Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells

Haruko Funaki1,2, Tadashi Yamamoto1, Yu Koyama1, Daisuke Kondo1, Eishin Yaoita1, Katsutoshi Kawasaki1, Hideyuki Kobayashi1, Shoichi Sawaguchi2, Haruki Abe2, and Itaru Kihara1

1 Department of Pathology, Institute of Nephrology and 2 Department of Ophthalmology, Niigata University School of Medicine, Niigata 951-8510, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
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References

Aquaporin (AQP) 5 gene was recently isolated from salivary gland and identified as a member of the AQP family. The mRNA expression and localization have been examined in several organs. The present study was focused on elucidation of AQP5 expression and localization in the eye, salivary gland, and lung in rat. RNase protection assay confirmed intense expression of AQP5 mRNA in these organs but negligible expression in other organs. To examine the mRNA expression sites in the eye, several portions were microdissected for total RNA isolation. AQP5 mRNA was enriched in cornea but not in other portions (retina, lens, iris/ciliary body, conjunctiva, or sclera). AQP5 was selectively localized on the surface of corneal epithelium in the eye by immunohistochemistry and immunoelectron microscopy using an affinity-purified anti-AQP5 antibody. AQP5 was also localized on apical membranes of acinar cells in the lacrimal gland and on the microvilli protruding into intracellular secretory canaliculi of the serous salivary gland. In the lung, apical membranes of type I pulmonary epithelial cells were also immunostained with the antibody. These findings suggest a role of AQP5 in water transport to prevent dehydration or to secrete watery products in these tissues.

aquaporin; water channel; messenger ribonucleic acid expression; immunohistochemistry; immunoelectron microscopy

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AQUAPORINS (AQP) are a family of water channels, which are water-selective transporting proteins with homology to major intrinsic protein (MIP) of lens (9). At present, 10 types of mammalian AQPs have been identified (13-15, 19-21, 33). AQP1 was the first to be isolated from human erythrocytes and was then demonstrated to occur widely in fluid-transporting epithelia and endothelia, including kidney and other tissues (3, 6, 25, 27, 31). AQP2 is a water channel that is exclusively present in collecting ducts in the kidney, and the translocation of this water channel from intracellular vesicles to the apical membranes is regulated by vasopressin (8, 23, 24, 35). AQP3 gene was isolated from kidneys and characterized as transporting water and small nonionic molecules such as urea and glycerol. AQP3 is localized on the basolateral membrane of collecting duct cells in kidneys and is found in the epithelial cells of digestive tract and in conjunctival epithelium in the eye (4, 7, 16, 22, 25). A mercury-insensitive water channel, AQP4 was demonstrated to occur abundantly in the brain and less abundantly in the eye, kidney, lung, and intestine (7, 10, 18, 25, 26). AQP5 gene was cloned from salivary gland cDNA (29). Northern blot analysis and in situ hybridization showed AQP5 mRNA expression in the salivary gland, eye, lacrimal gland, lung, and trachea (29). Immunocytochemical microscopy recently demonstrated the presence of AQP5 on the apical membrane of salivary gland, lung (11, 25), and lacrimal glands (17); however, localization of AQP5 in other organs, including eye, has not been examined. In the present study, we employed RNase protection assay and immunostaining for detection of AQP5 mRNA expression and localization in the eye and other systemic organs or tissues.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissues and RNA. Systemic organs (cerebrum, cerebellum, eye, lacrimal gland, submandibular salivary gland, heart, trachea, lung, liver, pancreas, small intestine, colon, spleen, lymph node, and kidney) were removed from adult Wistar-Kyoto rats. Eyes were segmentally dissected into cornea, iris/ciliary body, lens, retina, conjunctiva, and sclera, and the pulmonary bronchus was separated from the lung. A part of each organ was fixed in methyl-Carnoy fixative for 16 h, dehydrated in ethanol, and embedded in paraffin for immunohistochemistry. The eyes were quick frozen in n-hexane at -70°C for immunofluorescence microscopy. Small pieces of cornea and salivary gland were fixed with periodate-lysine-paraformaldehyde fixative for 4 h and embedded in glycol methacrylate resin. Total cellular RNA was purified from these organs and tissues by the acid guanidinium thiocyanate-phenol-chloroform method (2).

PCR cloning of AQP5. Nested, degenerate oligonucleotide primers employed in a previous study (29) were synthesized: sense primers were MDU-1 (5'-STBGGNCAYRTBAGYGGNGCNCA-3') and MDU-2 (5'-G<UNL>GGATCC</UNL>GCHCAYNTNAAYCCHGYNGTNAC-3'); antisense primers were MDD-1 (5'-GCDGRNSCVARDGANCGNGCNGG-3') and MDD-2 (5'-CG<UNL>GAATTC</UNL>GDGCDGGRTTNATNSHNSMNCC-3'), where <OVL>GGATCC</OVL> and <OVL>GAATTC</OVL> are BamH I and EcoR I sites, respectively. Rat salivary gland mRNA (1 µg) was reverse transcribed to cDNA using random hexamer primers and reverse transcriptase. The cDNA was amplified by PCR (30 cycles: 94°C, 1 min; 52°C, 1 min; and 72°C, 1 min) using 100 pmol of MDU-1 and MDD-1. The product was reamplified with 100 pmol of MDU-2 and MDD-2, and a band of ~300 bp of the PCR product was cut from an agarose gel after gel electrophoresis. The purified cDNA was ligated into pGEM-3Z vectors (Promega Japan, Tokyo, Japan) at the EcoR I/BamH I site. Rat AQP5 gene encoding bp 324-651 (328 bp) (29) was verified with an automated DNA sequencer (ABI 373A, Perkin-Elmer Japan, Urayasu, Japan), and the plasmid was linearized with BamH I.

RNase protection assay. RNase protection assay was done as reported previously (5, 30). In brief, 32P-labeled cRNA probes for AQP5 mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were synthesized by in vitro transcription, using the linearized plasmids inserted with AQP5 cDNA or GAPDH cDNA (114 bp, corresponding to bp 673-787) as a housekeeping gene (5, 34). The specific radioactivity of 32P-labeled cRNA probes was adjusted to 1 × 105 cpm/µl each in hybridization buffer (80% formamide, 40 mM PIPES, 0.4 M NaCl, and 1 mM EDTA). Ten micrograms of total cellular RNA isolated from various tissues were hybridized with the cRNA probes (1 × 105 cpm each) at 48°C for 16 h in 10 µl of hybridization buffer. Unhybridized probes were digested with RNase A (4.0 µg/ml) and RNase T1 (120 U/ml) mixture at 30°C for 1 h, and then the RNases were digested with proteinase K (0.5 mg/ml) at 37°C for 30 min. After phenol-chloroform extraction, the hybridized probes were precipitated with ethanol, denatured at 85°C for 3 min, and electrophoresed on 6% polyacrylamide gels. The dried gels were exposed to X-ray films (Fuji Photo Film, Kanagawa, Japan) for 16 h at -80°C.

Preparation of antibody. An oligopeptide corresponding to the COOH-terminal 15 amino acids of AQP5 (NH2-DHREERKKTIELTAH-COOH) with an added cysteine at the COOH-terminus was synthesized and conjugated with keyhole limpet hemocyanin as a carrier protein. The conjugate of 1 mg was emulsified with complete Freund's adjuvant and injected subcutaneously into New Zealand White rabbits three times at 2-wk intervals. One week after the last injection, the blood was collected to obtain antiserum (IBL, Fujioka, Japan). Anti-AQP5 antibody was affinity-purified by using an AQP5 synthetic peptide-conjugated column (Cellulofine, Seikagaku, Tokyo, Japan), and the reactivity of the antibody to AQP5 was examined by immunohistochemistry and Western blot analysis. The specificity of the immunostaining of tissues was verified by blocking of the staining after absorption of the antibody with the synthetic peptide. For absorption, 50 µg of the synthetic peptide (~10 times excess to IgG at molar ratio) was mixed with 1 ml of the affinity-purified anti-AQP5 antibody (0.5 mg IgG/ml) for 16 h at 4°C.

Western blotting. Rat salivary gland, whole eye, and cornea were homogenized in 8 M urea buffer [8 M urea, 50 mM Tris · HCl (pH 8.0), 1 mM dithiothreitol, and 1 mM EDTA] using a homogenizer (Polytron, Kinematica, Lucerne, Switzerland). The homogenates were kept at room temperature for 1 h and centrifuged for 30 min at 10,000 g at 4°C to remove cellular debris. Rat lung was homogenized in 50 mM Tris · HCl buffer (pH 7.4) and centrifuged for 10 min at 1,000 g to remove cellular debris. The supernatant was recentrifuged for 10 min at 40,000 g at 4°C to obtain the membrane fraction. Aliquots (~200 µg of total protein) of the supernatants were diluted in the 2× sample buffer [100 mM Tris · HCl (pH 6.8), 4% SDS, 10% beta -mercaptoethanol, and 20% glycerol], boiled for 5 min, separated by electrophoresis on a 4-20% gradient SDS-polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane. The membranes were preincubated for 2 h with blocking buffer (10% nonfat milk, 0.05% Tween 20, and 0.5% NaN3 in PBS) and incubated with preimmune serum (diluted 1:2,000 in blocking buffer, ~50 µg IgG/ml) or the affinity-purified antibody (diluted 1:10 in blocking buffer, 50 µg IgG/ml) at room temperature overnight. The membranes were then washed in several changes of washing buffer (0.05% Tween 20 in PBS), incubated for 1 h with goat anti-rabbit immunoglobulins conjugated to peroxidase-labeled dextran polymer (DAKO, Carpinteria, CA), which had been premixed with 0.02 volume of normal rat serum, and colored with diaminobenzidine.

Immunohistochemistry and immunofluorescence microscopy. The paraffin-embedded tissues were sectioned at a thickness of 4 µm, and the sections were deparaffined with xylene and then rehydrated through ethanol and distilled water. They were sequentially incubated with 1) normal goat serum (1:20 dilution) for 30 min, 2) affinity-purified anti-AQP5 antibody (1:3 dilution) or preimmune sera (1:400 dilution) or affinity-purified anti-AQP5 peptide antibody absorbed with synthetic AQP5 (1:3 dilution) for 16 h, and 3) goat anti-rabbit immunoglobulins conjugated to peroxidase-labeled dextran polymer (1:5 dilution) for 60 min. The peroxidase reaction products were colored with diaminobenzidine.

For localization of AQP5 in rat eyes, cryosections (4 µm thick) were fixed in acetone at 4°C for 5 min and incubated with the affinity-purified anti-AQP5 antibody (1:3 dilution), preimmune sera (1:400 dilution), or affinity-purified anti-AQP5 antibody preabsorbed with synthetic AQP5 (1:3 dilution) at 4°C overnight and then incubated with FITC-conjugated goat anti-rabbit IgG (Seikagaku Kogyo, Tokyo, Japan) for 30 min at 37°C.

Immunogold electron microscopy. Ultrathin sections of resin-embedded tissues collected on nickel grid meshes were incubated with 5% nonfat milk for 60 min and subsequently with affinity-purified anti-AQP5 antibody (1:3 dilution) overnight. After several washes with PBS, the sections were incubated with gold (10 nm)-labeled anti-rabbit IgG (1:20; Aurion, Wagrningen, The Netherlands) for 2 h. These sections were gently washed in PBS several times, postfixed with 2.5% glutaraldehyde, and washed again with distilled water twice. They were then counterstained with 2% aqueous uranyl acetate and 1% lead citrate and examined under an electron microscope (Hitachi, Ibaragi, Japan).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

mRNA expression in systemic organs. AQP5 mRNA expression was demonstrated to be intense in RNA obtained from the salivary gland and less intense in whole eye and lung by RNase protection assay (Fig. 1A). No or negligible AQP5 mRNA expression was detected in other organs, including the trachea and bronchus of the respiratory system (Fig. 1B). In the fractions of eye, the expression was extremely high in cornea RNA and was negligible in other compartments (Fig. 1B).


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Fig. 1.   Expression of aquaporin (AQP) 5 mRNA detected by RNase protection assay. RNA samples (10 µg each) were obtained from rat tissues. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. A: lane 1, cerebrum; lane 2, cerebellum; lane 3, eye; lane 4, submandibular salivary gland; lane 5, heart; lane 6, lung; lane 7, liver; lane 8, pancreas; lane 9, small intestine; lane 10, colon; lane 11, spleen; lane 12, lymph nodule; lane 13, kidney. B: lane 1, lung; lane 2, bronchus; lane 3, trachea; lane 4, whole eye; lane 5, cornea; lane 6, iris/ciliary body; lane 7, lens; lane 8, retina; lane 9, conjunctiva; lane 10, sclera.

Western blot analysis. The specificity or reactivity of the anti-AQP5 antibody was examined by Western blotting using solubilized salivary gland, lung, whole eye, and cornea samples. As shown in Fig. 2, ~27- and ~34-kDa bands were specifically stained in salivary gland and cornea samples with the affinity-purified anti-AQP5 antibody. The ~27-kDa band is presumed to be the nonglycosylated form of AQP5, and the ~34-kDa band is presumed to be the glycosylated form. These bands were less conspicuous in whole eye and lung samples.


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Fig. 2.   Western blot analysis. Two bands of ~27 and ~34 kDa, presumably corresponding to nonglycosylated and glycosylated forms of AQP5, respectively, were immunoblotted in each sample (lane 1, salivary gland; lane 2, lung; lane 3, whole eye; lane 4, cornea).

Immunohistochemistry. Immunohistochemistry and immunofluorescence microscopy using the affinity-purified AQP5 antibody demonstrated apparent staining in the eye, salivary gland, lung, and lacrimal gland but no staining in other organs.

In the eyes, the immunofluorescence staining was exclusively localized at the corneal epithelium (Fig. 3, A and B). The staining was removed when the antibody was preincubated with the synthetic AQP5 peptide (Fig. 3C). Among the corneal epithelium cells, wing cells composing the intermediate layer were more intensely stained, circumscribing the outline of the cell membranes. The staining was less intense in the superficial cells and columnar basal cells. Immunoelectron microscopy clearly demonstrated that AQP5 was present on the cell membranes of corneal epithelial cells (Fig. 4A). No specific staining was observed in the corneal epithelium with preimmune sera.


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Fig. 3.   Immunohistochemical localization of AQP5 protein in rat organs. A-C: by immunofluorescence microscopy, AQP5 is localized exclusively at corneal epithelium (asterisks) and not at corneal endothelium (arrows) or other tissue in rat eye (A, ×270; B, ×540). No staining is observed with antibody preincubated with synthetic AQP5 peptide (C, ×270). D-F: in salivary gland, specific staining of AQP5 is observed intensely along intercellular secretory canaliculi and less intensely on apical membrane but is not observed on basolateral membrane of serous gland (S) (D, ×270; E, ×540). No staining is shown in mucous glands (M) or striated ducts (arrows). Immunostaining is removed when anti-AQP5 antibody preabsorbed with synthetic AQP5 peptide is employed (F, ×270). G and H: in lung, antibody noticeably stains apical membrane of type I pulmonary epithelial cells (G, ×540) but not type II pulmonary epithelial cells (arrows), bronchi, or pulmonary arteries and veins. No specific staining is observed with antibody absorbed with synthetic AQP5 peptide (H, ×540). I and J: in lacrimal grands, apical membrane of acinar cells is stained with anti-AQP5 antibody (I, ×540). Staining is removed with synthetic AQP5 peptide-preabsorbed antibody (J, ×540).


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Fig. 4.   Immunogold electron microscopic localization of AQP5 in rat cornea and salivary gland. A: immunogold labeling for AQP5 is found on plasma membranes of corneal epithelium cells (×31,000). B: extensive gold labeling is observed on microvilli in intercellular secretory canaliculi (IC) of serous gland cells (×22,000).

In the salivary gland, acinar cells of the serous glands were specifically stained with the anti-AQP5 antibody by immunohistochemistry (Fig. 3, D and E). No staining was observed in acinar cells of the mucous glands or striated duct cells. In the serous glands, the immunostaining was intense along the intercellular secretory canaliculi and was less intense on the apical membrane of the acinar cells. AQP5 was not detected on the basolateral membrane of the serous gland acinar cells. The immunostaining observed at the secretory end pieces gradually disappeared at the transitional point to the intercalated duct. No specific staining was observed with preimmune serum or anti-AQP5 antibody preabsorbed with the synthetic AQP5 peptide (Fig. 3F). Immunoelectron microscopy confirmed these findings, showing intense gold labeling on the microvilli in the intercellular secretory canaliculi of the serous gland acinar cells (Fig. 4B).

Immunostaining for AQP5 was apparent on the apical membrane of type I pulmonary epithelial cells in the lung (Fig. 3G). The staining on the type I pulmonary epithelial cells was again absent when the anti-AQP5 antibody had been previously absorbed with the synthetic AQP5 peptide (Fig. 3H). No AQP5 was demonstrated in the type II pulmonary epithelial cells, bronchi, or pulmonary arteries and veins. In the trachea, no specific staining was observed with the anti-AQP5 antibody, compared with normal rabbit serum.

In the lacrimal gland, AQP5 was detected on the apical membrane of acinar cells (Fig. 3I). No specific staining was observed with anti-AQP5 antibody absorbed with the synthetic AQP5 peptide (Fig. 3J).

    DISCUSSION
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Abstract
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Materials & Methods
Results
Discussion
References

AQP5 mRNA expression in systemic organs has been examined by Northern blot and in situ hybridization (29) and by RNase protection assay (32). The expression of AQP5 mRNA in systemic organs observed in the present study was almost concordant with these previous studies (29, 32), in which AQP5 mRNA expression was demonstrated in the salivary gland, corneal epithelium, lacrimal gland, and lung. In contrast to these studies, AQP5 mRNA expression was not detected in the trachea by RNase protection assay in the present study. The reason for this discrepancy is unknown but may be explained by differences between the rat strains used (Wistar-Kyoto rats in the present study and Sprague-Dawley rats in the previous study). However, the finding of no AQP5 mRNA expression in trachea is also consistent with data obtained by the present immunohistochemistry and previous immunocytochemical (25) and in situ hybridization studies (29). Another possible explanation may be the presence of another AQP member in the trachea, the nucleotide sequence of which is closely homologous to that of AQP5 and is detected by cross-hybridization of Northern blotting.

The cellular and subcellular localization of AQP5 protein is informative for speculation on a role of AQP5 in water movement in these organs or tissues. A recent study clearly demonstrated localization of AQP5 at the apical side of the acinar cells in salivary gland and at the apical membrane of type I pulmonary epithelial cells in lung by use of a specific antibody against rat AQP5 (25). However, cellular and subcellular localization of AQP5 in the eye has not been revealed yet, although AQP5 mRNA expression is intense in the eye. To examine the AQP5 localization in rat eye and other organs, we prepared an antibody against rat AQP5 by immunizing rabbits with a synthetic peptide of rat AQP5 for immunohistochemistry, immunofluorescence, and immunoelectron microscopy. The antibody appeared to react to ~27- and ~34-kDa bands in protein samples prepared from cornea and salivary gland, which expressed AQP5 mRNA intensely. The ~27- and ~34-kDa bands are presumed to be the nonglycosylated and glycosylated forms of AQP5, respectively, as shown in other AQP family members (4, 24, 26, 27). Because no immunostaining was observed with this antibody in the kidney, where AQP1, AQP2, AQP3, and AQP4 have been demonstrated, the antibody was assumed to react to AQP5 specifically (data not shown). In addition, the anti-AQP5 antibody stained corneal epithelium in the eye, acinar cells in serous salivary gland, type I pulmonary epithelial cells, and acinar cells in the lacrimal gland, and the staining was removed when the preabsorbed antibody was used. These findings are concordant with AQP5 mRNA expression sites, indicating the reactivity and specificity of the AQP5 antibody to AQP5.

In the eye, several types of AQP have been demonstrated to be water channels: MIP (AQP0) in lens fiber (9); AQP1 in cornea, trabecular meshwork, canal of Schlemm, iris, ciliary body, and lens (28); AQP3 in conjunctival epithelium (7); and AQP4 in iris, ciliary body, and retinal nuclear layer (7, 10). The present study showed exclusive AQP5 mRNA expression in the cornea and negligible AQP5 mRNA expression in the other compartments of the eye. Immunoelectron microscopy clearly demonstrated that AQP5 was constitutively present on the cell membrane of corneal epithelial cells and on the microvilli of the intercellular canaliculi in salivary glands in the present study. The observations are consistent with the previous finding shown by in situ hybridization (29). Because AQP5 is phyrogenically close to AQP2 (29), AQP5 may be speculated to translocate in response to some stimuli, like AQP2 (23, 35). However, the ultrastructural findings demonstrated the presence of AQP5 on the cell membrane and its absence in the cytoplasm, suggesting that AQP5 is not reserved in the cytoplasm.

Corneal epithelium consists of three types of epithelial cells (superficial, wing, and basal cells), and AQP5 was demonstrated on the cell membranes of all these cells by immunohistochemistry and immunoelectron microscopy. The presence of water channel in corneal epithelium has been suggested by physiological data showing that unidirectional water permeability of frog corneal epithelium increased significantly when chloride ion was present (1, 12). Therefore, AQP5 is strongly presumed to play a role in the water permeability of the corneal epithelium, although dependency of AQP5 on chloride ion has not been elucidated. AQP1 is also present in cornea but is restricted to corneal endothelium (31); therefore, the corneal water permeability may not be accounted for by AQP1 alone. AQP1 likely provides the gate for water from the corneal stroma to the aqueous humor or vice versa, and AQP5 may play a pivotal role in water translocation through corneal epithelium. These observations suggest an integral role of AQP1 and AQP5 in the prevention of dehydration and the maintenance of transparency of cornea.

AQP5 was demonstrated to occur in the salivary gland in the present study, as previously shown (11, 25). However, the immunohistochemistry in the present study further clarified the exclusive localization of AQP5 in the apical membrane of serous gland cells but not of mucous gland cells. Furthermore, AQP5 was demonstrated by immunoelectron microscopy to be intensely expressed on the microvilli in the intercellular canaliculi of serous glands but not in the cytoplasm of the acinar cells. The serous glands are known to secrete water-rich fluid containing enzymatic proteins, whereas mucous glands secrete mainly mucin. Because the water-rich fluid is secreted in the intercellular canaliculi where AQP5 was abundantly present, it is reasonable to speculate that AQP5 plays an important role in the secretion. Concomitantly, the finding may indicate a possible presence of other AQP members in the basolateral membrane of serous gland cells, which makes it possible for water to translocate through these cells.

In lung, AQP5 mRNA expression was intense, and AQP5 was conspicuously localized in the type I pulmonary epithelial cells. The presence of AQP1 has been demonstrated in both the alveolar cells and capillary endothelial cells (6), suggesting a role of AQP1 in transalveolar water movement in the lung, primarily through the transcellular route under physiological conditions (6). AQP5 mRNA expression has been demonstrated on pulmonary walls by in situ hybridization (29) and on type I alveolar epithelial cells by immunohistochemistry (25). The present study confirmed the localization of AQP5 in the type I pulmonary epithelial cells, suggesting a role of AQP5 in transalveolar water movement in the lung that is the same as that of AQP1.

    ACKNOWLEDGEMENTS

We thank Kan Yoshida and Masaaki Nameta for expert technical assistance.

    FOOTNOTES

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture (Japan).

Address for reprint requests: T. Yamamoto, Dept. of Pathology, Institute of Nephrology, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata 951-8510, Japan.

Received 7 July 1997; accepted in final form 22 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Candia, O. A., and A. C. Zamudio. Chloride-activated water permeability in the frog corneal epithelium. J. Membr. Biol. 143: 259-266, 1995[Medline].

2.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

3.   Denker, B. M., B. L. Smith, F. P. Kuhajda, and P. Agre. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J. Biol. Chem. 263: 15634-15642, 1988[Abstract/Free Full Text].

4.   Ecelbarger, C. A., J. Terris, G. Frindt, M. Echevarria, D. Marples, S. Nielsen, and M. A. Knepper. Aquaporin-3 water channel localization and regulation in rat kidney. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F663-F672, 1995[Abstract/Free Full Text].

5.   Feng, L., W. W. Tang, D. J. Loakutoff, and C. B. Wilson. Dysfunction of glomerular fibrinolysis in experimental antiglomerular basement membrane nephritis. J. Am. Soc. Nephrol. 3: 1753-1764, 1993[Abstract].

6.   Folkesson, H. G., M. A. Matthay, H. Hasegawa, F. Kheradmand, and A. S. Verkman. Transcellular water transport in lung alveolar epithelium through mercury-sensitive water channels. Proc. Natl. Acad. Sci. USA 91: 4970-4974, 1994[Abstract].

7.   Frigeri, A., M. A. Gropper, C. W. Turck, and A. S. Verkman. Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes. Proc. Natl. Acad. Sci. USA 92: 4328-4331, 1995[Abstract].

8.   Fushimi, K., S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature 361: 549-552, 1993[Medline].

9.   Gorin, N. B., S. B. Yancey, J. Cline, J. P. Revel, and J. Horitz. The major intrinsic protein (MIP) of the bovine lens fiber membrane. Cell 39: 49-59, 1984[Medline].

10.   Hasegawa, H., T. Ma, W. Skach, M. A. Matthay, and A. S. Verkman. Molecular cloning of a mercurial-insensitive water channel expressed in selected water-transporting tissues. J. Biol. Chem. 269: 5497-5500, 1994[Abstract/Free Full Text].

11.   He, X., C.-M. Tse, M. Donowitz, S. L. Alper, S. E. Gabriel, and B. J. Baum. Polarized distribution of key membrane transport proteins in the rat submandibular gland. Pflügers Arch. 433: 260-268, 1997[Medline].

12.   Horwich, T., C. Ibarra, P. Ford, A. Zamudio, M. Parisi, and O. A. Candia. mRNA from frog corneal epithelium increases water permeability in Xenopus oocytes. Invest. Ophthalmol. Vis. Sci. 36: 2772-2774, 1995[Abstract].

13.   Ishibashi, K., M. Kuwahara, Y. Gu, Y. Kageyama, A. Tohsaka, F. Marumo, and S. Sasaki. Cloning and functional expression of a new water channel abundantly expressed in the testis also permeable to glycerol and urea. J. Biol. Chem. 272: 20782-20786, 1997[Abstract/Free Full Text].

14.   Ishibashi, K., M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki. Cloning and functional expression of a new aquaporin (AQP9) abundantly expressed in the peripheral leukocytes permeable to water and urea, but not to glycerol. Biochem. Biophys. Res. Commun. 244: 268-274, 1998[Medline].

15.   Ishibashi, K., M. Kuwahara, Y. Kageyama, A. Tohsaka, F. Marumo, and S. Sasaki. Cloning and functional expression of a new water channel abundantly expressed in the testis. Biochem. Biophys. Res. Commun. 237: 714-718, 1997[Medline].

16.   Ishibashi, K., S. Sasaki, K. Fushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y. Yamaguchi, T. Gojobari, and F. Marumo. Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells. Proc. Natl. Acad. Sci. USA 91: 6269-6273, 1994[Abstract].

17.   Ishida, N., S. Hirai, and S. Mita. Immunolocalization of aquaporin homologs in mouse lacrimal glands. Biochem. Biophys. Res. Commun. 238: 891-895, 1997[Medline].

18.   Jung, J. S., R. V. Bhat, G. M. Preston, W. B. Guggino, J. M. Baraban, and P. Agre. Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc. Natl. Acad. Sci. USA 91: 13052-13056, 1994[Abstract/Free Full Text].

19.   King, L. S., and P. Agre. Pathophysiology of the aquaporin water channels. Annu. Rev. Physiol. 58: 619-648, 1996[Medline].

20.   Knepper, M. A. The aquaporin family of molecular water channels. Proc. Natl. Acad. Sci. USA 91: 6255-6258, 1994[Free Full Text].

21.   Koyama, Y., T. Yamamoto, D. Kondo, H. Funaki, E. Yaoita, K. Kawasaki, N. Sato, K. Hatakeyama, and I. Kihara. Molecular cloning of a new aquaporin from rat pancreas and liver. J. Biol. Chem. 272: 30329-30333, 1997[Abstract/Free Full Text].

22.   Ma, T., A. Frigeri, H. Hasegawa, and A. S. Verkman. Cloning of a water channel homolog expressed in brain meningeal cells and kidney collecting duct that functions as a stilbene-sensitive glycerol transporter. J. Biol. Chem. 269: 21845-21849, 1994[Abstract/Free Full Text].

23.   Nielsen, S., C. L. Chou, D. Marples, E. I. Christensen, B. K. Kishore, and M. A. Knepper. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc. Natl. Acad. Sci. USA 92: 1013-1017, 1995[Abstract].

24.   Nielsen, S., S. R. DiGiovanni, E. I. Christensen, M. A. Knepper, and H. W. Harris. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc. Natl. Acad. Sci. USA 90: 11663-11667, 1993[Abstract].

25.   Nielsen, S., L. S. King, B. M. Christensen, and P. Agre. Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat. Am. J. Physiol. 273 (Cell Physiol. 42): C1549-C1561, 1997[Medline].

26.   Nielsen, S., E. A. Nagelhus, M. Amiry-Moghaddam, C. Bourque, P. Agre, and O. P. Pettersen. Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. J. Neurosci. 17: 171-180, 1997[Abstract/Free Full Text].

27.   Nielsen, S., B. L. Smith, E. I. Christensen, M. A. Knepper, and P. Agre. CHIP28 water channels are localized in constitutively water-permeable segments of nephron. J. Cell Biol. 120: 371-383, 1993[Abstract].

28.   Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385-387, 1992[Medline].

29.   Raina, S., G. M. Preston, W. B. Guggino, and P. Agre. Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J. Biol. Chem. 270: 1908-1912, 1995[Abstract/Free Full Text].

30.   Sambrook, J., F. F. Fritsch, and T. Maniatis. Extraction, purification, and analysis of messenger RNA from eukaryotic cells. In: Molecular Cloning. A Laboratory Manual (2nd ed.), edited by J. Sambrook, F. F. Fritsch, and T. Maniatis. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989, p. 71-78.

31.   Stamer, W. D., R. W. Snyder, B. L. Smith, P. Agre, and J. W. Regan. Localization of aquaporin CHIP in the human eye: implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance. Invest. Ophthalmol. Vis. Sci. 35: 3867-3872, 1994[Abstract].

32.   Umenishi, F., A. S. Verkman, and M. A. Gropper. Quantitative analysis of aquaporin mRNA expression in rat tissues by RNase protection assay. DNA Cell Biol. 15: 475-480, 1996[Medline].

33.   Verkman, A. S., A. N. van Hoek, T. Ma, A. Frigeri, W. R. Skach, A. Mitra, B. K. Tamarappoo, and J. Farinas. Water transport across mammalian cell membranes. Am. J. Physiol. 270 (Cell Physiol. 39): C12-C30, 1996[Abstract/Free Full Text].

34.   Yamamoto, T., S. Sasaki, K. Fushimi, K. Ishibashi, E. Yaoita, K. Kawasaki, H. Fujinaka, F. Marumo, and I. Kihara. Expression of AQP family in rat kidneys during development and maturation. Am. J. Physiol. 272 (Renal Physiol. 41): F198-F204, 1997[Abstract/Free Full Text].

35.   Yamamoto, T., S. Sasaki, K. Fushimi, K. Ishibashi, E. Yaoita, K. Kawasaki, F. Marumo, and I. Kihara. Vasopressin increases AQP-CD water channel in apical membrane of collecting duct cells in Brattleboro rats. Am. J. Physiol. 268 (Cell Physiol. 37): C1546-C1551, 1995[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(4):C1151-C1157
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