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Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, Università di Pavia, Italy;
* Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepathology, Mayo Medical School, Clinic and Foundation, Rochester, MN;
DIBIT, San Raffaele Scientific Institute, Milan, Italy; and
** Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Italy
3To whom correspondence should be addressed. E-mail: lumberto{at}unipv.it.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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38%; P < 0.05). Current results indicate that aquaporin-8 may play a major role in water movement through the colon by acting on the apical side of the superficial cells.
KEY WORDS: • aquaporins • water channel • small interfering RNA • colon
Water is an essential macronutrient because it must be introduced with foods to satisfy metabolic demand. The colonic epithelium plays a major role in salt and water transport and contributes to body fluid homeostasis. About 1.5–2 L of water (1) are absorbed daily by the colon against an osmotic gradient, producing dehydrated feces. Moreover, the colon may secrete water and electrolytes under certain physiologic and pathophysiologic conditions (2). In the past, the accepted paradigm was that fluid absorption takes place in the surface epithelium and fluid secretion in the crypt. In contrast, it is now established that colonic crypts can absorb as well as secrete so that most fecal consolidation is probably not due to crypt absorption (3,4).
Theoretically, water can cross the epithelia either by paracellular (i.e., across cell junctions) or transcellular (i.e., through the apical and basolateral membranes) routes. Transcellular transport may involve 3 different mechanisms: 1) diffusion across the lipid bilayer; 2) diffusion across the water channels i.e., aquaporins (AQPs)4 ; and 3) cotransport with ions and nutrients (5).
Because the colon is a tight epithelium characterized by high electrical resistance, it is conceivable that water flux occurs mainly by transporters and/or AQPs. As in other tissues of the gastrointestinal tract, AQPs were identified in the colon epithelium (6). AQP-2, -3, -4, and -8 were found along the large intestine (7–10), suggesting their possible involvement in water absorption and secretion. AQP-2 was detected in the apical membrane of superficial epithelial cells of the distal colon (9). AQP-3 and -4 were found in the basolateral membranes of superficial colonocytes (11), whereas AQP-8 was detected in the subapical cytoplasm of surface cells of the proximal colon (7,12). Although AQP-2, -3, -4, and -8 were shown to be present in the colon, the function and physiologic importance of these AQPs in water transport has not been addressed. Interestingly, studies on AQP-4 null mice demonstrated that fluid absorption was not impaired by AQP-4 deletion, although AQP-4 null mice had a slightly higher water content in feces and reduced water osmotic permeability compared with normal mice (13). A more recent investigation surprisingly found no significant impairment in colonic fluid absorption or fecal dehydration in AQP-8 null mice colon (14).
The purpose of this work was to study the expression and functional role of AQPs in water transport through colonic cells and to determine whether any differences existed between superficial (mature) and lower crypt cells (immature and proliferative).
For this study, we examined the expression of AQP-3, -4, and -8 mRNA and protein in colonocytes isolated along the crypt-lumen regions in the rat proximal colon using RT-PCR and immunoblotting. We performed functional experiments by assaying osmotically induced water permeability in freshly isolated cells in both the absence and presence of the well-known water channel inhibitor dimethyl sulfoxide (DMSO) to distinguish the contribution of AQP-3 and -8 to water permeability from that of AQP-4 (DMSO-insensitive). To determine the relative contribution of the AQP-8 toward the overall osmotic water permeability of isolated colonocytes, we used a well-established approach to selectively and specifically silence the AQP-8 gene by means of small interfering RNA (siRNA) (15,16).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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RT-PCR assay. Total cellular RNA was extracted from each cell fraction using the SV total RNA Isolation System (Promega). Single cDNA was synthesized from RNA (1 mg) using random hexamers and IMPROM II reverse transcriptase (Promega). Specific primers for rat ß-actin (7), AQP-3 (20), AQP-4 (sense, 5'-TTTCAAAGGCGTCTGGACTC-3'; antisense, 5'-TTCCATGAACCGTGGTGACT-3') and AQP-8 (sense, 5'-GGGATCTCTGGAGCCTGCATG-3'; antisense, 5'-CTGCTGCTGTCAGAGTGGCTC-3') were synthesized based on their published sequences. cDNA amplification was performed by Taq DNA polymerase (Promega) and for all PCR assays consisted of an initial denaturation of 5 min at 96°C followed by 30 s at 96°C, 30 s at different annealing temperatures (AQP-3, 56°C; AQP-4, 55°C; AQP-8, 61°C; ß-actin, 62°C), 30 s at 72°C, and an extension for 7 min at 72°C. Reverse transcription was always performed either in the presence or in the absence of reverse transcriptase enzyme. First, the sequences of AQP bands were checked using the Big dye terminator cycle sequencing kit (Applied Biosystem). PCR products were separated on a 3% Nusieve® (2:1) gel agarose, stained with ethidium bromide, and acquired with the Image Master VDS (Amersham Biosciences Europe). Densitometric analysis of the bands was performed by the Total Lab v 1.11 computer program (Amersham Biosciences Europe). The molecular weight of the PCR products was compared with the DNA molecular weight marker VIII (Roche Molecular Biochemicals).
Membrane preparation and immunoblotting. Freshly isolated colonocytes were homogenized with a Teflon glass Potter-Elvehjem homogenizer (Kontes) in a solution containing 100 mmol/L NaCl, 1 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 6.8, and 0.1 g/L phenylmethylsulfonyl fluoride. After centrifugation at 100,000 x g for 60 min at 4°C, the pellets were suspended in the homogenization buffer and treated as previously described (21). Solubilized proteins (70 µg) were subjected to 12.5% SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride membranes (Amersham Biosciences Europe) by electroelution. After 3 h of blocking with TBS containing 5% nonfat dry milk and 0.1% Tween, the membranes were incubated overnight with affinity-purified antibodies to AQP-3, -4, -8 (Alpha Diagnostic International) diluted 1:2000 in the blocking solution. The membranes were washed and incubated with peroxidase-conjugated goat anti-rabbit IgG (1:3000 in the blocking solution; Amersham Biosciences Europe) for 1 h. The bands were detected using the ECL-plus Western blotting detection system (Amersham Biosciences Europe) and analyzed as indicated above for PCR gels. Control experiments were performed using antibodies preadsorbed with a 20-fold molar excess of immunizing peptides (Alfa Diagnostic International). ChemiBlotTM Molecular Weight Markers were used to accurately estimate molecular weight and as a positive control for immunoblot (Chemicon International).
Immunohistochemistry. Rats were anesthetized with halothane and perfused intracardially with acetate-buffered 4% formalin. The colon was removed, postfixed for 1 h, and processed into paraffin. Serial paraffin sections (5 µm) were brought to water and treated with 3% hydrogen peroxide for 10 min at room temperature to block the endogenous peroxidases. After being washed for 5 min with PBS, sections were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Sections were incubated for 2 h at room temperature with affinity purified anti-AQP8 antibody diluted 1:2000 in PBS containing 1% BSA. After three 5-min washes with PBS containing 1% BSA, the sections were first incubated for 15 min at room temperature with biotinylated anti-rabbit IgG; after 3 washes with PBS containing 1% BSA, they were incubated for 15 min at room temperature with HRP-conjugated streptavidin (Universal DAKO LSAB® + kit, peroxidase, K0679, DakoCytomation). The reaction was visualized by incubation with the DakoCytomation 3,3'-diaminobenzidine chromogen solution. The sections were counterstained with hematoxylin and mounted in DPX (distyrene-plasticizer-xylene; Merck Eurolab). Control experiments were performed simultaneously using antibodies preadsorbed with immunizing peptide or omitting primary antibody. The immunostained slides were examined by light microscopy using an Olympus BX41, and digital images were captured with an Olympus Camedia C-5050 zoom digital camera (Olympus Italia).
Water permeability measurements.
Osmotic water permeability (Pf) was measured in a suspension of cells freshly isolated from the crypt-lumen regions of the proximal colon by the stopped-flow light scattering method (22,23). The experiments were performed at 25°C on a stopped flow apparatus (RX2000, Applied Photophysics) with a pneumatic drive accessory (DA.1, Applied Photophysics) straightforward coupled with a Varian Cary 50 spectrometer (Varian Australia). Scattered light intensity with a dead time of 6 ms was recorded at a wavelength of 450 nm. The time course of cell swelling caused by exposure to the hypotonic gradient (150 mosm/L) was measured for 60 s at the acquisition rate of 1 point/0.1 s. The initial rate constant (k) of cell volume changes was obtained by setting the time course light scattering with a single exponential equation (GraphPad Prism 4.00, 2003). The water permeability coefficient, Pf, was calculated as previously described by Wiener et al. (24) from the following equation:
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where 
C is the osmotic gradient, Vw the molar water volume, Vo the cell volume, and A is the cell surface area. Vo and A were obtained by light microscopy using 4.75-µm beads as an internal standard. Water transport was evaluated in: 1) normal untreated colonocytes; 2) cells treated for 5 min with 500 mmol/L DMSO; and 3) cells treated for 5 min with DMSO followed by 10 min of treatment with 5 mmol/L ß-mercaptoethanol (ß-ME).
In AQP-8 gene silencing experiments, osmotic water permeability was evaluated 24 h after the cells’ transfection by exposing colonocytes transfected with AQP8-siRNA or with scrambled siRNA to the hypotonic gradient (150 mosm/L). The time course of cell swelling was measured for 60 s by light microscopy using an Olympus BX41 while digital images were captured with an Olympus Camedia C-5050 zoom digital camera (Olympus Italia). Serial images were taken at 400X magnification and cell diameters measured in a randomized manner without knowledge of cell treatment using 4.75-µm beads as an internal standard. The cell surface area was evaluated by the Scion Image Beta 4.0.2 computer program (Scion Corporation) and the Pf values calculated as described above.
Osmotic water permeability in the gene-silencing experiments was not measured by the stopped-flow light scattering method because trypsinization of cells from culture matrix could damage the colonocytes.
Silencing of AQP-8 expression using siRNA. Cell fractions 3, 4, and 5 were pooled and seeded onto polystyrene tissue culture dishes (35 mm diameter) pretreated with collagen IV (6 mg/L) (Sigma C 0543). The growth medium was a DMEM/Nutrient mixture F-12 HAM supplemented with 10% fetal calf serum, 0.5 kU/L penicillin, and 50 mg/L streptomycin.
An siRNA duplex corresponding to nucleotides 165–185 of rat AQP-8 cDNA was chemically synthesized by a commercial vendor (Ambion). The siRNA duplex consisted of a 21-nucleotide sense strand (5'-CAUGGCUGACAGUUACCAUdTdT-3') and a 21-nucleotide antisense strand (5'-AUGGUAACUGUCAGCCAUGdTdT-3'). A scrambled siRNA (sense strand, 5'-UACUACCGUCGGAGUACAUdTdT-3'; antisense strand, 5'-AUGUACUCCGACGGUAGUAdTdT-3'), which showed no significant homology to any known protein (analyzed using BLAST), was used as a control.
After 24 h from seeding, cells were transiently transfected by incubation with a growth medium containing siRNA oligonucleotides or scrambled siRNA (20 nmol/L) and siPORTTM lipid siRNA transfection agent (Ambion, Europe), according to the manufacturer’s instructions. After 90 min, the incubation medium was diluted 1:1 (v:v) with fresh growth medium.
To determine the effectiveness of siRNA in silencing AQP-8 gene expression, 24 h after transfection, cells were harvested and analyzed for AQP-8 protein levels by immunoblotting.
Protein content. The protein content was determined using the method of Lowry et al. (25) with BSA as a standard.
Statistics. All data are expressed as means ± SEM. The significance of the differences of the means was evaluated using 1-way ANOVA followed by Newman-Keuls’s Q test, 2-way ANOVA followed by the Bonferroni post test, or Student’s t test. All statistical tests were carried out with statistical software programs (Primer of Biostatistics 1.0 (26); GraphPad Prism 4.00, 2003).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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mRNA and protein expression of AQPs. The presence of mRNA of AQP-3, -4, and -8 was explored by semiquantitative RT-PCR using specific primers for the different AQPs. The results of gel agarose electrophoresis of representative PCR reaction products are shown in Figure 2A. Single bands of the expected size of cDNA fragments were amplified. The sequences of the PCR products revealed complete matching with the published sequences for the related AQPs.
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Total membrane preparations from proximal colonocytes isolated along the crypt-lumen regions were analyzed by immunoblotting with affinity-purified antibodies against rat AQP-3, -4, and -8. Isolated colonocytes expressed the AQPs investigated with increasing intensity from the lower crypt toward the upper crypt regions (Fig. 2B). When antibodies were preadsorbed with large amounts of immunizing peptides, the protein bands disappeared completely, indicating the specificity of the reaction (not shown).
Two bands were observed in immunoblots probed with anti-AQP3 antibodies at 75 and 36 kDa, consistent with a dimer and a glycosylated monomeric form, respectively (29,30). Immunoblotting experiments for AQP-4 showed a 45-kDa glycosylated and a 30-kDa form, as previously reported (8,31,32). The AQP-8 protein pattern revealed a glycosylated monomeric form (33 kDa) and a dimeric form (60–68 kDa) (7,28,33).
Immunohistochemical localization of AQP-8 protein in the rat colon. The localization of AQP-8 protein in the colon was further investigated by immunohistochemistry because its cellular and subcellular distribution is presently unclear. In the proximal colon, the intensity of AQP-8 immunostaining decreased from the tip toward the base of the crypt (Fig. 3a). No labeling was detectable in the intercalated goblet cells (Fig. 3a and inset). At high magnification, the epithelial cells of the proximal colon showed strong AQP-8 immunostaining of the apical membranes (Fig. 3a inset; arrows). Intracellular staining was also observed, whereas basolateral membranes were negative. Immmunolabeled controls were negative (Fig. 3b).
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1. The upper and the lower crypt colonocytes exposed to the hypotonic buffer behaved as a functional osmometer, showing a sudden swelling recorded as a rapid decrease in light scattering (Fig. 4A). In control experiments, little or no signal change was observed in the absence of an osmotic gradient (data not shown). Figure 4A shows representative curves from light scattering measurements of the superficial and lower crypt cells from the proximal colon. The cells belonging to fractions 4 and 5 changed their volume slowly compared with fractions 1 and 2, as indicated by the slower variation of the light scattering signal. Water permeability was significantly higher in the upper compared with the lower crypt colonocytes (P < 0.05, 2-way ANOVA followed by the Bonferroni post test; Fig. 4B). In upper cell fractions, Pf values were 30% higher than in lower crypt fractions. The AQPs’ involvement in cell swelling was confirmed by the observation that cells pretreated with the AQP water channel inhibitor DMSO (28,34,35) significantly reduced their Pf values when exposed to a hypotonic environment (P < 0.05, 2-way ANOVA followed by the Bonferroni post test). This inhibitory effect was greater in the superficial cells (30–40%) than in the lower crypt cells (12–20%) of the proximal colon. The DMSO-induced inhibition of the osmotic water permeability was reversed by ß-ME treatment. Finally, the values of the DMSO-independent osmotic permeability were similar in both lower crypt and surface cells from the proximal colon (55 µm/s).
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First, we verified that the siRNA used was able to silence the AQP-8 gene. This was done by determining the levels of the AQP-8 protein by immunoblotting cultured colonocytes transfected with AQP8-siRNA (Silenced) or with scrambled siRNA (Control). Treatment with siRNA strongly reduced the endogenous AQP-8 protein level compared with controls (Fig. 5A). Quantification of immunoblots by densitometric analysis showed that AQP-8 protein levels were inhibited by AQP8-siRNA by 78.5% (P < 0.001, Student’s t test; Fig. 5B). These findings confirm the effectiveness of the siRNA approach in silencing the AQP-8 gene in colonocyte primary cultures.
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38% compared with control colonocytes (P < 0.05, 1-way ANOVA followed by Newman Keuls’s Q test; Fig. 5C). Moreover, AQP-8 knockdown colonocytes exhibited a water transport reduction similar to that obtained by incubating control cells with DMSO (33.5% inhibition; P < 0.05; Fig. 5C). These results demonstrate the direct involvement of AQP-8 in the overall water transport through isolated colonocytes and suggest a leading role for AQP-8 in DMSO-sensitive water permeability.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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60% of adult body weight with variations according to age and sex. The largest share of water in the human organism is introduced into the body through the intake of food and drink: for this reason water is considered an essential nutrient. Water has various essential biological functions; in the gastrointestinal tract, in particular, water is needed to digest and absorb nutrients.
The main function of the colonic epithelium is to contribute to body fluid homeostasis by salt and water transport and to produce dehydrated feces. Recent studies demonstrated that water absorption in the colon occurs in both surface and crypt epithelia (4). The main difference between the small and the large intestine is that in humans, the small intestine absorbs 9 L of water daily by a near-isosmotic mechanism, whereas colonic crypt epithelia absorb an additional 1.5 L of water even against high hydraulic resistance to produce a hypertonic adsorbate (3,6). Nevertheless, these differences in absorptive capacity (mechanisms) between the small and the large intestine cannot be ascribed to differences in AQP expression (1,6).
The mechanisms underlying water transport in the colon and the role of surface epithelial cells, in particular, are still not fully clear (1). To our knowledge, this is the first work assessing both the expression of AQPs and functional studies on water permeability in the surface and lower crypt cells from the proximal colon.
The cell fractions, obtained from the proximal colon by sequential time incubation, represent surface (fractions 1 and 2) and lower crypt (fractions 4 and 5) cell populations, respectively, as results from the thymidine kinase activity assay; this validates the cell fraction isolation procedure (Fig. 1).
The results clearly show that all of the AQP proteins investigated were expressed in the proximal colon with a distribution along the crypt-lumen regions decreasing from superficial to lower crypt colonocytes (Fig. 2). These findings are essentially in agreement with those previously reported (7,8,11). Indeed, the expression of AQP-3 and AQP-4 was demonstrated previously in the basolateral membrane of the colonic surface epithelium of rats (8,11). However, Koyama et al. (36) did not detect AQP-4 protein or AQP-4 mRNA in either the proximal or distal colon. The present results clearly demonstrate the presence of both AQP-3 and AQP-4, whose protein expression decreases progressively from the superficial to the lower crypt regions in the proximal colon (Fig. 2). The pattern of distribution along the crypt-lumen regions was similar for AQP-3 protein and transcript, whereas AQP-4 mRNA increased from the superficial to the lower crypt colonocytes. This reverse pattern of distribution for AQP-4 mRNA can be explained as an early transcription of the AQP-4 gene, as previously suggested for another AQP (11).
AQP-8 distribution in the colon has been studied but not clearly defined (7,12,36). Indeed, mRNA expression in the distal colon previously demonstrated by ribonuclease protection assay and in situ hybridization (36) was not confirmed by Calamita et al. (7). Our results (not shown) demonstrate that AQP-8 is distributed along the entire large intestine without significant differences between the proximal and distal portions. Moreover, immunohistochemical experiments (12) showed that in the colon, AQP-8 was expressed in both the superficial and lower crypt cells, whereas Calamita et al. (7) found consistent labeling only in superficial cells. Furthermore, according to Koyama et al. (36) and Calamita et al. (7) but not Elkjaer et al. (12), the AQP-8 protein localized is mainly in the superficial cells (Figs. 2and 3). The differences in AQP-8 distribution can be explained by the different experimental techniques utilized or the different conditions of the animals before killing (i.e., food deprived or fed). The results of our immunohistochemical studies on the subcellular localization of AQP-8 reveal particularly prominent expression in the apical membrane (Fig. 3a, inset), which agrees in part with the previously reported localization of the AQP-8 near to and at the apical membrane (7,12). These results strongly suggest that water movement in the proximal colon can utilize AQP-8. Because AQP-8 intracellular staining was also present (Fig. 3) (7,12), we speculate that a cAMP-induced redistribution of AQP-8 from intracellular vesicles to the plasma membrane similar to that already demonstrated in cultured hepatocytes may also occur (28). The membrane localization of AQP-8 was further confirmed by siRNA experiments. Indeed, primary cultured colonocytes silenced for the AQP-8 gene showed a significant reduction in water transport properties (Fig. 5). This suggests for the first time that AQP-8 under normal conditions may represent an important route for water transport through the apical membrane domain of rat colonic superficial cells.
The functional experiments performed in the presence of osmotically induced water fluxes showed that Pf values of the superficial cells were 30–40% higher than those of lower crypt cells (Fig. 4). Our functional observations agree well with the immunoblotting and RT-PCR results, which show higher AQP expression in superficial vs. lower crypt cells. The water permeability coefficient gradually decreased from fraction 1 to fraction 5; it was 100 µm/s in the surface cells and 60–70 µm/s in the lower crypt cells. These values indicate that in the surface cell populations, a large amount of water moves through AQP channels. Treatment with the water channel inhibitor DMSO, which blocks the AQP-3 and -8 (10,30,37,38), reduced the Pf by 30–40% in superficial cells but only by 12–21% in lower crypt cells. Thus, permeability inhibition due to DMSO is not the same in the upper and lower crypt colonocytes, even though AQP distribution along the crypt axis is the same. Indeed, we expected the same inhibition rate in the presence of a maintained ratio between the DMSO-sensitive and insensitive AQPs in superficial and lower crypt colonocytes. This finding could be explained by the presence of another not yet identified DMSO-insensitive water channel expressed more prominently in lower crypt colonocytes or, alternatively, by the higher diffusional water permeability of lower crypt cells. Interestingly, an increased ratio of cholesterol:phospholipid content in mature vs. immature proliferative cells that decrease membrane fluidity and permeability was already demonstrated in the rat small intestine (39).
To distinguish the contribution of different AQPs to overall osmotic water transport, we performed functional experiments in a primary culture colonocyte knockdown for AQP-8. The results clearly demonstrate that AQP-8 gene suppression is associated with a reduction in water transport similar to that obtained by DMSO treatment (Fig. 5). Therefore, this suggests that functioning AQP-8 can mediate most of the DMSO-sensitive water permeability of the colonocytes.
However, the different sensitivity of AQPs to DMSO and the relative permeability of the AQPs to water confirm that the DMSO-sensitive water permeability in isolated colonocytes is probably related almost entirely to the AQP-8 content. Indeed, the DMSO-sensitive AQP-3s have relative permeability to water (0.33) that is lower than that of the AQP-8 (1.35) (6). Overall, we speculate that transcellular water movement in rat colonocytes occurs mainly via AQP-8 in the apical pole and via AQP-3 and, especially, AQP-4 in the basolateral pole (8,11).
Recently, AQP-7 was also shown to exist in colonic tissue (40,41). Intense AQP-7 immunoreactivity was observed in the apical membrane of the superficial colonic cells, but intracellular staining was also present (41). Thus, its involvement in the DMSO-insensitive fluid transport in the colon could be advanced as a hypothesis.
In conclusion, our results strengthen the hypothesis of a pivotal role for AQPs in water transport in colonic superficial cells. The smaller numbers of water channels in the lower crypt cells confirm the ability of this epithelium to produce hypertonic absorbate, leading the luminal suction tension to dehydrate feces (3). An integrative hypothetical model of the channels and transporters responsible for colonic fluid and ion transport is depicted in Figure 6. Results summarized for ion movement are based on data previously reviewed (42,43). Transcellular water movement by surface epithelial colonocytes may occur via mechanisms involving AQP-7 and -8 at the luminal side and AQP-3 and -4 at the basolateral side. However, the intracellular presence of AQP-7 and -8 strongly suggests a regulated trafficking of these AQPs to the apical plasma membrane that should be further investigated (40).
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FOOTNOTES |
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2 Funded by COFIN 99 (GR, 9905021792_002) and by the University of Pavia FAR 2002.
4 Abbreviations used: AQP, aquaporin; ß-ME, ß-mercaptoethanol; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; Pf, osmotic water permeability; siRNA, small interfering RNA.
Manuscript received 24 May 2005. Initial review completed 29 June 2005. Revision accepted 13 July 2005.
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