) with ×60/1.42 Plan Apo N or ×100/1.40 Super-Plan APO objectives.
In-cell western assay
An in-cell western assay was performed using an Odyssey Infrared Imaging System (LICOR Biosciences, NE, USA). The cells were cultured at a density of 1.2 × 104 cells/well in 96-well culture plates and incubated overnight in complete culture medium. At 70% confluence, the cells were pre-treated with ROS inhibitors for 30 minutes and then exposed to hyperoxia for 24 hours. Cells were fixed with refrigerated 75% EtOH and stained with phosphorylated SPAK (Ser311) (diluted 1:200, OriGene, Rockville, MD) and beta-actin (diluted 1:200, Sigma Chemical Company, MO, USA) at 4°C overnight. Anti-rabbit IRDye® 680RD-labeled (1:5000) and anti-mouse IRDye® 800-labeled CW (1:5000) antibodies (LICOR Biosciences, NE, USA) were used as secondary antibodies at room temperature for 1 hour and were detected by the 700 and 800-nm channels, respectively.
Western blot analysis
Western blot analyses were performed based on a standard protocol with the relevant antibodies: claudin-18 (diluted 1:200, Thermo Fisher Scientific Inc, IL, USA), p-SPAK (diluted 1:1000, OriGene, MD, USA), phosphorylated-p38 (p-p38) (diluted 1:1000, Cell Signaling Technology, USA), total-p38 (T-p38) (diluted 1:1000, Cell Signaling Technology, USA), beta-actin (diluted 1:1000, Sigma Chemical Company, MO, USA) and GAPDH (diluted 1:1000, Thermo Fisher Scientific Inc, IL, USA).
Total RNA was isolated using an RNA-spin total RNA extraction kit (Intron Biotechnology, Korea) according to the manufacturer’s instructions. The synthesis of cDNA was performed with 2 µg of RNA using a High-Capacity cDNA Archive Kit (Applied Biosystems, CA, USA). Quantitative real-time PCR was performed for claudin-18 (Mm00517322_m1) and GAPDH (Mm99999915_g1) using TaqMan assays (Applied Biosystems, CA, USA). Each sample was analyzed in triplicate on a 96-well plate, which was centrifuged brieﬂy and placed in a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientiﬁc, MA, USA). The analysis was performed using the following program: 2 min at 50◦C, 10 min at 95◦C, and 40 cycles of 15 s at 95◦C and 1 min at 60◦C. The relative gene expression was calculated using the 2−ΔΔCT method.
We performed statistical analyses using GraphPad Prism v. 5.00 for Windows. All results are expressed as the mean ± standard deviation of the mean. There was no data point that was not included in the analysis. We used one-way analysis of covariance (ANOVA) to compare the differences between the study groups, and then Bonferroni's correction was employed for post-hoc comparisons. A p-value < 0.05 was considered significant.