1Departments of Pathology and 2Medicine, Emory University School of Medicine, Atlanta, Georgia 30322; and 3Third Department of Medicine, University of Kumamoto, Kumamoto, Japan
Submitted 14 October 2003 ; accepted in final form 16 February 2004
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ABSTRACT |
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urea transport; Caco-2
In this study, we provide the first description of a cloned intestinal urea transporter in the human colon and its characterization as a UT-B urea transporter.
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MATERIALS AND METHODS |
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Western blot analysis. Proteins (10 µg/lane) were separated on 10% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane (Gelman Scientific, Ann Arbor, MI). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) at room temperature for 30 min and then incubated with an antibody to the COOH terminus of the rat UT-B (27) (kindly provided by Dr. Jeff Sands, Division of Nephrology, Department of Medicine, Emory University School of Medicine, Atlanta, GA), an antibody that reacts with rat, mouse, and human UT-B, at a dilution of 1:1,000 overnight at 4°C. After three washes in TBS with 0.5% Tween (TBS-Tween), blots were incubated for 2 h with horseradish peroxidase-linked goat anti-rabbit IgG at a dilution of 1:5,000 (Amersham, Arlington Heights, IL) at room temperature. After two washes with TBS-Tween, the bound secondary antibody was visualized by chemiluminescence (ECL kit; Amersham).
Red blood cell membranes were prepared from human blood collected in heparinized tubes and centrifuged at 2,000 rpm for 10 min, the serum and buffy coat were removed, and cells were washed three times in 10 mM MgCl2. Cells were then washed with 5 mM Na2HPO4, centrifuged three times at 16,000 rpm for 10 min each, and solubilized in isolation buffer (10 mM triethanolamine, 250 mM sucrose, 1 µg/ml leupeptin, and 0.1 mg/ml phenylmethylsulfonyl fluoride, pH 7.6).
Glycosylation of the UT-B protein in the human colon, the rat outer medulla, and human red blood cell membranes was examined with N-glycosidase F, followed by separation on a 12.5% SDS-polyacrylamide gel, with the use of reagents and protocol provided in the PNGase F kit (New England BioLabs, Beverly, MA).
Immunolocalization. For immunohistochemistry, 5-µm sections of formalin-fixed, paraffin-embedded tissue were stained with the UT-B antibody at 1:2,000 dilution, followed by goat, horseradish peroxidase-conjugated anti-rabbit secondary antibody (Dako, Carpinteria, CA), as previously described (27). The primary antibody was omitted for negative control. For immunofluorescence, Caco-2 cells were grown onto eight-chamber sterile glass slides (Lab-Tek; Nalge Nunc, Naperville, IL), fixed with 3.7% formaldehyde, permeabilized with 3% Triton X-100, and stained at 37°C with the UT-B antibody (1:100), followed by FITC-conjugated goat anti-rabbit antibody at 1:40 dilution (Sigma, St. Louis, MO). Cells were also stained with a previously characterized primary rabbit polyclonal antibody to the COOH terminus of the rat UT-A1 urea transporter (an antibody that reacts with rat, mouse, and human UT-A1; generous gift of Dr. Jeff Sands) to test specificity (1:100 dilution). Madin-Darby canine kidney (MDCK) cells transiently transfected with the coding region of rat UT-A4, which has the same COOH terminus of UT-A1 and UT-A2 (9), subcloned into the pCDNA3 vector, were used as positive control for the UT-A antibody. The culture conditions and transfection procedures for the MDCK cells were as previously described (17).
Measurement of urea flux in Caco-2 cells.
Caco-2 cells (kindly provided by Dr. Asma Nusrat, Department of Pathology and Lab Medicine, Emory University School of Medicine, Atlanta, GA) were grown on plastic dishes or permeable membrane inserts (Corning, Marietta, GA) in 80% Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 15 mM HEPES, pH 7.4, 10% FBS, 100 IU/ml penicillin, 100 IU/ml streptomycin. Cells were grown to confluence on 1-cm2 permeable membrane support (Cluster 12 Transwell; Corning). Testing of transepithelial urea flux was usually conducted when cells reached transepithelial resistance close to 280 ·cm2 (277 ± 16
·cm2) (8). Assay of urea transport was conducted at 37°C in basic DMEM with 1 mM cold urea added. At the start of the experiment, the normal growth medium was replaced by medium containing 2 µl/ml [14C]urea (54 mCi/mmol; Amersham, Piscataway, NJ), with 0.5 ml on the apical side for apical-to basal flux direction and 1.5 ml on the basal side for basal-to-apical flux direction. Assay medium without tracer was placed in the opposite side of each membrane. Urea transfer in both directions was tested with or without phloretin. Samples of the medium in the basal (100 µl) or apical (33 µl) side of each well were collected after 1, 2, 5, 15, 30, 60 min, and, after addition of 3 ml scintillation fluid (National Diagnostics, Atlanta, GA), radioactivity was measured in a scintillation counter (6000 LS; Beckman, Fullerton, CA). The appearance of [14C]urea in either side of the well, calculated as nanomoles per square centimeter per minute (mean ± SD in 3 or 4 wells), was used to determine the transport of urea across the cell layer. Urea transport was measured in the presence of urea transporter inhibitors phloretin (0.6 mM), 1,3-dimethylurea (150 mM), and thiourea (150 mM) (Sigma), and the effect of individual inhibitors compared with control was analyzed by performing Student's t-test, with P < 0.05 indicative of statistically significant difference. Experiments were repeated two or three times.
RT-PCR and 3' rapid amplification of cDNA ends. Total RNA was purified with TriPure isolation reagent (Roche, Indianapolis, IN), and synthesis of cDNA was performed with 1 µg of total RNA as previously described (1). PCR amplification of the first-strand cDNA was performed with sense primer 5'-AGATAGCCATGGAGGACA-3' and antisense primer 5'-GTTCTCACAAAGGGCTTT-3', designed to amplify the coding sequence of UT-B (based on GenBank accession no. Y19039).
Antisense primers 5'-ATACTCGTGAAAAACAGCAG-3', 5'-ACATTCCAGTCTTAGTGCCA-3', 5'-TTAGTTGGTTTATGGGGTTT-3', 5'-TGCCTAAGCTGAGTATTTCA-3', 5'-CAATCTGACCTTTGCCTTCA-3', 5'-AAAGTGCTGGGATTACAGGC-3', and 5'-GATTTCACTCTTGTCGCCCA-3', designed from the 3' end sequence of the UT-B/Kidd mRNA sequence (GenBank accession no. NM_015865), in combination with sense primer 5'-GTGCATTCCAGGTGATTTAT-3', were used to amplify the 3' UTR sequence of the intestinal UT-B transcript and, after sequence analysis of the PCR products, to map the 3' end of the transcript.
The PCR products were gel purified and sequenced with an automatic DNA sequencing system (3100 Genetic Analyzer; Applied Biosystems, Foster City, CA). Sequence analysis was performed with the use of DNAStar/Lasergene software (DNAStar, Madison, WI).
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RESULTS |
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DISCUSSION |
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Evidence of mRNA expression was associated with demonstration of UT-B protein expression by Western blot hybridization and immunolocalization in human colonic epithelial cells. Of the proteins identified by the UT-B antibody, the 50-kDa protein is the closest to the expected molecular weight of the peptide encoded by the intestinal UT-B transcript. The 98-kDa protein has been noted before in the kidney, the brain, and the testis, and it is not clear whether it represents a complex of UT-B bound to a different protein (27). As Timmer et al. (27) previously noted, the pattern of glycosylation among the UT-B proteins expressed in the kidney or in other tissues may differ, and our findings in the colon seem to support these observations.
By immunohistochemistry, UT-B was detected in the superficial colonic epithelium, with a staining pattern indicating localization in the cytoplasm and the apical membrane of epithelial cells. Whether UT-B is expressed in the small intestine remains to be established. UT-B was detected in Caco-2 cells with a predominant localization to the cell membrane and a distribution similar to that described in cultured bovine aortic endothelial cells with the same antibody (27). The intensity of the staining in the colonic epithelium was weaker than it was in the human medullary vasa recta but definitely greater than that of red blood cells, whose membrane staining is barely detectable in the same tissue sections. These differences may reflect variations in the abundance of UT-B protein in different tissues, which may be determined by tissue-specific regulatory factors. We could not detect evidence of intestinal expression for mRNA or protein of the UT-A transporter, although its expression has been described in mouse colonic crypts (26).
The functional characterization of cloned UT-B erythrocyte urea transporter protein has been described previously and has been performed using the Xenopus oocyte expression system (12, 20, 30), in which the transport of urea mediated by UT-B was found to be sensitive to inhibition by phloretin, thiourea, 1,3-dimethylthiourea, and para-chloromercuribenzene sulfonate (pCMBS). In the present study, we assayed directly the transport of urea in cultured intestinal epithelial cells by using the Caco-2 cell line, which offers the possibility to study transepithelial movement of solutes when cells are grown on permeable support. Phloretin-inhibitable flux of urea was detected in the apical to basal and basal to apical directions, suggesting that passage of urea from the circulation into the intestinal lumen may be mediated by a carrier but also allowing for the possibility that transport of urea might occur in the opposite direction, as some studies have suggested (7, 14). The transport rate for urea across the Caco-2 cell monolayer appears to be significantly less than that estimated for erythrocytes (24). It is possible that more time might be required for urea to be transported across the polarized cells of the intestinal epithelium than to move through a single membrane enveloping the red blood cells, or that differences in membrane abundance and/or activity of UT-B in erythrocytes and intestinal epithelial cells might result in different rates of urea transfer.
Transport of urea in Caco-2 cells was significantly decreased in the presence of phloretin, thiourea, and 1,3-dimethylthiourea compared with control (commercial source for pCMBS was not found), and preliminary tests of pharmacological inhibition of urea transporter activity in Caco-2 cells are similar to those obtained in oocytes into which UT-B mRNA is injected. In our assay, the inhibitory effect of phloretin on urea transport was somewhat less than that reported previously in other studies. This may be accounted for in part by differences in the expression system used (e.g., measuring uptake in single oocytes or isolated cells vs. transepithelial transport). However, we cannot rule out the possibility that other carriers, such as aquaglyceroporins expressed in the colon, may contribute in part to urea transport in intestinal epithelial cells (6, 28). The substrate specificity of the UT-B intestinal urea transporter needs to be established more clearly, because studies in the oocyte expression system have indicated that in addition to urea, water and small solutes may be transported by the UT-B protein (12, 25, 30), and it is possible that, similarly to aquaporins, intestinal UT-B might also be involved in water absorption from the gut. Further studies are required to clarify the properties and physiological role of UT-B in the intestine.
The study of urea transporters has focused mostly on their involvement in the urine concentration mechanism. Aside from mild deficit in the ability to concentrate urine, no significant abnormalities have otherwise been reported in individuals who lack a functional UT-B transporter/Kidd antigen due to mutation in the Slc14a1 gene (11, 23), and a similar mild change in renal function has been described in UT-B knockout mice (29). Finding expression of urea transporters outside the kidney has raised new questions about the significance of urea in physiological processes in other tissues. It is tempting to hypothesize that regulation of urea transport in the intestine may occur in defined settings. For example, if urea moves physiologically from the circulation to the intestinal lumen, this could become a pathway through which to eliminate excess urea from the body when renal function is significantly impaired. It is not known whether long-term expression and abundance of urea transporters in the intestine may be upregulated in uremia or whether they may be affected by high or low content of proteins in the diet.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* H. Inoue and S. D. Jackson contributed equally to this work.
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