1 Centro de Estudios Científicos, Valdivia; 2 Facultad de Medicina, Instituto de Ciencias Biomédicas, Universidad de Chile; and 3 Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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Aquaporin-2 (AQP-2) is the vasopressin-regulated water channel expressed in the apical membrane of principal cells in the collecting duct and is involved in the urinary concentrating mechanism. In the rat distal colon, vasopressin stimulates water absorption through an unknown mechanism. With the hypothesis that AQP-2 could contribute to this vasopressin effect, we studied its presence in rat colonic epithelium. We used RT-PCR, in situ hybridization, immunoblotting, and immunocytochemistry to probe for AQP-2 expression. An AQP-2 amplicon was obtained through RT-PCR of colon epithelium RNA, and in situ hybridization revealed AQP-2 mRNA in colonic crypts and, to a lesser extent, in surface absorptive epithelial cells. AQP-2 protein was localized to the apical membrane of surface absorptive epithelial cells, where it colocalized with H+-K+-ATPase but not with Na+-K+-ATPase. AQP-2 was absent from the small intestine, stomach, and liver. Water deprivation increased the hybridization signal and the protein level (assessed by Western blot analysis) for AQP-2 in distal colon. This was accompanied by increased p-chloromercuriphenylsulfonic acid-sensitive water absorption. These results indicate that AQP-2 is present in the rat distal colon, where it might be involved in a water-sparing mechanism. In addition, these results support the idea that AQP-2, and probably other aquaporins, are involved in water absorption in the colon.
intestinal fluid absorption; immunofluorescence microscopy; in situ hybridization; reverse transcriptase-polymerase chain reaction; p-chloromercuriphenylsulfonic acid
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INTRODUCTION |
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THE SMALL AND LARGE INTESTINE are sites of abundant water transport in mammalian species. In humans, ~10 l of water are absorbed in these two segments of the gut. Indeed, the intestine served as the experimental model to establish what is today an accepted paradigm of water transport in fluid-absorbing epithelia such as the intestine and renal tubules (22). This model, in its updated version, considers the presence of an intercellular region of relative high osmotic pressure within the epithelium to which water rapidly flows across highly permeable membranes. The discovery of aquaporins (17), proteins conferring high water permeability to plasma membranes, has come to explain one key component of the paradigm.
Renal water excretion is under the influence of arginine vasopressin (AVP), which increases the osmotic water permeability of the apical membrane of collecting duct principal cells. This action is mediated by vasopressin V2-type basolateral receptors (10). AVP regulates AQP-2 in two ways: the first is a short-term or acute effect that involves the control of intracellular traffic of AQP-2, contained in subapical cytosolic vesicles (10, 16). The second is a transcriptional effect observed in rats after 24 h of water restriction. In this condition, the relative abundance of mRNA coding for AQP-2 and the protein are increased for the acute response (5, 19, 23).
The water-conserving action of AVP seems restricted to the kidney. However, there is evidence that the colon could be a target of action for AVP. In rat distal colon, it has been shown (1, 2, 26) that AVP stimulates water absorption. However, the mechanism by which AVP stimulates colonic transepithelial water transport is unknown. Regulation of water permeability similar to that in the kidney could account for the AVP-dependent increased water absorption in colonic epithelium.
Whether water channels exist in the intestinal tract and serve a purpose in osmotically driven transepithelial water transport has been a matter of debate (12, 22). Results (24) obtained with vesicles from brush-border and basolateral membranes of the small intestine have suggested an absence of water channels. Recent evidence (13), however, shows that several members of the aquaporin family are expressed in epithelial cells from the gastrointestinal tract. AQP-3, AQP-4, and AQP-8 transcript or protein have been demonstrated in the epithelium lining the small or large intestine, although there are discrepancies about their distribution in different intestinal segments and cell types. AQP-3 has been reported (7, 11, 18) to be located in the basolateral membrane of enterocytes from colonic surface and small intestinal villus. AQP-4 appears localized to the basolateral membrane of colonic epithelium (7), although it has also been reported (11) to be selectively localized in basolateral membranes of deep small intestinal glands, and its knockout in transgenic mice results in a decreased osmotic water permeability (28). AQP-8 transcript has been detected (11) in the columnar epithelial cells of jejunum and colon. No aquaporin has yet been shown to be present at the apical membrane of the small or large intestine.
Here we demonstrate that water absorption in the distal colon is increased in water deprivation and inhibited by aquaporin blockade. We have also investigated whether the large intestine expresses AQP-2 and whether its expression is regulated. AQP-2 transcript is indeed present in distal colon as demonstrated by RT-PCR and in situ hybridization. AQP-2 protein can be detected by Western blot analysis and is demonstrated to be on the apical side of surface colonocytes by immunohistochemistry. The results also suggest that the level of expression of colonic AQP-2 is increased by dehydration. This aquaporin could provide an apical membrane route for regulated transepithelial water transport in the colon.
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats (200-250 g) were kept in plastic cages and received food and water ad libitum. Water-deprived animals had no access to water but received food ad libitum and were controlled for body weight at the start and end of the experimental period. All experiments were done according to international regulations for animal care. Blood and urine samples were taken from animals anesthetized with pentobarbital sodium (60 mg/kg ip). Net fluid and Na+ transport from the colonic lumen was measured in vivo in anesthetized rats using 5% agarose gel cylinders inserted into the descending colon, as described previously (29). Briefly, the gel was cast into 6-mm-diameter cylinders, weighed, inserted into the distal colonic lumen, and secured by a ligature at least 1 cm distal to the gel. The gut was returned to the abdominal cavity and left for 1 h before removal. Body temperature was checked and maintained at 37°C throughout. Fluid absorption was measured from the weight difference after the 1-h period. Na+ concentration was measured by flame photometry after overnight extraction in 0.1 M HCl, and Na+ absorption was calculated by comparison with nonincubated gels. Fluxes are expressed per square centimeter of gel surface area.
Tissue preparation.
For RNA extraction, colonic mucosa was obtained by gentle scraping. For
in situ hybridization, tissues were embedded in freezing (OCT) medium
and dropped into liquid nitrogen. Cryosections (7 µm thick) were kept
at 80°C until use. For immunolocalization, sections were fixed in
Bouin's solution for 24 h at room temperature and then embedded
in paraffin.
RT-PCR and cloning. Extraction of total RNA and reverse transcription were performed as previously described for intestinal tissue (3). For PCR, the following sense and antisense primers were designed from rat AQP-2 cDNA sequence (8): sense, 5'-TCCACAACAACGCCACAGC-3', encoding amino acids 121-127; and antisense, 5'-GCACTTCACGTTCCTCCCA-3', encoding amino acids 246-248. The PCR profile was 35 cycles of the following: 30 s at 94°C, 45 s at 60°C, and 1 min at 72°C, followed by an extension of 7 min at 72°C. The PCR products were subcloned and sequenced. The 400-bp cDNA fragment of AQP-2 derived from colon was used to synthesize riboprobes for in situ hybridization.
In situ hybridization. Digoxigenin-labeled antisense and sense riboprobes were generated from the DNA fragment described above by using in vitro transcription with T7 and T3 RNA polymerase, respectively. The probes were used at a concentration of 10 ng/µl, as described previously (21). Tissue sections were observed and photographed on a Nikon Optiphot microscope.
Immunolocalization.
Immunohistochemistry with the peroxidase-antiperoxidase method was
carried out as described previously (27). For
immunofluorescence, rat kidneys and colon were perfused with
physiological saline and then removed. Kidney and colon sections were
frozen in liquid nitrogen and stored until use. Cryostat sections (7 µm) were blocked for 30 min with 2% BSA-0.5% Triton X-100 in PBS.
Purified antisera against AQP-2 (kindly provided by Dr. M. Knepper and
later purchased from Alomone Laboratories) was diluted 1:300, colonic
H+-K+-ATPase (a kind gift from Dr. T. DuBose)
to 1:150, and monoclonal antibody against the 1-subunit
of Na+-K+-ATPase (Upstate Biotechnology) to
1:300 in PBS. The sections were incubated with the antisera overnight
at 4°C. Goat antibodies against rabbit IgG coupled to Cy2 or Cy3 and
goat monoclonal antibodies against mouse IgG coupled to Cy3 (Jackson
ImmunoResearch) were used as secondary antibodies. In dual-labeling
studies, an intermediate blocking step with normal goat serum (diluted
1:20 in PBS) was performed. Confocal laser scanning microscopy was
performed on a LSM Zeiss system, and the software of the instrument was
used to merge images.
Immunoblotting. This was done with crude membrane fractions prepared from the tissues indicated. Renal medulla and cortex, dissected from kidney sections, and whole liver samples were homogenized in buffer containing 250 mM sucrose and 10 mM triethanolamine. Colonocytes, isolated as described previously (4), and small intestinal epithelium, obtained by gently scraping the mucosa with a glass slide, were homogenized with the same buffer. Homogenates were centrifuged at 2,000 g for 10 min at 4°C. The supernatants were spun down at 100,000 g for 1 h at 4°C, and pellets were resuspended. The protein concentration was determined by the Bradford assay. SDS-PAGE was performed using Laemmli buffers on 12% polyacrylamide minigels. Immunoblotting was performed with enhanced chemiluminescence to reveal antigen-antibody reaction. Quantification was carried out by densitometric scanning of the film, and data were expressed as the percentage of control results.
Statistics. Data are expressed as means ± SD. Differences between means were assessed by unpaired t-test, and P < 0.05 was considered to be significant.
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RESULTS |
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The effect of water deprivation on fluid absorption in the distal
colon was studied in rats kept for 96 h without access to water
but with free access to food. Figure
1C shows significantly increased plasma and urine osmolality in rats with water restriction. Consistent with the onset of a water-sparing mechanism, the ratio of
urine to plasma osmolality was also increased in thirsted rats. These
changes were accompanied by changes in the rate of fluid absorption
from the colonic lumen measured in vivo. There was a statistically
significant increase in water absorption in thirsted rats compared with
animals with free access to water (Fig. 1A). Water
absorption under both conditions was significantly reduced to similar
levels in the presence of p-chloromercuriphenylsulfonic acid
(PCMBS). The effect of the mercurial agent did not affect the rate of
Na+ absorption measured simultaneously (Fig.
1B).
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The presence of AQP-2 in colonic tissue was explored by RT-PCR. Figure
2A shows the gene for rat
AQP-2 in schematic form. The cDNA is also shown to display the position
of the primers used, which were designed to encompass three different
exons. Figure 2B shows that a single amplicon of ~0.4 kb
was obtained from kidney as expected (8). No amplification
product could be detected with RNA from the spleen or small intestine
or in the negative controls. An amplicon of the same size as that from the kidney was, however, detected with RNA from distal colonic mucosa.
Sequencing this amplicon revealed a complete identity with the
published sequence (8) for rat kidney AQP-2. This finding
is consistent with the presence of AQP-2 transcript in rat colon.
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AQP-2 expression in colon was also verified by Western blot analysis. Immunoblotting of membranes from kidney and colon of control and thirsted rats is shown in Fig. 2C. The analysis revealed a band of ~29 kDa in all cases plus an additional band of >40 kDa, perhaps corresponding to glycosylated and nonglycosylated forms of the protein.
To study the tissue location of the transcript for AQP-2 in colon, we
performed in situ hybridization. Figure
3A shows the localization of
AQP-2 mRNA in distal colon from a control rat. Considerable staining
could be observed only in the apical cytoplasm of epithelial cells from
colonic crypts. The intensity of the signal decreased from the base of
the crypt toward the mucosal surface. No hybridization was observed in
sections of tissue from the small intestine (Fig. 3A,
inset), liver (Fig. 3C), or stomach (Fig.
3D). Figure 3B shows that thirsting increased the
AQP-2 hybridization signal in colonic epithelium, with the staining spreading more toward the surface epithelium. Figure
3B, inset, depicts control in situ hybridization
with a sense probe in a thirsted rat colonic crypt, showing no
reaction.
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A considerable increase in the protein level was observed in colon from
dehydrated rats by immunoblotting (Fig. 2C). In three separate experiments, the increase in AQP-2 with thirsting, evaluated by densitometric scanning of the lower molecular weight bands, was
1.7 ± 0.2-fold. The expected augmentation in protein level was
also seen in kidney tissue, which increased 2.3 ± 0.3-fold. AQP-2
was not detected in the small intestine or liver of thirsted rats (Fig.
2C). The location of AQP-2 protein in colonic epithelium was
investigated by immunohistochemistry with a polyclonal antibody. As
shown in Fig. 4, immunolabeling for AQP-2
was present in the apical membrane of surface columnar epithelial
cells. Reaction was absent from goblet cells and crypt colonocytes. No
labeling was detected after preabsorbing with the peptide used in
preparing the antibody or in the small intestine (results not shown).
Immunhistochemistry for AQP-2 in thirsted rats also revealed an
increased reaction in the apical membrane of surface colonocytes
compared with control (Fig. 4B).
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To locate AQP-2 with better resolution within the epithelium,
double-labeling immunofluorescence with membrane proteins localized to
the apical or basolateral membrane was performed. Na+-
K+-ATPase and H+-K+-ATPase were
chosen, as they are known to be localized in the rat colon to the
basolateral and apical membranes, respectively. In initial experiments,
immunofluorescent staining with a H+-K+-ATPase
antibody gave a distribution of label consistent with the known
localization of this pump to the brush-border membrane of surface
enterocytes in the distal colon (Fig.
5J).
Simultaneous staining with a Na+-K+- ATPase
antibody gave the expected localization to the basolateral membranes in
both surface and crypt enterocytes (Fig. 5I). Expression of
H+- K+-ATPase and
Na+-K+-ATPase did not overlap (Fig.
5K). Figure 5, A-D, shows an
analogous experiment but performed with an anti-AQP-2 antibody
replacing the H+-K+-ATPase antibody. In
Fig. 5, A and D, the distribution of AQP-2 and
bright-field images are shown. AQP-2 is seen predominantly in what
appears to be surface enterocytes and in some crypt mouths cut
transversally. The label was predominantly at the apical surface of the
cells, although some background staining was also present over the
epithelial cells. In contrast, Na+-K+-ATPase
was confined to basolateral membranes of the epithelium as seen in Fig.
5B. To test the apical membrane distribution of the
aquaporin further, double labeling with
H+-K+-ATPase antibody was carried out. Figure
5, E-H, shows that both AQP-2 and
H+-K+-ATPase distribute mainly on the apical
aspect of surface colonic epithelium. Superposition (Fig.
5G) shows that the distribution of these two proteins had
significant overlap, as revealed from the emergence of yellow
labeling.
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DISCUSSION |
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The evidence presented here strongly suggests that AQP-2, the vasopressin-regulated water channel, is expressed in rat distal colon, an organ involved in water absorption and fecal dehydration. One possible way to control water absorption and thus water excretion is the mechanism that operates in the distal nephron, where AQP-2 water channels increase the osmotic water permeability of the apical plasma membrane of principal and inner medullary collecting duct cells. At the organism level, the consequence of this action is the reduction in renal water loss and the excretion of hyperosmotic urine. In fact, rats submitted to water restriction in this study showed increased plasma and urine osmolalities, and the ratio of urine to plasma osmolality was also increased compared with control rats. It is known that water restriction stimulates AVP secretion through an increase in plasma osmolality (19). In the rats used in this study, the increased urine osmolality and the ratio of urine to plasma osmolality are consistent with elevated levels of AVP.
In the present study, we demonstrate that dehydration elicits an increase in water absorption in distal colon. Both basal fluid absorption and that elicited by dehydration can be abolished by the mercurial agent PCMBS, suggesting a participation of aquaporin water channels in this process. The effect of PCMBS occurs without affecting the simultaneously measured rate of Na+ absorption, ruling out a general nonspecific effect. It is also interesting to notice that after dehydration the tonicity of the absorbate should turn from hypertonic to nearly isotonic. This might be due to the increased expression of AQP-2 in the apical membrane and suggests that significant transcellular water transport occurs. The inhibition of water absorption by PCMBS in control rats argues for a significant aquaporin-mediated basal water permeability. The in situ hybridization, immunoblotting, and immunohistochemical data also point to a constitutive expression of AQP-2, but other aquaporins could also account for this basal water permeability.
There is evidence suggesting that the epithelium of rat colon could be a target for vasopressin. In everted sacs of rat distal colon, AVP enhanced salt and water absorption (1, 2, 26), but the mechanism underlying this effect is unknown. We speculated that the mechanism of water transport regulation in the colon could be similar to that present in the kidney. We have, therefore, explored whether AQP-2 is expressed in colonic epithelium and regulated by the hydration state. Through RT-PCR using specific primers, we found evidence for the presence of the AQP-2 message in rat distal colon epithelium. Sequence analysis of the amplicon obtained from colon revealed a complete identity with the cDNA for rat kidney AQP-2 (8). The sense primer used corresponds to a sequence at the start of exon 2, and the antisense primer is in exon 4. Furthermore, the latter is part of the sequence that encodes amino acids in the cytosolic COOH-terminal tail, which is the most variable part among different members of the aquaporin family (20). Thus the fact that the amplicon encompasses three exons is consistent with the presence of the transcript rather than with the amplification of genomic DNA.
The message for AQP-2 in colon is present mainly in crypt epithelial colonocytes and less abundantly in surface colonocytes. Immunostaining for AQP-2, on the other hand, is consistent with the presence of AQP-2 protein in the apical membrane of columnar absorptive cells of the mucosal surface and its absence from goblet and crypt cells. The cellular localization of AQP-2 to the apical membranes of surface colonocytes is confirmed by its codistribution with H+-K+-ATPase and its separateness from Na+-K+-ATPase. The different distribution of AQP-2 message and protein along the crypt-villus axis is not uncommon in these cells that differentiate as they migrate from the site of cell division in the lower reaches of the crypts to the villus or surface epithelial location of mature enterocytes. Similar differing locations for protein and their messengers have been reported (9, 18) in intestinal epithelium, e.g., for AQP-3 and the SGLT1 cotransporter.
Dehydration in rats caused an increase in AQP-2 mRNA and protein of colonic epithelium that paralleled equivalent increases in renal tissue reported previously (10). It is accepted that AVP stimulation of AQP-2 gene transcription is involved in the renal effect. Another physiological signal independent of AVP might be involved in this effect, as suggested in studies (14) showing water deprivation-induced increase in renal AQP-2 mRNA under V2-receptor blockade. The same mechanisms could be responsible for the increased AQP-2 signal found in distal colon from dehydrated rats. This would enable the colon to increase water absorption and fecal dehydration (15, 29). The finding of AQP-2 in the distal colon is novel and raises the possibility that this organ may be a site of control of water absorption in the gastrointestinal tract and thus could act in concert with the kidney to conserve water during states of thirsting.
At the cellular level, several similarities exist between principal cells of the distal nephron and columnar absorptive cells in the distal colon. First, they express the three subunits of the epithelial Na+ channel in the apical membrane (6) and Na+-K+-ATPase in the basolateral membrane (25). The expression of these proteins is under the influence of aldosterone, which, as in the distal nephron, increases Na+ electrogenic transport. Second, AQP-3 and AQP-4, which are present in the basolateral membrane of principal cells (10), are also present in the same membrane domain of absorptive cells of the distal colon (7, 18). The presence of apical membrane AQP-2 in the distal colon would complete the analogy between the two cell types. Recently (11), AQP-8 mRNA has been localized in superficial colonic cells; however, the subcellular localization of this aquaporin remains to be established. At present, no other member of the aquaporin family has been localized to the apical membrane to account for the constitutive water absorption that occurs in the colon. Further study is necessary for better understanding of the physiological role of AQP-2 in colonic water absorption and its regulation.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. F. Aboitiz and E. Bustos for access to equipment and to Drs. T. DuBose, J. Codina, and M. Knepper for antibody gifts. H. Herrera gave valuable technical assistance.
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FOOTNOTES |
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This work was supported by FONDECYT Grant 2990008 and PUC (P. Gallardo). Institutional support to Centro de Estudios Científicos (CECS) by a group of Chilean private companies (CODELCO, Dimacofi, Empresas CMPC, MASISA SA, and Telefónica del Sur) and Fundación Andes (equipment grant) is also acknowledged. CECS is a Millennium Science Institute. F. V. Sepúlveda is an International Research Scholar of the Howard Hughes Medical Institute.
Address for reprint requests and other correspondence: P. Gallardo, ICBM, Facultad de Medicina, Universidad de Chile, Casilla 70058, Santiago, Chile (E-mail: pgallard{at}bitmed.med.uchile.cl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 March 2001; accepted in final form 2 May 2001.
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