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1Division of Renal Medicine, The Johns Hopkins Bayview Medical Center, Baltimore 21224; and 2Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Submitted 16 December 2003 ; accepted in final form 18 March 2004
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ABSTRACT |
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40- to 55- and 32-kDa bands in the ureter, bladder, and renal inner medulla, but not renal cortex. UT-B was localized by immunocytochemistry and was strongly expressed in all cell membranes (and to a limited extent in intracellular vesicles in the cytoplasm) of epithelial cells lining the rat bladder, ureter, and renal pelvis lumens except the apical membrane of the umbrella cells. It was also present in single-layer papillary surface epithelial cells. There was no difference in immunoblot expression of UT-B in the bladder or ureteral homogenates between groups of rats fed high- or low-protein or high- or low-sodium diets. Water restriction resulted in an increase in UT-B expression in ureters (49%, P = 0.001) but not in bladders (14%, P = not significant). The functional role of UT-B in the genitourinary tract epithelia is unknown. UT-B may participate in the regulation of epithelial cell volume and osmolality, in the dissipation of urea gradients, and in possible net urea transport across uroepithelia. Western blot analysis; immunocytochemistry; urea transport
In these and other studies, although the mechanism(s) of water and solute transport were not elucidated, net transport was usually in the direction of the concentration gradient. Although qualitatively consistent with a process of simple diffusion, the magnitude of the changes in the above studies seems surprisingly large, suggesting the possibility of facilitated or active transport. Transporters and channels capable of such transport have recently been found in mammalial urothelia, although their physiological roles remain to be defined. Thus an amiloride-sensitive, aldosterone-responsive sodium channel has been identified in urothelia of several mammalian species (reviewed in Ref. 10). Absorption via this channel was recently found to be inducible by increasing hydrostatic pressure, as occurs during bladder filling (23). The same investigators also demonstrated that hydrostatic pressure induced electroneutral chloride and potassium secretion into the mucosal compartment of modified Ussing chambers (23). Furthermore, with the use of immunoblotting and immunocytochemistry, both epithelial sodium channel (ENaC) subunits (16) and aquaporin (AQP) channels 1, 2, and 3 (17) have recently been localized to discrete sites in rat urothelia. We therefore sought to determine whether urea transporters might also be present.
Two urea transporter gene families, UT-A and UT-B, the former with multiple transporter isoforms, have been described. UT-A isoforms are found in several nephron sites (and in liver, heart, and testis) and are largely responsible for facilitated diffusion of urea in the loop of Henle and terminal connecting ducts where they play an important role in the urinary concentrating mechanism (reviewed in Refs. 1, 15). UT-B is the red blood cell (RBC) membrane facilitative urea channel containing the RBC Kidd antigen and is present in descending vasa recta endothelium where it participates in urea recycling and the urinary concentrating mechanism. UT-B protein has been detected in endothelial cells of various blood vessels (21) and in other tissues, notably in the testes and in the brain (3, 19). Recently, investigators reported localization of UT-B mRNA by in situ hybridization in rat papillary epithelia and pelvic transitional cell epithelia (20) and the (faint) presence of UTB protein in homogenized rat bladder subjected to Western blot analysis (19).
Taken together, the above studies suggest that 1) urea (as well as other solutes and water) can permeate mammalian urothelia; 2) under certain in vivo conditions, net transport of urea can occur; and 3) facilitative urea transporters might mediate this transport. The purpose of this study was to examine the expression and localization of UT-B in rat urothelial tissues and to determine whether UT-B, if present, might be chronically regulated by hydration status, or dietary salt or protein.
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METHODS |
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Animals.
The animals used in these studies were female Wistar rats (Harlan, Indianapolis, IN) weighing 225 g and maintained on an ad libitum intake of chow (Quality Lab Products) containing 14% protein until the day of experimental procedure or the start of one of the experimental diets.
Diets and resulting urine characteristics. Before experimental procedures, animals were either maintained on ad libitum standard chow or assigned to one of six treatment or dietary (Quality Lab Products) groups (all groups, n = 6): 1) 48-h water restriction accomplished by complete withdrawal of drinking water (in dehydrated animals, urine volume averaged 1.9 ml/day and urine osmolality averaged 3,785 mosmol/kgH2O.); 2) 48-h water loading, provoked by allowing rats 5% sucrose and water as the sole drinking fluid (in water-loaded animals, urine volume averaged 69 ml/day and urine osmolality averaged 283 mosmol/kgH2O.); 3) 7- to 10-day high-sodium (3.16%) diet [mean urine sodium concentration = 442 ± 36 (SE) meq/l]; 4) 7- to 10-day low-sodium (0.03%) diet (mean urine sodium concentration <1 meq/l); 5) 14- to 21-day high-protein (40%) diet [mean urine urea nitrogen concentration = 2,845 ± 471 (SE) mg/dl]; and 6) 14- to 21-day low-protein (6%) diet [mean urine urea nitrogen concentration = 611 ± 200 (SE) mg/dl].
Procedure for immunoblotting. To obtain tissue samples, rats were anesthetized with intraperitoneal Inactin and a midline abdominal incision was made. For immunoblotting, ureters and bladders (and in preliminary studies, kidneys) were rapidly removed, minced, and placed into an ice-cold isolation buffer solution composed of 250 mM sucrose and 10 mM triethanolamine adjusted to pH 7.6 with 1 N HCL. The solution contained the protease inhibitors leupeptin (1 µg/ml) and phenylmethylsulfonyl fluoride (0.1 mg/ml). Similarly, in preliminary studies, samples of cortex and inner medulla (including papilla) were obtained from each kidney, minced, and placed in ice-cold isolation buffer solution.
All minced tissues were homogenized in ice-cold isolation solution using a Tissumizer homogenizer (Tekmar, Cincinnati, OH). Tissues were homogenized with five bursts of five strokes of a microsaw tooth generator. Homogenates were centrifuged at 4°C at 3,000 g for 10 min to separate incompletely homogenized tissue. Aliquots of the supernatant were obtained for measurement of total protein concentration using a Pierce bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL). A quantity of 5x Laemmli buffer was added to the remainder of the supernatant in a ratio of one part buffer to four parts homogenate, and samples were then heated to 60°C for 15 min to solubilize proteins, aliquoted, and stored at –80°C until analyzed.
Antibodies. The affinity-purified polyclonal antibody to UT-B was raised in rabbits to a HPLC-purified synthetic peptide corresponding to the COOH-terminal 19 amino acids of hUT-B1 (13) and has been extensively characterized (18). The UT-B antibody was a generous gift from Dr. J. M. Sands (Atlanta, GA).
Electrophoresis and immunoblotting of membranes. SDS-PAGE was carried out on minigels of 10% polyacrylamide. The proteins were transferred to nitrocellulose membranes electrophoretically. After the membranes were blocked with 5% nonfat dry milk in phosphate buffer solution, the primary antibody was applied overnight, usually at a 1:3,000 dilution of antibody in phosphate buffer solution containing 0.2% bovine serum albumin. The blots were exposed for 1 h to secondary antibody (donkey anti-rabbit immunoglobin G conjugated with horseradish peroxidase, Amersham Pharmacia Biotech). Blots were developed with enhanced chemiluminescence agents (Amersham Pharmacia Biotech) before exposure to X-ray film to visualize sites of antigen antibody reaction. Where appropriate, controls were carried out using antibody preabsorbed overnight with the immunizing peptide.
For immunoblot comparisons of water-loaded to water-restricted animals, high- to low-dietary-sodium animals, and high- to low-dietary-protein animals, control minigels were run before Western blot analysis and were Coomassie stained to confirm equality of loading of each lane. For this purpose, several representative bands in each sample lane were quantified by densitometry and compared with analogous bands of other samples. Densitometry of Coumassie-stained gels and immunoblots was performed with a Molecular Dynamics Densitometer using ImageQuant, version 5.0, software. Before comparisons, dose (µg of sample loaded)-response (intensity of bands by densitometry) curves were obtained to ensure that loading doses fell in the linear response range.
Procedures for immunocytochemistry. Bladders and ureters of anesthetized rats were fixed for immunolocalization by immersion of the tissues in 4% freshly prepared paraformaldehyde in PBS for 1 h. Tissues were then immersed in a cryprotectant solution of 10% EDTA in 0.1 M Tris buffer, pH 7.4, for 1 h at 4°C and frozen on dry ice. Antibodies were immunolocalized on 8-µm frozen sections as previously described (17). Sections were incubated overnight at 4°C with primary antibodies diluted to 10 µl/ml. Secondary antibodies were species-specific goat anti-rabbit antibodies (Jackson Immunoresearch Labs, West Grove, PA) coupled to Alexa 488 and Alexa 568, respectively (Molecular Probes, Eugene, OR). Tissues thus treated were examined using standard immunofluorescent and confocal microscopy.
Light microscopy. For purposes of light microscopic orientation for immunocytochemistry, bladder and ureters were removed from a control rat and fixed in buffered formalin. Organs were embedded in paraffin blocks and sectioned at 3–5 µm; sections were affixed to glass slides stained with hematoxylin and eosin and examined by standard microscopy.
Statistics. Densitometry data are reported as means ± SE. Statistical comparisons were made by using an unpaired Student's t-test and corroborated using Wilcoxon rank sum testing.
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RESULTS |
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40- to 54-kDa band representing glycosylated UT-B protein species and a smaller band at 32 kDa representing the deglycosylated protein in the renal inner medulla but not in the renal cortex. We demonstrated identical bands in homogenates of bladder and ureter (Fig. 1A). For all tissues, preabsorption of the UT-B antibody with the immunizing peptide ablated the UT-B signals, as shown in Fig. 1B. As previously reported by Timmer and co-workers (18) in rat tissues except erythrocytes, and by Fenton et al. (3) using a different antibody in rat kidney inner medulla and testis [but not by Trinh-Trang-Tan et al. (19)], we found a strong 98-kDa band in immunoblots of bladder, ureter, and renal tissues (Fig. 1A). The significance of this 98-kDa band, which was largely eliminated when the antibody was preabsorbed with the immunizing peptide, is uncertain. It may represent a dimer of glycosylated UT-B or a complex containing UT-B (3, 18).
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Localization of UT-B in papillary surface epithelium. Confocal microscopy of renal papillary tissue (Fig. 3) demonstrates that UT-B is also found in the cytoplasm and cell membrane of the (single-cell) epithelial surface lining of rat papilla. UT-B expression in epithelial cells is much less intense than UT-B expression in descending vasa recta. As is the case for transitional epithelia, the UT-B signal is ablated when the antibody is preabsorbed with the immunizing peptide (not shown).
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98-kDa band in immunoblots of tissues of water-restricted and water-loaded rats.
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98-kDa bands in tissues of salt-loaded and salt-deprived rats.
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98-kDa bands between protein-loaded and -restricted rats.
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DISCUSSION |
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40- to 55-kDa band consistent with the size of the glycosylated protein species (18) and a narrower, fainter 31-kDa band consistent with the size of the nonglycosylated protein (18). The appearances of the 40- to 55-kDa and 31-kDa bands in immunoblots of the ureter and bladder are very similar to the analogous bands in the inner medulla in our study and to those in the inner and outer medulla and rat erythrocytes in previous studies (3, 18, 19), but apparently different from the sharper singlet or doublet bands previously demonstrated at 48 and 50 kDa for liver, brain, and testes (3, 18, 19). Whether the size and appearance of these bands reflect a functional difference in UT-B in these tissues is unknown. We confirm previous reports that UT-B is only faintly expressed, if at all, in the renal cortex (18, 19) and also demonstrate a strong 98-kDa band in all rat tissues studied, as reported in some (3, 18, 20), but not all (19), previous reports. The nature of the 98-kDa band is unclear; it does not deglycosylate when treated with PNGase, but the band is ablated when the antibody is preabsorbed with the immunizing peptide. It may represent a protein complex containing UT-B (20). Here, we localize UT-B to cell membranes of urothelia in the renal pelvis, ureter, and bladder except, notably, the apical (luminal) membrane of the so-called umbrella cells that line the lumen of mammalian GU tract. UT-B is also weakly detectable in cytoplasmic vesicles in these urothelial cells and is especially apparent in the cytoplasm of the umbrella cells. The presence of UT-B in urothelia and papillary surface epithelium was previously suggested by Tsukaguchi and co-workers (20), who described UT3 (a rat UT-B homolog) mRNA in papillary surface epithelium and renal pelvic urothelia using in situ hybridization in rat kidneys. In addition, we confirm the recent finding of Jung and co-workers (7) who demonstrated UT-B protein in the cell membranes, and possibly the cytoplasm, of the single-layer papillary surface epithelium.
Previous studies have not evaluated the regulation of UT-B in urothelia. We show that extremes of dietary protein over 14–21 days or dietary salt over 7–10 days have no effect of UT-B expression in urothelia. We also demonstrate that, compared with water loading, water restriction modestly but significantly upregulates UT-B expression in homogenates of rat ureter, although not in rat bladder. This is the first finding of a difference in response of bladder and ureteral tissues to regulatory stimuli, because past studies of urothelial transporters [AQPs (17); ENaC, Spector D, Mulchandani H, Wade JB, unpublished observations] demonstrated similar responses of transporter abundance to dietary salt and water manipulations in both ureteral and bladder tissues. It is possible that the presence of constituently expressed UT-B protein in the abundant vascular supply and in red cells perfusing the bladder muscle layers was so great in quantity that it obscured modest changes in regulated UT-B protein located in the thin epithelial lining of the bladder.
The possible role(s) of UT-B in urothelial tissue is unknown. Initial studies described UT-B in RBC membranes and the endothelia of descending vasa recta where UT-B participates in the renal concentrating mechanism by helping establish high inner medullary urea concentrations (reviewed in Ref. 15). The strong presence of UT-B in some sites (spleen, bone marrow, thymus, lung) might simply reflect sequestration of red cells (20) or widespread UT-B presence in blood vessels (21). Its presence in other sites may reflect a role in mediating urea transport across the membranes of cells that synthesize urea, as in the case of liver cells and cells in endothelia, seminiferous vessels, and other tissues that contain arginase and where ureagenesis occurs as a byproduct of polyamine synthesis, resulting in increased intracellular urea concentrations (21). Similarly, urothelial UT-B may operate in urothelia to dissipate high, potentially damaging (24), concentrations of urea that enter across the apical membrane of the "umbrella cells" from the GU lumen. Exposure of urothelial cells to high concentrations of urea might occur as a result of leakage or diffusion of urea across the apical membrane barrier or as a consequence of a urea transport system (potentially including other UT transporters in the apical membrane), possibly regulated, across the urothelia layer. In these regards, upregulation of UT-B protein might be particularly advantageous during antidiuresis, when urine urea concentrations are particularly high and any leakage or diffusion of urea across the apical membrane is more likely to result in high concentrations of urea in epithelial cells. UT-B transporters would allow urea to quickly traverse from the underlying layers of epithelial cells into the capillary network located in the stromal layer immediately below the urothelia.
Although no urea transporters have yet been identified in the apical membrane of surface umbrella cells, urea diffusion across the apical membrane lipid bilayer might normally occur across the 10% of the apical membrane of umbrella cells not covered by uroplakins, the dense paracrystalline array of proteins representing a major ionic and solute permeability barrier of urothelia (5). Alternatively, increased diffusion or leakage of urea might occur as umbrella cells slough off (normally at a slow rate) from underlying epithelia or during the course of cycles of contractions/relaxations due to peristalsis and bladder filling and emptying, when insertion and release of apical membrane uroplakins occur, allowing solute entry into the underlying urothelial cells.
Alternatively, exposure of urothelial cells to high urea concentrations might be the result of a urea transporting system (in contrast to leakage or diffusion), because in vivo studies indicate that net transport of urea may occur under certain circumstances (9, 22). Such a system might include other urea transporters such as UT-A, which have not, to this date, been reported in urothelia. If net urea transport occurs, UT-B, possibly in a regulated fashion, could play a role in such transport. Because urea excretion is the major means of elimination of mammalian nitrogenous waste, the biological advantage or purpose of urea reabsorption across GU epithelia is not clear. On the other hand, in the hibernating bear, nitrogenous wastes reabsorbed from urothelia are apparently recycled by gut bacteria into new amino acids that are subsequently reabsorbed through the intestine and used as a source for protein manufacture (12). Similar nitrogen cycling has been shown in other mammals including humans (2), and urothelial urea reabsorbtion might thus serve the purpose of nitrogen conservation under certain circumstances.
Finally, UT-B may also play a role, in conjunction with the other known urothelial transporters, AQPs and ENaC, in urothelial cell volume and tonicity regulation (17). Given that urothelial cells are likely exposed to urine constituents whether by leakage, diffusion, or regulated transport, defense of urothelial cell volume and tonicity would be especially important. Support for this notion comes from recent data demonstrating that organic osmolytes are present in urothelia and are upregulated in thirsted rats (8). Interestingly, many of these osmolytes are known to "stabilize" enzymatic reactions perturbed by urea (24).
In summary, these studies demonstrate that the rat urinary bladder, ureter, and renal pelvis express UT-B in discrete membrane and cytoplasmic locations. Moreover, these experiments suggest that the abundance of UT-B may be upregulated by water restriction, at least in the ureter. Although the functional role of UT-B in uroethelia remains to be established, UT-B may play a role in dissipation of high urea concentrations in urothelial cells, in regulation of urothelial cell volume and osmolality, and potentially in net urea transport across uroepithelia, thus influencing the amount of urea in the final urine.
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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.
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