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17β-Estradiol administration attenuates seawater aspiration-induced acute lung injury in rats
Pulmonary Pharmacology & Therapeutics 24 (2011) 673e681
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17b-Estradiol administration attenuates seawater aspiration-induced acute lung injury in rats Qixin Fan a, c,1, Pengtao Zhao b,1, Jiahuan Li a, Xiaoyan Xie a, Min Xu b, Yong Zhang a, Deguang Mu a, Wangping Li a, Ruilin Sun a, Wei Liu a, Yandong Nan a, Bo Zhang b, Faguang Jin a, *, Zhichao Li b, ** a b c
Department of Respiratory Medicine, Tangdu Hospital, Fourth Military Medical University, Xi’an, PR China Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi’an, PR China Department of Respiratory Medicine, Shaanxi Provincial Corps Hospital, Chinese People’s Armed Police Force, Xi’an, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2011 Received in revised form 8 July 2011 Accepted 14 July 2011
There is very little evidence on the value of administering estrogen in cases of seawater drowning which can induce acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Therefore, this study aimed to investigate whether 17b-estradiol (E2) treatment can attenuate seawater aspiration-induced ALI in rats. In the experiment, ALI was induced by endotracheal instillation of seawater (4 mL/kg) and the rats were then given intraperitoneal injection of E2 (5 mg/kg) 20 min after seawater instillation. Finally, the changes of arterial blood gases which contained hydrogen ion concentration (pH), arterial oxygen tension (PaO2) and arterial carbon dioxide tension (PaCO2) were measured and the measurement of extravascular lung water (EVLW) was observed. The pulmonary histological changes were evaluated by hematoxylineeosin stain. The expression of aquaporins (AQPs) 1, AQP5, and estrogen receptor-b (ERb) was measured by western blotting and immunohistochemical methods. The results showed that compared with normal saline water, seawater aspiration induced more serious ALI in rats which was markedly alleviated by E2 treatment. Meanwhile, the ERb in lung tissues was activated after E2 administration. The seawater aspiration group also presented with severe pulmonary edema which was paralleled with over expressed AQP1 and AQP5. However, the up-regulation of AQP1 and AQP5 was suppressed by the administration of E2, resulting in an attenuation of lung edema. In conclusion, E2 treatment could effectively attenuate seawater aspiration-induced acute lung injury in rats by the downregulation of AQP1 and AQP5. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Aquaporins Acute lung injury 17b-Estradiol Estrogen receptor b Seawater aspiration
1. Introduction Worldwide, some half a million people die each year from drowning, and for each death, there are one to four drowning incidents serious enough to warrant hospitalization [1]. Water aspiration can induce acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), which are characterized by an acute inflammatory process in airspaces and lung parenchyma. These syndromes are manifestations of the loss of barrier function of the alveolar epithelial and pulmonary capillary endothelial cells and thus prompt and aggressive resuscitation attempts are crucial for optimal survival [2]. The sites of drowning
* Corresponding author. Tel./fax: þ86 29 84777425. ** Corresponding author. Tel./fax: þ86 29 84774548. E-mail addresses: [email protected] (F. Jin), [email protected] (Z. Li). 1 These authors contributed to the article equally. 1094-5539/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2011.07.002
are numerous, with the majority cases occurring in freshwater. However, pulmonary injuries induced by seawater drowning, such as lung edema, hypoxemia and inflammatory reaction, are more serious compared to that induced by freshwater drowning [2]. Several studies have shown that administration of estrogen attenuated ALI/ARDS [3e6]. Speyer et al. [6] reported that estrogen suppresses lung inflammation responses in mice. Very recently, Doucet et al. [3] have demonstrated that 17b-estradiol (E2) administration ameliorated lung injury in ovarectomized female rats. Estrogen promotes lung function by supporting alveoli in rodents but probably in humans also [7]. However, there is very little evidence of whether E2 plays an important role in seawater-induced ALI/ARDS. In addition, pulmonary edema is a common feature in early seawater aspiration-induced ALI and careful attention must be taken to the patient’s fluid status [8]. Aquaporins (AQPs), a family of trans-membrane water channel proteins, play a major role in transcellular and trans-epithelial water movement [9]. Previous studies
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have shown that AQP1 and AQP5 contribute to the formation of edema in rat’s lung [10]. In this study, there are mainly two objectives. Firstly, we aimed to evaluate the protective effect of E2 on seawater-induced ALI in rats. Secondly, we attempted to explore the potential mechanism of it, focusing on the changes of AQP1 and AQP5 in particular. 2. Materials and methods 2.1. Animal preparation Male SpragueeDawley rats of clean grade were used in the experiment after one week of adaptive feeding in laboratory, weighing 220 20 g. The animals were obtained from the Animal Center of the Fourth Military Medical University (Xi’an, China). All experiments were approved by the Animal Care and Use Committee of the Fourth Military Medical University and were in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 8523, revised 1985). 2.2. Drugs and reagents Seawater (osmolality 1300 mmol/L, pH 8.2, specific weight 1.05, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl2 2.447 g/L, CaCl2 1.141 g/L, KCl 0.725 g/L, NaHCO3 0.202 g/L, and NaBr 0.083 g/L) was prepared according to the major composition of the East China Sea provided by Chinese Ocean Bureau. A stock solution of 17b-estradiol (E2, Sigma Chemical Co. Pharmaceuticals) was made (5 mg/mL; 100% ethanol). Anti-AQP1, anti-AQP5 and anti-estrogen receptor-b (ERb) polyclonal antibodies were purchased from Chemicon International Inc. (Chemicon, Massachusetts, USA) and anti-b-actin monoclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). SP immunohistochemistry kit was purchased from Zymed Laboratories (USA). All the chemicals and reagents used were from standard commercial sources.
immunohistochemistry studies in each group, and the results would be presented below. 2.4. Arterial blood gas analysis (ABGs) In order to investigate the effect of estrogen on seawater aspiration-induced lung injury, the changes in ABGs which include those of hydrogen ion concentration (pH), arterial oxygen tension (PaO2) and arterial carbon dioxide tension (PaCO2) were measured with a blood gas analyzer. ABGs in different groups were performed at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater instillation. 2.5. Measurement of extravascular lung water (EVLW) The EVLW was measured by a modified method as described by Pearce et al. [11]. Briefly, the left lower lung was placed in a tared container for the determination of water. The lung was homogenized on ice after the addition of an equal weight of NS. A portion of the homogenate was dried to a constant weight (at 60 C for 72 h), and the fractional water content was calculated. Another portion of the homogenate was centrifuged at 12,000 g for 3 h at 4 C to obtain a clear red supernatant. A correction for the residual blood in the lung was made after determination of the hemoglobin concentration in the supernatant and the body by the standard cyanomethemoglobin method. EVLW ¼ (wet lung weight dry lung weight residual blood weight in the lung)/wet lung weight. 2.6. Lung morphology For lung histological studies, the right lower lungs were removed and fixed with 10% formalin, and then embedded in paraffin wax, sliced, and stained with hematoxylineeosin. Microscopic evaluation was performed to characterize lung injury, according to the degree of neutrophil infiltration, hemorrhage and edema in the interstitial and alveolar space.
2.3. Seawater instillation-induced acute lung injury model 2.7. Immunohistochemistry study of AQP1, AQP5 and ERb Seawater Group (SG): The rats were anesthetized with 20% urethane (1.0 g/kg) intraperitoneally. A catheter was inserted into the right carotid artery to administer drugs and obtain blood samples. The rats were maintained in the supine position during experiments with the head elevated 30 degrees. A 1 mL syringe was gently inserted into the trachea approximately 1.5 cm above the carina. Following a 20 min stable baseline period, 4 mL/kg body weight of seawater was instilled within 4 min into both lungs. Normal Saline Group (NS): The rats received the same volume of normal saline instead of seawater, and the other treatment conditions were the same as the SG’s. 17b-Estradiol Group (EG): 5 mg/kg E2 was injected intraperitoneally 20 min after seawater instillation as in SG, and the other treatment conditions were the same as the SG’s. Naive Group (NG): Neither seawater nor normal saline, or E2 was given in this group. And the other treatment conditions were the same as the SG’s. The rats were exsanguinated after euthanasia by aortic transection at the indicated time points. They were then heparinized and blood samples were taken for ABGs at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater instillation. EVLW and pulmonary histological changes were investigated to assess the degree of lung injury, and the parameters would be presented below. At different time points, the rats’ thorax was opened rapidly and lungs were processed separately for western blotting and
Immunohistochemistry was performed to evaluate the constitutive expression of AQP1, AQP5 and ERb during the development and recovery of ALI, as well as the effects of E2 on the expression of AQP1 and AQP5. Several studies have demonstrated that estrogen bind to and activate estrogen receptors which in turn up-regulate the expression of many genes [12]. The rat ER exists as two subtypes, ER alpha and ER beta. However, ERb has a moderate to high expression in lung [13]. Therefore, we performed immunohistochemistry and western blotting to determine the changes of ERb in the lung tissues after E2 administration. Slices of rats lung tissues were obtained as previously described. Sections (5 mm) were deparaffinized, rehydrated in graded alcohol, and blocked by incubating in 0.3% H2O2 for 30 min. Antigen retrieval was performed by treating the slides in citrate buffer in a microwave for 10 min. The slides were incubated for 1 h with normal goat serum, and then incubated in a moist chamber with anti-AQP1 (1:300), anti-AQP5 (1:200) and anti-ERb (1:50) at 4 C overnight. After a complete wash in phosphate buffer solution (PBS), the tissues were incubated in biotin-labeled goat anti-rabbit (1:2000, Sigma) for 30 min at 37 C, rinsed with PBS, and incubated with avidinebiotinperoxidase complex for 30 min at 37 C. The signal was detected using diaminobenzidine (DAB) as the chromogen for 8 min. Then the lung tissues were counterstained with hematoxylin, dehydrated, air-dried and mounted. Goat serum was used as the primary antibody for negative controls for all assays.
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2.8. Western blotting for AQP1, AQP5 and ERb To examine AQP1, AQP5 and ERb expression, lung tissues were harvested and extracted with lysis buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 400 mM NaCl, 10% glycerol, 0.5% NP-40, 5 mM sodium fluoride, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol). The lysates were centrifuged at 12,000 rpm for 30 min at 4 C, and then the supernatants were collected. Equal amounts of protein (50 mg) were separated by SDS-PAGE, transferred to nitrocellulose membrane at 100 V for 2.5 h at low temperature, and blocked with 5% skim milk for 2 h. Subsequently, the membranes were incubated with anti-AQP1 (1:600), anti-AQP5 (1:300), anti-ERb (1:100) and
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anti-b-actin (1:2000) individually at 4 C overnight. After repeated washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000, Sigma), and bands visualized by using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). The results were expressed as the ratio to b-actin level in the same protein samples. 2.9. Statistical analysis All data were presented as means SD of three independent experiments in triplicate. These data were statistically analyzed through the following tests for multiple comparisons: two-way repeated measures analysis of variance (ANOVA) followed by Dunnett’s test for multiple time point observation, Student’s unpaired t-test for single time point observation, and Wilcoxon Utest for histological data. A statistical difference was accepted and considered significant if p < 0.05. 3. Results 3.1. Effects of E2 treatment on ABGs As shown in Fig. 1, seawater aspiration caused a significant decrease in pH (Fig. 1A) and PaO2 (Fig. 1B) and an increase in PaCO2 (Fig. 1C). It was found that the most serious respiratory failure occurred at the time point of 1 h after seawater exposure. The hypoxemia, hypercapnia and metabolic acidosis were the common clinical symptoms of seawater aspiration. However, these pulmonary injuries induced by seawater aspiration were strongly reversed by treatment of E2 at the time point of 2 h, and the effect of the treatment reached its peak at the time point of 4 h after seawater instillation. Therefore the 4 h time point was considered suitable for the following studies (EVLW and pulmonary histological studies). The respiratory failure induced by seawater exposure was more serious than that induced by normal saline aspiration (Fig. 1A, B and C). 3.2. Effect of E2 treatment on extravascular lung water (EVLW) The EVLW was significantly lower in the EG than in the SG (0.515 0.108 vs. 0.697 0.128, p < 0.01, Fig. 2) at the time point of 4 h after seawater instillation. Furthermore, the EVLW was also lower in the NS group than in the SG (0.601 0.097 vs. 0.697 0.128, p < 0.01, Fig. 2).
Fig. 1. The time course of response in respiratory failure parameters in naive group (NG), seawater group (SG), 17b-estradiol (E2) group (EG) and normal saline (NS) group. (A) pH, (B) PaO2 and (C) PCO2. At the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater instillation, blood samples were obtained from the right carotid artery and were measured by a blood gas analyzer. The measurements of pH, PaO2 and PCO2 were then made to assess the extent of respiratory failure. Data are presented as means SD (n ¼ 6), *p < 0.05, **p < 0.01 vs. SG.
Fig. 2. Effects of 17b-estradiol (E2) on seawater aspiration-induced lung edema. At 4 h time point after seawater aspiration, lungs were separated and the ratios of extravascular lung water (EVLW) were determined to assess tissue edema in seawater group (SG), E2 group (EG) and normal saline (NS) group. Data are presented as means SD (n ¼ 6), *p < 0.01 vs. SG.
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Fig. 3. Effects of 17b-estradiol (E2) on seawater aspiration-induced lung histopathological changes (hematoxylineeosin stain; magnification: 400). Histopathological examination of the lungs was carried out at 4 h time point after seawater aspiration. (A) Naive Group (NG). (B) Seawater Group (SG). Hemorrhage, edematous changes of alveolar walls, swelling of alveolar epithelial cells and thickened alveolar wall were observed. (C) E2 Group (EG). Lung injury was significantly alleviated and less damage was observed compared with the SG. (D) Normal saline (NS) Group. The alveolar architecture and histological changes were the minimal in NS Group.
3.3. Histopathology Lung histology injury was also performed on lung tissues at the 4 h time point after seawater administration. As the sections of
hematoxylineeosin stained showed, normal lung tissue structures existed in the NG group (Fig. 3A). Seawater aspiration at 4 h time point induced prominent lesions, such as focal hemorrhage, distortion, and alveolar thickening (Fig. 3B). In contrast, these changes were
Fig. 4. Immunohistochemistry was performed to evaluate the constitutive expression of ERb after seawater instillation. The lung tissues of rats were carried out at 4 h time point following seawater aspiration (DAB stain, 200). (A) Naive Group (NG). (B) Seawater Group (SG). (C) E2 Group (EG). (D) Normal saline (NS) Group.
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expression of ERb at the 4 h time point following seawater instillation in rat lung tissues (Fig. 4C). After E2 treatment, the most serious respiratory failure was significantly reversed at the time point of 2 h, and the effect reached peak at the 4 h time point following from seawater instillation. ERb expression was investigated by western blotting, and the results showed that E2 treatment significantly increased the expressions of ERb by seawater instillation at the time points of 2 h and 4 h (p < 0.05) (Fig. 5A and B).
3.5. Expression of AQPs after seawater aspiration-induced lung injury
Fig. 5. Effects of 17b-estradiol (E2) on ERb in seawater aspiration-induced lung injury. (A) After protein quantitation, western blotting was performed to investigate the ERb content at 2 h or 4 h time point after seawater administration in Seawater Group (SG) and E2 Group (EG). (B) The ratios of ERb protein to b-actin were obtained by density scanning of the film using a Scion image analysis system. Data are presented as means SD (n ¼ 6). *p < 0.01 vs. SG.
Expressions of AQP1 and AQP5 protein levels were evaluated by western blotting at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater aspiration in response to seawater exposure (Fig. 6). The levels of AQP1 and AQP5 were detectable in NG which indicated that AQPs did exist in normal lungs. After seawater administration, the levels of AQP1 and AQP5 increased. The protein levels of AQP1 and AQP5 began to increase
ameliorated by E2 treatment (Fig. 3C). Furthermore, the alveolar architecture and histological changes were minimal in NS (Fig. 3D). 3.4. The expression of ERb on E2 treatment after seawater aspiration As revealed in immunohistochemistry staining, it was seen that ERb was expressed predominantly in the microvascular endothelium and alveoli epithelium in normal rats as reported in the previous report [47] (Fig. 4A). E2 administration induced the
Fig. 6. The expression of AQP1 and AQP5 at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater aspiration. (A) Western blotting was performed to investigate the AQP1 and AQP5 contents from the beginning to 6 h after seawater administration. (B) The ratios of AQP1 and AQP5 protein to b-actin were obtained by density scanning of the western blotting band using an image analysis system. Naive group (NG), Seawater group (SG). Data are presented as means SD (n ¼ 6). *p < 0.05, **p < 0.01 vs. NG.
Fig. 7. Effects of 17b-estradiol (E2) on AQP1 and AQP5 in seawater aspiration-induced lung injury. (A) After protein quantitation, western blotting was performed to investigate AQP1 and AQP5 contents at 2 h or 4 h time point in seawater group (SG) and E2 group (EG). (B) The ratio of AQP1 protein to b-actin. (C) The ratio of AQP5 protein to b-actin. All the ratios were obtained by density scanning of the western blotting band using an image analysis system. Data are presented as means SD (n ¼ 6). *p < 0.05 vs. SG.
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Fig. 8. Immunohistochemistry was performed to evaluate the constitutive expression of AQP1 after seawater instillation. The lung tissues of rats were carried out at 4 h time point following seawater aspiration (DAB stain, 200). (A) NG; (B) SG; (C) EG; (D) NS Group.
Fig. 9. Immunohistochemistry was performed to evaluate the constitutive expression of AQP5 after seawater instillation. The lung tissue was carried out at 4 h time point following seawater aspiration (DAB stain, 200). (A) NG; (B) SG; (C) EG; (D) NS Group.
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soon after seawater aspiration peaked at 2e4 h, and dropped at 6 h (Fig. 6A and B). 3.6. Effects of E2 on AQP1 and AQP5 expressions in seawater aspiration-induced ALI We also evaluated the effect of E2 on AQP1 and AQP5 protein expressions by immunohistochemistry and western blotting. AQP1 and AQP5 expressions were examined on lung samples collected from NG, SG and EG. We chose 2 h and 4 h as the time points as the expressions of AQP1 and AQP5 were at their highest value after seawater exposure. The data showed that E2 administration inhibited the elevation of AQP1 and AQP5 induced by seawater instillation at the time points of 2 h and 4 h (p < 0.05) (Fig. 7A, B and C). Immunohistochemistry was performed to evaluate the constitutive expression of AQP 1 and AQP5 at the time point of 4 h after seawater instillation. It was observed that AQP1 was expressed predominantly in the microvascular endothelium and AQP5 was expressed predominantly in the lung epithelium of the NG group (Figs. 8A and 9A). Moreover, an enhanced AQP1 and AQP5 staining was found and the distribution of AQPs-positive cells was greater in the lungs after seawater aspiration at the time point of 4 h (Figs. 8B and 9B). Compared with SG, E2 treatment observably decreased the expression of AQP1 and AQP5 in lung tissues at the time point of 4 h (Figs. 8C and 9C).
Fig. 10. The expression of AQP1 and AQP5 in seawater group (SG) and normal saline (NS) group. (A) After protein quantitation, western blotting was performed to investigate the AQP1 and AQP5 contents at 4 h time point in the SG and NS group. (B) The ratios of AQP1 and AQP5 protein to b-actin were obtained by density scanning of the western blotting band using an image analysis system. Data are presented as means SD (n ¼ 6). *p < 0.01 vs. SG.
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3.7. The expression of AQP1 and AQP5 in SG and NS groups Next, western blotting and immunohistochemistry were applied to evaluate the AQP1 and AQP5 protein levels at the time point of 4 h after seawater/normal saline instillation. In NS group, the normal saline didn’t induce obvious increase of AQP1 and AQP5 (Figs. 8D and 9D) in lung tissues and the staining was close to NG (Figs. 8A and 9A). The western blotting data also showed that compared with SG, the expressions of AQP1 and AQP5 were lower in NS group (Fig. 10A and B). 4. Discussion In this study, we investigated the role of estrogen for early seawater instillation-induced ALI with a rat model. The results showed that intratracheal instillation of seawater (4 mL/kg) impaired arterial blood gas in a short time with a significant decrease in pH and PaO2 and an increase in PaCO2, and induced serious pulmonary edema and histopathologic changes in lung tissues. Meanwhile, E2 treatment effectively inhibited the seawater-induced lung injury. Numerous clinical and experimental studies have suggested that females tolerate trauma-hemorrhage and sepsis better than males [14,15]. Sex differences in certain aspects of lung function in experimental animals have been documented [16e18]. Female rats were more resistant to shock [19,20] and carrageenan-induced lung injury [21] than their male counterparts. In addition, females are better able to adjust to hypobaria [22] and more resistant to infection than males [23e26]. When studying lung physiology and disease, it is important to consider sex and hormonal status as modifying factors, because estrogen plays a major role in the lung both physiologically and pathophysiologically in animals [27]. Previous results have shown that E2 was effective for the treatment of pulmonary diseases [3,6,28,29]. The evidence presented here also indicates that E2 therapy was effective for early ALI. Like all steroid hormones, estrogens readily diffuse across the cell membrane. Once inside the cell, they bind to and activate estrogen receptors which in turn up-regulate the expression of many genes [12]. In recent years, newer concepts of sex steroid hormone receptor signaling have emerged, including rapid cellular activation pathways that do not involve the direct alteration of gene transcription [30]. Huang-Ping Yu et al. demonstrated that estrogen attenuated lung injury by the activation of ERa, ERb or both receptors [31]. Doucet’s recent work has shown that tissues with higher densities of ERb than ERa, such as the lung, are better protected from trauma hemorrhagic shock-induced injury by ERb than ERa agonists [3]. Interestingly, our results showed that ERb increased in 17b-Estradiol Group (EG) indicating that the protective effect of estrogen on ALI was conducted by ERb. Seawater is a hyperosmolar fluid and its NaCl concentration is 3e3.5％, about 3 fold of that of physiologic saline [32]. Our results showed that compared with normal saline water, seawater aspiration induced more serious ALI in rats which was characterized by more severe blood gas index, pulmonary edema and histopathologic changes. These differences suggested that the hyperisotonic pressure might play an important role in seawater aspirationinduced lung injury in rat. Our data showed that AQP1 and AQP5 increased after seawater endotracheal instillation, but no obvious changes were found in NS group. This is in agreement with other studies which have shown that hypertonicity could induce the expression of AQP1 and AQP5 [33,34]. Additional indirect evidence indicated that the regulation of AQP expression in an adult lung by osmotic stress was one of physiologic roles in respiratory physiology [35]. The findings in our study also indicated that hyperisotonic pressure might be an
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important activator of AQP1 and AQP5 in rat airway epithelium. However, the role of AQP1 and AQP5 in ALI is still controversial. AQP1 and AQP5 expressions were shown to be reduced in rodent lungs following adenoviral infection [36], lipopolysaccharideinduced lung injury [37], and exposure to TNF-a [38]. Meanwhile, AQP5 expression was found increased after bleomycin exposure [39]. Therefore, further experiments are needed to study the role of AQP1 and AQP5 in ALI. Phenotype analysis of transgenic knockout mice lacking AQPs has suggested a role from them in the lungs and airways. AQP1 and AQP5 may provide the principal route for osmotically driven water transport between airspace and capillary compartments. However, alveolar fluid clearance (AFC) in the neonatal and adult lung does not appear to be affected by the deletion of AQPs, as well as lung fluid accumulation in experimental models of lung injury [40e44]. This evidence indicates that AQP1 and AQP5 contribute to the formation of edema, but not the response to AFC in rat lungs. Our results showed that the expression of AQP1 and AQP5 significantly increased in the lung tissues of the rats after seawater instillation and reached its peak at the time points of 2 h and 4 h. Osmotically driven water transport across cell membranes is the principle mechanism of fluid transport [45]. The increased AQPs expression could cause more water from cellular and vascular compartments to flow into interstitium and alveolar spaces because of the hyperosmolality of seawater, which may increase the pleural effusion and pulmonary edema. Coincidentally, the time course of EVLW was paralleled with the change of AQP1 and AQP5 expressions after either seawater or normal saline aspiration, which further confirmed that the up-regulation of AQPs expression contributed to the development of lung edema. There was little information regarding the influence of gender hormones on pulmonary edema. In an early study, Farhat et al. [46] reported that estradiol treatment prevented the increase in lung wet to dry weight ratio. In the present model of lung injury, we found that E2 administration could alleviate seawater aspirationinduced pulmonary edema. This effect may be attributed to the observed decrease in extravascular lung water. Furthermore, E2 treatment reversed the up-regulation of AQP1 and AQP5 expressions induced by seawater aspiration. The inhibition of AQP1 and AQP5 could reduce the water exosmosis from cellular and vascular compartments to interstitial after seawater aspiration, leading to the recovery from severe pulmonary edema. This finding indicated that E2 might be useful to treat seawater drowning by downregulating the increased AQP1 and AQP5.
5. Conclusions In conclusion, data in this study showed that compared with normal saline water, seawater aspiration can induce more serious ALI in rats and that E2 treatment could alleviate seawater-induced ALI in rat lung and the effect of E2 may be mediated via the activation of ERb. Meanwhile, seawater aspiration could induce more severe pulmonary edema with AQP1 and AQP5 being up-regulated. The up-regulated AQP1 and AQP5 were suppressed by administration of E2, resulting in an attenuation of lung edema. These results suggest a new mechanism of seawater-induced ALI, and highlighted the important role of AQP1 and AQP5 during the formation of seawater-induced pulmonary edema. This study suggests that E2 treatment may be beneficial to patients with seawater drowning-induced ALI. However, further investigations on the effect of E2 on clinical medicine are called for. Competing interests The authors declare that they have no competing interests.
Acknowledgments This work was supported by grants from the Military Key Projects in the 11th Five-year Plan of PR China (Project No. 08G102), Shaanxi Province Key Projects (Project No. 2008K14-08). References [1] Brenner Ruth A. Prevention of drowning in infants, children, and adolescents. Pediatrics 2003;112(2):440e5. [2] Salomez F, Vincent JL. Drowning: a review of epidemiology, pathophysiology, treatment and prevention. Resuscitation 2004;63(3):261e8. [3] Doucet D, Badami C, Palange D, Bonitz RP, Lu Q, Xu DZ, et al. Estrogen receptor hormone agonists limit trauma hemorrhage shock-induced gut and lung injury in rats. PLoS One 2010;5(2):e9421. [4] Kan WH, Hsu JT, Schwacha MG, Choudhry MA, Bland KI, Chaudry IH. Estrogen ameliorates trauma-hemorrhage-induced lung injury via endothelial nitric oxide synthase-dependent activation of protein kinase G. Ann Surg 2008; 248(2):294e302. [5] Hsieh YC, Frink M, Hsieh CH, Choudhry MA, Schwacha MG, Bland KI, et al. Downregulation of migration inhibitory factor is critical for estrogenmediated attenuation of lung tissue damage following trauma-hemorrhage. Am J Physiol-Lung C 2007;292(5):L1227e32. [6] Speyer CL, Rancilio NJ, McClintock SD, Crawford JD, Gao H, Sarma JV, et al. Regulatory effects of estrogen on acute lung inflammation in mice. Am J Physiol-Cell Ph 2005;288(4):C881e90. [7] Massaro D, Massaro GD. Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice. Am J Physiol-Lung C 2004;287(6):L1154e9. [8] Ames Nancy. Caring for the patient with complex respiratory disorders. 36:1079e126. [9] Agre Peter, King Landon S, Yasui Masato, Guggino Wm B, Ottersen Ole Petter, Fujiyosh Yoshinori, et al. Aquaporin water channels e from atomic structure to clinical medicine. J Physiol 2002;542(1):3e16. [10] Verkman AS. Seawater exposure-induced lung injury. Resp Physiol Neurobiol 2007;159:324e30. [11] Pearce ML, Yamashita J, Beazell J. Measurement of pulmonary edema. Circ Res 1965;16(5):482e8. [12] Whitehead SA, Nussey S. Endocrinology: an integrated approach. Oxford: BIOS, Taylor & Francis; 2001. [13] Kuiper GGJM, Carlsson B, Grien K, Enmark E, Haggblad J, Nilsson S, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors a and b. Endocrinology 1997;138(3):863e70. [14] George RL, McGwin Jr G, Metzger J, Chaudry IH, Rue III LW. The association between gender and mortality among trauma patients as modified by age. J Trauma 2003;54:464e71. [15] Deitch EA, Livingston DH, Lavery RF, Monaghan SF, Bongu A, Machiedo GW. Hormonally active women tolerate shock-trauma better than do men: a prospective study of over 4000 trauma patients. Ann Surg 2007;246:447e53. [16] Carey MA, Card JW, Bradbury JA, Moorman MP, Haykal-Coates N, Gavett SH, et al. Spontaneous airway hyperresponsiveness in estrogen receptor-alphadeficient mice. Am J Respir Crit Care Med 2007;175:126e35. [17] Reinhard C, Eder G, Fuchs H, Ziesenis A, Heyder J, Schulz H. Inbred strain variation in lung function. Mamm Genome 2002;13:429e37. [18] Bates JHT, Mitzner W. Point:counterpoint lung impedance measurements are/ are not more useful than simpler measurements of lung function in animal models of pulmonary disease. J Appl Physiol; April 12, 2007. doi:10.1152/ japplphysiol.00369.02007. [19] Adams Jr CA, Magnotti LJ, Xu DZ, Lu Q, Deitch EA. Acute lung injury after hemorrhagic shock is dependent on gut injury and sex. Am Surg 2000;66: 905e13. [20] Caruso JM, Xu DZ, Lu Q, Dayal SD, Deitch EA. The female gender protects against pulmonary injury after trauma hemorrhagic shock. Surg Infect (Larchmt) 2001;2:231e40. [21] Cuzzocrea S, Mazzon E, Sautebin L, Serraino I, Dugo L, Calabro G, et al. The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat. Mol Med 2001;7:478e87. [22] McMurtry IF, Frith CH, Will DH. Cardiopulmonary responses of male and female swine to simulated high altitude. J Appl Physiol 1973;35:459e62. [23] Offner PJ, Moore EE, Biffl WL. Male gender is a risk factor for major infections after surgery. Arch Surg 1999;134:935e40. [24] Schröder J, Kahlke V, Staubach KH, Zabel P, Stuber F. Gender differences in human sepsis. Arch Surg 1998;133:1200e5. [25] Wichmann MW, Inthorn D, Andress HJ, Schildberg FW. Incidence and mortality of severe sepsis in surgical intensive care patients: the influence of patient gender on disease process and outcome. Intens Care Med 2000;26: 167e72. [26] Angele MK, Schwacha MG, Ayala A, Chaudry IH. Effect of gender and sex hormones on immune responses following shock. Shock 2000;14:81e90. [27] Carey Michelle A, Card Jeffrey W, Voltz James W, Germolec Dori R, Korach Kenneth S, Zeldin Darryl C. The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies. Am J Physiol-Lung C 2007;293:L272e8.
Q. Fan et al. / Pulmonary Pharmacology & Therapeutics 24 (2011) 673e681 [28] Yang SJ, Chen HM, Hsieh CH, Hsu JT, Yeh CN, Yeh TS, et al. Akt pathway is required for oestrogen-mediated attenuation of lung injury in a rodent model of cerulein-induced acute pancreatitis. Injury 2010;13 [Epub ahead of print]. [29] Carey MA, Card JW, Voltz JW, Germolec DR, Korach KS, Zeldin DC. The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies. Am J Physiol-Lung C 2007;293(2):L272e8. [30] Mendelsohn ME, Karas RH. Molecular and cellular basis of cardiovascular gender differences. Science 2005;308:1583e7. [31] Yu H-P, Hsieh Y-C, Suzuki T, Shimizu T, Choudhry MA, Schwacha MG, et al. Salutary effects of estrogen receptor-b agonist on lung injury after traumahemorrhage. Am J Physiol-Lung C 2006;290(5):L1004e9. [32] Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000;342(18):1334e49. [33] Umenishi Fuminori, Schrier Robert W. Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene. J Biol Chem 2003; 278(18):15765e70. [34] Hoffert Jason D, Leitch Virginia, Agre Peter, Hoffert JD. Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway. J Biol Chem 2000;275(12):9070e7. [35] King LS, Nielsen S, Agre P, Brown RH. Decreased pulmonary vascular permeability in aquaporin-1-null humans. Proc Natl Acad Sci U S A 2002;99: 1059e63. [36] Towne JE, Harrod KS, Crane CM, Menon AG. Decreased expression of aquaporin (AQP)1 andAQP5 in mouse lung after acute viral infection. Am J Respir Cell Mol Biol 2000;22:34e44. [37] Jiao G, Li E, Yu R. Decreased expression of AQP1 and AQP5 in acute injured lungs in rats. Chin Med J (Engl) 2002;115:867e963.
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[38] Towne JE, Krane CM, Bachurski CJ, Menon AG. Tumor necrosis factor-a inhibits aquaporin 5 expression in moue lung epithelial cells. J Biol Chem 2001;276: 18657e64. [39] Gabazza EC, Kasper M, Ohta K, Keane M, D’Alessandro-Gabazza C, Fujimoto H, et al. Decreased expression of aquaporin-5 in bleomycin-induced lung fibrosis in the mouse. Pathol Int 2004;54:774e80. [40] Bai C, Fukuda N, Song Y, Ma T, Matthay Ma, Verkman AS. Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice. J Clin Invest 1999;103(4): 555e61. [41] Verkman AS. Physiological importance of aquaporins: lessons from knockout mice. Curr Opin Nephrol Hypertens 2000;9:517e22. [42] Verkman AS, Matthay MA, Song Y. Aquaporin water channels and lung physiology. Am J Physiol-Lung C 2000;278:L867e79. [43] Verkman AS, Yang B, Song Y, Manley GT, Ma T. Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice. Exp Physiol 2000;85:S233e41. [44] Zemans Rachel L, Matthay Michael A. Bench-to-bedside review: the role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury. Crit Care 2004;8:469e77. [45] Borok Z, Lubman RL, Danto SI, Zhang XL, Zabski SM, King LS, et al. Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5. Am J Respir Cell Mol Biol 1998;18:554e61. [46] Farhat MY, Chen MF, Bhatti T, Iqbal A, Cathapermal S, Ramwell PW. Protection by oestradiol against the development of cardiovascular changes associated with monocrotaline pulmonary hypertension in rats. Br J Pharmacol 1993; 110(2):719e23. [47] Saunders PT, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinology 1997;154:R13e6.
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Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt
17b-Estradiol administration attenuates seawater aspiration-induced acute lung injury in rats Qixin Fan a, c,1, Pengtao Zhao b,1, Jiahuan Li a, Xiaoyan Xie a, Min Xu b, Yong Zhang a, Deguang Mu a, Wangping Li a, Ruilin Sun a, Wei Liu a, Yandong Nan a, Bo Zhang b, Faguang Jin a, *, Zhichao Li b, ** a b c
Department of Respiratory Medicine, Tangdu Hospital, Fourth Military Medical University, Xi’an, PR China Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi’an, PR China Department of Respiratory Medicine, Shaanxi Provincial Corps Hospital, Chinese People’s Armed Police Force, Xi’an, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2011 Received in revised form 8 July 2011 Accepted 14 July 2011
There is very little evidence on the value of administering estrogen in cases of seawater drowning which can induce acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Therefore, this study aimed to investigate whether 17b-estradiol (E2) treatment can attenuate seawater aspiration-induced ALI in rats. In the experiment, ALI was induced by endotracheal instillation of seawater (4 mL/kg) and the rats were then given intraperitoneal injection of E2 (5 mg/kg) 20 min after seawater instillation. Finally, the changes of arterial blood gases which contained hydrogen ion concentration (pH), arterial oxygen tension (PaO2) and arterial carbon dioxide tension (PaCO2) were measured and the measurement of extravascular lung water (EVLW) was observed. The pulmonary histological changes were evaluated by hematoxylineeosin stain. The expression of aquaporins (AQPs) 1, AQP5, and estrogen receptor-b (ERb) was measured by western blotting and immunohistochemical methods. The results showed that compared with normal saline water, seawater aspiration induced more serious ALI in rats which was markedly alleviated by E2 treatment. Meanwhile, the ERb in lung tissues was activated after E2 administration. The seawater aspiration group also presented with severe pulmonary edema which was paralleled with over expressed AQP1 and AQP5. However, the up-regulation of AQP1 and AQP5 was suppressed by the administration of E2, resulting in an attenuation of lung edema. In conclusion, E2 treatment could effectively attenuate seawater aspiration-induced acute lung injury in rats by the downregulation of AQP1 and AQP5. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Aquaporins Acute lung injury 17b-Estradiol Estrogen receptor b Seawater aspiration
1. Introduction Worldwide, some half a million people die each year from drowning, and for each death, there are one to four drowning incidents serious enough to warrant hospitalization [1]. Water aspiration can induce acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), which are characterized by an acute inflammatory process in airspaces and lung parenchyma. These syndromes are manifestations of the loss of barrier function of the alveolar epithelial and pulmonary capillary endothelial cells and thus prompt and aggressive resuscitation attempts are crucial for optimal survival [2]. The sites of drowning
* Corresponding author. Tel./fax: þ86 29 84777425. ** Corresponding author. Tel./fax: þ86 29 84774548. E-mail addresses: [email protected] (F. Jin), [email protected] (Z. Li). 1 These authors contributed to the article equally. 1094-5539/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2011.07.002
are numerous, with the majority cases occurring in freshwater. However, pulmonary injuries induced by seawater drowning, such as lung edema, hypoxemia and inflammatory reaction, are more serious compared to that induced by freshwater drowning [2]. Several studies have shown that administration of estrogen attenuated ALI/ARDS [3e6]. Speyer et al. [6] reported that estrogen suppresses lung inflammation responses in mice. Very recently, Doucet et al. [3] have demonstrated that 17b-estradiol (E2) administration ameliorated lung injury in ovarectomized female rats. Estrogen promotes lung function by supporting alveoli in rodents but probably in humans also [7]. However, there is very little evidence of whether E2 plays an important role in seawater-induced ALI/ARDS. In addition, pulmonary edema is a common feature in early seawater aspiration-induced ALI and careful attention must be taken to the patient’s fluid status [8]. Aquaporins (AQPs), a family of trans-membrane water channel proteins, play a major role in transcellular and trans-epithelial water movement [9]. Previous studies
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have shown that AQP1 and AQP5 contribute to the formation of edema in rat’s lung [10]. In this study, there are mainly two objectives. Firstly, we aimed to evaluate the protective effect of E2 on seawater-induced ALI in rats. Secondly, we attempted to explore the potential mechanism of it, focusing on the changes of AQP1 and AQP5 in particular. 2. Materials and methods 2.1. Animal preparation Male SpragueeDawley rats of clean grade were used in the experiment after one week of adaptive feeding in laboratory, weighing 220 20 g. The animals were obtained from the Animal Center of the Fourth Military Medical University (Xi’an, China). All experiments were approved by the Animal Care and Use Committee of the Fourth Military Medical University and were in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 8523, revised 1985). 2.2. Drugs and reagents Seawater (osmolality 1300 mmol/L, pH 8.2, specific weight 1.05, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl2 2.447 g/L, CaCl2 1.141 g/L, KCl 0.725 g/L, NaHCO3 0.202 g/L, and NaBr 0.083 g/L) was prepared according to the major composition of the East China Sea provided by Chinese Ocean Bureau. A stock solution of 17b-estradiol (E2, Sigma Chemical Co. Pharmaceuticals) was made (5 mg/mL; 100% ethanol). Anti-AQP1, anti-AQP5 and anti-estrogen receptor-b (ERb) polyclonal antibodies were purchased from Chemicon International Inc. (Chemicon, Massachusetts, USA) and anti-b-actin monoclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). SP immunohistochemistry kit was purchased from Zymed Laboratories (USA). All the chemicals and reagents used were from standard commercial sources.
immunohistochemistry studies in each group, and the results would be presented below. 2.4. Arterial blood gas analysis (ABGs) In order to investigate the effect of estrogen on seawater aspiration-induced lung injury, the changes in ABGs which include those of hydrogen ion concentration (pH), arterial oxygen tension (PaO2) and arterial carbon dioxide tension (PaCO2) were measured with a blood gas analyzer. ABGs in different groups were performed at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater instillation. 2.5. Measurement of extravascular lung water (EVLW) The EVLW was measured by a modified method as described by Pearce et al. [11]. Briefly, the left lower lung was placed in a tared container for the determination of water. The lung was homogenized on ice after the addition of an equal weight of NS. A portion of the homogenate was dried to a constant weight (at 60 C for 72 h), and the fractional water content was calculated. Another portion of the homogenate was centrifuged at 12,000 g for 3 h at 4 C to obtain a clear red supernatant. A correction for the residual blood in the lung was made after determination of the hemoglobin concentration in the supernatant and the body by the standard cyanomethemoglobin method. EVLW ¼ (wet lung weight dry lung weight residual blood weight in the lung)/wet lung weight. 2.6. Lung morphology For lung histological studies, the right lower lungs were removed and fixed with 10% formalin, and then embedded in paraffin wax, sliced, and stained with hematoxylineeosin. Microscopic evaluation was performed to characterize lung injury, according to the degree of neutrophil infiltration, hemorrhage and edema in the interstitial and alveolar space.
2.3. Seawater instillation-induced acute lung injury model 2.7. Immunohistochemistry study of AQP1, AQP5 and ERb Seawater Group (SG): The rats were anesthetized with 20% urethane (1.0 g/kg) intraperitoneally. A catheter was inserted into the right carotid artery to administer drugs and obtain blood samples. The rats were maintained in the supine position during experiments with the head elevated 30 degrees. A 1 mL syringe was gently inserted into the trachea approximately 1.5 cm above the carina. Following a 20 min stable baseline period, 4 mL/kg body weight of seawater was instilled within 4 min into both lungs. Normal Saline Group (NS): The rats received the same volume of normal saline instead of seawater, and the other treatment conditions were the same as the SG’s. 17b-Estradiol Group (EG): 5 mg/kg E2 was injected intraperitoneally 20 min after seawater instillation as in SG, and the other treatment conditions were the same as the SG’s. Naive Group (NG): Neither seawater nor normal saline, or E2 was given in this group. And the other treatment conditions were the same as the SG’s. The rats were exsanguinated after euthanasia by aortic transection at the indicated time points. They were then heparinized and blood samples were taken for ABGs at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater instillation. EVLW and pulmonary histological changes were investigated to assess the degree of lung injury, and the parameters would be presented below. At different time points, the rats’ thorax was opened rapidly and lungs were processed separately for western blotting and
Immunohistochemistry was performed to evaluate the constitutive expression of AQP1, AQP5 and ERb during the development and recovery of ALI, as well as the effects of E2 on the expression of AQP1 and AQP5. Several studies have demonstrated that estrogen bind to and activate estrogen receptors which in turn up-regulate the expression of many genes [12]. The rat ER exists as two subtypes, ER alpha and ER beta. However, ERb has a moderate to high expression in lung [13]. Therefore, we performed immunohistochemistry and western blotting to determine the changes of ERb in the lung tissues after E2 administration. Slices of rats lung tissues were obtained as previously described. Sections (5 mm) were deparaffinized, rehydrated in graded alcohol, and blocked by incubating in 0.3% H2O2 for 30 min. Antigen retrieval was performed by treating the slides in citrate buffer in a microwave for 10 min. The slides were incubated for 1 h with normal goat serum, and then incubated in a moist chamber with anti-AQP1 (1:300), anti-AQP5 (1:200) and anti-ERb (1:50) at 4 C overnight. After a complete wash in phosphate buffer solution (PBS), the tissues were incubated in biotin-labeled goat anti-rabbit (1:2000, Sigma) for 30 min at 37 C, rinsed with PBS, and incubated with avidinebiotinperoxidase complex for 30 min at 37 C. The signal was detected using diaminobenzidine (DAB) as the chromogen for 8 min. Then the lung tissues were counterstained with hematoxylin, dehydrated, air-dried and mounted. Goat serum was used as the primary antibody for negative controls for all assays.
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2.8. Western blotting for AQP1, AQP5 and ERb To examine AQP1, AQP5 and ERb expression, lung tissues were harvested and extracted with lysis buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 400 mM NaCl, 10% glycerol, 0.5% NP-40, 5 mM sodium fluoride, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol). The lysates were centrifuged at 12,000 rpm for 30 min at 4 C, and then the supernatants were collected. Equal amounts of protein (50 mg) were separated by SDS-PAGE, transferred to nitrocellulose membrane at 100 V for 2.5 h at low temperature, and blocked with 5% skim milk for 2 h. Subsequently, the membranes were incubated with anti-AQP1 (1:600), anti-AQP5 (1:300), anti-ERb (1:100) and
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anti-b-actin (1:2000) individually at 4 C overnight. After repeated washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000, Sigma), and bands visualized by using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). The results were expressed as the ratio to b-actin level in the same protein samples. 2.9. Statistical analysis All data were presented as means SD of three independent experiments in triplicate. These data were statistically analyzed through the following tests for multiple comparisons: two-way repeated measures analysis of variance (ANOVA) followed by Dunnett’s test for multiple time point observation, Student’s unpaired t-test for single time point observation, and Wilcoxon Utest for histological data. A statistical difference was accepted and considered significant if p < 0.05. 3. Results 3.1. Effects of E2 treatment on ABGs As shown in Fig. 1, seawater aspiration caused a significant decrease in pH (Fig. 1A) and PaO2 (Fig. 1B) and an increase in PaCO2 (Fig. 1C). It was found that the most serious respiratory failure occurred at the time point of 1 h after seawater exposure. The hypoxemia, hypercapnia and metabolic acidosis were the common clinical symptoms of seawater aspiration. However, these pulmonary injuries induced by seawater aspiration were strongly reversed by treatment of E2 at the time point of 2 h, and the effect of the treatment reached its peak at the time point of 4 h after seawater instillation. Therefore the 4 h time point was considered suitable for the following studies (EVLW and pulmonary histological studies). The respiratory failure induced by seawater exposure was more serious than that induced by normal saline aspiration (Fig. 1A, B and C). 3.2. Effect of E2 treatment on extravascular lung water (EVLW) The EVLW was significantly lower in the EG than in the SG (0.515 0.108 vs. 0.697 0.128, p < 0.01, Fig. 2) at the time point of 4 h after seawater instillation. Furthermore, the EVLW was also lower in the NS group than in the SG (0.601 0.097 vs. 0.697 0.128, p < 0.01, Fig. 2).
Fig. 1. The time course of response in respiratory failure parameters in naive group (NG), seawater group (SG), 17b-estradiol (E2) group (EG) and normal saline (NS) group. (A) pH, (B) PaO2 and (C) PCO2. At the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater instillation, blood samples were obtained from the right carotid artery and were measured by a blood gas analyzer. The measurements of pH, PaO2 and PCO2 were then made to assess the extent of respiratory failure. Data are presented as means SD (n ¼ 6), *p < 0.05, **p < 0.01 vs. SG.
Fig. 2. Effects of 17b-estradiol (E2) on seawater aspiration-induced lung edema. At 4 h time point after seawater aspiration, lungs were separated and the ratios of extravascular lung water (EVLW) were determined to assess tissue edema in seawater group (SG), E2 group (EG) and normal saline (NS) group. Data are presented as means SD (n ¼ 6), *p < 0.01 vs. SG.
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Fig. 3. Effects of 17b-estradiol (E2) on seawater aspiration-induced lung histopathological changes (hematoxylineeosin stain; magnification: 400). Histopathological examination of the lungs was carried out at 4 h time point after seawater aspiration. (A) Naive Group (NG). (B) Seawater Group (SG). Hemorrhage, edematous changes of alveolar walls, swelling of alveolar epithelial cells and thickened alveolar wall were observed. (C) E2 Group (EG). Lung injury was significantly alleviated and less damage was observed compared with the SG. (D) Normal saline (NS) Group. The alveolar architecture and histological changes were the minimal in NS Group.
3.3. Histopathology Lung histology injury was also performed on lung tissues at the 4 h time point after seawater administration. As the sections of
hematoxylineeosin stained showed, normal lung tissue structures existed in the NG group (Fig. 3A). Seawater aspiration at 4 h time point induced prominent lesions, such as focal hemorrhage, distortion, and alveolar thickening (Fig. 3B). In contrast, these changes were
Fig. 4. Immunohistochemistry was performed to evaluate the constitutive expression of ERb after seawater instillation. The lung tissues of rats were carried out at 4 h time point following seawater aspiration (DAB stain, 200). (A) Naive Group (NG). (B) Seawater Group (SG). (C) E2 Group (EG). (D) Normal saline (NS) Group.
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expression of ERb at the 4 h time point following seawater instillation in rat lung tissues (Fig. 4C). After E2 treatment, the most serious respiratory failure was significantly reversed at the time point of 2 h, and the effect reached peak at the 4 h time point following from seawater instillation. ERb expression was investigated by western blotting, and the results showed that E2 treatment significantly increased the expressions of ERb by seawater instillation at the time points of 2 h and 4 h (p < 0.05) (Fig. 5A and B).
3.5. Expression of AQPs after seawater aspiration-induced lung injury
Fig. 5. Effects of 17b-estradiol (E2) on ERb in seawater aspiration-induced lung injury. (A) After protein quantitation, western blotting was performed to investigate the ERb content at 2 h or 4 h time point after seawater administration in Seawater Group (SG) and E2 Group (EG). (B) The ratios of ERb protein to b-actin were obtained by density scanning of the film using a Scion image analysis system. Data are presented as means SD (n ¼ 6). *p < 0.01 vs. SG.
Expressions of AQP1 and AQP5 protein levels were evaluated by western blotting at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater aspiration in response to seawater exposure (Fig. 6). The levels of AQP1 and AQP5 were detectable in NG which indicated that AQPs did exist in normal lungs. After seawater administration, the levels of AQP1 and AQP5 increased. The protein levels of AQP1 and AQP5 began to increase
ameliorated by E2 treatment (Fig. 3C). Furthermore, the alveolar architecture and histological changes were minimal in NS (Fig. 3D). 3.4. The expression of ERb on E2 treatment after seawater aspiration As revealed in immunohistochemistry staining, it was seen that ERb was expressed predominantly in the microvascular endothelium and alveoli epithelium in normal rats as reported in the previous report [47] (Fig. 4A). E2 administration induced the
Fig. 6. The expression of AQP1 and AQP5 at the time points of 0 min (the NG was designed as 0 min), 1 h, 2 h, 4 h and 6 h after seawater aspiration. (A) Western blotting was performed to investigate the AQP1 and AQP5 contents from the beginning to 6 h after seawater administration. (B) The ratios of AQP1 and AQP5 protein to b-actin were obtained by density scanning of the western blotting band using an image analysis system. Naive group (NG), Seawater group (SG). Data are presented as means SD (n ¼ 6). *p < 0.05, **p < 0.01 vs. NG.
Fig. 7. Effects of 17b-estradiol (E2) on AQP1 and AQP5 in seawater aspiration-induced lung injury. (A) After protein quantitation, western blotting was performed to investigate AQP1 and AQP5 contents at 2 h or 4 h time point in seawater group (SG) and E2 group (EG). (B) The ratio of AQP1 protein to b-actin. (C) The ratio of AQP5 protein to b-actin. All the ratios were obtained by density scanning of the western blotting band using an image analysis system. Data are presented as means SD (n ¼ 6). *p < 0.05 vs. SG.
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Fig. 8. Immunohistochemistry was performed to evaluate the constitutive expression of AQP1 after seawater instillation. The lung tissues of rats were carried out at 4 h time point following seawater aspiration (DAB stain, 200). (A) NG; (B) SG; (C) EG; (D) NS Group.
Fig. 9. Immunohistochemistry was performed to evaluate the constitutive expression of AQP5 after seawater instillation. The lung tissue was carried out at 4 h time point following seawater aspiration (DAB stain, 200). (A) NG; (B) SG; (C) EG; (D) NS Group.
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soon after seawater aspiration peaked at 2e4 h, and dropped at 6 h (Fig. 6A and B). 3.6. Effects of E2 on AQP1 and AQP5 expressions in seawater aspiration-induced ALI We also evaluated the effect of E2 on AQP1 and AQP5 protein expressions by immunohistochemistry and western blotting. AQP1 and AQP5 expressions were examined on lung samples collected from NG, SG and EG. We chose 2 h and 4 h as the time points as the expressions of AQP1 and AQP5 were at their highest value after seawater exposure. The data showed that E2 administration inhibited the elevation of AQP1 and AQP5 induced by seawater instillation at the time points of 2 h and 4 h (p < 0.05) (Fig. 7A, B and C). Immunohistochemistry was performed to evaluate the constitutive expression of AQP 1 and AQP5 at the time point of 4 h after seawater instillation. It was observed that AQP1 was expressed predominantly in the microvascular endothelium and AQP5 was expressed predominantly in the lung epithelium of the NG group (Figs. 8A and 9A). Moreover, an enhanced AQP1 and AQP5 staining was found and the distribution of AQPs-positive cells was greater in the lungs after seawater aspiration at the time point of 4 h (Figs. 8B and 9B). Compared with SG, E2 treatment observably decreased the expression of AQP1 and AQP5 in lung tissues at the time point of 4 h (Figs. 8C and 9C).
Fig. 10. The expression of AQP1 and AQP5 in seawater group (SG) and normal saline (NS) group. (A) After protein quantitation, western blotting was performed to investigate the AQP1 and AQP5 contents at 4 h time point in the SG and NS group. (B) The ratios of AQP1 and AQP5 protein to b-actin were obtained by density scanning of the western blotting band using an image analysis system. Data are presented as means SD (n ¼ 6). *p < 0.01 vs. SG.
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3.7. The expression of AQP1 and AQP5 in SG and NS groups Next, western blotting and immunohistochemistry were applied to evaluate the AQP1 and AQP5 protein levels at the time point of 4 h after seawater/normal saline instillation. In NS group, the normal saline didn’t induce obvious increase of AQP1 and AQP5 (Figs. 8D and 9D) in lung tissues and the staining was close to NG (Figs. 8A and 9A). The western blotting data also showed that compared with SG, the expressions of AQP1 and AQP5 were lower in NS group (Fig. 10A and B). 4. Discussion In this study, we investigated the role of estrogen for early seawater instillation-induced ALI with a rat model. The results showed that intratracheal instillation of seawater (4 mL/kg) impaired arterial blood gas in a short time with a significant decrease in pH and PaO2 and an increase in PaCO2, and induced serious pulmonary edema and histopathologic changes in lung tissues. Meanwhile, E2 treatment effectively inhibited the seawater-induced lung injury. Numerous clinical and experimental studies have suggested that females tolerate trauma-hemorrhage and sepsis better than males [14,15]. Sex differences in certain aspects of lung function in experimental animals have been documented [16e18]. Female rats were more resistant to shock [19,20] and carrageenan-induced lung injury [21] than their male counterparts. In addition, females are better able to adjust to hypobaria [22] and more resistant to infection than males [23e26]. When studying lung physiology and disease, it is important to consider sex and hormonal status as modifying factors, because estrogen plays a major role in the lung both physiologically and pathophysiologically in animals [27]. Previous results have shown that E2 was effective for the treatment of pulmonary diseases [3,6,28,29]. The evidence presented here also indicates that E2 therapy was effective for early ALI. Like all steroid hormones, estrogens readily diffuse across the cell membrane. Once inside the cell, they bind to and activate estrogen receptors which in turn up-regulate the expression of many genes [12]. In recent years, newer concepts of sex steroid hormone receptor signaling have emerged, including rapid cellular activation pathways that do not involve the direct alteration of gene transcription [30]. Huang-Ping Yu et al. demonstrated that estrogen attenuated lung injury by the activation of ERa, ERb or both receptors [31]. Doucet’s recent work has shown that tissues with higher densities of ERb than ERa, such as the lung, are better protected from trauma hemorrhagic shock-induced injury by ERb than ERa agonists [3]. Interestingly, our results showed that ERb increased in 17b-Estradiol Group (EG) indicating that the protective effect of estrogen on ALI was conducted by ERb. Seawater is a hyperosmolar fluid and its NaCl concentration is 3e3.5％, about 3 fold of that of physiologic saline [32]. Our results showed that compared with normal saline water, seawater aspiration induced more serious ALI in rats which was characterized by more severe blood gas index, pulmonary edema and histopathologic changes. These differences suggested that the hyperisotonic pressure might play an important role in seawater aspirationinduced lung injury in rat. Our data showed that AQP1 and AQP5 increased after seawater endotracheal instillation, but no obvious changes were found in NS group. This is in agreement with other studies which have shown that hypertonicity could induce the expression of AQP1 and AQP5 [33,34]. Additional indirect evidence indicated that the regulation of AQP expression in an adult lung by osmotic stress was one of physiologic roles in respiratory physiology [35]. The findings in our study also indicated that hyperisotonic pressure might be an
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important activator of AQP1 and AQP5 in rat airway epithelium. However, the role of AQP1 and AQP5 in ALI is still controversial. AQP1 and AQP5 expressions were shown to be reduced in rodent lungs following adenoviral infection [36], lipopolysaccharideinduced lung injury [37], and exposure to TNF-a [38]. Meanwhile, AQP5 expression was found increased after bleomycin exposure [39]. Therefore, further experiments are needed to study the role of AQP1 and AQP5 in ALI. Phenotype analysis of transgenic knockout mice lacking AQPs has suggested a role from them in the lungs and airways. AQP1 and AQP5 may provide the principal route for osmotically driven water transport between airspace and capillary compartments. However, alveolar fluid clearance (AFC) in the neonatal and adult lung does not appear to be affected by the deletion of AQPs, as well as lung fluid accumulation in experimental models of lung injury [40e44]. This evidence indicates that AQP1 and AQP5 contribute to the formation of edema, but not the response to AFC in rat lungs. Our results showed that the expression of AQP1 and AQP5 significantly increased in the lung tissues of the rats after seawater instillation and reached its peak at the time points of 2 h and 4 h. Osmotically driven water transport across cell membranes is the principle mechanism of fluid transport [45]. The increased AQPs expression could cause more water from cellular and vascular compartments to flow into interstitium and alveolar spaces because of the hyperosmolality of seawater, which may increase the pleural effusion and pulmonary edema. Coincidentally, the time course of EVLW was paralleled with the change of AQP1 and AQP5 expressions after either seawater or normal saline aspiration, which further confirmed that the up-regulation of AQPs expression contributed to the development of lung edema. There was little information regarding the influence of gender hormones on pulmonary edema. In an early study, Farhat et al. [46] reported that estradiol treatment prevented the increase in lung wet to dry weight ratio. In the present model of lung injury, we found that E2 administration could alleviate seawater aspirationinduced pulmonary edema. This effect may be attributed to the observed decrease in extravascular lung water. Furthermore, E2 treatment reversed the up-regulation of AQP1 and AQP5 expressions induced by seawater aspiration. The inhibition of AQP1 and AQP5 could reduce the water exosmosis from cellular and vascular compartments to interstitial after seawater aspiration, leading to the recovery from severe pulmonary edema. This finding indicated that E2 might be useful to treat seawater drowning by downregulating the increased AQP1 and AQP5.
5. Conclusions In conclusion, data in this study showed that compared with normal saline water, seawater aspiration can induce more serious ALI in rats and that E2 treatment could alleviate seawater-induced ALI in rat lung and the effect of E2 may be mediated via the activation of ERb. Meanwhile, seawater aspiration could induce more severe pulmonary edema with AQP1 and AQP5 being up-regulated. The up-regulated AQP1 and AQP5 were suppressed by administration of E2, resulting in an attenuation of lung edema. These results suggest a new mechanism of seawater-induced ALI, and highlighted the important role of AQP1 and AQP5 during the formation of seawater-induced pulmonary edema. This study suggests that E2 treatment may be beneficial to patients with seawater drowning-induced ALI. However, further investigations on the effect of E2 on clinical medicine are called for. Competing interests The authors declare that they have no competing interests.
Acknowledgments This work was supported by grants from the Military Key Projects in the 11th Five-year Plan of PR China (Project No. 08G102), Shaanxi Province Key Projects (Project No. 2008K14-08). References [1] Brenner Ruth A. Prevention of drowning in infants, children, and adolescents. Pediatrics 2003;112(2):440e5. [2] Salomez F, Vincent JL. Drowning: a review of epidemiology, pathophysiology, treatment and prevention. Resuscitation 2004;63(3):261e8. [3] Doucet D, Badami C, Palange D, Bonitz RP, Lu Q, Xu DZ, et al. Estrogen receptor hormone agonists limit trauma hemorrhage shock-induced gut and lung injury in rats. PLoS One 2010;5(2):e9421. [4] Kan WH, Hsu JT, Schwacha MG, Choudhry MA, Bland KI, Chaudry IH. Estrogen ameliorates trauma-hemorrhage-induced lung injury via endothelial nitric oxide synthase-dependent activation of protein kinase G. Ann Surg 2008; 248(2):294e302. [5] Hsieh YC, Frink M, Hsieh CH, Choudhry MA, Schwacha MG, Bland KI, et al. Downregulation of migration inhibitory factor is critical for estrogenmediated attenuation of lung tissue damage following trauma-hemorrhage. Am J Physiol-Lung C 2007;292(5):L1227e32. [6] Speyer CL, Rancilio NJ, McClintock SD, Crawford JD, Gao H, Sarma JV, et al. Regulatory effects of estrogen on acute lung inflammation in mice. Am J Physiol-Cell Ph 2005;288(4):C881e90. [7] Massaro D, Massaro GD. Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice. Am J Physiol-Lung C 2004;287(6):L1154e9. [8] Ames Nancy. Caring for the patient with complex respiratory disorders. 36:1079e126. [9] Agre Peter, King Landon S, Yasui Masato, Guggino Wm B, Ottersen Ole Petter, Fujiyosh Yoshinori, et al. Aquaporin water channels e from atomic structure to clinical medicine. J Physiol 2002;542(1):3e16. [10] Verkman AS. Seawater exposure-induced lung injury. Resp Physiol Neurobiol 2007;159:324e30. [11] Pearce ML, Yamashita J, Beazell J. Measurement of pulmonary edema. Circ Res 1965;16(5):482e8. [12] Whitehead SA, Nussey S. Endocrinology: an integrated approach. Oxford: BIOS, Taylor & Francis; 2001. [13] Kuiper GGJM, Carlsson B, Grien K, Enmark E, Haggblad J, Nilsson S, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors a and b. Endocrinology 1997;138(3):863e70. [14] George RL, McGwin Jr G, Metzger J, Chaudry IH, Rue III LW. The association between gender and mortality among trauma patients as modified by age. J Trauma 2003;54:464e71. [15] Deitch EA, Livingston DH, Lavery RF, Monaghan SF, Bongu A, Machiedo GW. Hormonally active women tolerate shock-trauma better than do men: a prospective study of over 4000 trauma patients. Ann Surg 2007;246:447e53. [16] Carey MA, Card JW, Bradbury JA, Moorman MP, Haykal-Coates N, Gavett SH, et al. Spontaneous airway hyperresponsiveness in estrogen receptor-alphadeficient mice. Am J Respir Crit Care Med 2007;175:126e35. [17] Reinhard C, Eder G, Fuchs H, Ziesenis A, Heyder J, Schulz H. Inbred strain variation in lung function. Mamm Genome 2002;13:429e37. [18] Bates JHT, Mitzner W. Point:counterpoint lung impedance measurements are/ are not more useful than simpler measurements of lung function in animal models of pulmonary disease. J Appl Physiol; April 12, 2007. doi:10.1152/ japplphysiol.00369.02007. [19] Adams Jr CA, Magnotti LJ, Xu DZ, Lu Q, Deitch EA. Acute lung injury after hemorrhagic shock is dependent on gut injury and sex. Am Surg 2000;66: 905e13. [20] Caruso JM, Xu DZ, Lu Q, Dayal SD, Deitch EA. The female gender protects against pulmonary injury after trauma hemorrhagic shock. Surg Infect (Larchmt) 2001;2:231e40. [21] Cuzzocrea S, Mazzon E, Sautebin L, Serraino I, Dugo L, Calabro G, et al. The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat. Mol Med 2001;7:478e87. [22] McMurtry IF, Frith CH, Will DH. Cardiopulmonary responses of male and female swine to simulated high altitude. J Appl Physiol 1973;35:459e62. [23] Offner PJ, Moore EE, Biffl WL. Male gender is a risk factor for major infections after surgery. Arch Surg 1999;134:935e40. [24] Schröder J, Kahlke V, Staubach KH, Zabel P, Stuber F. Gender differences in human sepsis. Arch Surg 1998;133:1200e5. [25] Wichmann MW, Inthorn D, Andress HJ, Schildberg FW. Incidence and mortality of severe sepsis in surgical intensive care patients: the influence of patient gender on disease process and outcome. Intens Care Med 2000;26: 167e72. [26] Angele MK, Schwacha MG, Ayala A, Chaudry IH. Effect of gender and sex hormones on immune responses following shock. Shock 2000;14:81e90. [27] Carey Michelle A, Card Jeffrey W, Voltz James W, Germolec Dori R, Korach Kenneth S, Zeldin Darryl C. The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies. Am J Physiol-Lung C 2007;293:L272e8.
Q. Fan et al. / Pulmonary Pharmacology & Therapeutics 24 (2011) 673e681 [28] Yang SJ, Chen HM, Hsieh CH, Hsu JT, Yeh CN, Yeh TS, et al. Akt pathway is required for oestrogen-mediated attenuation of lung injury in a rodent model of cerulein-induced acute pancreatitis. Injury 2010;13 [Epub ahead of print]. [29] Carey MA, Card JW, Voltz JW, Germolec DR, Korach KS, Zeldin DC. The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies. Am J Physiol-Lung C 2007;293(2):L272e8. [30] Mendelsohn ME, Karas RH. Molecular and cellular basis of cardiovascular gender differences. Science 2005;308:1583e7. [31] Yu H-P, Hsieh Y-C, Suzuki T, Shimizu T, Choudhry MA, Schwacha MG, et al. Salutary effects of estrogen receptor-b agonist on lung injury after traumahemorrhage. Am J Physiol-Lung C 2006;290(5):L1004e9. [32] Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000;342(18):1334e49. [33] Umenishi Fuminori, Schrier Robert W. Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene. J Biol Chem 2003; 278(18):15765e70. [34] Hoffert Jason D, Leitch Virginia, Agre Peter, Hoffert JD. Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway. J Biol Chem 2000;275(12):9070e7. [35] King LS, Nielsen S, Agre P, Brown RH. Decreased pulmonary vascular permeability in aquaporin-1-null humans. Proc Natl Acad Sci U S A 2002;99: 1059e63. [36] Towne JE, Harrod KS, Crane CM, Menon AG. Decreased expression of aquaporin (AQP)1 andAQP5 in mouse lung after acute viral infection. Am J Respir Cell Mol Biol 2000;22:34e44. [37] Jiao G, Li E, Yu R. Decreased expression of AQP1 and AQP5 in acute injured lungs in rats. Chin Med J (Engl) 2002;115:867e963.
681
[38] Towne JE, Krane CM, Bachurski CJ, Menon AG. Tumor necrosis factor-a inhibits aquaporin 5 expression in moue lung epithelial cells. J Biol Chem 2001;276: 18657e64. [39] Gabazza EC, Kasper M, Ohta K, Keane M, D’Alessandro-Gabazza C, Fujimoto H, et al. Decreased expression of aquaporin-5 in bleomycin-induced lung fibrosis in the mouse. Pathol Int 2004;54:774e80. [40] Bai C, Fukuda N, Song Y, Ma T, Matthay Ma, Verkman AS. Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice. J Clin Invest 1999;103(4): 555e61. [41] Verkman AS. Physiological importance of aquaporins: lessons from knockout mice. Curr Opin Nephrol Hypertens 2000;9:517e22. [42] Verkman AS, Matthay MA, Song Y. Aquaporin water channels and lung physiology. Am J Physiol-Lung C 2000;278:L867e79. [43] Verkman AS, Yang B, Song Y, Manley GT, Ma T. Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice. Exp Physiol 2000;85:S233e41. [44] Zemans Rachel L, Matthay Michael A. Bench-to-bedside review: the role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury. Crit Care 2004;8:469e77. [45] Borok Z, Lubman RL, Danto SI, Zhang XL, Zabski SM, King LS, et al. Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5. Am J Respir Cell Mol Biol 1998;18:554e61. [46] Farhat MY, Chen MF, Bhatti T, Iqbal A, Cathapermal S, Ramwell PW. Protection by oestradiol against the development of cardiovascular changes associated with monocrotaline pulmonary hypertension in rats. Br J Pharmacol 1993; 110(2):719e23. [47] Saunders PT, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinology 1997;154:R13e6.