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Expression and localization of aquaporins
in rat gastrointestinal tract
YU KOYAMA,1 TADASHI YAMAMOTO,2 TATSUO TANI,1 KOUEI NIHEI,1
DAISUKE KONDO,2 HARUKO FUNAKI,2 EISHIN YAOITA,2 KATSUTOSHI KAWASAKI,2
NOBUAKI SATO,1 KATSUYOSHI HATAKEYAMA,1 AND ITARU KIHARA2
2Department of Pathology, Institute of Nephrology, and 11st Department of Surgery,
Niigata University School of Medicine, 1–757 Asahimachi-dori, Niigata 951–8510, Japan
messenger ribonucleic acid; in situ hybridization; immunohistochemistry
RECENTLY, A FAMILY of water channels (aquaporin, AQP)
has been identified as molecules that locate on plasma
membrane of various cell types and increase its water
permeability in mammals (12). AQP1 (CHIP28, channelforming intrinsic protein of 28 kDa) was first found as a
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homologous protein to MIP (major intrinsic protein of
bovine lens) in erythrocytes (12, 28) and the proximal
tubule and thin descending limb of Henle in the kidney
and then various epithelial and endothelial cells in
systemic organs (23, 24). AQP2 is exclusively expressed
in the apical membrane and intracellular vesicles of the
collecting duct principal cells in the kidney and is
demonstrated to be regulated by vasopressin (5, 22, 34).
AQP3 (1, 10, 17) and AQP4 (6, 11) are present in many
organs, but AQP5 and AQP6 are selectively localized in
the salivary gland, eye, and lung (30) and in the kidney
(18), respectively. Recently, AQP7 (7, 14), AQP8 (9, 13,
19), and AQP9 (8) were isolated from testis and liver
libraries as new members of AQP. AQP7 is intensely
expressed in the testis, kidney, and heart (7); AQP8 in
the liver, pancreas, salivary gland, and testis (9, 13);
and AQP9 in the liver and leukocytes (8).
The gastrointestinal tract, especially the small and
large intestines, is a main organ for water entrance to
the body. A large volume of water moves and is absorbed
in the gastrointestinal tract every day, estimated as 7.5
liters in the small intestine and 1.5 liters in the large
intestine in humans (25, 27). Although electrolytes
have been demonstrated to cross the absorptive epithelial cells of these tissues by various transporters located on the cell membranes (transcellular route), the
pathway of water transport in the gastrointestinal
tract has not received much attention and water has
been presumed to pass the epithelial cell layer by the
paracellular route through tight junctions (26, 27).
However, recent studies showing the existence of AQP
on the epithelial cells in the gastrointestinal tract
suggested their roles in the water absorption or secretion there (10–13), although the precise cellular localization and the portions of mRNA expression of the
AQP family in the gastrointestinal tract have not been
examined intensively to date. In the present study, we
examined expression and localization of AQP1, AQP2,
AQP3, AQP4, AQP5, and AQP8 in the rat gastrointestinal tract by RNase protection assay and by in situ
hybridization and immunohistochemistry.
MATERIALS AND METHODS
Tissue and RNA preparation. Tissues (esophagus, upper
and lower portions of stomach, jejunum, middle portion of
small intestine, ileum, and proximal and distal colons) were
removed from Wistar-Kyoto rats (3 mo old) and were frozen at
⫺80°C in n-hexane. Total cellular RNA was also isolated from
these tissues by a modified acid guanidinium thiocyanate
phenol-chloroform extraction method (TRIzol, GIBCO BRL,
Life Technologies, Rockville, MD).
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
C621
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Koyama, Yu, Tadashi Yamamoto, Tatsuo Tani, Kouei
Nihei, Daisuke Kondo, Haruko Funaki, Eishin Yaoita,
Katsutoshi Kawasaki, Nobuaki Sato, Katsuyoshi
Hatakeyama, and Itaru Kihara. Expression and localization of aquaporins in rat gastrointestinal tract. Am. J.
Physiol. 276 (Cell Physiol. 45): C621–C627, 1999.—A family
of water-selective channels, aquaporins (AQP), has been
demonstrated in various organs and tissues. However, the
localization and expression of the AQP family members in the
gastrointestinal tract have not been entirely elucidated. This
study aimed to demonstrate the expression and distribution
of several types of the AQP family and to speculate on their
role in water transport in the rat gastrointestinal tract. By
RNase protection assay, expression of AQP1–5 and AQP8 was
examined in various portions through the gastrointestinal
tract. AQP1 and AQP3 mRNAs were diffusely expressed from
esophagus to colon, and their expression was relatively
intense in the small intestine and colon. In contrast, AQP4
mRNA was selectively expressed in the stomach and small
intestine and AQP8 mRNA in the jejunum and colon. Immunohistochemistry and in situ hybridization demonstrated
cellular localization of these AQP in these portions. AQP1 was
localized on endothelial cells of lymphatic vessels in the
submucosa and lamina propria throughout the gastrointestinal tract. AQP3 was detected on the circumferential plasma
membranes of stratified squamous epithelial cells in the
esophagus and basolateral membranes of cardiac gland epithelia in the lower stomach and of surface columnar epithelia in
the colon. However, AQP3 was not apparently detected in the
small intestine. AQP4 was present on the basolateral membrane of the parietal cells in the lower stomach and selectively in the basolateral membranes of deep intestinal gland
cells in the small intestine. AQP8 mRNA expression was
demonstrated in the absorptive columnar epithelial cells of
the jejunum and colon by in situ hybridization. These findings
may indicate that water crosses the epithelial layer through
these water channels, suggesting a possible role of the
transcellular route for water intake or outlet in the gastrointestinal tract.
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AQP FAMILY IN GASTROINTESTINAL TRACT
NaCl, 10 mM Tris · HCl, pH 8.0, for 10 min at room temperature. Then they were hybridized with digoxigenin-labeled
sense or antisense probe (1 ng/ml) or 35S-labeled sense and
antisense AQP8 cRNA probes (1 ⫻ 106 counts per min per
section) overnight at 55°C. After a washing in 2⫻ salinesodium citrate (SSC), 1 mM EDTA at room temperature, the
sections were treated with 20 µg/ml of RNase A for 30 min at
room temperature, followed by washing in 0.1⫻ SSC, 1 mM
EDTA at 55°C for 2 h and washing in 0.5⫻ SSC at room
temperature. Thereafter, they were incubated at 4°C overnight with anti-digoxigenin antibody (Boehringer Mannheim,
1:200 dilution) and colored with nitro blue tetrazolium-5bromo-4-chloro-3-indolyl phosphate. After color development,
the sections were counterstained with Kernechtrot stain
solution (Muto Pure Chemicals, Tokyo, Japan) for 20 min at
room temperature, dehydrated with ethanol, and then
mounted with ENTELLAN (Merck, Darmstadt, Germany).
Alternatively, the sections hybridized with 35S-labeled sense
and antisense AQP8 cRNA probes were exposed to autoradiographic emulsion (NR-M2, Konica, Tokyo, Japan) for 5–7
days in the dark at 4°C, developed, and counterstained with
hematoxylin.
RESULTS
AQP mRNA expression in gastrointestinal tract.
RNase protection assay showed ubiquitous expression
of AQP1 and AQP3 mRNA along the gastrointestinal
tract, although the expression intensity varied; AQP1
mRNA expression was abundant in the middle portion
of the small intestine, ileum, and the proximal colon
and was much less in other portions of the rat gastrointestinal tract (Fig. 1A). AQP3 mRNA expression was
intense in the lower portion of the stomach, middle
portion of the small intestine, ileum, and both proximal
and distal colon, whereas the expression was less in the
esophagus, upper portion of stomach, and jejunum (Fig.
1B). In contrast to AQP1 and AQP3 mRNA expression,
AQP4 mRNA expression was relatively selective: abundant in the lower portion of the stomach and the ileum,
but faint in the esophagus and the jejunum, and
negligible in the upper portion of the stomach or colon
(Fig. 1C). AQP8 mRNA was exclusively expressed in
the jejunum and colon (Fig. 1D). No AQP2 or AQP5
mRNA was detected in the rat gastrointestinal tract
(data not shown). The quantitative data on the AQP
mRNA expression are summarized in Table 1.
Localization of AQP mRNA and protein. By immunohistochemistry, AQP1 was demonstrated on the endothelial cells of the lymphatic vessels in the submucosa and
lamina propria and capillary endothelial cells in the
smooth muscle layer throughout the gastrointestinal
tract (Fig. 2A). AQP3 was localized on the circumferential plasma membranes of stratified squamous epithelial cells of the esophagus (Fig. 2, B and C) and those of
the upper portion of the stomach (data not shown). In
the lower portion of the stomach, the localization of
AQP3 was restricted in the basolateral membrane of
columnar epithelia of cardiac glands in the vicinity of
the junction to the upper portion (Fig. 2D). In the small
intestine from jejunum to ileum, AQP3 immunostaining was not evident, although minimal or trace immunoreactive AQP3 was present in the columnar epithelia
in the villi and crypt (Fig. 2E). In contrast, AQP3
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RNase protection assay. AQP1 (356 bp, ⫹226 to ⫹581) and
AQP3 (377 bp, ⫹256 to ⫹632) cDNA fragments were cloned
from rat colon RNA, AQP4 (330 bp, ⫹302 to ⫹631) cDNA
fragments were from rat ileum RNA, and AQP5 cDNA (328
bp, ⫹324 to ⫹651) fragments were from rat salivary gland
RNA by a PCR-based cloning method using the nested,
degenerate oligonucleotide primers for AQP family as reported previously (30). The PCR products were subcloned into
pGEM T vectors (Promega Japan, Tokyo, Japan), and their
sequences were verified by an automated DNA sequencer
(Perkin Elmer, Foster City, CA). Partial fragments of rat
AQP2 cDNA (309 bp, ⫹1 to ⫹309), AQP8 cDNA (315 bp, ⫹701
to ⫹1015 ), and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were inserted in pSPORT1 vector
(GIBCO BRL), pGEM11Z (Promega), and pGEM3Z, respectively, as reported previously (13, 33). These plasmids were
linearized with appropriate restriction enzymes and used as
templates for in vitro transcription of ␣-32P-labeled antisense
cRNA probes.
The RNase protection assay was carried out as follows (13,
33): 10 µg of each RNA sample were hybridized with 1 ⫻ 105
counts/min each of the AQP probe combined with the GAPDH
probe in 10 µl of hybridization buffer (80% formamide, 40 mM
PIPES, 0.4 M NaCl, 1 mM EDTA) overnight at 48°C. Then,
unhybridized probes were digested with RNase A and RNase
T1 at 30°C for 1 h, and the ribonucleases were digested with
proteinase K at 37°C for 30 min. After phenol-chloroform
extraction, the hybridized probes were precipitated with
ethanol, denatured at 85°C, and electrophoresed on 6%
polyacrylamide gels. The dried gels were exposed to X-ray
films for 3 days (Fuji Photo Film, Kanagawa, Japan).
For quantitation of the autoradiography bands, the RNase
protection assay was repeated five times using different RNA
samples, the X-ray films were optically scanned (HP ScanJet
3C, Hewlett-Packard, Greeley, CO), and the density of each
band was analyzed by a computerized densitometry (Power
Macintosh 9500/132, Apple Computer, Cupertino, CA) using
the National Institute of Health (NIH) Image software (version 1.59, NIH Division of Computer Research and Technology, Bethesda, MD). The data were represented as ratios
(AQP mRNA/GAPDH mRNA band density) as reported previously (20, 33).
Immunohistochemistry and in situ hybridization. For immunohistochemistry, the tissues were fixed with methylCarnoy’s fixative (60% methanol, 30% chloroform, 10% acetic
acid), embedded in paraffin, and sectioned at 4 µm. They
were sequentially incubated with rabbit anti-rat AQP1 antibody (Chemicon, Temecula, CA), anti-rat AQP3 antibody
purified by an affinity chromatography (10), anti-rat AQP4
antibody (Chemicon), or normal rabbit serum, and then
incubated with goat anti-rabbit immunoglobulins conjugated
to peroxidase labeled polymer (EnVision, DAKO, Kyoto,
Japan), colored by diaminobenzidine reaction and counterstained with hematoxylin.
Localization of AQP3, AQP4, and AQP8 mRNA-expressing
cells was examined by in situ hybridization using digoxigeninlabeled cRNA probes for the rat AQP3, AQP4, and AQP8 or
35S-labeled cRNA probes for the rat AQP8 transcripted in
vitro from the linearized rat AQP3 (377 bp, ⫹256 to ⫹632),
AQP4 (330 bp, ⫹302 to ⫹631), and AQP8 cDNA template (809
bp, ⫹81 to ⫹889). Sense and antisense digoxigenin- or
35S-labeled probes were prepared according to the manufacturer’s protocol (Boehringer Mannheim, Mannheim, Germany)
and as reported previously (13). The tissues of the rat
gastrointestinal tract were cryosectioned at 10-µm thickness,
and the sections were fixed in 4% paraformaldehyde in PBS
and treated with 3 µg/ml of proteinase K (Promega) in 0.5 M
AQP FAMILY IN GASTROINTESTINAL TRACT
C623
mRNA expression was apparently localized in the crypt
epithelia of ileum and the middle of small intestine by
in situ hybridization (Fig. 2, F and G). In the colon, the
basolateral membrane of surface columnar epithelial
cells was apparently immunostained with the antiAQP3 antibody (Fig. 2H).
By immunohistochemistry, AQP4 was localized on
the basolateral membranes of the parietal cells in
oxyntic glands of the lower stomach, but not in the chief
cells (Fig. 3A). The basolateral membranes of the crypt
cells in the small intestine (from jejunum to ileum),
which were present at the bottom of the crypts, were
labeled with the anti-AQP4 antibody (Fig. 3B). In situ
hybridization also showed AQP4 mRNA expression at
the comparable sites; gastric glands and deep crypt
cells of the small intestine (Fig. 3, C and D). However,
Table 1. Quantitative evaluation of AQP family mRNA
expression in rat gastrointestinal tract
AQP1
AQP3
AQP4
AQP8
Esophagus
2.4 ⫾ 0.9 5.7 ⫾ 2.5 0.5 ⫾ 0.2
Stomach (upper portion) 5.4 ⫾ 1.6 7.4 ⫾ 1.6
Stomach (lower portion) 3.2 ⫾ 0.9 12.7 ⫾ 1.4 10.7 ⫾ 1.4
Jejunum
2.2 ⫾ 1.1 2.3 ⫾ 0.4 1.2 ⫾ 0.4 10.2 ⫾ 2.1
Small intestine (middle) 16.6 ⫾ 2.2 14.4 ⫾ 3.9 3.2 ⫾ 1.3
Ileum
16.4 ⫾ 1.3 14.2 ⫾ 1.4 7.3 ⫾ 2.4
Proximal colon
10.3 ⫾ 2.0 15.5 ⫾ 2.1
5.2 ⫾ 2.6
Distal colon
7.4 ⫾ 1.2 21.7 ⫾ 3.2
8.7 ⫾ 2.9
Values are means ⫾ SD of ratios (AQP/GAPDH mRNA densitometric unit) ⫻ 100; n ⫽ 5 samples. AQP, aquaporins; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
no or negligible immunostaining for AQP4 was detectable in the esophagus, the upper portion of the stomach, and the colon (data not shown).
The localization of AQP8 mRNA-expressing cells was
examined in the rat gastrointestinal tract by in situ
hybridization. The absorptive columnar epithelial cells
were prominently labeled in the jejunum (Fig. 4, A, B,
E, and F) and colon (Fig. 4, C, D, G, and H) with both
digoxigenin- and 35S-incorporated antisense cRNA
probes for AQP8 mRNA but not with the sense probes.
No signals were detected in the esophagus, stomach,
and ileum with the antisense AQP8 probes (data not
shown).
DISCUSSION
The epithelial layer of the gastrointestinal tract
system serves as entrance and barrier for water and
nutrients from outside to inside the body. Two routes,
paracellular and transcellular, are speculated for solutes across the barrier (26, 27). On the other hand,
water has been presumed to move between epithelial
cells (paracellular route) by interpretation of electrophysiological studies, although transcellular water
movement was not completely denied (26, 27). The
histological characteristics of the interepithelial junction are leaky in the small intestine and moderately
leaky in the colon, supporting this presumption. Thus
the structural stability or rigidity of the tight junction
sealing adjacent epithelial cells has been said to correspond to the structure that determines the permeability of water through the paracellular route. In the
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Fig. 1. Expression of aquaporin (AQP) mRNA in rat gastrointestinal tract examined by ribonuclease protection
assay. A: AQP1. B: AQP3. C: AQP4. D: AQP8. Lane 1, esophagus; lane 2, upper stomach; lane 3, lower stomach; lane
4, jejunum; lane 5, middle portion of small intestine; lane 6, ileum; lane 7, proximal colon; and lane 8, distal colon.
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AQP FAMILY IN GASTROINTESTINAL TRACT
Fig. 3. Immunohistochemistry and in situ hybridization for AQP4 in rat gastrointestinal tract. Immunoreactive
AQP4 is present on basolateral membranes of parietal cells in lower portion of stomach (A, ⫻240) and deep crypt
epithelia in ileum (B, ⫻240). Antisense probe for AQP4 hybridizes to AQP4 mRNA expression in deep crypt epithelia
of ileum (C, ⫻240) but no significant signals are seen in ileum with sense AQP4 probe hybridization (D, ⫻240).
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Fig. 2. Immunohistochemistry and in situ hybridization for AQP1 and AQP3 in rat gastrointestinal tract. AQP1
colored brown by immunoperoxidase staining is localized on endothelial cells of lymphatic vessels in esophagus
(A, ⫻120). Squamous epithelia in esophagus are also labeled with anti-AQP3 antibody (B, ⫻360) and not stained
with control rabbit serum (C, ⫻360). AQP3 is demonstrated on basolateral membranes of cardiac gland epithelia in
lower stomach (D, ⫻240), whereas AQP3 is questionable in small intestine (E, ⫻240). In situ hybridization using
antisense probe for AQP3 shows AQP3 mRNA expression in villous epithelia in ileum (F, ⫻360) but hybridization
with sense probe for AQP3 to same site is negligible (G, ⫻360). Basolateral membranes of surface columnar
epithelial cells are immunostained with anti-AQP3 antibody in colon (H, ⫻120).
AQP FAMILY IN GASTROINTESTINAL TRACT
C625
kidney, 99% of the volume of water filtered from the
glomerulus is reabsorbed at the proximal tubules, thin
descending limbs of Henle, and the distal tubules. The
tight junction of proximal tubular epithelia and the
epithelia of thin descending limbs of Henle are shallow
and discontinuous, which is characteristic for leaky
epithelia, suggesting that water moves between the
intercellular junction (12). However, AQP1 was localized on the apical and basolateral membranes of the
epithelial cells along these nephron segments (28), and
a transcellular route for water reabsorption at these
nephron segments is now highly predicted (12).
Recently AQP1, AQP3, and AQP4 mRNA expression
was demonstrated in the gastrointestinal tract, and the
transcellular route of water for absorption and secretion was suggested (10–13). However, AQP1 was localized on the endothelial cell membrane of intestinal
lymphatic vessels and not in the absorptive epithelia of
the gastrointestinal tract as shown by a previous study
(23), suggesting a role of AQP1 for movement of water
between interstitial fluid and lymphatic fluid in the
digestive tract.
In contrast, AQP3, AQP4, and AQP8 were localized in
the epithelia of several portions of the gastrointestinal
tract. AQP3 was demonstrated on basolateral membranes of cardiac gland cells in the lower stomach and
surface colonic epithelia, which may suggest an involvement of AQP3 in water absorption in these portions (3).
Although AQP3 mRNA expression apparently has been
demonstrated in the small intestine previously (3) and
was confirmed by the present study, the cellular localization of AQP3 was hardly identified in this portion by
immunostaining. Translation from the AQP3 mRNA to
protein may be interfered with or AQP3 epitopes may
be masked by unidentified mechanisms. The intense
expression of AQP3 mRNA and questionable presence
of the protein may indicate that the AQP3 mRNA in the
small intestine is reserved for emergent demand of
AQP3 protein production in some pathological conditions. Further intensive studies need to be done to
clarify the mysterious discrepancy between the AQP3
mRNA expression and AQP3 immunodetection in the
small intestine.
Interestingly, the present study demonstrated novel
expression of AQP3 mRNA and immunolocalization of
AQP3 on the circumferential plasma membrane of
stratified squamous epithelial cells in the esophagus.
The esophagus has not been regarded as a water-
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Fig. 4. Localization of AQP8 mRNA in rat jejunum (A, B, E, F) and colon (C, D, G, H) detected by in situ
hybridization using digoxigenin-labeled cRNA probes (A-D) and 35S-labeled cRNA probes (E-H). Villous epithelia of
jejunum have strong signals for AQP8 mRNA when hybridized with antisense probes (A, ⫻120 and E, ⫻360) but not
with sense probes (B, ⫻120, F, ⫻360). In colon, AQP8 mRNA expression is detected in surface epithelia with
antisense probes (C, ⫻120 and G, ⫻240) but not with sense probes (D, ⫻120 and H, ⫻240).
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AQP FAMILY IN GASTROINTESTINAL TRACT
tion of AQP8 has not been defined. Gastric cardiac
gland cells and parietal cells also possess AQP3 and
AQP4, respectively, only on the basolateral membranes. The possible presence of other AQP on the
apical membranes of these cells needs to be searched.
No prominent phenotypic abnormalities in intake of
water or nutrient have been observed in the AQP1deficient human subjects (29) and AQP1 or AQP4
knockout mice in physiological conditions (16). These
observations may indicate compensatory redundancy
of AQP in the gastrointestinal tract at a single cell level
if water channels play a pivotal role in water intake and
food digestion.
Although a possible role of water channels in water
absorption or secretion in the gastrointestinal tract is
predicted by the present study, the major route, transcellular or paracellular, for water to cross the gastrointestinal epithelium is still obscure. Transcellular water
movement has been denied in the small intestine
because no significant water permeability was observed in the membrane fraction of small intestinal
epithelia (31, 32). This observation may be partly
comparable to our present findings that immunoreactive AQP3 and AQP4 were restricted in the deep gland
cells in small intestine and AQP8 mRNA expression
was also restricted in the jejunum. The role of AQP in
water permeability in each portion of the small intestine needs to be examined intensively in the future. In
addition, pathological conditions related to water intake and outlet such as diarrhea or malabsorption may
be determined in part by the expression or the amounts
of AQP family members in the gastrointestinal tract.
The possible role(s) and involvement of AQP in physiological and pathological conditions of the gastrointestinal tract remain to be studied.
We thank Drs. K. Ishibashi and S. Sasaki, Tokyo Medical and
Dental University, for providing the anti-rat AQP3 antibody. We
thank Kan Yoshida for technical assistance.
This work was supported in part by a Grant-in-Aid for Science
Research from the Ministry of Education, Science, Sports and
Culture, Japan (09470237).
This work was presented in part at the 33rd annual meeting of the
Japanese Society of Surgical Metabolism and Nutrition in Kohchi,
Japan (July 1996).
Address for reprint requests: T. Yamamoto, Dept. of Pathology,
Institute of Nephrology, Niigata Univ. School of Medicine, 1–757
Asahimachi, Niigata 951–8510, Japan (E-mail: tdsymmt@ med.niigata-u.ac.jp).
Received 9 February 1998; accepted in final form 15 December 1998.
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involvement of AQP8 in the enormous water movement
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membrane for the intracellular water transport through
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members that are present in the apical membranes of
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in a cooperative manner with AQP4 and AQP3 in the
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AQP FAMILY IN GASTROINTESTINAL TRACT
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