Slov Vet Res 2024 | Vol 61 No 1 | 29 Localization of Aquaporin-1 in the Small and Large Intestines of Geese (Anser anser) Key words geese; intestine; aquaporin-1 Ebru Karadag Sari1, Buket Bakir2*, Gizem Fidan Arslan3, Sevda Elis Yildiz4 1Department of Histology and Embryology, Faculty of Veterinary Medicine, Kafkas University, 36100 Kars, 2Department of Histology and Embryology, Faculty Of Veterinary Medicine, Namık Kemal University, 59100 Tekirdağ, 3Department of Histology and Embryology, Health Science Institute, Kafkas University, 36100 Kars, 4Department Of Midwifery, Faculty Of Health Sciences, Kafkas University, 36100 Kars, Turkey *Corresponding author: buketbakir@nku.edu.tr Abstract: Aquaporins are selective water channels that serve transportation of wa- ter across cell membranes, which play a vital role in all cells. In this study, using the immunohistochemical method, the authors intended to investigate the localization of Aquaporin-1 in the small and large intestines of geese. In this study, small and large in- testine tissue samples taken from healthy adult geese (Anser anser) (n = 10) were used as materials. After fixation for 24 hours at 10% formaldehyde, the tissue samples were passed through graded series of ethanol and xylol and embedded in paraffin. Mallory's modified triple-staining method was used to examine the general structure of the in- testine. The Avidin-Biotin-Peroxidase Complex (ABC) method was applied to determine the immunoreactivity of Aquaporin-1. The apical parts of crypt epithelial cells showed strong Aquaporin-1 immunoreactivity in the duodenum and moderate Aquaporin1 im- munoreactivity in the jejunum and ileum. Strong Aquaporin-1 immunoreactivity was de- termined in vascular endothelial cells in the duodenum, jejunum, and ileum, and weak immunoreactivity was found in smooth muscle cells. However, a weak Aquaporin-1 im- munoreactivity was detected only in the smooth muscle cells of the cecum and rectum but not in vascular endothelial cells and crypt epithelial cells. The intestine tissue regu- lates salt transport and hydrostatic pressure differences, enabling the transportation of water. It was suggested that the duodenum and jejunum sections in particular are permeable to high levels of water for balancing the osmotic pressure of the intestinal content. Consequently, with this study, Aquaporin-1 immunoreactivity was detected in the crypt epithelial cells, smooth muscle cells, and vascular endothelium of the small intestines of geese. Received: 23 February 2023 Accepted: 6 July 2023 Slovenian Veterinary Research DOI 10.26873/SVR-1711-2023 UDC 598.252.2:616.345:577:616-092 Pages: 29–35 Original Research Article Introduction Water passes through the plasma membrane by simple diffusion. Channels consisting of specialized membrane proteins are required for the rapid and intense passage of water (1). Aquaporins (AQPs), a family of water channel pro- teins, are small hydrophobic and integral membrane chan- nel proteins that facilitate the transportation and velocity of water and are responsible for water balance regulation by ensuring continuous and rapid permeability of water with low activation energy throughout the epithelial cells (2, 3). Water molecules that pass through AQP channels move very quickly. In one second, 109 water molecules pass through the AQP channel. The speed of water that transit through the AQP channel is even faster than the catalase enzyme, which is known metabolically as the fastest. This speed is a high speed for metabolic events (4). Aquaporins have been reported to be hydrophobic proteins with six transmembrane domains whose molecular weight ranges from 28 kDa (unglycosylated form) to 40-50 kDa (glycosylated form), and they are mostly found as a ho- motetramer (5). Depending on their permeability, AQPs in mammals are divided into three groups 1): Water-selective Aquaporins (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8), 2); Aquaglyceroproteins which mediate the passage 30 | Slov Vet Res 2024 | Vol 61 No 1 of glycerol, urea, and some neutral molecules besides wa- ter (AQP3, AQP7, AQP9, and AQP10), and unorthodox or super-aquaporins (AQP11 and AQP12) (6, 7). AQP 1, 2, 4, and 5 show the widespread distribution in every tissue and organ where water is crucial (8). At least eleven varieties of Aquaporin are expressed in various tissues in the gastrointestinal tract. AQP1 is expressed in duodenum, ileum, large intestine, liver, pancreas and gallbladder, AQP2 in small intestine, AQP3 in small intestine, colon and liver, AQP4 in duodenum and colon, AQP5 in duodenum and pan- creas, AQP7 in small intestine and colon, AQP8 in large in- testine, liver, pancreas, and gallbladder, AQP9 in duodenum, ileum and liver, AQP10 in small intestine, AQP11 in small intestine, colon and liver and AQP12 in pancreas (9, 10). Aquaporin-1 (AQPl) was first discovered by chance during studies of the human red cell Rh protein as a homologous protein to MIP (Major Intrinsic Protein of Bovine lens) and was labelled as CHIP28 (Channel Forming Intrinsic Protein of 28 kDa). After that CHIP was designated Aquaporin-1 (ab- breviated AQPl) by the Human Genome Committee (11, 12). AQP1 was determined to have important roles in physiolog- ical processes such as water homeostasis, neuro-homeo- stasis, digestion, body temperature regulation, and repro- duction by contributing to fluid release and fluid absorption in the body (13). It has been suggested that AQP1 may be involved in angiogenesis, wound healing, organ regenera- tion, and tumor metastasis (14). A positive correlation has been established between endometrial adenocarcinoma progression and AQP1, microvascular density, as well as vascular endothelial growth factor (VEGF) (15). Chicken ceca and rectum were determined to have AQP4 immunoreactivity (16). Also, jejunum, ileum, and colon have ck-AQP5 mRNA (17), and the lower intestinal tract of a spar- row has AQ1 distribution (18), but no study of AQ1 immu- noreactivity in the small and large intestines of geese was encountered in the literature. In this study it was aimed to determine immunolocalization of Aquaporin-1, that is im- portant in terms of physiological and pathological roles it assumes in the small and large intestines of geese which has economic importance. Material and Methods Animal Material Tissue samples were harvested in compliance with an approved Kafkas University Animal Care and Use Committee Protocol (No. 2018/04, dated 26.04.2018 and coded KAÜHADYEK/2018-049) for this study. The small and large intestine tissue samples taken from 10 female geese (Anser anser) at the age of 8 months that local breeders slaughtered for consumption purposes were used as materials. Histological Procedure The small and large intestinal tissue samples were fixed for 24 hours in a 10% formaldehyde solution. Afterwards, they were dehydrated (ethanol), cleared (xylol), and embedded in paraffin. Paraffin blocks were cut into 5-μm thick sections on a rotary microtome (LIECA) and stained with Mallory's modified triple staining to examine the general structure of the tissues. Immunohistochemical procedure The Avidin-Biotin Peroxidase complex (ABC) technique was used to determine the localization of Aquaporin 1 (AQP 1) immunohistochemically in small and large intestine tis- sues. 4-μm cross-sections were fixed to lamellas covered with chrome alum gelatin and were subjected to deparaf- finization and dehydration. They were then incubated for 20 minutes in the solution of hydrogen peroxide in methanol (3%) to prevent endogenous peroxide activity. Then they were kept in the microwave oven at 600 watts for 20 min- utes within a sodium citrate buffer (pH 6.0) solution to re- lease antigenic receptors. Sections incubated for 10 min- utes with Blocking Solution A (Invitrogen-Histostatin Plus Bulk Kit) were kept at room temperature for 1 hour after dripping the Aquaporin 1 primary antibody [1/500] (abcam: ab9566) without a PBS wash. The sections were then in- cubated for 30 minutes with the biotinylated secondary antibody and 30 minutes in Streptavidin Peroxidase solu- tion. To demonstrate the antibody reaction, the DAB (3.3 ′ -Diaminobenzidine) chromogen solution was added to the cross-sections, and they were examined with a light micro- scope. The reaction was stopped with PBS by checking the condition of immunoreactivity. Distilled water-washed sec- tions were subjected to Harris hematoxylin stain for reverse staining and were dehydrated and covered with entellan. The evaluation was made by two independent observers using the semi-quantitative method by taking the degree of staining in the cross-sections as a criterion. Depending on the staining properties, the slides were scored within the range of 0–3 during their evaluation: no immunoreactivity 0(-), weak immunoreactivity 1(+), moderate immunoreac- tivity 2(++), and strong immunoreactivity 3(+++). To deter- mine whether immunohistochemical staining is specific, the sections were subjected to an immunohistochemical staining procedure without adding a primary antibody (neg- ative control), provided that all processes were identical. The preparations prepared for histological and immunohis- tochemical examinations were then photographed and as- sessed under the light microscope (Olympus Bx53 JAPAN). Statistical analysis The data were analyzed with the IBM Statistical Package for Social Sciences (SPSS) program. In analysis, minimum- maximum values, mean, and standard deviation were used and median was calculated to evaluate the data. Slov Vet Res 2024 | Vol 61 No 1 | 31 Table 1: Statistical analysis of Aquaporin-1 immunoreactivity in geese small and large intestine Cells N Min Max Mean±SD Median Duodenum Crypt epithelial cells 8 2 3 2,56±0,42 2,50 Vascular endothelial cells 8 2 3 2,56±0,42 2,50 Smooth muscle cells 8 0 2 1,1875±0,65 1 Jejenum Crypt epithelial cells 8 1 3 2,06±0,62 2 Vascular endothelial cells 8 2 3 2,625±0,44 2,75 Smooth muscle cells 8 0 2 1,0625±0,582 1 Ileum Crypt epithelial cells 8 1 3 1,88±0,69 2 Vascular endothelial cells 8 2 3 2,63±0,44 2,75 smooth muscle cells 8 0,5 2 1,0625±0,49 1 Cecum Crypt epithelial cells 8 0 1 0,25±0,38 0 Vascular endothelial cells 8 0 1 0,19±0,37 0 Smooth muscle cells 8 0 2 1,0625±0,67 1 Rectum (Colon) Crypt epithelial cells 8 0 1 0,31±0,46 0 Vascular endothelial cells 8 0 1 0,25±0,46 0 Smooth muscle cells 8 0,5 2 1,0625±0,49 1 Figure 1: Goose intestine tissue. A; İleum, B; Cecum. Mallory's modified triple staining. A and B; Bar: 200 μm, original magnification, X10 32 | Slov Vet Res 2024 | Vol 61 No 1 Results The normal histological structure of the small and large in- testines of geese is shown in Figure 1. The apical parts of crypt epithelial cells in the duodenum showed strong, and the apical parts of crypt epithelial cells in the jejunum and il- eum showed moderate AQP1 immunoreactivity. In the duo- denum, jejunum, and ileum, strong AQP1 immunoreactivity was determined in vascular endothelial cells and weak im- munoreactivity in smooth muscle cells. Weak AQP1 immu- noreactivity was detected in the smooth muscle cells of the Figure 2: Goose small intestine tissue. AQ1 immunoreactivity. A, B; duodenum, C, D; jejenum E, F; ileum. The Avidin-Biotin-Peroxidase Complex (ABC) method. B; Bar: 50 μm, original magnification, X40, A, D, F; Bar: 100 μm, original magnification, X20, C, E; Bar: 200 μm, original magnification, X10 Slov Vet Res 2024 | Vol 61 No 1 | 33 cecum and rectum but no immunoreactivity was observed in either the crypt epithelial cells or the vascular endothelial cells (Table 1 and Figures 3). Discussion In the transportation of water, the intestines are the second most important organ after the kidneys. Water transport occurs in the digestive tract because of hydrostatic pres- sure and osmotic pressure caused by the transport of salt. Much of the transported water is used to regulate saliva, gastric juice, bile, pancreatic fluid, and intestinal fluid, and to adjust water and ion balance (19, 20). The feed material is ingested, moisturized, ground into small particles, acidified, and attacked by endogenous enzymes in the digestive tract of poultry similar to other animal species (21). In poultry, the intestine is an important organ where enzymatic digestion takes place, and nutrients are absorbed via numerous ion channels and transporters present on the apical intestinal epithelial border. Materials consisting of indigestible food and waste are mixed up with urine in the cloaca and are excreted from the body as feces (22). Intestine composed of duodenum, jejunum, ileum and ceca, rectum (colon), and cloaca. The wall structure of the poultry intestines consists of mucosa, submucosa, muscu- laris, and serosa layers, like the mammalian intestine (23). AQP-1 gene sequences of chicken, human, and toad exhibit 94%, 88%, and 78% homology, respectively (18). Specific AQP1 labeling was seen in the endothelia of central lacte- als in the villi of the porcine small intestine (24). In the calf of adult buffalo, AQP-1 was detected in the endothelium, Figure 3: Goose large intestine tissue. AQ1 immunoreactivity. A; cecum, B, C, D; rectum (colon). The Avidin-Biotin-Peroxidase Complex (ABC) method. A; Bar: 50 μm, original magnification, X40, B; Bar: 500 μm, original magnification, X4, C; Bar: 100 μm, original magnification, X20, D; Bar: 200 μm, original magnification, X10 34 | Slov Vet Res 2024 | Vol 61 No 1 enterocytes, lymphoid tissue, and enteric neurons of both the small and large intestines (25). AQP1 was demonstrat- ed on endothelial cells of lymphatic vessels in the submu- cosa and lamina propria and capillary endothelial cells in the smooth muscle layer throughout the rat gastrointesti- nal tract and villus intestinalis and crypt epithelium cells, vascular endothelium, erythrocytes and connective tissue within the small intestine and serosa layer, vascular endo- thelium and erythrocytes in the large intestines of the mice (26, 27). Strong AQP4-immunoreactivity was demonstrated in a fi- ber network in the enteric plexus in chicken ceca and rec- tum (16). In poultry, ck-AQP5 mRNA was found in the crypt cells of the jejunum, ileum, and colon, but not in the cells that cover the villi (17). Goose testis and vas deferens capil- laries were reported to have AQP-1 immunoreactivity in en- dothelial cells (28). In bird and mammal kidneys, AQP-1,2 and 4 were expressed (29). In this study it was determined that there was AQP1 immunoreactivity in crypt epithelial cells, vascular endothelial cells, and smooth muscle cells in geese duodenum, jejenum and ileum like rat (26), mice (27), and porcine (24) whereas only smooth muscle cells showed a reaction in cecum and rectum. AQP1 distribution was determined on the apical membrane of the enterocytes, especially in the crypts, and on the cell membrane of erythrocytes of bottlenose dolphin’s small in- testine. Strong immunostaining was reported in the apical membrane of enterocytes in the mid and bottom regions of the crypt, also comparatively moderate immunostaining was demonstrated at the apical membrane and cytoplasm of enterocytes in the villi and upper region of the crypt (30). It was observed the apical parts of crypt epithelial cells in the duodenum showed strong AQP1 immunoreactivity, and the apical parts of crypt epithelial cells in the jejunum and ileum showed moderate AQP1 immunoreactivity. Also, strong AQP1 immunoreactivity was determined in vascular endothelial cells in the duodenum, jejunum, and ileum and a weak immunoreactivity in smooth muscle cells of geese intestine. The presence of AQP-1 in the distal rectum of sparrows was reported in large intestines from the ceca to copro- deum with limited distribution. It was suggested that the AQP-1 was present within the cecae especially in the lam- ina propria and in the mucosa and the muscularis of the proximal rectum and in the epithelium, the lamina propria and muscularis of the distal rectum of house sparrows (18). In this study it was seen that a weak Aquaporin-1 immuno- reactivity was detected only in the smooth muscle cells of the geese cecum and rectum. Furthermore, identification of AQP-1 in the mucosa of the large intestine suggested that AQP1 may play a role in water transportation, while lo- calization of AQP-1 in the distal rectal epithelium drew more attention to the importance of retrograde peristalsis for wa- ter conservation (18). In conclusion, indicating that Aquaporin 1 immunoreactiv- ity was observed especially in the apical membranes of crypt enterocytes in geese small intestines as in dolphins (30) and that Aquaporin 1 was localized in endothelial cells of lymph vessels in lamina propria and in the submucosa from the esophagus to the colon of rats (26) suggests that Aquaporin 1 release may be similar in poultry and mam- malian intestines. Acknowledgements This study was presented as an oral presentation at the 2nd International Congress on Advances in Veterinary Sciences & Technics (ICAVST-2017) in Barcelona. Ethical approval. Ethics Committee approval was obtained from the Experimental Animal Ethics Committee of Kafkas University (No. 2018/04, dated 26.04.2018 and coded KAÜ- HADYEK/2018-049) for this research. Conflict of interest. There are no conflicts of interest to be declared by the authors. Author’s Contribution. EKS conceptualized the study. meth- odology, E.K.S., B.B., G.F.A., and S.E.Y; software, E.K.S, G.F.A. and S.E.Y; validation, E.K.S., B.B., G.F.A., and S.E.Y; formal analysis, E.K.S., B.B., G.F.A., and S.E.Y; resources, E.K.S. and B.B. ; data curation, E.K.S., B.B., G.F.A., and S.E.Y; writing—original draft preparation, E.K.S., B.B., G.F.A., and S.E.Y; writing—review and editing, E.K.S., B.B., G.F.A., and S.E.Y; visualization, E.K.S., B.B., G.F.A., and S.E.Y; supervi- sion, E.K.S., B.B. and S.E.Y; All authors have contributed to the final version of the manuscript. All authors approved the final manuscript. Funding. The authors received no financial support for the research, authorship, and/or publication of this article. References 1. Smith BL, Agre P. Eriythrocyte Mr 28,000 transmembrane protein ex- ists as a multisubunit oligomer similar to channel proteins. J Biol Chem 1991; 266: 6407–15. 2. Huang HF, He RH, Sun CC, Zhang Y, Meng QX, Ma YY. 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