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 OPEN ACCESS
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. Copyright © 2013 SciRes. OPEN ACCESS
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). OPEN ACCESS
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 Copyright © 2013 SciRes. OPEN ACCESS
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. REFERENCES [1] King, L.S. and Agre, P. (1996) Pathophysiology of the aquaporin water channels. Annual Review of Physiology, 58, 619-648. doi:10.1146/annurev.ph.58.030196.003155 [2] Preston, G.M., Carrol, T.P., Guggino, W.B. and Agre, P. (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science, 256, 385- 387. doi:10.1126/science.256.5055.385 [3] Fushimi, K., Uchida, S., Hara, Y., Hirata, Y., Marumo, F. and Sasaki, S. (1993) Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature, 361, 549-552. doi:10.1038/361549a0 [4] Ishibashi, K., Sasaki, S., Fushimi, K., Uchida, S., Kuwa- hara, M., Saito, H., Furukawa, T., Nakajima, K., Yama- guchi, Y. and Marumo, F. (1994) Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water ex- pressed at the basolateral membrane of kidney collecting duct cells. Proceeding of the National Academy of Sci- ences of the United States of America, 91, 6269-6273. [5] Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B., Baraban, J.M. and Agre, P. (1994) Molecular characteri- zation of an aquaporin cDNA from brain candidate os- moreceptor and regulator of water balance. Proceeding of the National Academy of Sciences of the United States of America, 91, 13052-13056. [6] Raina, S., Preston, G.M., Guggino, W.B. and Agre, P. (1995) Molecular cloning and characterization of an aq- uaporin cDNA from salivary, lacrimal and respiratory tis- sues. Journal Biological Chemistry, 270, 1908-1912. doi:10.1074/jbc.270.4.1908 [7] Ma, T., Yang, B., Kuo, W.L. and Verkman, A.S. (1996) cDNA cloning and gene structure of a novel water chan- nel expressed exclusively in human kidney: Evidence for a gene cluster of aquaporins at chromosome locus 12q13. Genomics, 35, 543-550. doi:10.1006/geno.1996.0396 [8] Ishibashi, K., Kuwahara, M., Gu, Y., Kageyama, Y., Tohsaka, A., Suzuki, F., Marumo, F. and Sasaki, S. (1997) Cloning and functional expression of a new water chan- nel abundantly expressed in the testis permeable to water, glycerol and urea. Journal Biological Chemistry, 272, 20782-20786. doi:10.1074/jbc.272.33.20782 Copyright © 2013 SciRes. OPEN ACCESS
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