Role of aquaporins in lung liquid physiology

Aquaporins (AQPs) are small, integral membrane proteins that facilitate water transport across cell membranes in response to osmotic gradients. Water transport across epithelia and endothelia in the peripheral lung and airways occurs during airway hydration, alveolar fluid transport and submucosal gland secretion. Several AQPs are expressed in the lung and airways: AQP1 in microvascular endothelia, AQP3 and AQP4 in airway epithelia, and AQP5 in type I alveolar epithelial cells, submucosal gland acini, and a subset of airway epithelial cells. Phenotype analysis of transgenic knockout mice lacking AQPs has defined their roles in the lung and airways. AQP1 and AQP5 provide the principal route for osmotically driven water transport between airspace and capillary compartments; however, alveolar fluid clearance in the neonatal and adult lung is not affected by their deletion, nor is lung fluid accumulation in experimental models of lung injury. In the airways, though AQP3 and AQP4 facilitate osmotic water transport, their deletion does not impair airway hydration, regulation of airway surface liquid, or fluid absorption. In contrast to these negative findings, AQP5 deletion in submucosal glands reduced fluid secretion by >50%. The substantially slower fluid transport in the lung compared to renal and secretory epithelia probably accounts for the lack of functional significance of AQPs in the lung and airways. Recent data outside of the lung implicating the involvement of AQPs in cell migration and proliferation suggests possible new roles for lung AQPs to be explored.     

Anti-asthmatic agents alleviate pulmonary edema by upregulating AQP1 and AQP5 expression in the lungs of mice with OVA-induced asthma

Ovalbumin (OVA)-induced asthma in mouse lungs causes changes in the mRNA and protein levels of aquaporins (AQPs). AQP expression was examined in the presence of various anti-asthmatic agents, including dexamethasone, ambroxol, and terbutaline. The influence of these agents on OVA-induced airway inflammation was also evaluated. The mRNA expression levels of AQP1, 4, and 5 were significantly reduced and that of AQP3 was significantly increased 24h after the last OVA exposure. The protein levels of AQP1, 3, and 5 mirrored the mRNA expression profiles, but AQP4 did not exhibit any changes. Only the mRNA and protein expression levels of AQP1 and AQP5 were significantly increased by these three anti-asthmatic agents. Dexamethasone and ambroxol improved the eosinophil infiltration, mucus secretion, and pulmonary edema caused by OVA, but terbutaline only alleviated pulmonary edema. These results indicate that AQP1 and AQP5 are closely related to pulmonary edema but not to eosinophil infiltration or mucus secretion during asthma. Anti-asthmatic agents could alleviate pulmonary edema through upregulating the expression of AQP1 and AQP5 in mouse lungs that have OVA-induced asthma.     

Expression and localization of epithelial aquaporins in the adult human lung

Aquaporins (AQPs) facilitate water transport across epithelia and play an important role in normal physiology and disease in the human airways. We used in situ hybridization and immunofluorescence to determine the expression and cellular localization of AQPs 5, 4, and 3 in human airway sections. In nose and bronchial epithelia, AQP5 is expressed at the apical membrane of columnar cells of the superficial epithelium and submucosal gland acinar cells. AQP4 was detected in basolateral membranes in ciliated ducts and by in situ in gland acinar cells. AQP3 is present on basal cells of both superficial epithelium and gland acinus. In these regions AQPs 5, 4, and 3 are appropriately situated to permit transepithelial water permeability. In the small airways (proximal and terminal bronchioles) AQP3 distribution shifts from basal cell to surface expression (i.e., localized to the apical membrane of proximal and terminal bronchioles) and is the only AQP identified in this region of the human lung. The alveolar epithelium has all three AQPs represented, with AQP5 and AQP4 localized to type I pneumocytes and AQP3 to type II cells. This study describes an intricate network of AQP expression that mediates water transport across the human airway epithelium.     

Bronchiolar expression of aquaporin-3 (AQP3) in rat lung and its dynamics in pulmonary oedema

Aquaporins (AQPs) are water channel proteins that permit osmotically driven water movement. To determine their dynamics in pulmonary oedema, we examined the expression of mRNA and protein for AQP1, AQP3, AQP4, and AQP5 in the lungs of normal and thiourea-treated rats. In the thiourea group, lung water content increased significantly (vs. controls) with the peak at around 4 h. Semi-quantitative RT-PCR showed that AQP3 mRNA in the thiourea group rose significantly, peaking at around 4–8 h. The expression of AQP1, AQP4, AQP5, ENaC and CFTR mRNA each decreased significantly some time after the peak in lung water content. Immunoblot analysis showed that glycosylated AQP3 protein was increased 4–10 h after treatment. Expression of the other AQP proteins was not significantly altered, except for that of AQP4. Immunohistochemical examination revealed that AQP1 was expressed in endothelia, AQP3 in the basal cells of the large airways and in cuboidal cells in the bronchioles, AQP4 in the basolateral membrane of airway cells and AQP5 in type-I pneumocytes. Our results suggest that AQP3 is expressed not only in large airways, but also in bronchioles, and is related to water movement in pulmonary oedema.     

Immunohistochemical Localization of the Aquaporins AQP1, AQP3, AQP4, and AQP5 in the Mouse Respiratory System

Aquaporins are membrane water channel proteins that function mainly in water transfer across cellular membranes. In our present study, we investigated the immunohistochemical distribution of aquaporin 1 (AQP1), AQP3, AQP4, and AQP5 in the mouse respiratory system by immunofluorescence, immunoperoxidase, and immunoelectron microscopy. AQP3, AQP4, and AQP5 are expressed in epithelial cells, whereas AQP1 is expressed in subepithelial connective tissues and capillaries. In the airway surface epithelia from the nasal cavity to the intrapulmonary bronchioles, AQP5 was found to be mainly localized to the luminal side and both AQP3 and AQP4 to the abluminal side. In the alveolar epithelium, AQP5 is localized to the apical membranes of both type I and type II alveolar cells. Compared with the previous studies on the rat respiratory system, in which AQP5 is restricted to the alveolar type I cells and absent from the airway surface epithelia, we found that AQP5 in the mouse is much more widely distributed throughout the surface epithelia. These results suggest that AQP5 has a critical role in water-handling, such as the maintenance of airway surface liquid and clearance of alveolar fluid in the mouse respiratory system.     

Tanshinone IIA ameliorates seawater exposure-induced lung injury by inhibiting aquaporins (AQP) 1 and AQP5 expression in lung

Aquaporins (AQPs), a family of transmembrane water channels, mediate physiological response to changes of fluid volume and osmolarity. It is still unknown what role of AQPs plays in seawater drowning-induced acute lung injury (ALI) and whether pharmacologic modulation of AQPs could alleviate the severity of ALI caused by seawater aspiration. In our study, the results from RT-PCR and Western blotting showed that intratracheal installation of seawater up-regulated the mRNA and protein levels of AQP1 and AQP5 in lung tissues. Furthermore, we found that treatment of tanshinone IIA (TIIA, one of the main active components from Chinese herb Danshen) significantly reduced the elevation of AQP1 and AQP5 expression induced by seawater in rats, A549 cells and primary alveolar type II cells. Treatment of TIIA also improved lung histopathologic changes and blood-gas indices, and reduced lung edema and vascular leakage. These findings demonstrated that AQP1 and AQP5 might play an important role in the development of lung edema and lung injury, and that treatment with TIIA could significantly alleviate seawater exposure-induced ALI, which was probably through the inhibition of AQP1 and AQP5 over-expression in lungs.     

Immunolocalization of water channel aquaporins in human knee articular cartilage with intact and early degenerative regions

Aquaporins (AQPs), a family of water channel proteins expressed in various cells and tissues, serve as physiological pathways of water and small solute transport. Articular cartilage is avascular tissue with unique biomechanical structure, a major component of which is “water”. Our objective is to investigate the immunolocalization and expression pattern changes of AQPs in articular cartilage with normal and early degenerative regions in the human knee joint, which is the joint most commonly involved in osteoarthritis (OA). Two isoforms (AQPs 1 and 3) of AQPs were examined by immunohistochemical analyses using isoform-specific antibodies with cartilage samples from OA patients undergoing total knee arthroplasty. AQP 1 and AQP 3 were expressed in human knee articular cartilage and were localized in chondrocytes, both in the intact and early degenerative cartilage regions. Compared to the intact cartilage, both AQP 1 and AQP 3 immunopositive cells were observed at the damaged surface area in the degenerative region. These findings suggest that these AQPs play roles in metabolic water regulation in articular cartilage of load bearing joints and that they are responsible for OA onset.     

Decreased expression of aquaporin (AQP)1 and AQP5 in mouse lung after acute viral infection

Intratracheal infection of mice with adenovirus is associated with subsequent pulmonary inflammation and edema. Water movement through the air space-capillary barrier in the distal lung is facilitated by aquaporins (AQPs). To investigate the possibility that distal lung AQPs undergo altered regulation under conditions of aberrant fluid handling in the lung, we analyzed messenger RNA (mRNA) and protein expression of AQPs 1 and 5 in the lungs of mice 7 and 14 d after infection with adenovirus. Here, we demonstrate that AQP1 and AQP5 show decreased expression following adenoviral infection. Northern blot analysis showed significantly decreased mRNA levels of AQP1, which is expressed in the capillary endothelium, and AQP5, which is expressed in alveolar epithelium, in the lungs of mice both 7 and 14 d after infection. Immunoblotting studies demonstrated significantly reduced levels of AQP1 and AQP5 protein after infection as well. In addition, mRNA expression of the alpha subunit of the epithelial sodium channel was reduced in the lungs of mice 7 and 14 d after adenoviral infection. In contrast, mRNA expression of the alpha1 subunit of the Na,K-adenosine triphosphatase in the lung was unaltered. Immunohistochemical analysis demonstrated that the decreases in AQP1 and AQP5 expression were not localized to regions of overt inflammation but were found throughout the lung. Thus, this study provides the first report of AQP gene regulation in an in vivo model of pulmonary inflammation and edema. Decreased AQP1 and AQP5 levels during adenoviral infection suggest a role for AQP1 and AQP5 in the abnormal fluid fluxes detected during pulmonary inflammation.     

Downregulation of aquaporin 5 induced by vector-based short hairpin RNA and its effect on MUC5AC gene expression in human airway submucosal gland cells

The aquaporins (AQPs) are a family of homologous water channels expressed in many epithelial and endothelial cells, however no reliable and non-toxic inhibitors of AQPs have been reported yet. Our researchers have analyzed the changes of AQP5 expression induced by vector-based short hairpin RNA (shRNA) in the human airway submucosal gland cell line (SPC-A1) and observed its regulation on the expression of MUC5AC gene. Localizations of AQP5 and MUC5AC in SPC-A1cells were detected by Immunofluorescence. AQP5 mRNA was significantly reduced by 75.1% one day after transfection with specific shRNA, named shAQP5. However, the significant suppression of AQP5 protein did not appear until day 5 after transfection. MUC5AC mRNA was remarkably increased by 119.9% On day 3 after shAQP5 transfection, while comparable MUC5AC protein changes were not found in SPC-A1 cells with flow cytometry analysis. These results indicate that vector-based shRNA could be used as a potential tool to inhibit the expression of AQP5. This is the first investigation providing evidence demonstrating the regulation of the mucin gene by AQP5 gene silencing.     

Aquaporins—new players in cancer biology

The aquaporins (AQPs) are small, integral-membrane proteins that selectively transport water across cell plasma membranes. A subset of AQPs, the aquaglyceroporins, also transport glycerol. AQPs are strongly expressed in tumor cells of different origins, particularly aggressive tumors. Recent discoveries of AQP involvement in cell migration and proliferation suggest that AQPs play key roles in tumor biology. AQP1 is ubiquitously expressed in tumor vascular endothelium, and AQP1-null mice show defective tumor angiogenesis resulting from impaired endothelial cell migration. AQP-expressing cancer cells show enhanced migration in vitro and greater local tumor invasion, tumor cell extravasation, and metastases in vivo. AQP-dependent cell migration may involve AQP-facilitated water influx into lamellipodia at the front edge of migrating cells. The aquaglyceroporin AQP3, which is found in normal epidermis and becomes upregulated in basal cell carcinoma, facilitates cell proliferation in different cell types. Remarkably, AQP3-null mice are resistant to skin tumorigenesis by a mechanism that may involve reduced tumor cell glycerol metabolism and ATP generation. Together, the data suggest that AQP expression in tumor cells and tumor vessels facilitates tumor growth and spread, suggesting AQP inhibition as a novel antitumor therapy.     

AQP1 mediates water transport in the carotid body

In this study, we explored the presence of aquaporins (AQPs), a family of membrane water channel proteins, in carotid body (CB) type I chemoreceptor cells. The CB is a polymodal chemoreceptor whose major function is to detect changes in arterial O₂ tension to elicit hyperventilation during hypoxia. The CB has also been proposed to function as a systemic osmoreceptor, thus we hypothesized that the presence of AQPs in type I cell membrane may confer higher sensitivity to osmolarity changes and hence accelerate the activation of chemoreceptor cells. We detected the expression of AQP1, AQP7, and AQP8 in the CB and confirmed the location of AQP1 in type I cells. We have also shown that inhibition of AQP1 expression clearly reduced type I cell swelling after a hyposmotic shock, demonstrating that AQP1 has a major contribution in transmembrane water movement in these chemoreceptor cells. Interestingly, CB AQP1 expression levels change during postnatal development, increasing during the first postnatal weeks as the organ matures. In conclusion, in this study, we report the novel observation that AQPs are expressed in the CB. We also show that AQP1 mediates water transport across the cell membrane of type I cells, supporting the contribution of this protein to the osmoreception function of the CB.     

脑水通道蛋白的分布、功能及调控机制

背景：在哺乳动物脑中主要表达的水通道蛋白是水通道蛋白1、水通道蛋白4和水通道蛋白9,其他的仅为零星表达。目前国内外尚未见到系统分析维持脑正常生理功能的水通道蛋白分布、功能及其调节机制的综述报道。 目的：综述近年国内外维持脑正常生理功能的水通道蛋白分布、功能及其调节机制的研究进展。 方法：应用计算机检索1980年1月至2013年7月PubMed数据库、中国期刊全文数据库有关脑正常生理功能维持中水通道蛋白分布、功能及其调节机制的文章,英文检索词“AQP1, AQP4,AQP9, function, brain, adjusting mechanism”;中文检索词“水通道蛋白,功能,脑,调节机制”。共检索到163篇相关文献,85篇文献符合纳入标准。 结果与结论：近年来,有大量学者对脑水通道蛋白的表达、功能及其调节机制进行了较深层次的研究,具体归纳为如下3个方面：①水通道蛋白1主要表达于脑室脉络丛参与脑脊液的形成;在其他类型的细胞中,气体小分子CO2,NO,NH3 及O2也可通过水通道蛋白1。②水通道蛋白4主要表达在胶质细胞足突、胶质界膜以及室管膜中,帮助水进出脑组织,并加速胶质细胞迁移及改变神经活动。③水通道蛋白9主要分布于星形胶质细胞及儿茶酚胺等神经元中,主要功能是参与脑内能量代谢。水通道蛋白被认为是对脑中水运输提供关键路径的主要水通道,有关水通道蛋白分布、功能及调控机制的研究对于攻克脑相关疾病起重要作用。水通道蛋白在维持脑正常生理及相关疾病中表达的调节机制尚未明晰,相关分子信号通路尚待更加深入、系统地研究。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Use of aquaporins 1 and 5 levels as a diagnostic marker in mild-to-moderate adult-onset asthma

Characteristic features of asthma include airway inflammation and hyperactivity, mucus hypersecretion, mucosal edema, and airway remodeling. These features could be due to pathological water transport across pulmonary epithelia and aquaporins (AQPs) have recently been isolated as key proteins in fluid transportation in the human respiratory tract. We aimed to evaluate the role of aquaporins in the pathogenesis of asthma and their possible use a diagnostic marker of the disease. A total of 110 hospitalized and outpatients with mild to moderate adult-onset asthma were invited to participate in this study and 34 submitted an induced sputum sample adequate for analysis. The amount of AQP1, AQP5 and MUC5AC were measured with ELISA assay. The amount of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17 in both serum and sputum were measured with Cytometry Bead Array (CBA kit). Our results suggest that sputum AQP5, AQP1 and MUC5AC are all in a good correlation (r=0.498 between AQP5 and AQP1, r=0.529 and r=0.661 between MUC5AC and AQP5 or AQP1, respectively, all P<0.05). The AUC value for AQP1 and AQP5 to diagnose asthma were 0.729 and 0.745, respectively. In conclusion, water homeostasis plays an important role in maintaining adequate fluid transportation within the lung and is involved in the pathogenesis of asthma. Our results suggest that AQP may influence pulmonary physiology that their dysfunction can contribute to pulmonary pathogenesis, such as asthma. Moreover, their quantification could serve as biomarkers for the diagnosis of asthma.     

Immunolocalization of water channel aquaporins in the nasal olfactory mucosa

Aquaporins (AQPs), membrane water channel proteins expressed in various tissues and organs, serve in the transfer of water and small solutes across the membrane. We raised antibodies to AQPs using isoform-specific synthetic peptides and surveyed their expression in the rat nasal olfactory and respiratory mucosae. AQP1, AQP3, AQP4, and AQP5 were detected by immunohistochemical and immunoblotting analyses. AQP1 was expressed in the endothelial cells of blood vessels and the surrounding connective tissue cells in the olfactory and respiratory mucosae. AQP1 may be involved in water transfer across the blood vessel wall. In the olfactory epithelium, no AQP was detected in the olfactory sensory cells. Instead, AQP3 was abundant in the olfactory epithelium, where it was localized in the supporting cells and basal cells. Expression of AQP3 was mostly restricted to the basal cells in the respiratory epithelium. In marked contrast, AQP4 was abundant in the respiratory epithelium, but its abundance was limited to the basal cells in the olfactory epithelium. In the Bowman's gland, AQP5 was localized in the apical membrane in the secretory acinar cells, whereas AQP3 and AQP4 were found in the basolateral membrane. Similar localization was seen in its duct cells. These results showed a distinct localization pattern for AQPs in the olfactory epithelium. AQP3 and AQP4 in the supporting cells and basal cells may play an important role in generating and maintaining the specific microenvironment around the olfactory sensory cells. AQP3, AQP4, and AQP5 in the Bowman's gland may serve in the secretion to generate the microenvironment at the apical surface of the olfactory dendrites for odorant reception.     

Cell locations for AQP1, AQP4 and 9 in the non-human primate brain

The presence of three water channels (aquaporins, AQP), AQP1, AQP4 and AQP9 were observed in normal brain and several rodent models of brain pathologies. Little is known about AQP distribution in the primate brain and its knowledge will be useful for future testing of drugs aimed at preventing brain edema formation. We studied the expression and cellular distribution of AQP1, 4 and 9 in the non-human primate brain. The distribution of AQP4 in the non-human primate brain was observed in perivascular astrocytes, comparable to the observation made in the rodent brain. In contrast with rodent, primate AQP1 is expressed in the processes and perivascular endfeet of a subtype of astrocytes mainly located in the white matter and the glia limitans, possibly involved in water homeostasis. AQP1 was also observed in neurons innervating the pial blood vessels, suggesting a possible role in cerebral blood flow regulation. As described in rodent, AQP9 mRNA and protein were detected in astrocytes and in catecholaminergic neurons. However additional locations were observed for AQP9 in populations of neurons located in several cortical areas of primate brains. This report describes a detailed study of AQP1, 4 and 9 distributions in the non-human primate brain, which adds to the data already published in rodent brains. This relevant species differences have to be considered carefully to assess potential drugs acting on AQPs non-human primate models before entering human clinical trials.     

肺脏的水孔蛋白家族

90年代以来 ,作为膜上水分子通道的水孔蛋白(ａｑｕａｐｏｒｉｎｓ ,ＡＱＰｓ)家族克隆成功[1,2 ] ,对自由水被动跨膜转运机制做出更加形象而深入的解释。肺脏的许多生理功能都有水分子运动的参与 ,同时许多肺脏疾病 ,如哮喘、肺水肿和急性呼吸窘迫综合征等也涉及肺内水运动平衡的紊乱。因此肺内水孔蛋白的分布及其病理生理意义日益受到重视。1　水孔蛋白的结构与功能特点水孔蛋白属于膜主体内在蛋白 (ｍａｊｏｒｉｎｔｅｒｎａｌｐｒｏｔｅｉｎ ,ＭＩＰ)家族的成员 ,目前在人类和哺乳动物身上共发现 10个亚型 ,广泛分布于多个组织器官 ,具…     

Decreases in rat brain aquaporin-4 expression following intracerebroventricular administration of an endothelin ETB receptor agonist

Aquaporins (AQPs) comprise a family of water channel proteins, some of which are expressed in brain. Expressions of brain AQPs are altered after brain insults, such as ischemia and head trauma. However, little is known about the regulation of brain AQP expression. Endothelins (ETs), vasoconstrictor peptides, regulate several pathophysiolgical responses of damaged nerve tissues via ET_B receptors. To show possible roles of ET_B receptors in the regulation of brain AQP expression, the effects of intracerebroventricular administration of an ET_B agonist were examined in rat brain. In the cerebrum, the copy numbers of AQP4 mRNAs were highest among AQP1, 3, 4, 5 and 9. Continuous administration of 500 pmol/day Ala^1,3,11,15-ET-1, an ET_B selective agonist, into rat brain for 7 days decreased the level of AQP4 mRNA in the cerebrum, but had no effect on AQP1, 3, 5 and 9 mRNA levels. The level of AQP4 protein in the cerebrum decreased by the administration of Ala^1,3,11,15-ET-1. Immunohistochemical observations of Ala^1,3,11,15-ET-1-infused rats showed that GFAP-positive astrocytes, but not neurons, activated microglia or brain capillary endothelial cells, had immunoreactivity for AQP4. These findings indicate that activation of brain ET_B receptors causes a decrease in AQP4 expression, suggesting that ET down-regulates brain AQP4 via ET_B receptors.     

水通道蛋白-4在大鼠大脑中的定位研究

目的 观察水通道蛋白-4(AQP4)在大脑实质和室管膜、脉络丛、最后区、神经垂体、腺垂体和松果体中的分布，为研究脑组织中水分子运输和平衡以及内分泌腺体的分泌和调节机制提供形态学基础。方法 免疫组织化学漂洗法(LAB-SA、BCIP／NBT法)。结果 AQP4主要分布于软脑膜、脑室和导水管系统的室管膜、脉络丛等与脑脊液(CSF)直接接触的组织中及脑实质的血管周围；在脑实质内可见阳性细胞体；在海马的锥体细胞层、齿状回的颗粒细胞层和多形细胞层细胞呈阳性；在脑室周围器官如神经垂体、最后区(AP)及松果体和腺垂体(包括中间部)各种腺细胞膜表面均可见AQP4的阳性标记。结论 AQP4不仅与水分子的转运及平衡调节有关、同时与电解质代谢和内分泌活动有关，还可能与下丘脑的神经内分泌活动有关。     

Alterations of AQP2 expression in trigeminal ganglia in a murine inflammation model

Aquaporins (AQPs) are small membrane channel proteins involved in osmoregulation. To date, only AQP1, AQP2, AQP4 and AQP9 have been found in the nervous system. Generally, they are involved in water movement in nervous tissue, nevertheless, recent data would suggest the involvement of AQPs in neurotransmission. In this work, we have evaluated the expression of AQP1 and AQP2 in the trigeminal ganglia of mice in an animal model of perioral acute inflammatory pain using immunohistochemistry and immunoblotting analysis. Our data have shown for the first time, the alteration of AQP2 expression in trigeminal ganglia in acute inflammatory pain showing increased and intracellular redistribution of AQP2 mainly in small-sized neurons and Schwann cells. Apart from this, the AQP1 expression remained unaltered. On the whole, these data support the hypothesis that AQP2 is involved in pain transmission in the peripheral nervous system.