Open Journal of Gastroenterology, 2013, 3, 253-258 OJGas
http://dx.doi.org/10.4236/ojgas.2013.35043 Published Online September 2013 (http://www.scirp.org/journal/ojgas/)
Enhanced aquaporin 8 expression after subtotal
colectomy in rat
Masato Nakano1, Yu Koyama1*, Hitoshi Nogami1, Tadashi Yamamoto2, Toshifumi Wakai1
1Division of Digestive & General Surgery, Niigata University Graduate School of Medical & Dental Sciences, Niigata, Japan
2Division of Renal Pathology, Niigata University Graduate School of Medical & Dental Sciences, Niigata, Japan
Email: *yukmy@med.niigata-u.ac.jp
Received 11 July 2013; revised 12 August 2013; accepted 26 August 2013
Copyright © 2013 Masato Nakano et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Background: Aquaporins (AQPs), the family of wa-
ter-selective channels, are localized in various organs
and tissues, including the gastrointestinal (GI) tract.
However, the roles of AQPs in the GI tract remain
unclear. Materials and Methods: Male SD rats were
subjected to subtotal colectomy (Group C, n = 22) or
a sham operation (Group S, n = 16) and were sacri-
ficed on postoperative days 7, 14, and 28. Total RNAs
from the distal ileum and rectum were extracted.
Quantitative RT-PCR was performed to measure
AQP8 mRNA expression. For light-microscopy or im-
munohistochemistry, paraffin-embedded sections of 4
µm were prepared with H-E staining or anti-AQP8
antibody reaction. Mann-Whitney U-test was per-
formed to compare the AQP8 distributions between
the two groups, and the statistical significance was
defined as p < 0.05. Results: AQP8 mRNA expression
was enhanced in both the ileum and rectum in Group
C at day 7. AQP8 protein expression was consis-
tently observed in the ileum and rectum. The villus
length in the ileum of Group C was significantly
greater than that of Group S at days 7 and 14. Con-
clusion: Enhanced AQP8 mRNA expression in the
subtotal colectomy model suggests that AQP8 plays
an important role in maintaining the intestinal fluid
balance.
Keywords: Aquaporin 8; Subtotal Colectomy; mRNA;
Immunohistochemistry
1. INTRODUCTION
Water-selective channels (aquaporins; AQPs) have been
identified as molecules located mainly on the plasma
membrane of variou s cell types and increasing water p er-
meability. The mammalian water channel, AQP1, was
first identified as a protein homologous to the major in-
trinsic protein of the bovine lens in erythrocytes [1,2],
and was also shown to be present in red blood cells, renal
proximal convoluted tubules and the thin descending
limb of Henle [1,2]. Homology cloning techniques have
been used to clone AQPs, which have been cloned from
various organs: AQP2 [3] and AQP3 [4] from the kidney,
AQP4 from the brain [5], AQP5 from the salivary gland
[6], AQP6 from the k idney [7], AQP7 fr om the testis [8],
AQP8 from the pancreas and liver [9], AQP9 from the
liver [10], and AQP10 from the duodenum and jejunum
[11].
Previous studies have demonstrated the expression of
several AQP types in the gastrointestinal (GI) tract, sug-
gesting their participation in water absorption or secretion
[1,4,5,9]. We have demonstrated both mRNA and protein
expression of AQP1, AQP3, AQP4, and AQP8 in the rat
GI tract [12,13]. However, the functions of the AQPs in
the GI tract have not been well elucidated. In the present
study, we examined the mRNA expression and protein
expression of AQP8 in rat subtotal colectomy models in
order to investigate the possible functions of AQP8 in the
GI tract during postoperative adaptation t o s ubt ot al co lec -
tomy.
2. MATERIALS & METHODS
2.1. Experimental Animals
All experim ents and surgical procedures con formed to the
guidelines for the proper care and use of laboratory ani-
mals issued by the Public Health Serv ice, National Insti-
tutes of Health. Male Sprague-Dawley (SD) rats weighing
approximately 200 g were purchased from Charles River
Japan (Yokohama, Japan). Throughout the experimental
period, except for 12 h prior to the surgical procedures,
the animals were allowed free access to standard lab rat
chow and tap water. Their condition and weight were
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M. Nakano et al. / Open Journal of Gastroenterology 3 (2013) 253-258
254
recorded daily.
2.2. Subtotal Colectomy Model
The rats were divided into two groups: Group C (n = 22)
was subjected to subtotal colectomy with primary anas-
tomosis and Group S (n = 16), a sham operation. They
were denied food for 12 h and were anesthetized by in-
traperitoneal injection of pentobarbital. In Group C, a
short segment of the ileum and the entire large intestine
was resected from 1 cm proximal to the ileocecal valve to
2 cm proximal to the anal verge, and ileoproctal anasto-
mosis was performed. In Group S, the ileum was divided
at 1 cm proximal to the ileocecal valve, and the rectum
was divided at 2 cm proximal to the anal verge. Subse-
quently, anastomosis was performed without removal of
the small or large intestine. All bowel anastomoses were
completed with eight interrupted 5-0 silk sutures. The
abdominal wound was closed in two running layers, using
2-0 silk. Both groups were allowed free access to standard
lab rat chow and tap water after the operation.
The animals in both Group C and Group S were sacri-
ficed at postoperative days 7 (n = 8 and n = 6, respec-
tively), 14 (n = 7 and n = 5, respectively), and 28 (n = 7
and n = 5, respectively). Total RNAs were isolated from
the ileum and the remnant rectum by a modified acid
guanidiniumthiocyanate phenol-chloroform extraction
method using TRIzol (GIBCO BRL, Life Technologies,
Rockville, MD, USA), as described previously [12].
2.3. PCR Cloning of Rat Aquaporin 8
Rat AQP8 (315 bp; + 701 ~ +101 5) cDNA fr agments were
obtained from the rat ileum and remnant rectum by the
PCR-based cloning method using primers for AQP8 as
reported previously [9,12]. The PCR products were sub-
cloned into pGEM 11Z (Promega Japan Inc., Tokyo,
Japan) and their sequences were verified using an auto-
mated DNA sequencer (Perkin Elmer, Foster City, CA,
USA). A partial fragment of rat glyceraldehyde-3-phos-
phate dehydrogenase (GAPDH) cDNA of 123 bp was
inserted in pGEM 3Z (Promega).
The plasmid with the rat AQP8 and GAPDH gene in-
serts was linearized with appropriate restriction enzymes
and used as the template for quantitative RT-PCR.
2.4. Quantitative RT-PCR
The total RNA (1 µg) of each sample was reverse-tran-
scribed at 42˚C for 1 h by using an oligo (dT) primer and
Superscript II reverse transcriptase (GIBCO BRL) in a
volume of 20 µl. Each of these transcripts was used as a
template for multiplex quantitative PCR to measure
AQP8 mR NA an d G APDH e xpressi on in a singl e wel l b y
using an AB I PRISM 7700 seque nce detect ion instr ument
(PE Biosystems Japan, Chiba, Japan).
The AQP8-specific primers were 5’-GGCAGGTGGT
GGGATCTCT-3’ and 5’-GCCTAATGAGCAGTCCCA
CAA-3’, and the fluorogenic probe was 5’-TGGATC
TACTGGCTGGGCCCACTC-3’. A GAPDH TaqMan
Rodent GAPDH Control Reagent VIC TM Probe (PE
Biosystems Japan) was used to amplify rat GAPDH for
use as an internal control.
The amplification reactions m ixture (50 µl) contained a
reverse-transcript (1 µl), 1 × TaqMan Universal PCR
Master Mix (PE Biosystems), 900 nM of each AQP8
primer, 250 nM of the AQP8 fluorogenic probe, 100 nM
of rodent GAPDH primers, and 250 nM of the rodent
GAPDH fluorogenic probe. All quan titative 2-step -PCR
reactions we re perfor med accor ding to the manufacturer’s
instructions under the following thermocycler conditions:
50˚C holdi ng for 2 mi n, 95˚C holdi ng for 10 m in followed
by 40 cycles at 95˚C for 15 seconds, and at 60˚C for 1
min. Template-negative controls were run on each PCR
plate. A calibrator reverse-transcript sample was ampli-
fied in parallel on all plates in order to allow a comparison
of the samples run at different times. The data were ana-
lyzed using Se quence Detecti on Software (PE B iosystems
Japan), and the values were represented as the mean
(SEM) of ratios (AQP/GAPDH mRNA amplicon) ×
100%.
2.5. Immunohistochemistry
Since the results of mRNA quantification revealed en-
hanced AQP8 expression in the ileum and the remnant
rectum of the Group C animals, immunohistochemistry
was performed as described previously [13]. In brief, the
rat ileum and rectum samples were cut and fixed with
methyl-Carnoy’s fixative (60% methanol, 30% chloro-
form, 10% acetic acid) overnight, dehydrated with etha-
nol, embedded in paraffin, and sectioned at 4 µm. The
slide-sectioned tissues were deparaffinated with xylene
and ethanol, hydrated in distilled water, and then blocked
with normal goat serum (1:20 dilution) for 1 h. After a
three-time rinse with PBS, the slides were incubated with
an anti-AQP8 antibody (2.0 µg/ml) (Alpha Diagnostic
Intl. Inc., San Antonio, TX, USA) for 1 h at 37˚C; this
was followed by overnight incubation at 4˚C, rinsing
three times with PBS, and incubationed with goat anti-
rabbit immunoglobulins conjugated to a peroxidase-la-
beled polymer (En Vision, DAKO, Kyoto, Japan), and
coloring by diaminobenzidine reaction. After rinsing
with distilled water, the slides were counterstained with
hematoxylin for observation.
2.6. Morphologic Measurements
To measure the villus length of the ileum and the crypt
depth of the remnant rectum and the ileum, rat ileum and
rectum samples were cut and fixed as described above.
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M. Nakano et al. / Open Journal of Gastroenterology 3 (2013) 253-258
Copyright © 2013 SciRes.
255
The slide-sectioned tissues were deparaffinated with xy-
lene and ethanol, hydrated in distilled water, and then
immersed in a Hematoxylin-Eosin solution for 15 min.
The measurements were performed 5 times in each slide
section, and the values were described as the mean ± SD.
AQP8 mRNA expression in the ileum and remnant rec-
tum of Group C rats at days 7 (p < 0.01); however, this
was not observed at days 14 and 28 (Figure 2). In the
ileum at days 7, the AQP8 mRNA expression level of
Group C was almost 4 times that of Group S. In the
remnant rectum, it was almost 3 times that of Group S.
The level, however, returned to normal at days 14 and
28.
2.7. Statistical Analysis
Mann-Whitney U-test was performed to compare the
distributions between the two groups. A two-tailed p
value of <0.05 was considered to indicate statistical sig-
nificance.
3.3. Immunohistochemistry
Immunohistochemistry was used to detect the AQP8
protein expression in the apical membrane of the ileum
in Group C and the apical membrane of the remnant rec-
tum in both groups (Figure 3). In the ileum of Group S,
no protein expression was detected. AQP8 mRNA ex-
pression in the ileum was low at days 14 and 28, but
AQP8 protein in the ileum was maintained through the
experimental period.
3. RESULTS
3.1. Overall Animal Health
During the current experimental series, the perioperative
lethality was 40%. Twenty-five rats were sacrificed due
to bleeding from the mesenterium, ileus or peritonitis.
The weight of the Group C rats decreased and diarrhea
was observed until day 7. The weight o f the Group S rats
decreased until day 3; however, it gradually increased
after day 4. Moreover, no diarrhea was observed. The
rats in Group C showed a temporary decrease in weight
from 210.5 ± 14.9 g to 165.8 ± 20.0 g, 7 days after the
operation. The other group showed a temporary decrease
from 200.2 ± 8.2 g to 192.7 ± 8.4 g, 3 days after the op-
eration. The weight loss afte r surgery was more severe in
Group C as compared to Group S (Group C: 210.5 ± 14.9
3.4. Morphological Measurements
The villus length of the ileum and crypt depth of the il-
eum and rectum were measured. The results are shown in
Table 1. The villu s length of the ileum on days 7 and 14
of Group C was significantly greater than that of Group
S (p < 0.002 and 0.02, respectively). The crypt depth
remained unchanged (Table 2).
4. DISCUSSION
to 165.8 ± 20.0, Group S: 200.2 ± 8.2 to 192.7 ± 8.4).
However, it recovered gradually in both groups, although
the postoperative weight on days 7, 14, and 28 was sig-
nificantly lesser in Group C than in Group S (Figure 1).
Since the discovery of AQP1, 12 mammalian AQPs have
been recognized, and the clinical or physiological im-
portance of AQPs has been shown or suggested; AQP0
mutations have been identified in some forms of con-
genital cataracts [14]. AQP1-null individuals have shown
limited ability to maximally concentrate urine under wa-
ter-deprived conditions [15] and have shown decreased
pulmonary vascular permeability [16]. AQP2 mutations
Watery stool was observed for 7 days post operatio n in
Group C, but n ot in Group S.
3.2. Quantitative RT-PCR
Significant quantitative RT-PCR analyses enhanced
Figure 1. Weight of experimental animals. The rats in Group C showed a temporary decrease in weight from 210.5 ± 14.9 g
to 165.8 ± 20.0 g at 7 days post operation. Group S showed a temporary decrease from 200.2 ± 8.2 g to 192.7 ± 8.4 g at 3
days post operation. The weight on postoperative days 7, 14, and 28 were significantly lesser in Group C than in Group S (p
< 0.001 on days 7 and 14, and p < 0.01 on day 28).
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M. Nakano et al. / Open Journal of Gastroenterology 3 (2013) 253-258
256
Figure 2. AQP8 mRNA expression. AQP8 mRNA expression
in the ileum (A). B, AQP8 mRNA expression in the remnant
rectum (B). AQP8 mRNA expression was enhanced in the il-
eum and the remnant rectum of Group C rats at day 7 (*p <
0.01).
(A)
(B)
Figure 3. AQP8 protein expression. A, AQP8 protein expres-
sion in the ileum (A). AQP8 protein expression in the remnant
rectum (B). AQP8 protein expression (arrow) was detected in
the apical membrane of the ileum of Group C and the apical
membrane of the remnant rectum of both groups. The top line
(a, c, e) is a specimen from Group C and the bottom line (b, d,
f), from Group S. The right column (a, b) is day 7; the middle
column (c, d), day 14; and the left column (e, f), day 28.
cause clinical congenital diabetes insipidus [17], and
AQP3 and AQP4 seem to play a role in urinary concen-
tration in knockout mice studies [18,19]. Abnormalities
in AQP5 distribution have been shown to be related to
Sjogren’s syndrome [20]. Although several experimental
models using transgenic mice lacking AQPs have been
examined previously [21], the functions of AQPs in di-
gestive organs remain unclear.
Previously, we have shown the expression, distribution,
and localization of AQPs in rat digestive organs, includ-
ing the GI tract: Generalized expression of AQP1 and
AQP3 mRNA is observed widely along the gastrointes-
tinal tract; AQP4 mRNA is expressed selectively in the
lower portion of the stomach and small intestine; and
AQP8 mRNA is expressed more selectively in the jeju-
num and colon [12,13 ]. Recently we cloned AQP1 0 fro m
the human jejunum. AQP10 was selectively expressed in
the upper sites of the small intestine, such as the duode-
num and jejunum in humans [11]; however, AQP10 has
not been iden tified yet in rats.
Purdy et al. [22] reported a change in AQP3 expres-
sion in the ileostomy model. We have previously shown
enhanced expression of AQP8 mRNA after colectomy in
rats. Here, we decided to construct a rat colectomy model
to elucidate the function of AQP8 and intestinal adapta-
tion.
We performed a quantitative investigation using Real-
Time PCR to elucidate the changes in AQP family gene
expression of mRNA in the rat gastrointestinal tract after
bowel removal. Our result revealing a prominent en-
hancement in AQP8 mRNA expression in the ileum and
remnant rectum of the subtotal colectomy rats suggest
that AQP8 in the ileum and remnant rectum is usefu l for
water absorption.
We also used immunohistochemistry to confirm the
enhancement of the AQP8 protein in the rectum of the
subtotal colectomy rats being in accordance with the en-
hancement of AQP8 mrnaexpression. Our results showed
prominent AQP8 protein on the colu mnar epithelial cells
of the ileum and the remnant rectum of the subtotal
colectomy rats; in the sham-operated rats, the staining
was negligible. However, AQP8 mrnaexpression was
normalized on days 14 and 28, suggesting that AQP8
protein expression in Group C was enhanced even on
days 28. This suggests that the half-life of the AQP8
protein would continue after a decrease in AQP8 mRNA
expression, and the AQP8 protein would function for
water absorption.
Morphologic measurements showed an elongated vil-
lus length and crypt depth of the ileum on days 7 and 14
in Group C. On days 28, there was no significant differ-
ence in the villus length between the groups. No signifi-
cant difference was observed in the crypt depth of the
rectum. Willis et al. [23] reported an increased villus
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M. Nakano et al. / Open Journal of Gastroenterology 3 (2013) 253-258 257
Table 1. Villus length and crypt depth of ileum.
Day 7 Day 14 Day 28
Group C Group S Group C Group S Group C Group S
Vi l lus (µm) 345.1 ± 36.7** 265.4 ± 36.3** 357.2 ± 112.8** 240.9 ± 143.3** 346.9 ± 53.9 307.4 ± 37.7
Crypt (µm) 193.6 ± 41.7* 131.7 ± 21.8* 208.3 ± 50.2* 154.5 ± 34.1* 203.4 ± 42.9 157.6 ± 29.8
Group C, subtotal colectomized rat; Group S, s ham operated rat; **p < 0.005; *p < 0.05.
Table 2. Crypt depth of rectum.
Day 7 Day 14 Day 28
Group C Group S Group C Group S Group C Group S
Crypt (µm) 315.7 ± 47.8 309.1 ± 53.5 305.6 ± 73.1 290.0 ± 29.8 303.9 ± 47.4 283.5 ± 34.6
Group C, subtotal colectomized rat; Group S, sham opera te d rat.
length and density after colectomy in rats. They con-
cluded that the increased ileal mucosal surface is proba-
bly responsible for the elevation in electrolyte and glu-
cose absorption. Our results suggested that the elongated
villi would assist AQP8 in water absorption in the sub to-
tal colectomy model. We considered that most p art of the
adaptation of the ileum would be completed after 14
days.
Continuous diarrhea and weight loss were observed in
Group C. The diarrhea continued and the bodyweight
continued to decrease until 7 days after the operation,
possibly suggesting that bodyweight loss and/or diarrhea
would accelerate the adaptation of the intestine after
colectomy in rats.
In summary, in the ileum and rectum in Group C,
AQP8 mRNA expression was enhanced on days 7 and
AQP8 protein expression was enhanced on days 7, 14
and 28. The villus length of the ileum was increased on
days 7 and 14 in Group C. These results suggested that
intestinal adaptation after subtotal colectomy occurred
after 7 posto perative days.
5. CONCLUSIONS
The physiological roles of water channels in water
transportation through the epithelial cell layer in the GI
tract remain unclear. However, our results suggest an
important role of AQP8 in the maintenance of intestinal
fluid balance, and for the adaptation to postoperative
conditions.
AQP8 may facilitate water movement through the
epithelium of the ileum and the remnant rectum after
subtotal colectomy.
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