INTRODUCTION

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DISCOVERY OF THE AQUAPORIN family of water channel proteins has provided a molecular explanation for rapid water movements across the plasma membranes of cells (2). Aquaporin-1 (AQP1) is a constitutively activated water channel (27) and has been identified in multiple tissues, including red blood cells, renal proximal tubules, and capillary endothelium (7, 25, 26, 29), whereas AQP2 is restricted to renal collecting duct, where it is regulated by vasopressin (6, 14, 22). AQP3 is present in the basolateral plasma membranes of the renal collecting duct (9, 13). AQP4 is highly abundant in perivascular glial cells and in ependymal cells (13, 23). The Aqp5 cDNA was isolated from rat salivary gland (28).

Movements of water in the distal lung, airways, oral cavity, and nasopharynx are normally involved in clearance of alveolar fluid, airway humidification, and generation of oral and nasal secretions, whereas abnormal movements of water are found in clinical disorders such as pulmonary edema, freshwater drownings, cystic fibrosis, asthma, allergic rhinitis, and Sjögren's syndrome (17). Aquaporins may participate in these processes, but understanding of these proteins in lung, airways, oral cavity, and nasopharynx is incomplete. Developmental immunoblots have defined the expression patterns of aquaporins in lung and nasopharynx; however, cellular and subcellular sites of expression are incompletely resolved. AQP1 is present in the pulmonary visceral pleura and in peribronchiolar capillary endothelium, where expression is induced by corticosteroids, whereas AQP1 is sparsely present in alveolar capillary endothelium (18); AQP3 and AQP4 have been described in basolateral membranes of tracheal epithelia (13); AQP5 mRNA is strongly expressed in lung, but its cellular location is undefined (28). Moreover, no aquaporin has been defined in alveolar epithelium (17), and expression of aquaporins has yet not been assessed in nasopharynx.

In an accompanying study (19), affinity-purified rabbit antibodies to AQP1, AQP3, AQP4, and AQP5 were shown to react specifically with 28- to 30-kDa polypeptides on immunoblots of respiratory and glandular tissues. Here we use the same antibodies for a comprehensive analysis of the cellular and subcellular distribution of AQP1, AQP3, AQP4, and AQP5 in the lung and upper airways to provide insight into the roles of aquaporins in the complex physiology of the respiratory tract and glandular tissues.

	MATERIALS AND METHODS

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Antibodies. Anti-peptide antibody specific for AQP5 was prepared and characterized as described in the accompanying study (19). The synthetic peptide (NH₂-CEPEEDWEDHREERKKTIELTAH-COOH) corresponding to the COOH-terminus of AQP5 was cross-linked to keyhole limpet hemocyanin and injected into New Zealand White rabbits (Lofstrand Laboratories, Gaithersburg, MD). Polyclonal anti-AQP5 immunoglobulin G (IgG) was affinity purified from serum, using Sulfolink coupling gel (Pierce) conjugated with 2-4 mg of the synthetic peptide. As a negative labeling control, anti-AQP5 was preincubated with a 100-fold excess of the immunizing peptide at 4°C for 24 h. Polyclonal, affinity-purified rabbit antibodies to AQP1 (anti-AQP1) that react with the 4-kDa COOH-terminal domain of the protein were previously described (29). Affinity-purified peptide-derived antibodies against AQP3 (kindly provided by Dr. Mark Knepper, National Institutes of Health, Bethesda, MD) and AQP4 have previously been characterized in detail (9, 23, 30).

Preparation of tissues for immunocytochemistry. Adult male Wistar rats were anesthetized, heparinized, and perfusion-fixed through the right atrium with 4 or 8% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Tissues were postfixed for 2 h in the same fixative, infiltrated for 30 min with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and rapidly frozen in liquid N₂. These blocks were used for cryosectioning or freeze substitution with embedding in Lowicryl HM20. With use of an automatic freeze substitution system (AFS, Reichert, Vienna, Austria), samples were sequentially equilibrated over 3 days in 0.5% uranyl acetate in methanol, at temperatures gradually decreasing from 80°C to 70°C, and then rinsed in pure methanol for 24 h at 70°C to 45°C (21, 22, 25). At 45°C, the samples were serially infiltrated with Lowicryl HM20 and methanol 1:1, then 2:1, and finally with pure Lowicryl HM20, before ultraviolet polymerization for 2 days at 45°C and 2 days at 0°C.

Immunohistochemistry. For light microscopy, cryosections were placed on gelatin-coated glass slides and processed as described (22, 24, 25). After preincubation with phosphate-buffered saline containing 1% bovine serum albumin or 0.1% skimmed milk and 0.05 M glycine, the sections were incubated with anti-aquaporin antibodies overnight at 4°C. The use of the affinity-purified antibodies against AQP1, AQP3, AQP4, and AQP5 for immunocytochemistry has previously been described (9, 22-26, 30). The following concentrations (in µg/ml) were used: 0.1-0.2 anti-AQP1, 0.5 anti-AQP3, 1 anti-AQP4, and 0.5-2 anti-AQP5. The labeling was visualized with peroxidase-conjugated secondary antibody (P448, 1:100, DAKO A/S, Glostrup, Denmark). Sections were counterstained with Meier counterstain.

Immunoelectron microscopy. Ultrathin Lowicryl sections (60-80 nm) or ultrathin cryosections (80 nm) were obtained with a Reichert Ultracut FSC ultracryomicrotome. The sections were incubated with affinity-purified anti-AQP5 or anti-AQP3 (described in Antibodies) and labeling was visualized with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10, BioCell Research Laboratories, Cardiff, UK). The sections were stained with uranyl acetate for 10 min or with uranyl acetate for 10 min followed by lead citrate for 15 s, and examined in a Philips CM100 electron microscope.

Immunolabeling controls. The following controls confirmed specificity of light and electron microscopic studies: 1) incubation with protein A-purified preimmune rabbit IgG, 2) adsorption of anti-AQP5 (0.1 µg/ml) with the immunizing AQP5 peptide (10 µg/ml), and 3) incubations without primary antibody or without primary and secondary antibody.

	RESULTS

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Cellular and subcellular distribution of aquaporins in distal lung. Immunocytochemical studies performed on 0.9-µm cryosections of distal lung using affinity-purified antibodies revealed that AQP5 is expressed in alveolar type I pneumocytes (Fig. 1, A and B). AQP5 was not detected in type II pneumocytes, vascular endothelium, or interstitial cell types (Fig. 1, A and B), and specificity was demonstrated by nonimmune control (Fig. 1C). Immunoelectron microscopy also demonstrated that AQP5 is present only in the apical membrane of type I pneumocytes (Fig. 2, A and D), with no expression in type II cells (Fig. 2C) or capillary endothelium (Fig. 2, A and D). Specificity was established by nonimmune controls (Fig. 2, B and E), and AQP1, AQP3, and AQP4 were not detected in alveolar epithelium of rat (not shown).

View larger version (111K): [in this window] [in a new window]   Fig. 1.   Immunocytochemical localization of aquaporins in rat lung. Cryosections (0.9 µm) of distal rat lung (A-C), bronchus (D, F, and H), and trachea (E, G, and I) were labeled with affinity-purified antibodies and visualized with peroxidase-conjugated secondary antibody. A and B: type I pneumocytes exhibit aquaporin-5 (AQP5) immunolabeling (arrows), whereas endothelial cells of capillaries (A, arrowheads) and venules (B, arrowheads), myocytes, and fibroblasts are unlabeled. ×1,000. C: immunolabeling control using nonimmune immunoglobulin G (IgG) in place of anti-AQP5 reveals absence of labeling. ×480. D: section of bronchus reveals no labeling with anti-AQP3. ×480. E: basal cells of ciliated, pseudostratified tracheal epithelium exhibit strong immunolabeling for AQP3 (arrows). Except for occasional sites that most likely represent sectioning artifact, basolateral and apical membrane of surface epithelial cells (arrowhead) are unlabeled. ×1,000. Sections of bronchus (F) and trachea (G) demonstrate expression of AQP4 on lateral (arrows) and basal plasma membranes of surface cells in pseudostratified columnar epithelium but are distinct from those labeled with anti-AQP3 (E); apical membranes are unlabeled. ×1,000. Bronchus (H) and trachea (I) exhibit no labeling with anti-AQP5. ×480.

View larger version (68K): [in this window] [in a new window]   View larger version (79K): [in this window] [in a new window]   View larger version (47K): [in this window] [in a new window]   Fig. 2.   Immunoelectron microscopic localization of AQP5 in distal rat lung. Ultrathin cryosections (A-C) or ultrathin Lowicryl sections (D and E) were labeled with affinity-purified anti-AQP5 and visualized with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles. A: type I pneumocytes exhibit labeling with anti-AQP5 in apical plasma membranes (arrows); basolateral plasma membranes are unlabeled (arrowheads). Capillary endothelial cell (EN) is unlabeled. ×60,000. EP, epithelial cells. B: control with nonimmune IgG reveals absence of immunolabeling. Arrowheads, basolateral plasma membranes. ×60,000. C: section of type II pneumocyte with lamellar granule (G) reveals negligible labeling with anti-AQP5. ×60,000. D: Lowicryl section of type I pneumocyte showing anti-AQP5 labeling of apical plasma membrane (arrows) with no labeling of basolateral membrane (arrowheads) or adjacent endothelial cell. ×60,000. E: control using nonimmune IgG reveals absence of immunolabeling. ×60,000. Note that majority of immunogold particles in A and D appear to overlie apical plasma membrane, whereas only a single immunogold particle is found in C and most likely represents background staining.

Cellular and subcellular distribution of aquaporins in bronchus and trachea. AQP4 is present in basolateral membranes of bronchial epithelium (Fig. 1F) and tracheal epithelium (Fig. 1G), whereas AQP3 is restricted to only the tracheal epithelium (Fig. 1, D and E), consistent with the distribution found by immunoblotting (19). The AQP3 and AQP4 antibodies label distinctly different cells in the ciliated, pseudostratified tracheal epithelium. AQP3 appears only in the basal cells of the tracheal epithelium, where it is heavily expressed (Fig. 1E). In contrast, AQP4 is present in the basolateral plasma membranes of the ciliated columnar cells that reach the epithelial surface and is not expressed in basal cells (Fig. 1G). This labeling pattern was confirmed by immunoelectron microscopy revealing abundant AQP3 in basal cells (Fig. 3, A and B) but absence of AQP3 in surface epithelial cells. AQP5 was not detected in either bronchial or tracheal surface epithelium (Fig. 1, H and I).

View larger version (157K): [in this window] [in a new window]   Fig. 3.   Immunoelectron microscopic localization of AQP3 in rat trachea. Ultrathin Lowicryl sections were labeled with affinity-purified anti-AQP3 and visualized with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles. A: plasma membranes of basal cells (BC) in tracheal epithelium exhibit extensive AQP3 immunolabeling (arrows), but surface epithelial cells (SE) displayed no labeling. Immunolabeling of basal cells is notable in plasma membrane processes interdigitating with unlabeled processes of surface epithelial cells. B: basal plasma membrane of basal epithelial cells facing basement membrane also display heavy anti-AQP3 labeling (arrows). Surface epithelial cells are unlabeled. ×75,000.

Cellular and subcellular distribution of aquaporins in nasopharynx and conchus. Complex patterns of aquaporin distribution were found at these sites. As in trachea, AQP3 is present in the basal cells of the nasopharyngeal epithelium (Fig. 4A). In nasal conchus, however, anti-AQP3 strongly labels the basolateral membrane of surface epithelial cells (Fig. 5A'). Immunoelectron microscopy confirmed extensive anti-AQP3 labeling of basolateral plasma membranes in conchal surface epithelial cells (Fig. 6). Most of the surface epithelial cells are labeled with anti-AQP3 (Figs. 5A' and 6), but closer inspection revealed a few adjacent cells not surrounded by an anti-AQP3-labeled plasma membrane, appearing as occasional large cells with two nuclei (Fig. 5A'); these may correspond to the AQP4-labeled cells (Fig. 5B). Immunoelectron microscopy confirmed the absence of AQP3 immunolabeling in these conchal epithelial cells (Fig. 6). As in trachea, AQP4 is present in basolateral membranes of ciliated surface epithelial cells in nasopharynx (Fig. 4B); goblet cells were not immunolabeled. Cells at the base of the pseudostratified columnar epithelium of nasopharynx are predominantly unlabeled by anti-AQP4 (Fig. 4B). AQP1 was not detected in the surface epithelium of nasopharynx or conchus, but AQP1 is abundant in the capillaries and venules beneath the nasopharyngeal epithelium (Fig. 4D), as well as in the capillaries and venous sinuses of the nasal conchus (Fig. 5D).

View larger version (164K): [in this window] [in a new window]   Fig. 4.   Immunocytochemical localization of aquaporins in rat nasopharyngeal surface epithelium (A-D) and subepithelial glands (E-G). Cryosections (0.9 µm) were labeled with affinity-purified antibodies and visualized with peroxidase-conjugated secondary antibody. A: basal cells of pseudostratified ciliated nasopharyngeal epithelium are heavily labeled with anti-AQP3 (arrowheads). Except for occasional cells that most likely represent sectioning artifact, basolateral and apical membrane of ciliated surface epithelium (arrows) are unlabeled. ×1,000. B: lateral plasma membranes of nasopharyngeal surface epithelial cells strongly express AQP4 (arrows); basal cells (arrowheads) of pseudostratified epithelium are predominantly unlabeled. ×1,000 C: AQP5 is not expressed in nasopharyngeal epithelium. ×1,000 D: AQP1 is not expressed in nasopharyngeal surface epithelium, but strong immunolabeling is seen in both apical and basolateral plasma membranes of endothelial cells in capillaries and venules (arrows). Subepithelial fibroblasts (arrowheads) also express AQP1. ×1,000. E: glands beneath nasopharyngeal epithelium exhibit no labeling with anti-AQP3. ×480. F: immunolabeling control for G using nonimmune IgG. ×480. G: distinct labeling with anti-AQP5 is noted in apical plasma membrane of glandular epithelial cells (arrows), with no labeling of basolateral domains. ×1,000. This distribution was confirmed on multiple other sections (not shown).

View larger version (141K): [in this window] [in a new window]   Fig. 5.   Immunocytochemical localization of aquaporins in rat conchus epithelium and glands. Cryosections (0.9 µm) were immunolabeled with affinity-purified antibodies and visualized with peroxidase-conjugated secondary antibodies. A and A': AQP3 is strongly expressed in basal and lateral plasma membranes (arrows) of most conchus surface epithelial cells (A') and glandular epithelial cells (A). Apical plasma membranes of conchus surface epithelium are not immunolabeled (horizontal arrowheads, A'), and a few cells did not label with anti-AQP3 (vertical arrowheads). ×480. B: AQP4 is expressed in basolateral membrane of only occasional cells in conchus surface epithelium and in glandular epithelial cells (arrows). ×480. C: AQP5 is expressed in apical membrane of select clusters of cells within conchus epithelium (arrows). ×480. D: conchus surface epithelium is not labeled with anti-AQP1; endothelial cells in capillaries and venules (arrows) exhibit strong labeling. ×480. E: AQP4 immunolabeling is present in basolateral membrane (arrows) of clusters of epithelial glandular cells within surface epithelium with no labeling of apical membrane. ×1,000. F: parallel section to (E) reveals expression of AQP5 in apical plasma membrane (arrows) of glandular cell clusters. ×1,000. G: heavy AQP3 immunolabeling is visible in basolateral membrane of conchal subepithelial glandular cells (small arrows); most cells in acinus are labeled, but apical membrane is unlabeled (large arrows). ×1,000. H: AQP4 immunolabeling is visible in basolateral (small arrows) but not apical (large arrow) plasma membrane of some subepithelial glandular cells of conchus. Some cells are not labeled (arrowheads). ×1,000.

View larger version (151K): [in this window] [in a new window]   Fig. 6.   Immunoelectron microscopic localization of AQP3 in rat conchal surface epithelium. Ultrathin Lowicryl sections were labeled with affinity-purified anti-AQP3 and visualized with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles. Plasma membranes of surface epithelial cells of conchal epithelium exhibit extensive AQP3 immunolabeling (arrows). Few cells within epithelium (marked with asterisks) display no labeling. Labeling of surface epithelial cells is seen also in plasma membrane processes interdigitating with other surface epithelial cells. ×75,000.

Cellular and subcellular distribution of aquaporins in nasal and salivary glands. In nasal conchus, AQP3 is strongly expressed in the basolateral membrane of intraepithelial (Fig. 5A) and subepithelial glandular cells (Fig. 5, A and G). AQP4 is expressed on the basolateral membrane of some epithelial cells in nasal conchus (scattered intraepithelial glands, Fig. 5, B and E). AQP4 is present on a larger number of the subepithelial glandular cells (Fig. 5H) but is not present in all cells of subepithelial glands. Thus, as in trachea, AQP3 and AQP4 are expressed in nonoverlapping sites in nasopharynx and conchus (Figs. 4 and 5). In contrast, submandibular salivary glands did not label with anti-AQP3 or anti-AQP4 (not shown); thus a mechanism for basolateral water transport remains to be identified in salivary glands.

View larger version (198K): [in this window] [in a new window]   Fig. 7.   Immunocytochemical localization of AQP5 in rat submandibular salivary gland epithelium. Cryosections (0.9 µm) were labeled with affinity-purified antibodies and visualized with peroxidase-conjugated secondary antibodies. A: AQP5 is confined to apical plasma membrane domains of apical surface of glandular epithelium (arrows). No labeling is present in basolateral aspects of cells. B: no labeling is seen (arrows) in control for which nonimmune IgG was used as primary antibody. C and D: in addition to immunolabeling of apical plasma membrane of secretory glandular epithelial cells (arrows), prominent labeling is also seen over apical plasma membrane domains of cells forming secretory canaliculi and very initial part of secretory duct (arrowheads). A-D: ×1,000.

	DISCUSSION

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Fluid homeostasis in the lung and upper airway is complex, since myriad anatomic and physiological factors influence the disposition of water. Characterization of the relevant molecules is essential to understand the regulation of water transport in the respiratory tract. In this study, we demonstrate distinct, nonoverlapping distributions of AQP1, AQP3, AQP4, and AQP5 from the alveolus to salivary glands and nasopharynx in rat (Table 1).

The alveolar membrane has long been considered a tight epithelium, restricting the movement of water and solute (11). Cellularity of the membrane is maintained by differentiation of type II pneumocytes into type I cells (1). Although AQP5 is specifically expressed in type I pneumocytes, its absence in type II cells suggests a differentiation-specific signal, consistent with recent description of AQP5 expression in a type II cell culture model (33). Water permeability was shown to be greater than solute permeability in the alveolar membrane, suggesting that water crosses the membrane by a transcellular rather than paracellular path (10). Mercury-inhibitable movement of water from the vascular space to the airspace was observed in sheep, and it was proposed that AQP1 in type I pneumocytes mediates water movement into the alveolus (12). Our demonstration of abundant AQP5 in type I cells implicates this molecule in transalveolar water movement, whereas AQP1 is not abundant in alveolar capillary endothelium of rat (18). Alterations in AQP5 expression or function may participate in the pathogenesis or resolution of alveolar edema; likewise, AQP5 may mediate the rapid absorption of water noted to occur in the lungs of freshwater drowning victims. Other questions about alveolar fluid dynamics still remain. It is uncertain whether salt transporters are expressed in type I pneumocytes (20), and it is unknown whether AQP5 in apical membranes of type I cells is complemented by an unidentified water channel in the basolateral membrane (Fig. 8A).

View larger version (43K): [in this window] [in a new window]   Fig. 8.   Schematic diagrams representing aquaporins in rat airway and glandular tissues. A: alveolus contains AQP5 (red) in apical membranes of type I pneumocytes (P1); AQP1 (green) is sparsely present in underlying capillary endothelium. Although a pathway is defined linking airspace and vascular space, no known aquaporin has been identified in basolateral membrane of type I pneumocytes. P2, type II pneumocytes. B: pseudostratified epithelium of trachea and nasopharynx contains two populations of cells, basal cells that contain AQP3 (yellow) and columnar cells that reach airway surface and contain AQP4 (blue). Subepithelial capillaries and fibroblasts contain abundant AQP1. No known aquaporin has been identified in apical membrane of surface epithelium. C: secretory glands contain AQP5 in apical membranes but not in basolateral membranes of acinar cells. Salivary glands do not contain a known aquaporin in basolateral domains (not represented), whereas glandular cells of nasopharynx and conchus contain AQP3 in basolateral membranes of some acinar cells and AQP4 in basolateral membranes of other acinar cells. Secretory canaliculi and intercalated cells forming very initial part of secretory duct contain abundant AQP5 but not AQP3 or AQP4 (not represented).

AQP3 and AQP4 have been identified in tracheal and bronchial epithelium (13), but our studies establish that these proteins have distinct, nonoverlapping distributions in the ciliated, pseudostratified columnar epithelium of trachea (Fig. 8B). AQP3 is found only in basal cells of tracheal epithelium; these cells are thought to serve as stem cells for the tissue and to anchor the epithelium to the basement membrane (16). AQP3 is not expressed in lung distal to the trachea. In contrast, AQP4 resides in the basolateral membranes of columnar cells reaching the airway surface in trachea and bronchi. The functional significance of this compartmentalization remains to be elucidated. AQP5 was identified in trachea by immunoblotting (19) but was not visualized by histochemical analysis of tracheal epithelium. We believe that AQP5 is expressed in glandular epithelium in trachea, as in nasopharynx, but the relative paucity of subepithelial glands in rat trachea (16) makes histological assessment problematic.

We also demonstrate that AQP3, AQP4, and AQP5 have abundant, nonoverlapping expression in the ciliated, pseudostratified columnar epithelium and subepithelial glands of nasopharynx and nasal conchus. Curiously, the patterns of expression of AQP3 and AQP4 are not identical. Cultured nasal epithelial cells have been reported to rapidly change volume when hypertonic solutions were applied to the luminal membrane (32), suggesting the existence of water channels at the apical surface. We did not identify any of the known water channels in the apical membrane of nasopharyngeal or conchal surface epithelium, although AQP5 is present in the apical membrane of submucosal glandular epithelium (Fig. 8C). Lack of a known water channel at the apical membrane of nasopharyngeal surface epithelium indicates that either a still unknown aquaporin resides at that location or another mechanism may be responsible for water transport at that site (Fig. 8B).

AQP1 is expressed on the apical and basolateral membrane of vessels throughout the respiratory system. We previously demonstrated abundant AQP1 in the bronchial circulation of the rat (18), a distribution consistent with expression in subepithelial vessels of the trachea shown here. Additionally, we now find abundant AQP1 expression in subepithelial capillaries and venous sinusoids in nasopharynx, vessels that in large part determine the resistance to airflow through the nose (8). Because nasal capillaries and sinusoids are believed to be fenestrated, AQP1 might play a role in cell volume regulation in addition to its role in transcellular water movement (Figs. 8, A and B). The presence of AQP3 and AQP4 in the basolateral membranes of surface epithelium of airways from trachea, nasopharynx, and conchus suggests that these cells may experience rapid increases or decreases in cell volumes without large transcellular water flow (31). Alternatively, the presence of aquaporins in the basolateral plasma membranes of respiratory epithelial cells may give this cellular surface high water permeability, thereby preventing extensive changes in cell volume during periodic loss of water from the apical plasma membrane from airway humidification during inspiration and expiration.

Although specific functions remain speculative, the potential physiological and pathophysiological significance of aquaporins in the respiratory tract is considerable. Adequate gas exchange necessitates tight control of water in the distal lung and alveolar space. Altered expression or function of AQP5 or AQP1 in distal lung may contribute to generation or amelioration of pulmonary edema. Alterations of the airway surface layer may affect mucociliary transport and may also play a role in the pathogenesis of exercise- or cold-induced asthma (3). It is tantalizing to postulate that secondary alterations in aquaporin expression or function could contribute to the pathogenesis of cystic fibrosis (4) or provide a therapeutic mode for altering the viscosity of airway secretions. Abundant expression of aquaporins in the nasopharynx strongly suggests their participation in normal physiological processes, such as humidification of inspired air, but also suggests that alterations in their function or expression will contribute to the pathogenesis of nasal congestion and rhinorrhea.

Appropriate management of fluid in vascular, interstitial, and airspace compartments is essential for normal function of the respiratory system throughout its length. Each of the aquaporin water channel proteins present in the lung has a unique distribution and ontogeny, which suggests functional specialization. In parallel to the extensive investigation of solute transport in the lung (5), investigation of water channels in this system is needed to fully understand the physiology and pathophysiology of the respiratory tract.