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 |
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 |
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 |
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'-
GCHCAYNTNAAYCCHGYNGTNAC-3'); antisense primers were MDD-1
(5'-GCDGRNSCVARDGANCGNGCNGG-3') and MDD-2 (5'-
GDGCDGGRTTNATNSHNSMNCC-3'),
where
and
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%
-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 |
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 |
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.
 |
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