Introduction

Pulmonary arterial hypertension (PAH) is a devastating disease that will ultimately lead to right-heart failure and is characterized by pulmonary-selective vascular remodeling and elevated pulmonary artery pressure [1, 2]. PAH is diagnosed when the mean pulmonary arterial pressure is greater than 25 mmHg at rest. However, despite considerable progress toward the understanding of PAH, there is no treatment capable of decreasing the long-term morbidity and mortality rate of patients. This may be attributed to a lack of knowledge regarding the mechanism underlying the development of PAH [3, 4].

PAH is associated with pathological changes including an increase in pulmonary arterial smooth muscle cell (PASMC) proliferation that plays a key role in the development of pulmonary remodeling, as well as an infiltration of inflammatory cells [5–9]. These inflammatory cells, specifically macrophages, have been detected in PAH patients and animal models of PAH [10–13], and thus, the recruitment and activation of macrophages may be involved in PAH development [14–18]. Furthermore, PASMC proliferation has been reported to be macrophage-dependent, although the signaling pathway determining macrophage function during PAH development remains unidentified [19, 20].

Serum–glucocorticoid regulated kinase 1 (SGK1) belongs to the protein kinase A, G, and C (AGC) family of serine–threonine kinases [21]. Under physiological conditions, the majority of cells express low levels of SGK1. However, the expression is much higher under certain pathophysiological conditions, such as in the presence of excess glucocorticoids or mineralocorticoids, inflammation, cell shrinkage, or ischemia [22]. These increases in the expression and activation of SGK1 have been identified in various disorders, including pulmonary fibrosis, diabetic nephropathy, obstructive nephropathy, and liver cirrhosis [22]. We have previously reported that SGK1 expression and activation in macrophages or smooth muscle cells contribute to angiotensin II (Ang-II)-induced cardiac fibrosis or neointima formation in vein grafts [23, 24]. Another study reported that SGK1 plays an important role in redox-sensitive regulation of tissue factor by thrombin and is involved in pulmonary vascular remodeling [25]. However, the expression of SGK1 and the role of this protein kinase in the inflammatory processes associated with PAH remain unclear.

In this present study, MCT-induced PAH animal models were used to determine the role and distribution of SGK1 in PAH development. SGK1 knockout mice and an SGK1 inhibitor were used to further evaluate effects of SGK1 on PAH progression. These models were also used to determine the role of SGK1 in macrophage-dependent PASMC proliferation.

Materials and Methods

Antibodies and Reagents

The primary antibodies against F4/80, α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), phospho-SGK1 and SGK1 were from Abcam (Cambridge, MA, USA); and the antibodies against bromodeoxyuridine (BrdU) and IgG were from Santa Cruz Biotechnology (Paso Robles, CA, USA). ChemMate TM EnVision System/DAB Detection Kits were from Dako (Glostrup, Denmark). MCT (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.1 mol/L HCl, neutralized with 0.1 mol/L NaOH to pH 7.4, and sterilized through a 0.22 μm disk filter. EMD638683 (an SGK1 inhibitor described previously [26]) was synthesized by Haoyuan (Shanghai, China) and dissolved in 1 % dimethyl sulfoxide (DMSO). Platelet-derived growth factor-BB (PDGF-BB) was from PeproTech EC (London, UK).

Animals and Treatment

Sprague–Dawley rats were obtained from the Jackson Laboratory. SGK1 knockout (SGK1−/−) mice were supplied by Florian Lang (Tuebingen, Germany). Mice and rats were housed under specific pathogen-free conditions with a 12:12 h light/dark cycle. All animal care and experimental protocols complied with the Animal Management Rule of the Ministry of Health, People’s Republic of China (Documentation No. 55, 2001), and the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996) and were approved by the Animal Care and Use Committee of Capital Medical University. All animals were maintained with standard laboratory chow and water ad libitum.

PAH was induced in 2-month-old male Sprague–Dawley rats by administering a single subcutaneous injection of MCT (60 mg/kg, n = 12) as reported previously [27]. Rats in the control group were given the vehicle saline (0.5 mL, subcutaneously, n = 12). Six rats in each group were given the SGK1 inhibitor EMD638683 (20 mg/kg) intragastrically once daily starting 2 days prior to MCT treatment [26]. Rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). Right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling were evaluated 21 days after MCT injection. At the age of 10–12 weeks, male SGK1−/− mice and their wild-type (WT) littermates were given MCT in doses of 60 mg/100 g body weight once a week for 8 consecutive weeks by subcutaneous injection to induce PAH [28]. There were eight mice per group. Mice were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) on day 8 after the last MCT administration. Then RVSP, right ventricular hypertrophy, and pulmonary vascular remodeling were evaluated.

Hemodynamic Measurements and Lung Histochemistry

Right ventricular systolic pressure and right ventricular hypertrophy were measured using a modified version of a previously reported method [29]. Briefly, a 23-gauge needle filled with heparinized saline was connected to a pressure transducer and inserted via the diaphragm into the right ventricle (RV). The RVSP was then recorded and measured using a BL-420F Biological Function Experimental System (Chengdu Technology and Market, Sichuan, China). Right ventricular hypertrophy was evaluated using a right ventricular hypertrophy index (RVHI), which measures the wet weight ratio of RV to left ventricle (LV) with septum (S).

After anesthesia, the rats and mice were perfused with PBS through the LV. Lung tissues were removed, fixed in formalin, embedded in paraffin, and sectioned at 5-μm intervals. H&E staining was performed to facilitate visualization of intrapulmonary vessels. To quantitate pulmonary arterial wall thickness, medial wall thickness % (MWT%) (the ratio of the average medial thickness divided by the average vessel radius) was assessed in 30 muscular arteries with external diameter of 50–100 μm per lung section, using Image Pro Plus 3.0 (Eclipse 80i/90i; Nikon, Tokyo, Japan) [29].

Immunohistochemistry and Immunofluorescence Analysis

Immunostaining was performed on lung sections that were permeabilized in 0.3 % Triton X-100 (Sigma-Aldrich) in PBS. Protein was detected by incubating the sections with a primary anti-SGK1 antibody (1:200). Frozen lung tissue sections were incubated with an anti-F4/80 antibody (1:100) and an anti-PCNA antibody (1:200) antibody, followed by incubation with an HRP-conjugated secondary antibody (Dako) and finally reaction with 3,3′-diaminobenzidine. Tissues were visualized using a Nikon 80i microscope, and images were acquired using DS-cooled camera and NIS-Elements Br 3.0 software (Melville, NY, USA) [30].

For immunofluorescence, frozen lung sections (7 μm) were incubated overnight with primary antibodies against F4/80 (1:100) and SGK1 (1:200) at 4 °C and for 1 h with FITC or tetramethylrhodamine isothiocyanate-conjugated secondary antibodies at room temperature (Jackson Immuno Research Laboratories). Sections were viewed using a confocal fluorescence microscope (Leica, Heidelberg, Germany) and a Nikon Labophot 2 microscope [24].

Isolation of Pulmonary Artery Smooth Muscle Cells and Macrophages

Pulmonary artery smooth muscle cells (PASMCs) were isolated from rat distal pulmonary arteries according to a method described previously [29]. The isolated PASMCs were cultured in a 6-well plate (1 × 105 cells per well) containing 1,500 μL of DMEM supplemented with 10 % FBS, 10 mmol/L l-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin and incubated for 3–4 days in 5 % CO2 at 37 °C. One day prior to co-culture, the medium was replaced with DMEM supplemented with 0.3 % FBS to arrest growth.

Bone marrow-derived macrophages (BMDMs) were isolated from the tibias and femurs of 3-month-old WT and SGK1−/− mice as described previously [24]. Briefly, bone marrow was flushed from the femur and tibia and purified through Ficoll-Paque gradient. Cells were then incubated in complete culture medium supplemented with M-CSF (50 ng/mL, Pepro Tech, Rocky Hill, NJ, USA), while half of the BMDMs isolated from WT mice were pre-treated with 50 μmol/L EMD638683, and all BMDMs were incubated for 3 days [26].

Co-culture of PASMCs with Macrophages and PASMC Proliferation Assay

Cells were co-cultured in a 24-well transwell chambers with 0.2 μm-pore-size membranes, with 1 × 104 macrophages seeded in each upper chamber and 2 × 104 PASMCs seeded in each lower chamber. The medium in the co-culture system was DMEM supplemented with 3 % FBS.

After 5 days of co-culture, PASMC proliferation was assessed by BrdU incorporation (10 μmol/L) which was added to the culture medium 4 h before the end point. BrdU was detected by denaturing the cellular DNA and then performing immunofluorescence using a monoclonal anti-BrdU antibody (1:200) overnight at 4 °C. This was followed by an Alexa 488-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, CA, USA) at 37 °C for 30 min. BrdU-positive cells were measured using MetaXpress software (Molecular Devices, Sunnyvale, CA, USA). Treatment of PASMCs with 25 ng/mL platelet-derived growth factor-BB (PDGF-BB) served as a positive control. Fresh cell culture medium (DMEM) with 3 % FBS served used as a negative control.

RNA Analysis

RNA was extracted from whole lungs or macrophages using the Trizol reagent method (Invitrogen), and first-strand cDNA was synthesized with SuperScript II (Invitrogen). The mRNA levels of gene expression were analyzed using qRT-PCR, which was performed with 2 × SYBR master mix (Takara, Otsu, Shiga, Japan) using the Bio-Rad iCycler iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The following primers were used in the PCR: SGK1, fwd: 5′-GGACAGTGGACTGGTGGTG-3′, rev: 5′-GCTGTGTTCCGGCTGTAGA-3′; β-actin, fwd: 5′-TCATCACTATTGGCAACGAGC-3′, rev: 5′-AACAGTCCGCCTAGAAGCAC-3′; and tubulin, fwd: 5′-TCTAACCCGTTGCTATCATGC-3′, rev: 5′-GCCATGTTCCAGGCAGTAG-3′.

Western Blot Analysis

SGK1 and p-SGK1 protein expression was analyzed in BMDMs from SGK1−/− mice and WT mice treated with M-CSF and incubated for 3 days. The macrophages were then lysed with lysis buffer and analyzed by Western blot, as described previously [31]. Protein expression was detected using an anti-phospho-SGK1 antibody (1:1,000) and an anti-SGK1 antibody (1:1,000) followed by incubation with an IR dye-conjugated secondary antibody (1:5,000, Rockland Immunochemicals, Gilbertsville, PA, USA) for 1 h. Images were quantified using an Odyssey infrared imaging system (LI-COR Biosciences Lincoln, NE, USA). Protein content was normalized to the level of GAPDH. All experiments were performed at least 3 times.

Statistical Analysis

Data are here expressed as mean ± SEM. Data analysis was performed using GraphPad software (GraphPad Prism version 5.00 for Windows, GraphPad Software). Statistical analysis was evaluated using an unpaired two-tailed Student’s t test or ANOVA, and P < 0.05 was considered statistically significant.

Results

SGK1 Expression is Increased in PAH

To evaluate the role of SGK1 in the development of PAH, rats were injected with MCT or saline, and the level of SGK1 expression in the lung was determined using real-time RT-PCR and immunohistochemistry (IHC). The mRNA level of SGK1 was upregulated approximately 3.2-fold in the lungs of MCT-injected rats relative to that in the control group (Fig. 1a). IHC analysis showed that SGK1-positive cells had infiltrated into the alveolar space of lungs from MCT-treated rats (Fig. 1b, c).

Fig. 1
figure 1

MCT increases SGK1 expression in lung tissues. Eight-week-old male Sprague–Dawley rats were injected subcutaneously with MCT (60 mg/kg) or saline. a Real-time RT-PCR analysis for mRNA expression of SGK1 in lungs 21 days after MCT injection. *P < 0.05 versus saline control. b Immunohistochemical analysis of SGK1 expression in lung sections with saline injection or MCT injection. Magnification, ×400. Scale bar 25 μm. c Quantitative representation of SGK1-positive cells in 30 randomly selected microscopic fields. Data are graphed as mean ± SEM for n = 6 rats per group. ***P < 0.001

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SGK1 Knockout (SGK1−/−) Mice are Resistant to MCT-Induced PAH and Pulmonary Vascular Remodeling

To determine whether SGK1 plays a role in PAH, MCT was administered to both WT and SGK1 knockout mice. After 8 weeks of MCT treatment, the RVSP, RVHI, and histological changes in pulmonary vasculature (MWT%) were evaluated in both SGK1−/− mice and WT mice. RVSP was elevated from 21.2 ± 2.6 mmHg in WT control (WT/SAL) mice to 34.0 ± 4.1 mmHg in WT/MCT mice (P < 0.05; n = 8), but RVSP was significantly lower in SGK1−/− mice than in WT mice after MCT treatment (Fig. 2a). There was no significant difference in the RVSP between WT control mice (WT/SAL) and SGK1−/− control mice (SGK1−/−/SAL, 20.80 ± 2.08 vs. 21.20 ± 2.60 mmHg; P = 0.9; n = 8). As shown in Fig. 2b, RVHI was significantly increased in WT mice with MCT treatment (0.23 ± 0.02 in WT/MCT vs. 0.17 ± 0.02 in WT/SAL; P < 0.05; n = 8), but no such increase was observed in SGK1−/− mice. After administration of MCT, SGK1−/− mice (SGK1−/−/MCT, 0.16 ± 0.02) showed significantly less in RVHI than WT mice (WT/MCT, 0.23 ± 0.02; P < 0.05; n = 8). No significant difference was found between SGK1−/−/SAL and WT/SAL mice (0.16 ± 0.03 vs. 0.17 ± 0.02; P > 0.05; n = 8).

Fig. 2
figure 2

Knockout of SGK1 attenuates MCT-induced pulmonary arterial hypertension. WT and SGK1−/− mice were injected subcutaneously with saline (control) or MCT (600 mg/kg) every week for 8 weeks. Then the hemodynamics of pulmonary vascular was assessed. a Changes in RVSP. b Changes in RVHI. Results are expressed as mean ± SEM. n = 8. *P < 0.05 versus control. # P < 0.05 versus MCT injection of WT mice

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PAH causes pulmonary vascular remodeling [5–8]. For this reason, this remodeling was evaluated by measuring MWT%. MWT% in vessels of <100 μm was significantly increased in WT/MCT compared with WT/SAL mice (28.2 ± 3.1 vs. 14.3 ± 1.4; P < 0.01; n = 8), but no difference was found between SGK1−/−/MCT- and SGK1−/−/SAL-treated mice (15.8 ± 2.5 vs. 13.7 ± 2.4; P > 0.05; n = 8) (Fig. 3). MWT% was significantly lower in SGK1−/−/MCT than in WT/MCT mice (15.8 ± 2.5 vs. 28.2 ± 3.1; P < 0.05; n = 8) (Fig. 3). Taken together, these results indicated that SGK1-deficient mice were protected against MCT-induced pathological changes in the pulmonary vasculature of mice.

Fig. 3
figure 3

Knockout of SGK1 reduces MCT-induced pulmonary vascular remodeling. Pulmonary vascular remodeling was assessed after 8 weeks of injection with saline (control) or MCT in WT and SGK1−/− mice. a Representative images of lung sections of WT and SGK1−/− mice injected with saline (control) or MCT. Original magnification, ×400. Scale bar 25 μm. b Changes in medial wall thickness % (MWT%) in the lung sections of WT and SGK1−/− mice injected with saline (control) or MCT. Results are expressed as mean ± SEM. n = 8. **P < 0.01 versus control. # P < 0.05 versus MCT injection in WT mice

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Inhibition of SGK1 Activity Ameliorates Right Ventricle Systole Pressure, Right Ventricle Hypertrophy, and Vascular Remodeling in MCT-Induced PAH of Rats

To determine whether inhibition of SGK1 activity prevents progression to PAH, the SGK1 inhibitor, EMD638683, was administered to rats prior to MCT treatment. The resultant pulmonary hemodynamic change and pulmonary vascular remodeling were investigated. EMD638683 or vehicle (DMSO) was administrated daily, starting 2 days before MCT or saline injection. Hemodynamic characteristics showed that EMD638683 treatment attenuated RVSP (15.8 ± 2.5 vs. 28.2 ± 3.1 mmHg; P < 0.05; n = 6) and RVHI (0.27 ± 0.02 vs. 0.41 ± 0.06; P < 0.05; n = 6) compared to vehicle-dosed controls, as shown in Fig. 4. As measured on day 22, MCT was associated with significantly greater medial thickness in the small pulmonary arteries than in controls (71.3 ± 5.1 vs. 23.8 ± 3.9; P < 0.05; n = 6). Also on day 22, this increase was less pronounced in the EMD638683-supplemented group than in the group that received MCT alone (47.0 ± 6.4 vs. 71.3 ± 5.1; P < 0.05; n = 6) (Fig. 5). In this way, inhibition of SGK1 was found to decrease the progression to PAH and thus prevent RVSP, RVHI, and pulmonary vascular remodeling.

Fig. 4
figure 4

SGK1 inhibitor EMD638683 ameliorates progression of MCT-induced PAH in rats. Eight-week-old male Sprague–Dawley rats were injected subcutaneously with saline (control) or MCT (60 mg/kg). Two days earlier, half of the control rats and MCT-injected rats were treated with EMD638683 (20 mg/kg, i.g.) once daily. Control rats received the same volume of vehicle (DMSO). Pulmonary arterial hypertension and pulmonary vascular remodeling were assessed 3 weeks after MCT injection. a Changes in RVSP. b Changes in RV/LV + S (RVHI). Results are expressed as mean ± SEM. n = 6. *P < 0.05 versus control. # P < 0.05 versus MCT injection in rats treated with DMSO

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Fig. 5
figure 5

SGK1 inhibitor (EMD638683) prevents progression of MCT-induced pulmonary vascular remodeling in rats. Pulmonary vascular remodeling was assessed 21 days after injection of saline (control) or MCT in rats pre-treated with EMD638683 or DMSO. a Representative images of lung sections in rats. Original magnification, ×400. Scale bar 50 μm. b Changes in medial wall thickness % (MWT%) in the lung sections of rats. Results are expressed as mean ± SEM. n = 6. *P < 0.05 versus control. ***P < 0.001 versus control. # P < 0.05 versus MCT injection of rats treated with DMSO

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Deficiency or Inhibition of SGK1 Diminish Macrophage Infiltration and Reduce Pulmonary Vascular Cell Proliferation in MCT-Induced PAH

PASMC proliferation is a key event in pulmonary arterial remodeling [9]. Inflammation has been found to exacerbate pulmonary vascular remodeling during PAH [32]. For these reasons, the ability of SGK1 to regulate pulmonary inflammation and vascular cell proliferation in MCT-induced PAH was investigated. There was more macrophage recruitment in the MCT-treated groups of rats, and this effect was suppressed by EMD638683 treatment (Fig. 6a, b). Similarly, SGK1−/− mice showed a less macrophage infiltration after MCT treatment than WT mice (Fig. 6c, d). The expression of PCNA in PASMCs was higher in the MCT-treated group compared to control group, and this increase was diminished by EMD638683 (Fig. 7).

Fig. 6
figure 6

EMD638683 treatment and SGK1 knockout reduced macrophage infiltration in lungs of rats with MCT-induced PAH. Immunohistochemical analysis of the infiltration of F4/80-positive macrophages in lung sections injected with saline or MCT in rats treated with DMSO or EMD638683 and lung sections injected with saline or MCT in WT mice or SGK1−/− mice. a Representative images of lung sections of rats. Arrows indicate F4/80-positive cells. Original magnification, ×400. Scale bar 50 μm. b Quantitative representation of macrophage infiltration. The number of macrophages in 30 randomly selected microscopic fields was quantified using Image-Pro discovery software. Results are expressed as mean ± SEM. n = 6. *P < 0.05 versus control. ***P < 0.001 versus control. # P < 0.05 versus MCT injection of rats treated with DMSO. c Representative images of lung sections of mice. Arrows indicate F4/80-positive cells. Original magnification, ×400. Scale bar 50 μm. d Quantitative representation of macrophage infiltration. The number of macrophages in 30 randomly selected microscopic fields was quantified using Image-Pro discovery software. Results are expressed as mean ± SEM. n = 8. *P < 0.05 versus control. ***P < 0.001 versus control. ### P < 0.001 versus WT mice injected with MCT

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Fig. 7
figure 7

EMD638683 treatment improved pulmonary vascular proliferation in PAH. Histological illustrations of MCT-induced increases in the numbers of PCNA-positive cells in the pulmonary arterial walls of rats. a Representative images of lung sections of rats. Original magnification, ×400. Scale bar 50 μm. b Relative changes in the numbers of PCNA-positive cells in pulmonary arterial walls in a control group treated with DMSO. Arrows indicate PCNA-positive cells. Results are expressed as mean ± SEM. n = 6. ***P < 0.001 versus control. ## P < 0.01 versus MCT injection of rats treated with vehicle

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SGK1 Expression and Activation is Essential for Macrophage-Induced PASMC Proliferation

It has been shown that SGK1 is highly expressed in macrophages found in the hearts of mice with hypertension [24]. For this reason, the expression of SGK1 was examined in macrophages that infiltrate the lungs of rats injected with MCT. The results showed that SGK1 expression was markedly upregulated in macrophages after MCT injection (Fig. 8). Because macrophages can promote smooth muscle cell proliferation, the role of macrophage SGK1 in PASMC proliferation was evaluated [19, 20]. As expected, SGK1 expression was high in macrophages derived from WT mice (Fig. 9a). Next, PASMCs from WT mice were co-cultured with BMDMs from WT or SGK1−/− mice, and PASMC proliferation was determined. As shown in Fig. 9b, co-culture with WT macrophages significantly increased the proliferation of PASMC, while co-culture of SGK1−/− macrophages and WT macrophages treated with SGK1 inhibitor EMD638683 failed to stimulate PASMC proliferation (Fig. 9c).

Fig. 8
figure 8

SGK1 expression is markedly upregulated in macrophages after MCT injection. Double immunofluorescence analysis of macrophage (anti-F4/80) and SGK1 expression in lung sections of rats 21 days after MCT or saline injection. The samples were immunostained with anti-F4/80 (red) and anti-SGK1 (green). Nuclei were counterstained with DAPI (blue). Original magnification, ×400. Scale bar 50 μm (Color figure online)

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Fig. 9
figure 9

SGK1 expression and activation regulate macrophage-mediated PASMC proliferation. BMDMs derived from SGK1−/− mice and WT mice were treated with M-CSF for 3 days. a Western blot analysis of SGK1 and p-SGK1 in BMDMs derived from SGK1−/− mice and WT mice. b Diagrammatic representation of co-culture experiments. Macrophages were located in the upper chambers of transwell inserts, and PASMCs were seeded in the bottom. The medium in the co-culture system was replaced with DMEM supplemented with 3 % FBS. After 5 days of co-culture, PASMC proliferation was assessed using BrdU incorporation assays. c The relative number of BrdU-positive cells was calculated and normalized to the average BrdU incorporation in PASMCs cultured with DMEM. Platelet-derived growth factor-BB (PDGF-BB; 25 ng/mL) served as a positive control, and DMEM served as a negative control. **P < 0.01 versus blank control (DMEM). ***P < 0.001 versus blank control (DMEM). # P < 0.05 versus co-culture with BMDMs derived from WT mice

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Discussion

The data presented here indicate a novel role for SGK1 in the regulation of the inflammatory and proliferative response during PAH. The expression of pulmonary SGK1 was higher in MCT-induced PAH than that in control groups, and SGK1 deficiency or inhibition ameliorated PAH response to MCT injection. Macrophages that expressed SGK1 were able to promote PASMC proliferation, which was lost with SGK1-deficient mice or inhibition with EMD638683.

SGK1 is a downstream effector of the phosphoinositide-3 kinase cascade and is sensitive to various stimuli, such as growth factors like TGF-β [22], steroids and peptide hormones like mineralocorticoids [22], and cytokines like IL-6 [33]. It can be activated by PPAR-γ [34] and mTOR [23]. Some of these stimuli have been reported to participate in the development of MCT-induced PAH. Under certain pathophysiological conditions, upregulated SGK1 expression has been detected in lung tissue [25, 35]. These results are consistent with those of the present study: The level of SGK1 expression was significantly higher in the lungs of PAH animals that had been exposed to MCT than in control group animals (Fig. 1).

BelAiba et al. [25] showed that SGK1 activates NFκB in cultivated PASMCs and that SGK1 may contribute to thrombin-induced vascular remodeling in PAH. While the role of SGK1 in the development of PAH has not been fully defined, the contribution of SGK1 to the PAH response in MCT-induced PAH models was evaluated in SGK1−/− mice and rats treated with an SGK1-specific inhibitor EMD638683 (Figs. 2, 3, 4, 5).

EMD638683 was first described by Ackermann et al. [26]. They tested the effects of EMD638683 on 69 protein kinases by using a biochemical assay. And the activity of SGK1, SGK2, and SGK3 was decreased more than other kinases in the presence of EMD638683. Ackermann et al. [26] also proved the inhibitory effect of EMD638683 on SGK1-dependent phosphorylation of NDRG1 (N-Myc downstream-regulated gene 1) in human cervical carcinoma HeLa cells. In addition, EMD638683 has already been referred to as an SGK1 inhibitor in several other studies [36–38]. As SGK1 was the only isoform of SGK to be reported expressed in lung tissue [25, 35, 39], the effects we observed using EMD638683 could belong to a specific inhibitory effect on SGK1 in lungs.

Deficiency or inhibition of SGK1 reduced the degree of macrophage infiltration in lung tissue (Fig. 6). This is consistent with our previous study in a model of Ang-II-induced cardiac inflammation, we showed that the macrophage infiltration was significant decreased in heart tissue of SGK1 knockout mice compared with WT mice [24]. As we reported before, the possible reasons may involve the effects of survival of infiltrated macrophages, polarization of macrophages, and activation NFκB by SGK1 [24, 40].

The present study also showed that SGK1 expression took place primarily in the alveolar macrophages during MCT-induced PAH, but there was less SGK1 expression in PASMCs (Figs. 1, 8). This suggests that SGK1 may participate in macrophage-mediated inflammation in the pathogenesis of MCT-induced PAH. It has been reported that SGK1 regulates M2 macrophage differentiation by activating the STAT3 pathway and contributing to Ang-II-induced cardiac inflammation [24]. In the present study, SGK1 was found to regulate macrophage activation in the development of PAH. Macrophages are recognized as a type of inflammatory cell with a pro-proliferation function [19, 20] and have been shown to induce PASMC proliferation during PAH [14]. The present study demonstrated the pro-proliferative effect of macrophages on PASMCs with activation of SGK1 (Fig. 9). These results suggest that expression and activation of SGK1 enhanced the pulmonary vascular remodeling by regulating macrophage-mediated inflammation.

Taken together, the findings collected in the present study show the importance of SGK1 expression and activation to inflammation-associated PAH. SGK1 plays a significant role in the development of MCT-induced PAH, and administration of the SGK1 inhibitor, EMD638683, was found to attenuate inflammation-associated PAH. These findings highlight an option for therapeutic intervention of PAH.