Expression, localization, and regulation of aquaporin-1 to -3 in rat urothelia

David A. Spector1, James B. Wade2, Russell Dillow1, Deborah A. Steplock3, and Edward J. Weinman3,4

1 Division of Renal Medicine, The Johns Hopkins Bayview Medical Center, Baltimore 21224; and Departments of 2 Physiology and 3 Medicine, University of Maryland School of Medicine, and 4 Department of Veterans Affairs, Baltimore, Maryland 21201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although mammalian urothelia are generally considered impermeable to constituents of urine, in vivo studies in several species indicate urothelial transport of water and solutes under certain conditions. This study investigates the expression, localization, and regulation of aquaporin (AQP)-1, -2, and -3 in ureteral and bladder tissues in 48-h dehydrated and water-loaded female Wistar rats. Immunoblots of homogenates of whole ureter and bladder identified characteristic ~28- and 35- to 44-kDa bands for AQP-1, -2, and -3. AQP-1 was localized to capillary and arteriole endothelial cells, whereas AQP-2 and -3 circumferentially lined the epithelial cell membranes except for the apical membrane of the epithelial cells adjacent to the lumens of both ureter and bladder. AQP-2 was also present in epithelial cell cytoplasm. Dehydration resulted in 160-200% increases of AQP-3 signal and 24-49% increases of AQP-2 signal but no change in AQP-1 signal on immunoblots of homogenates of ureters and bladders. AQPs in genitourinary tract urothelia likely play a role in the regulation of epithelial cell volume and osmolality and may play a role in bulk water movement across urothelia.

Western blot analysis; immunocytochemistry; water transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS GENERALLY ASSUMED THAT the mammalian lower urinary tract is an impermeable transit and storage system and that micturated urine is identical to that excreted by kidneys (7). In fact, recent in vitro studies have documented very low urothelial permeability to water, ions, and nonelectrolytes and demonstrated that a major permeability barrier resides in the apical luminal membrane of the epithelial cells ("umbrella cells") lining the bladder lumen (reviewed in Ref. 15; 19).

Although urothelial permeability to water and solutes may be low, it is finite, and the possibility that urine might be significantly modified in transit by the lower urinary tract is plausible given the large urothelial surface area and long elapsed time between egress of urine from collecting ducts and micturation. Indeed, significant net transport (both lumen to blood and blood to lumen) of solutes, including urea, creatinine, sodium, potassium, and chloride, usually down their respective blood/urine gradients, has been demonstrated in vivo in rabbits and rats and studies of perfused dog ureter (14, 29) and bladder (8, 9, 14, 21, 27). Furthermore, a number of studies have shown that rabbit and guinea pig urinary bladder epithelium contain an aldosterone-stimulated, amiloride-inhibited sodium transporter (2, 16), which has recently been identified by Northern blot analysis as the epithelial sodium channel (22). The epithelial sodium channel is known to be responsible for salt and fluid transport across epithelia of many tissues (6).

Although isotopic movement of D2O or isotopically labeled water across mammalian urothelia has been reported, net transport of water has been more difficult to demonstrate. Johnson et al. (12) reported a 45-69% egress of D2O and H2O18 from a solution left in dog bladder for up to 2.5 h; however, no net change in volume was demonstrated over this period. Studies in sheep also showed no net change in volume of urine remaining in bladder for 3 h (18). On the other hand, Fellows and Turnbull (4) demonstrated net transurothelial water influx into rabbit bladders instilled with hypertonic Ringer solution and further demonstrated a high ratio of osmotic water permeability (PF) to diffusional water permeability (PD), suggesting the possibility that water channels mediated at least some transepithelial water flow (26). Net water influx was demonstrated in rats in which bladders were instilled with 0.3 ml of isotonic or hypertonic NaCl. (9). In dogs in which ureters and bladders were perfused with urine via a catheter placed in the renal pelvis, there was an increase in retrieved urine volume from the distal ureter and bladder, indicating water influx into the lumen (14). The volume of water influx was inversely related to the perfusion flow rate. The most dramatic example of net urothelial water (and solute) transport is exemplified by hibernating bears that are capable of reabsorbing their entire daily urine production over the 3-4 mo of winter hibernation (20). Although isotope studies showed reabsorption of 14C urea and D2O from bladder during hibernation, the mechanism(s) were not investigated.

Recently, a large family of transmembrane channel proteins called aquaporins (AQPs) have been identified in diverse plant and animal tissues. These proteins serve to regulate transepithelial water movement and body fluid hemostasis and, in some cases (AQP-3, -7, and -9), may also serve as channels for small nonelectrolytes such as urea. Ten AQP isoforms have been identified to date in mammalian tissues, with AQP-1 to -4 playing major roles in nephron water transport. Of these, AQP-2 and -3, located in the collecting duct cells, have been shown to be regulated by circulating vasopressin and hydration status (24).

AQPs have also been demonstrated in endothelial and epithelial cells in nonrenal mammalian tissues, and, recently, a "faint band" representing AQP-3 was reported in Northern blots of homogenates of rat urinary bladder (11). Furthermore, although unable to demonstrate glycerol intrinsic protein (AQP-3) on immunoblots of homogenates of urinary bladder, Frigeri et al. (5) localized glycerol intrinsic protein by immunocytochemistry to transitional epithelium of the lower urinary tract.

Taken together, the above studies suggest that 1) water (and solutes) can permeate mammalian urothelia; 2) under certain in vivo conditions, net transport of water can occur; and 3) AQPs might mediate at least a portion of this transport. The purpose of this study was to examine the expression and localization of AQP-1 to -3 in rat ureter and bladder and determine whether these AQPs, if present, might be chronically regulated by hydration status.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

The animals used in these studies were female Wistar rats (Harlan, Indianapolis, IN), weighing ~275 g and maintained on an ad libitum intake of Teklad Rodent Diet containing 24% protein (Harlan) until the day of study.

Animal Treatments

All experiments were conducted in conformity with Guiding Principles for Research Involving Animals and Human Beings. Before study, animals underwent either 48-h dehydration accomplished by withdrawal of drinking water or 48-h water diuresis accomplished by giving rats 5% sucrose and water as the sole drinking fluid. In dehydrated animals, urine volume averaged 1.9 ml/day, and urine osmolality was 3,785 mosmol/kgH2O. In water diuretic animals, urine volume averaged 69 ml/day, and urine osmolality was 283 mosmol/kgH2O.

To obtain tissue samples, rats were anesthetized with intraperitoneal Inactin, and a midline abdominal incision was made. For immunoblotting, kidneys, ureters, and bladders were rapidly removed, and ureters and bladders were 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 g/ml) and phenylmethylsulfonyl fluoride (0.1 mg/ml). Similarly, samples of cortex and inner medulla (including papilla) were obtained from each kidney, minced, and placed in ice-cold isolation buffer solution.

Tissue Preparation

All minced tissues were homogenized in ice-cold isolation solution with a Tissumizer homogenizer (Tekmar, Cincinnati, OH). Tissues were homogenized with five bursts of five strokes of a microsawtooth 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 by using a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL) A quantity of 5× 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.

In experiments designed to localize the AQP signal to the epithelial cell layer of bladder, bladders were removed from rats as described in Animal Treatments, placed on iced petri dishes, and opened with iris scissors to expose the lumen. The epithelial cell layer was scrapped twice with a straight-edged scalpel blade, and the accumulated cells were washed into a microcentrifuge tube with a total of 60 liters iced isolation solution. Laemmli buffer was added in a 1:4 ratio, and the samples were treated as described above. For comparison, the remainder of bladders from which the epithelial cells were scraped were processed identically to whole bladders as described above.

Antibodies

Antibodies to AQP-1 (L266), -2 (L414), and -3 (L762) and their corresponding immunizing peptides were generously provided by Dr. Mark A. Knepper (National Institutes of Health, Bethesda, MD). These antibodies have been extensively characterized (1, 3, 24). They were raised in rabbits to synthetic peptides (18-24 amino acids) corresponding to regions in the COOH terminus of the respective water channels and were affinity purified with columns on which the immunizing peptides were immobilized. Antibody LC45 was raised in chicken to the same peptide used to produce antibodies to AQP-3 (24).

Electrophoresis and Immunoblotting of Membranes

SDS-PAGE was carried out on minigels of 12% polyacrylamide. The proteins were electrophoretically transferred to nitrocellulose membranes. After the membranes were blocked with 5% nonfat dry milk in phosphate-buffered solution, the primary antibody was applied overnight, usually at a 1:1,000 dilution of antibody (AQP-1 at 0.59 mg/ml IgG, AQP-2 at 0.11 mg/ml IgG, and AQP-3 at 0.16 mg/ml IgG) in phosphate-buffered solution containing 0.2% bovine serum albumin. The blots were exposed for 1 h to secondary antibody (donkey anti-rabbit IgG 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 by using affinity-purified antiserum preabsorbed with the immunizing peptide. For AQP-1, the complete AQP-1 protein was used for preabsorbtion.

For comparisons of water-loaded with dehydrated animals, control minigels were run before Western blotting and were stained with Coomassie blue to confirm equality 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 Coomassie blue-stained gels and immunoblots was performed with a densitometer (Molecular Dynamics) with ImageQuant, version 5.0 software. Before comparisons, dose (mg sample loaded)-response (intensity of bands by densitometry) curves were obtained to ensure that loading doses fell in the linear response range for the AQP being examined.

RT-PCR for AQP-2

Bladder, ureter , and kidney (inner medulla) samples were collected by using aseptic techniques and extensively rinsed with sterile PBS. The tissues were cut to a maximum thickness of 0.5 cm, submerged in 5 vol of RNAlater (Ambion), incubated overnight at 4°C, and stored at -20°C. Total RNA was isolated by using a RNAqueous-4PCR kit (Ambion). Two-step RT-PCR using a RETROscript kit (Ambion) was performed with both oligo(dT) and random decamers. First-strand synthesis was carried out in a 20-µl reaction volume containing 1-2 µg total RNA, 2 µl RT buffer [(in mM) 500 Tris · HCl (pH 8.3), 750 KCl, 30 MgCl2, 50 dithiothreitol], 4 µl of 2.5 mM 2-deoxynucleotide 5'-triphosphate mix, 1 µl RNAase inhibitor, and 1 µl (100 U) Moloney murine leukemia virus-RT. Reaction tubes were incubated at 44°C for 1 h and terminated by incubation at 92°C for 10 min followed by placement on ice. PCR was performed using 1-5 µl of the cDNA samples in the presence of 2.5 mM 2-deoxynucleotide 5'-triphosphate mix, 10 mM Tris · HCl (pH 8.3), 5 mM KCl, 1.5 mM MgCl2, 0.25 µM each of primer, and 1 U/µl of SuperTaq (Ambion). Samples were denatured at 94°C for 5 min and held at 80°C until the addition of Taq. The temperature and times of the PCR reaction were 94°C for 1 min, 76°C for 1 min, and 72°C for 1 min for 5 cycles; 94°C for 1 min, 73°C for 1 min, and 72°C for 1 min for 5 cycles; and 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 30 cycles. Final extension was performed at 72°C for 5 min. Samples were visualized by loading 10 µl of each sample on a 1% Tris, acetic acid, EDTA-agarose-ethinium bromide gel. Expected product sizes were 593 kb for AQP-2 and 361 kb for rig/S15, a "housekeeping" gene that encodes a small ribosomal subunit protein. PCR primers for AQP-2 were 5'-GTGGCATGCCTGGTGGGTTGCCATGTCTCCTTCC-3' (sense) and 5'-TTGCTGCCGCGAGGCAGGCTCTGAGGAGAGTG-3' (antisense). The primers for rig/S15 were 5'-TTCCGCAAGTTCACCTACC-3' (sense) and 5'-CGGGCCGGCCATGCTTTACG-3' (antisense).

Immunocytochemistry

Fixation of tissue and immunocytochemistry. Bladders of anesthetized rats were fixed for immunolocalization by perfusion of 0.1 ml of 2% freshly prepared paraformaldehyde in PBS into the lumen and immersion of bladders in this fixative for 5 min. In some instances, the lumen-perfusion step was not performed. Ureters were immersion fixed in the same fixative for 5 min. Tissue was immersed in a cryoprotectant solution of 10% EDTA in 0.1 M Tris buffer, pH 7.4, for 1 h at 4°C. Antibodies were immunolocalized on frozen sections as previously described (29). Sections were incubated overnight at 4°C with primary antibodies diluted to 10 l/ml. Double labeling of AQP-1 and -3 was performed using rabbit anti-AQP-1 (L266) and chicken anti-AQP-3 (LC45). Secondary antibodies were species-specific donkey anti-rabbit and donkey anti-mouse 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, the bladder and ureter were removed from a dehydrated rat and fixed in buffered formalin. Organs were embedded in paraffin blocks and sectioned at 3-5 µm, and sections were affixed to glass slides stained with hematoxylin and eosin and were examined by standard microscopy.

Statistics

Densitometry data are reported as means ± SE. Statistical comparisons were made with unpaired Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AQP-1

AQP-1 immunoblot results for homogenates of rat renal cortex, inner medulla, bladder, and ureteral tissues are shown in Fig. 1. In all tissues, two bands were observed, as demonstrated previously in renal tissue homogenates (19): one sharp band at ~28 kDa, representing the nonglycosylated protein, and one broad band at 35-44 kDa, representing the mature glycosylated protein. Preincubation of antibody with a CHIP (AQP-1) protein resulted in complete ablation of the 35- to 44-kDa band and almost complete ablation of the 28-kDa band (Fig. 1).


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Fig. 1.   A: immunoblot of aquaporin (AQP)-1 in homogenates of renal cortex, inner medulla (In Med), ureter, and bladder of Wistar rats. Quantities of 10, 10, 10, and 20 µg of total protein were loaded onto the gel lanes. B: immunoblot of AQP-1 antibody preabsorbed with CHIP 28 (AQP-1) protein in homogenates of the same tissues as in A. Quantities of 20, 20, and 21 µg total protein were loaded into the gel lanes for renal cortex, inner medulla, and bladder.

AQP-2

AQP-2 immunoblot results for homogenates of renal cortex, inner medulla, bladder, and ureteral tissues are shown in Fig. 2. In all tissues, two bands (29 and ~36-45 kDa) were observed and were similar to those previously reported in renal tissues (19). The glycosylated bands in ureteral and bladder tissue were not as broad as those in inner medulla, and the nonglycosylated bands were not as prominent. Preincubation of antibody with the immunizing peptide resulted in complete ablation of both bands (Fig. 2B). To localize the site of AQP-2 within the bladder, immunoblots of lysates of bladder epithelial cells obtained by scraping the bladder lumen were performed (data not shown). Narrow ~44- to 48-kDa AQP-2 bands were present in the epithelial cell lysates, and broader bands were present in the residual bladder wall tissue, indicating the presence of AQP-2 in epithelial cells and possibly in the submucosa.


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Fig. 2.   A: immunoblot of AQP-2 in homogenates of renal cortex, inner medulla, ureter, and bladder of Wistar rats. Quantities of 20, 2, 20, and 30 µg total protein were loaded into the gel lanes. B: immunoblot of AQP-2 antibody preabsorbed with the immunizing peptide in identical homogenates of tissues as in A. Quantities of 21, 19, and 19 µg of total protein were loaded into the gel lanes.

AQP-3

AQP-3 immunoblotting results for homogenates of renal cortex, inner medulla, ureteral, and bladder tissues are shown in Fig. 3. As was the case for other AQPs, two bands (~26 and 35-44 kDa) were present, representing the nonglycosylated and glycosylated isoforms, respectively. The immunoblots of whole bladder homogenates yielded fainter bands representing AQP-3 than other tissues yielded, despite relatively large protein loading, presumably because of the relatively large quantity of muscle tissue (relative to epithelial cell) protein in the whole bladder homogenates. Incubation of the AQP-3 antibody with the immunizing peptide ablated the bands in all tissues examined (Fig. 3B). To localize the site of AQP-3 within the bladder, immunoblots of lysates of scrapped epithelial cells and of homogenates of the remaining bladder walls from which the cells were scrapped were performed. Intense bands characteristic of AQP-3 were present in the epithelial cell lysates, but there was only a faint trace of the glycosylated AQP-3 isoform in bladder wall homogenates (data not shown).


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Fig. 3.   A: immunoblot of AQP-3 in homogenates of renal cortex, inner medulla, ureter, and bladder of Wistar rats. Quantities of 20, 15, 20, and 20 µg of total protein were loaded into the gel lanes. B: immunoblot of AQP-3 antibody preabsorbed with its immunizing AQP-3 peptide in identical homogenates of tissues as in A. Quantities of 20, 8, and 50 µg protein were loaded into the gel lanes for inner medulla, ureter, and bladder, respectively.

RT-PCR for AQP-2. RT-PCR for AQP-2 yielded the single expected 593-bp product in kidney, bladder, and ureteral tissues as shown in Fig. 4. Omitting the reverse transcriptase from the RT reaction solution confirmed that the mRNAs were free from contaminating genomic DNA. (Fig. 4, lane 4).


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Fig. 4.   Expression of AQP-2 mRNA in rat renal inner medulla, bladder, and ureteral tissues analyzed by RT-PCR. A single expected 593-bp product was detected in all tissues. No PCR product was obtained when RT was omitted (lane 4).

Immunocytochemistry

To localize the sites of expression of the AQPs, immunocytochemical studies of bladder and ureter were performed. Immunocytochemical labeling for AQP-1 was positive in vascular endothelial cells of medium and small blood vessels, including radial arterioles coursing toward the lumen through adventitial tissues, and in capillary networks supplying the basal epithelial cells of both bladder (Fig. 5E, green) and ureter (Fig. 5B, green). A faint AQP presence was detected in circular smooth muscle cells. (Fig. 5B). No AQP-1 was seen in epithelial cells. Preincubation of the AQP-1 antibody with CHIP (AQP-1) protein resulted in complete ablation of staining in ureteral and bladder tissues (data not shown).


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Fig. 5.   A: AQP expression in rat urothelia. For purposes of orientation, light microscopic views of hematoxylin- and eosin-stained tissues are shown for ureter (A) and bladder (D). Confocal microscopy of immunocytochemical colocalization of AQP-1 (green) and -3 (red) expression are shown in rat ureter (B; higher power, C) and bladder (E; higher power, F). In both tissues, AQP-1 was present in endothelial cells of capillaries and small arteries, and AQP-3 was present only in epithelial cell membranes. Faint AQP-1 was also present in smooth muscle cells of ureter (M) and bladder (not shown). AQP-3 was not present on the apical membrane of the cells lining bladder and ureteral lumens (arrows). Arrowheads, subepithelial capillaries; S, submucosa.

Immunocytochemical labeling for AQP-3 was strong in bladder and ureteral epithelia, as shown in Fig. 5 (red). AQP-3 was not found in endothelial or muscle cells or in adventitia. Preincubation of AQP-3 antibody with the immunizing peptide ablated the labeling in bladder epithelia (Fig. 6).


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Fig. 6.   Immunocytochemical localization of AQP-3 in rat bladder. A: AQP-3 was present only in the epithelial cells. Arrows, serosa; L, lumen. B: AQP-3 signal was ablated when AQP-3 antibody was preabsorbed with the immunizing peptide.

Under higher magnification using confocal microscopy, AQP-3 labeling in the urothelia seemed limited to the cell membranes and was not evident in cytoplasm (Fig. 5, C and F). Staining was most intense in the basilar cell layer of the epithelia and was not apparent on the luminal or apical membrane side of the large umbrella cells lining the epithelial surface. Colocalization studies of AQP-3 and -1 demonstrated that the network of capillaries containing a signal for AQP-1 abuts the basilar epithelial cell layer of the AQP-3- labeled urothelia in both bladder and ureter.

Immunocytochemical labeling for AQP-2 was strong in bladder and ureteral epithelia, as shown for bladder in Fig. 7. Under high magnification using confocal microscopy, intense AQP-2 labeling in the urothelia was present in epithelial cell membranes, including the basolateral membranes of the umbrella cells lining the lumen. The labeling seemed to extend approximately to the sites of the tight junctions but was not present in the luminal membrane of the umbrella cells (Fig. 7A). In most views, AQP-2 labeling seemed more intense in the membranes of cells located closer to the lumen than in the membranes of the basal epithelial cells, in contrast to the cell membrane labeling of AQP-3, which was most intense in cell membranes of the basal epithelial cells. In addition to labeling of cell membranes, there was faint AQP-2 intracytoplasmic labeling in most epithelial cells, including the luminal umbrella cells (Fig. 7A, arrows). Preincubation of AQP-2 antibody with the immunizing peptide ablated AQP-2 labeling in epithelial cell membranes and cytoplasm (Fig. 7B).


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Fig. 7.   High-power confocal microscopy of immunocytochemical localization of AQP-2 and -3 in bladder epithelia. A: AQP-2 (green) staining was intense in epithelial cell membranes and weaker in cell cytoplasm (arrows). Faint AQP-2 was present in submucosal tissue. S, submucosa. B: in the same tissue treated with both AQP-3 antibody (red), and AQP-2 antibody incubated with its immunizing peptide, AQP-2 (green) was ablated.

Effect of Hydration Status on AQP Levels in Ureteral, Bladder, and Inner Medullary Homogenates

AQP-1. Western blotting results for AQP-1 for homogenates of whole bladder and whole ureter of dehydrated and water-loaded rats are shown in Fig. 8. There was no detectable difference in AQP-1 expression between dehydrated and water-loaded rats, and semiquantitative densitometry indicated a 9 ± 4% decrease in AQP-1 expression in bladders and a 15 ± 8% increase in AQP-1 expression in ureters of dehydrated rats compared with those of water-loaded rats (both P = not significant; Fig. 9).


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Fig. 8.   Immunoblot of AQP-1 in homogenates bladder (A) and ureter (B) of water-loaded and dehydrated Wistar rats. Each lane represents results from 1 animal. Quantities of 20 and 10 µg total protein were loaded onto the gel lanes for bladder and ureter, respectively.



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Fig. 9.   Percent change in AQP-1, -2, and -3 water channel expression in whole bladder and whole ureter homogenates of water-loaded and dehydrated Wistar rats, as determined by densitometry of Western blots. Values are expressed as percent change (means ± SE) of dehydrated animals relative to the mean for water-loaded animals. *P < 0.05, statistically significant.

AQP-2. Immunoblotting results for AQP-2 in homogenates of whole bladder and whole ureter of dehydrated and water-loaded rats are shown in Fig. 10. Semiquantitative densitometry demonstrated a ~250% increase in AQP-2 expression in inner medullas of all dehydrated animals, as others have previously described (19). (Blots and densitometry are not shown.) There also was a significant, albeit smaller, increase in AQP-2 signal in homogenates of dehydrated ureters and bladders. Semiquantitative densitometry indicated a 49 ± 7% increase (P < 0.01) of total AQP-2 expression in ureters and a 24 ± 7% increase (P = 0.01) of total AQP-2 expression in bladders of dehydrated rats compared with those of water-loaded rats (Fig. 9).


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Fig. 10.   Immunoblot of AQP-2 in homogenates of bladder (A) and ureter (B) of water-loaded and dehydrated Wistar rats. Each lane represents results from 1 animal. Quantities of 20 and 15 µg total protein were loaded onto the gel lanes for bladder and ureter, respectively. Blot was overexposed to demonstrate the lower band.

AQP-3. Immunoblotting results for AQP-3 for homogenates of whole bladder and whole ureter of dehydrated and water-loaded rats are shown in Fig. 11. There was also a significant upregulation of AQP-3 expression in both bladder and ureter in response to dehydration. Figure 9 illustrates changes in expression levels of AQP-3 in bladder and ureter as determined by densitometry. Compared with water-loaded animals, dehydration increased total AQP-3 expression by 199 ± 50% in bladder (P < 0.01) and by 163 ± 38% in ureter (P < 0.01).


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Fig. 11.   Immunoblot of AQP-3 in homogenates of bladder (A) and ureter (B) of water-loaded and dehydrated Wistar rats. Each lane represents 1 animal. Quantities of 25 and 10 µg total protein were loaded onto the gel lanes for bladder and ureter, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates the presence of AQP-1, -2, and -3 water channels in rat ureteral and bladder tissues. AQP-1 localizes to the endothelial cells of capillaries and arterioles in both rat ureter and bladder, notably in the capillary network abutting the basilar epithelial cells of urothelia in both organs. By contrast, we find AQP-2 and -3 solely in the urothelia, where they seem to lie circumferentially in the epithelial cell membrane of all cells except on the apical (luminal) surface of the so-called umbrella cells that form the luminal cell layer of both rat ureter and bladder. The membrane staining for AQP-2 is stronger in epithelial cells located near the lumen, in contrast to the membrane staining for AQP-3, which is stronger in epithelial cells located in the basal layers. AQP-2, but not AQP-3, is apparent in epithelial cell cytoplasm. Because the renal pelvis and proximal urethra are lined by identical cells that form a contiguous epithelial cell surface with the rat bladder and ureter (7), it would seem likely that AQP-1, -2, and -3 might be present in those organs as well.

As previously described in renal tissue (24), we show that AQP-1 and -3 in urothelial tissue comprise two bands: a relatively sharp lower band at ~26-28 kDa, representing the nonglycosylated protein, and a broader band at 35-44 kDa, representing the mature glycosylated protein. However, in contrast to the appearance of the bands of AQP-1 and -3 and to the appearance of AQP-2 in renal tissues, AQP-2 protein in urothelial tissues migrates as a strong, relatively narrow band at ~38-40 kDa and only a faint band at ~26-28 kDa. We cannot explain the apparent relative increase in glycosylated AQP-2 protein abundance in urothelia. However, by contrast, it is interesting that a dramatic reduction in the percentage of glycosylated AQP-2 has been reported in rat vas deferens (in which site AQP-2 was shown to be a vasopressin-independent constitutive apical membrane protein) (23). Although the general role of glycosylation of proteins is not clear, it is possible that the glycosylation state of AQP-2 determines its intracellular targeting and trafficking and/or modifies its functional role. Regardless of its function, AQP-2 presence in urothelia is supported by our finding of AQP mRNA using RT-PCR.

We further demonstrate dehydration-induced upregulation of AQP-3 and -2, but not AQP-1, expression in both ureteral and bladder tissue. The 160-200% upregulation of AQP-3 that we found in urothelia is similar to the reported upregulation of AQP-3 in renal homogenates after water deprivation (19, 22). In contrast, the modest 24-49% increase in AQP-2 in ureteral and bladder tissues of dehydrated rats is considerably less than the 100-300% upregulation of AQP-2 in homogenates of inner medulla after water deprivation that was reported by others (19) and confirmed in our study.

On the other hand, AQP-1 expression in ureteral and bladder endothelial cells was not regulated by the animal's hydration status. This is in keeping with other studies showing that in vascular endothelium, renal cortex, and inner and outer medulla, AQP-1 is not regulated by hydration status or circulating arginine vasopressin (AVP) (24).

The mechanism whereby ureteral and bladder AQP-3 and -2 are upregulated by dehydration is not known. One possibility would be a direct effect of increased levels of circulating vasopressin on urothelial cells in a manner analogous to its effect on principal cells of the collecting duct. Vasopressin is known to increase AQP-2 in collecting duct cells by means of cAMP-induced transcription of the AQP-2 gene. In this regard, it is noteworthy that AVP binding sites have been identified in rat bladder (25). Incubation of bladder tissue with AVP, however, did not increase cAMP levels, suggesting a non-vasopressin-2 receptor interaction. Vasopressin receptors were also identified in rabbit bladder and urethra (10). In this study, binding sites were identified in the submucosal tissue underlying and adjacent to the urothelium but not in the urothelial cells per se.

It is possible that the increases in AQP-2 and -3 expression are the result of exposure of epithelial cells to urine of higher osmolality or ionic strength resulting from dehydration. It has recently been reported that organic osmolytes, including inositol, glycerophosphocholine, sorbitol, and betaine, are present in the urinary bladder; levels are much higher in urothelium than in adjacent muscle tissue; and epithelial concentrations, in particular, increase dramatically in thirsted rats (13). Subsequently, it was shown that the sorbitol content of porcine urothelial cells is regulated by changes in sorbitol release and aldose reductase activity, just as in the embryologically related collecting duct cells (17).

Although these studies were not designed to study the physiological role of AQP channels in bladder and ureter, it seems probable that AQPs play at least a contributing role in the regulation of epithelial cell volume and osmolality. These cells and cells in the submucosa underlying the epithelia are likely to be rendered intermittently hypertonic as a consequence of absorbed solutes from hypertonic urine via passive (urea, potassium) or active (sodium) transport processes or as a consequence of water loss from epithelial cells into the hypertonic urine (13). The AQP-1 in submucosal capillaries abutting the basal layer of the urothelia would complement the AQP-2 and -3 present circumferentially in epithelial cell membranes in providing a continuous transport pathway for water required to regulate cell tonicity or volume. Furthermore, because AQP-3 is capable of transporting small molecules including urea (11), it is possible that AQP-3 functions to dissipate urea absorbed from hypertonic urine.

AQPs may also play a role in net water transport across urothelia in either direction. Although the apical membrane of the umbrella cells has been shown to constitute a significant permeability barrier in vitro (reviewed in Ref. 15; 19), in vivo studies suggest that under certain circumstances, bulk water flow may occur across urothelia (14, 20). If bulk water transport occurs, AQP-2 and -3, which this study shows are present in a regulated fashion, could play a role in such transport. The apparent absence of AQP-2 and -3 on the apical membrane in our study suggests that another AQP or another mechanism of water transport must be operative across this surface.

In summary, these studies demonstrate that the rat urinary bladder and ureter express AQP-1, -2, and -3 in discrete locations. Moreover, these experiments indicate the abundance of AQP-2 and -3, but not AQP-1, is increased by 48 h of water deprivation. It is suggested that AQPs may play a regulatory role in urothelial cell volume and osmolality and in determining the composition of final urine.


    ACKNOWLEDGEMENTS

The authors thank Dr. M. A. Knepper for generously supplying advice and AQP antibodies and peptides, Jie Lie and Richard Coleman for technical assistance, and David Kurniawan and Patricia King for photographic and manuscript assistance.


    FOOTNOTES

This work was supported in part by the National Kidney Foundation of Maryland (D. A. Spector), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-32839 (J. B. Wade) and DK-55881 (E. J. Weinman), and the Research Service, Department of Veterans Affairs (E. J. Weinman).

Address for reprint requests and other correspondence: D. A. Spector, Johns Hopkins Bayview Medical Ctr., Div. of Renal Medicine, B2N, 4940 Eastern Ave., Baltimore, MD 21224 (E-mail: dspector{at}jhmi.edu).

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.

First published January 29, 2002;10.1152/ajprenal.00136.2001

Received 1 May 2001; accepted in final form 2 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 282(6):F1034-F1042