Bladder permeability barrier: recovery from selective injury of surface epithelial cells

John Lavelle1, Susan Meyers2, Richard Ramage2, Sheldon Bastacky3, Debra Doty2, Gerard Apodaca2, and Mark L. Zeidel2

1 Department of Urology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and Departments of 2 Medicine, 2Cell Biology and Physiology, and 3 Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mammalian bladder maintains high electrochemical gradients between urine and blood, permitting the kidney to modify body chemistries through urinary excretion. To perform this function, the urothelium maintains a tight permeability barrier. When this barrier is damaged, leakage of urine components into the underlying bladder layers results, with symptoms of cystitis. In these studies, we develop a model of selective urothelial injury using protamine sulfate (PS) and define the process by which this epithelium recovers from damage. Exposure to PS (10 mg/ml), but not vehicle, caused a rapid fall in transepithelial resistance as well as striking increases in water and urea permeabilities. These changes were accompanied by necrosis and sloughing of sheets of umbrella cells, as seen by scanning and transmission electron microscopy. Over the 72 h after PS exposure, barrier function recovered, with transepithelial resistance and water and urea permeabilities returning to normal values. After loss of umbrella cells, the underlying intermediate cells underwent rapid maturation, as evidenced by increased expression of uroplakins and gradual formation of well-defined tight junctions. At day 5 after PS exposure, barrier function was restored and the surface cells exhibited normal-appearing tight junctions and normal labeling for uroplakins and zonula occludens 1. However, the cells remained smaller than umbrella cells until day 10 after exposure, when normal size was restored. These studies develop for the first time a controlled model of selective urothelial damage and demonstrate a characteristic process by which barrier function is restored and underlying intermediate cells develop into mature umbrella cells. This model will be useful in defining the mechanisms that regulate repair of urothelial damage.

water; urea; barrier epithelia; cystitis; apoptosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE KIDNEY MAINTAINS A CONSTANT body fluid composition by excreting a urine that differs markedly from plasma in osmolality, electrolyte and nonelectrolyte (e.g., urea and ammonia) concentrations, and pH (15). In mammals, the bladder may store this urine for prolonged periods without permitting highly permeable molecules such as water, ammonia, urea, and protons from crossing the bladder and equilibrating with the blood. The bladder permeability barrier, which maintains these steep electrochemical gradients, is located in the apical membrane of the superficial layer of epithelial cells, the so-called umbrella cells (3, 7, 15). Underlying these cells in the epithelium are intermediate cells and basal cells.

The permeability barrier consists of three components: the apical membrane, tight junctions, and an active trafficking mechanism that moves membrane from subapical vesicles into the apical membrane in response to stretching or filling of the bladder (7, 11, 15). The apical membrane contains a group of four related transmembrane proteins: the uroplakins, which, together with a specialized lipid structure (1, 10), exhibit exceptionally low permeabilities to water; small nonelectrolytes; and protons (13, 14). The tight junctions between the surface epithelial cells block transepithelial ion flux so successfully that the epithelium can maintain transepithelial resistances (TERs) of 20,000 Omega  · cm2 (5, 6, 10, 12). Under basal conditions, umbrella cells express large quantities of uroplakins and exhibit well-developed tight junctions between cells.

A number of conditions lead to disruption of the bladder permeability barrier, with leakage of urine constituents into the underlying cell layers. These include bacterial infection, exposure to noxious chemicals, and the dysplasia of tumor growth (5, 15). In addition, we have recently shown that interstitial cystitis, a chronic painful condition of unknown cause, is associated in cats with disruption of the permeability barrier (4). Because bacterial, chemical, and interstitial cystitis are relatively common conditions, it appears that the bladder epithelium frequently suffers significant damage in disease. Although bladder epithelial damage is common, little is known about how the epithelium repairs itself and the role of nonepithelial factors, such as nociceptor nerve fibers, underlying bladder muscle, and white blood cells, in the repair of bladder epithelial damage. A mechanistic understanding of the repair of bladder epithelium after injury may provide important clues as to how to treat different forms of cystitis, especially interstitial cystitis, which are presently refractory to therapy.

An ideal model of urothelial injury would involve selective damage to the surface urothelial or umbrella cells. On the basis of its potential to damage the surface glycosaminoglycan layer of urothelial cells, protamine sulfate (PS) has been instilled into bladders in vivo and the effects on bladder function have been evaluated (5, 8, 9, 12). Further studies in rabbit bladder demonstrated that PS can induce nonselective pores in the apical membrane, leading to irreparable cell damage (5, 12). In the present study, we have adjusted the conditions of PS exposure in rats to selectively damage the umbrella cell layer. We have defined the functional and structural effects of the damage and have characterized in detail the process by which the epithelium recovers.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Unless specified otherwise, all chemicals were obtained from Sigma (St. Louis, MO) and were of reagent grade. [14C]urea and [3H]water were obtained from DuPont-New England Nuclear (Wilmington, DE). All animal studies were carried out with the approval of the University of Pittsburgh Animal Care and Use Committee and maintained according to the standards set forth in the Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996). The media used in an Ussing chambers was a modified Ringer solution containing (in mM) 111.2 NaCl, 25 NaHCO3, 5.8 KCl, 2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11.1 glucose. The solution was maintained at 37°C and at pH 7.5 with constant bubbling with 95% O2-5% CO2.

Induction of urothelial damage with PS. Female Sprague-Dawley rats (275-350 g) were maintained on a standard diet (Lab Diet 5P00, PMI Nutritional, Brentwood, MO) with free access to water in all studies. PS diluted into PBS (0.6 ml) or PBS alone was instilled transurethrally into the bladder via a lubricated 3 French Tom Cat catheter (Sherwood Medical, St. Louis, MO) for 15 min while the animal was maintained under light halothane anesthesia. The PS or PBS was instilled as a bolus over 30-45 s and maintained for 15 min in the bladder before emptying. At varying time points after PS or PBS exposure, animals were euthanized and the bladders were excised. The bladder was placed immediately into Ringer solution and then placed on a rack with the epithelium facing downward. The bladder was then stretched on a 0.73-cm2 ring and held in place by several pins that were inserted away from the area through which the permeability was to be measured. The tissue was then mounted between two halves of an Ussing chamber as described (3, 4, 7, 11), and the chamber was filled with Ringer solution. Temperature was maintained at 37°C, and the hemichambers were constantly stirred. Electrical measurements of transmembrane resistance were performed to determine epithelial membrane integrity. The membranes were allowed to stabilize for ~1 h before addition of the isotope and measurement of permeability.

Permeability measurements. These were performed as described (3, 4, 7, 11). [14C]urea (0.25 µCi/ml) and [3H]water (1 µCi/ml) were added to the apical (luminal) side of the membrane, and both hemichambers were then sampled (2 × 100 µl/hemichamber) at 15-min intervals throughout the experiment. After 1 h of baseline measurements, nystatin (185 µM) was added to the apical side to increase permeability, because nystatin is a nondiscriminate pore former in the apical membrane. To determine the contribution of unstirred layers to the measured permeabilities, the apical membrane was destroyed by the addition of 100 µl of Triton X-100 1 h after nystatin had been added. In all experiments, addition of nystatin and Triton X-100 abolished TER. Corrections were made for removing the sample volume by the addition of replacement fresh Ringer solution, and the calculated fluxes were corrected for these modest dilutional effects.

Calculation of permeabilities and statistical analysis. Results for each rat were on the basis of the mean of four flux measurements during each experimental stage. Flux rates were obtained before and after permeabilization of the epithelium with nystatin and Triton X-100. The values obtained after permeabilization were used to estimate the permeability of the unstirred layer. Diffusive water and urea permeability coefficients (PD) were calculated from the isotopic fluxes by using the flux equation
P<SUB>D</SUB><IT>=&PHgr;/</IT>(<IT>A</IT>)(<IT>&Dgr;C</IT>) (1)
where Phi  is the flux of the tracer across the membrane and is calculated from the net increase of the tracer in the basolateral side, A is the area of the apical membrane and is calculated from the capacitance measurements, and Delta C is the concentration gradient for the isotope across the membrane and is calculated from the mean concentration of the isotope in each chamber for the sampling period. In all flux measurements, the flux rate was linear (R > 0.98). Corrections for the dilution by the replacement of the previous sample with nonradioactive solution were made for the flux calculations as described (3, 4, 7, 11).

To determine the water or urea permeability of the apical membrane, it is necessary to estimate not only the resistance to the flux exerted by the unstirred layer but also the resistance exerted by the postapical membrane structures (intracytosolic membranes, basolateral membranes, basement membranes, connective tissue, and the remnants of the muscle layer). The relationship of the PD(Measured) to PD(AM) (PD of the apical membrane) and PD(TUL) (PD of the total unstirred layer, including the bulk-phase unstirred layer and the postapical membrane structures) can be expressed in the following equation
1/P<SUB>D(Measured)</SUB><IT>=</IT>1<IT>/P</IT><SUB>D(AM)</SUB><IT>+</IT>1<IT>/P</IT><SUB>D(Total UL)</SUB> (2)
PD(Total UL) can be expressed as
P<SUB>D(Total UL)</SUB><IT>=D/&dgr;</IT> (3)
where D is the free diffusion coefficient of the isotope in water and delta  is the equivalent thickness of water layer to the thickness of the total unstirred layer (3, 4, 7, 11). Equations 2 and 3 can be written for water, permitting calculation of PD(AM) for water from PD(Total UL) for water.

To determine PD(Total UL) for water in rabbit bladder epithelium, we used two approaches (7). The first approach consisted of measuring the permeability coefficient of the lipophilic molecule butanol. The second approach involved measuring fluxes after permeabilization of the epithelium with nystatin and Triton X-100. In these earlier studies, both methods gave identical values for the contribution of the unstirred layer to permeability. We have therefore used the permeabilization method in these studies. The electrical resistance values represent the means of four to five recordings taken during the course of each section of the experiment. Results are expressed as means ± SE except where explicitly stated. Statistical significance is taken at <5% probability (P < 0.05) on the basis of analysis with Student's t-test.

Light microscopy of urothelium. All images are representative of similar results obtained in at least three bladders. Tissue was fixed in formalin, stained with hematoxylin and eosin, and examined by using standard light microscopy.

Scanning electron microscopy of urothelium. Urothelium, still attached to the ring, was cut into blocks that were fixed in 2.0% (vol/vol) glutaraldehyde and 2.0% (wt/vol) paraformaldehyde in 200 mM Na cacodylate, pH 7.4, 1 mM CaCl2 , and 0.5 mM MgCl2 for 2-4 h at 4°C. The samples were then washed three times for 45 min in PBS and dehydrated for 15 min in a graded series of ethanol: 30% (vol/vol) in PBS and 50, 70, and 95% (vol/vol) in water. The samples were then incubated three times for 45 min each in absolute ethanol. The dehydrated samples were dried, sputter coated with gold-palladium, and viewed in a Jeol JSM T300 scanning electron microscope at 20 kV. Images were captured on Kodak type 52 film (Kodak, Rochester, NY), scanned on a Linotype-Hell Saphir Ultra II scanner, contrast corrected in Photoshop 5.0 (Adobe), assembled in FreeHand (Macromedia, San Francisco, CA), and output on a Kodak 8650PS dye sublimation printer (3, 4, 11). All images are representative of similar results obtained on at least three bladders.

Transmission electron microscopy of urothelium. Bladder epithelium was fixed as described above and then cut into small blocks. The samples were washed three times over a 15-min period with 100 mM Na cacodylate buffer, pH 7.4, and treated with 1% OsO4 (wt/vol), 100 mM Na cacodylate, pH 7.4, for 90 min at room temperature. After several water rinses the samples were stained en bloc overnight with 0.5% uranyl acetate in water. Samples were dehydrated in a graded series of ethanol, embedded in the epoxy resin LX-112 (Ladd, Burlington, VT), and sectioned with a diamond knife (Diatome, Fort Washington, PA). Sections, silver to pale gold in color, were mounted on butvar-coated copper grids, contrasted with uranyl acetate and lead citrate, and viewed at 80 kV in a Jeol 100 CX electron microscope. Images were printed and processed as described in Scanning electron microscopy of urothelium. All images are representative of similar results obtained in at least three bladders.

Immunofluorescence analysis. Tissue was stretched mucosal side down on a ring, and the smooth muscle layers were then carefully removed by dissection. Fixation and indirect immunofluorescence as well as labeling of nuclei with propidium iodide were performed as described (3, 4, 11). Primary antibodies included a mouse monoclonal antibody directed against uroplakin III (11), a rabbit polyclonal antiserum directed against zonula occludens 1 (ZO-1; Zymed, South San Francisco, CA). Secondary antisera included goat anti-mouse conjugated with FITC and goat anti-rabbit conjugated with Texas red (Jackson ImmunoResearch, Fort Wayne, PA). All images are representative of similar results obtained in at least three bladders.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Development of the injury model. The goal of these studies was to develop a model in which the initial injury was confined to the surface umbrella cells, leaving the underlying epithelial cells, the basement membrane, and the muscularis layers intact. This required catheterization of the urethra and instillation of 0.6 ml of volume containing the injurious agent. Multiple studies established that instillation of 0.6 ml of PBS ensured that the entire bladder surface was exposed and resulted in the least injury, as evaluated by permeability and TER measurements. To determine the proper conditions for exposure to PS in vivo, we placed control urothelia into an Ussing chamber and exposed the apical surface to varying concentrations of PS for 15 min. Bladders were placed in the chamber and allowed to stabilize for 1 h. They were then exposed to PS or to vehicle for 15 min. At the end of this time, the buffer in the mucosal side of the chamber was removed and replaced with fresh, prewarmed, and preoxygenated buffer. As shown in Fig. 1, 10 mg/ml of PS strikingly reduced TER (Fig. 1A) and increased water and urea permeabilities (Fig. 1, B and C). Because lower concentrations of PS were not uniformly effective, we performed all in vivo exposures of the bladder with this concentration of PS.


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Fig. 1.   Effect of protamine sulfate (PS; 10 mg/ml) on permeability barrier of rat bladder urothelium. Rat bladders were placed in an Ussing chamber, and values for transepithelial resistance (TER; A), water permeability (Pd water; B), and urea permeability (Pd urea; C) were obtained during a 1-h incubation period. After this period, PS was added to the apical chamber, and the same parameters were measured over the next 1-h period. Values are means ± SE for 7 different bladders. * P < 0.001 for control vs. PS periods.

Time course of damage to and recovery of the bladder permeability barrier. Figure 2 shows the immediate and long-term effects of PS on the function of the permeability barrier. PBS instillation leads to no statistically significant change in TER (Fig. 2A) or urea permeability (Fig. 2C) over the entire time course, although this exposure does decrease water permeability somewhat at 1 and 24 h (Fig. 2B). PS leads to a striking decrease in TER at days 1 and 2, with recovery by day 3 (Fig. 2A). Similarly, PS increases water permeability (Pd water) at 1 h, days 1 and 2, with gradual declines in permeability to control levels during days 3-5 after PS exposure (Fig. 2B). PS also increases urea permeability strikingly at day 1, with recovery to control values during day 2 (Fig. 2C). These results demonstrate that exposure to PS in vivo causes a clear-cut disruption of the bladder permeability barrier that starts within 1 h of exposure and recovers during days 2-5. The response to PS at 1 h was more pronounced when the bladders were exposed in the chambers than when they were exposed in vivo. It is likely that the exposure to PS is more complete in the Ussing chamber than in vivo because the bladders are stretched out before placement in the chambers, exposing the surface more effectively to PS. In addition, PS is better mixed in the chambers than in the intact bladder because of constant stirring and the mixing effect of the gas bubbles. Finally, PS is diluted somewhat in vivo by ongoing urine production.


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Fig. 2.   Time course of effects of PBS or PS instillation in vivo on TER (A), water (B), and urea (C) permeabilities of bladders excised and studied in an Ussing chamber. In all cases, PS or PBS was instilled at time 0 (C), and, at varying times thereafter, bladders were excised and mounted in the Ussing chamber. Values are means ± SE of measurements on 6-12 bladders. * P < 0.05, points on the PS time course vs. those on the PBS time course.

Effects of PS on bladder epithelium and underlying layers: light microscopy. Figure 3 shows hematoxylin and eosin sections of bladder epithelium exposed to PBS or PS, at varying times after the exposure. Exposure to PBS produced no detectable change in structure (Fig. 3A). At 1 h post-PS, only epithelial cells appeared damaged, whereas the underlying submucosa and muscularis appeared entirely normal (Fig. 3B). The damage was patchy, with some areas showing minimal to no apparent damage. Where the urothelium was damaged, however, there was no apparent injury to the underlying muscularis. During the next 3 days after PS exposure, bladder wall edema and inflammation became evident (Fig. 3C). By day 7 after exposure, the entire bladder wall appeared normal (Fig. 3D). These results indicate that PS exposure causes an initial selective injury to the urothelium and that subsequent inflammation and edema occur after the PS has been removed from the bladder.


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Fig. 3.   Light microscopy of bladders exposed to PS. A: control bladder. Note the multilayered urothelium that sits on the subcellular matrix. B: 1 h after PS exposure. Note the loss of the upper layer of urothelial cells. C: day 3 after PS exposure. Urothelium has reappeared, and underlying layers show some edema. D: day 7 post-PS exposure. Urothelium and underlying layers appear grossly normal.

Effects of PS on ultrastructure of urothelial cells: scanning and transmission electron microscopy. Figures 4-6 show scanning electron micrographs of bladders exposed to PBS (Fig. 4) or PS (Figs. 5 and 6) at varying time points after exposure. The urothelium exposed to PBS exhibited normal ridges along the surface of the umbrella cells, with well-defined tight junctions between the cells (Fig. 4, B, D, and F). Note that the average cell size is ~40-100 µm across. At all time points after PBS exposure, the urothelium maintained this appearance.


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Fig. 4.   Structural analysis of the apical surface of rat urothelium. Scanning electron microscopy of normal bladder epithelial surface (A and B) and control bladder tissue at 1 (C and D) and 48 h (E and F) after 15-min instillation of 0.7 ml PBS are shown. Note the presence of abundant ridges and well-defined borders between cells.



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Fig. 5.   Structural analysis of rat urothelium after 15-min exposure to 0.7 ml PS (10 mg/ml). A and B: bladder was fixed immediately after exposure. There are clear-cut areas that are denuded of umbrella cells (asterisks). At 1 (C and D) and 2 h (E and F) after exposure, umbrella cells continue to be shed. D and F: junctional complexes (arrows) between intermediate cells can be discerned. Arrows, denuded areas.



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Fig. 6.   Structural analysis of rat urothelium at prolonged periods after PS exposure. At 24 (A and B) and 48 h (C and D) after PS exposure, it appears that intermediate cells are developing an angular surface membrane pattern that differs from that of normal umbrella cells. These cells are also smaller in diameter (10-40 µm) than umbrella cells (100 µm). After day 5 (E and F), the surface pattern more closely resembles that of mature umbrella cells, although the cell size remains reduced. After day 10 (G and H), the cells appear to have a normal surface pattern and are nearly the size of mature umbrella cells (70-100 µm).

Exposure of the urothelium to PS led to prompt destruction of surface umbrella cells, leading to exposure of underlying intermediate cells (Fig. 5). At the earliest time points, it was difficult to resolve individual intermediate cells (Fig. 5B). By 1 h, the borders of the intermediate cells became discernable (Fig. 5D). These intermediate cells were smaller than the umbrella cells and initially did not display the characteristic ridges of the umbrella cells. In addition, the junctions between intermediate cells appeared indistinct. By 2 h, it was easier to distinguish individual cells and the cell surface appeared more rough. At days 1 and 2 after PS exposure (Fig. 6, A-D), the surface cells remained smaller than normal umbrella cells and began to display ridges, although the ridges appeared to be less well developed than those of mature umbrella cells. At this stage of recovery from injury, the junctions between cells were now easily discerned. By day 5, the surface epithelial cells had a normal appearance, but they remained smaller than normal umbrella cells (Fig. 6, E and F). By day 10, the surface epithelial cells had all the characteristic features of umbrella cells, including large size (70-110 µm), surface ridges, and well-defined junctions between the cells (Fig. 6, G and H).

Figure 7 shows transmission electron micrographs of bladders exposed to PBS or PS at varying time points after exposure. The umbrella cells exposed to PBS (Fig. 7, A and B) revealed the characteristic scalloped appearance of the asymmetrical unit membrane, large numbers of subapical vesicles (arrows, Fig. 7B), and well-developed tight junctions between cells (arrowheads, Fig. 7). These features did not change in the time course after exposure to PBS. By contrast, exposure to PS led to rapid death of umbrella cells, with disruption of tight junctions and sloughing of cells, leading to exposure of the underlying intermediate cells. In the initial hour (Fig. 7, C and D), some umbrella cells appeared darkened, and it appears that these eventually sloughed off, on the basis of the scanning electron microscopy studies. By day 2 (Fig. 7, E and F), smaller intermediate-appearing cells were on the surface, and they exhibited poorly developed tight junctions (arrowhead, Fig. 7F) and some subapical membrane vesicles (arrows, Fig. 7F).


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Fig. 7.   Transmission electron microscopy of urothelium exposed to PBS (A and B) or PS (C-F). Urothelium exposed to PBS exhibits a characteristic ridging of the apical membrane, well-developed tight junctions between adjacent umbrella cells (arrowhead, B), and numerous subapical vesicles (arrows, B). At 1 h after PS exposure (C and D), some urothelial cells darken. At 24 h after PS exposure (E and F), immature-appearing intermediate cells have reached the surface. They show poorly developed tight junctions (arrowhead, F) but show the presence of subapical vesicles (arrows) that resemble those of mature umbrella cells.

Expression of uroplakins and ZO-1 after PS injury. Because uroplakins represent a major protein component of the apical membrane, uroplakin III provides a convenient marker for the presence of appropriate apical membrane proteins in mature umbrella cells. In addition, the high resistance of the urothelium indicates an important role for the tight junctions between cells. ZO-1, a major structural protein of the tight junctions, serves as a marker for the presence of appropriate proteins in the tight junctions of mature umbrella cells. Confocal microscopy was performed at varying time points after exposure to PBS or PS (Figs. 8 and 9). To assist in localization of cell layers, nuclei were labeled with propidium iodide (red); control experiments showed that omission of propidium iodide led to labeling of tight junctions with ZO-1 antisera and no nuclear labeling. Views of the apical region of umbrella cells (Figs. 8, A, C, and E, and 9, A, C, and E) or of the cells underlying the surface cells (Figs. 8, B, D, and F, and 9, B, D, and F) are shown. At 1 h after exposure to PBS, there was abundant surface labeling of umbrella cells with uroplakin (green, Fig. 8A), and a well-defined distribution of ZO-1 in the tight junctions between cells (red, Fig. 8A). Views of the intermediate cells underlying the umbrella cells revealed punctate labeling of uroplakin in these cells (Fig. 8B) at very low fluorescence intensities compared with those observed in surface views of umbrella cells. Note that ZO-1 labeling is not present between intermediate cells, indicating the lack of well-defined tight junctions. Immediately after PS exposure (Fig. 8, C and D), it is apparent that many umbrella cells have sloughed off, and there is no apparent uroplakin labeling of the newly exposed umbrella cells shown in this micrograph (the labeling is from the umbrella cell in Fig. 8C). In other fields (not shown), the uroplakin labeling was detected immediately after PS exposure, but it was always faint. At 1 h after PS exposure, uroplakin expression is observed in the newly exposed intermediate cells (Fig. 8E). ZO-1 labeling is present between these surface cells, but the labeling is punctate and unlike the smooth pattern observed in control umbrella cells (Fig. 8, E vs. A). The intermediate cells underlying these new surface cells exhibit punctate uroplakin labeling (Fig. 8F).


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Fig. 8.   Distribution of uroplakin, ZO-1, and nuclei in rat urothelial cells. Rat bladders were instilled by catheter for 15 min with either PBS (control) or PS in PBS, excised, stripped of their muscle layers, and then fixed and stained for detection by indirect immunofluorescence. Optical sections were obtained with a laser confocal microscope. Three sequential sections were merged to show the structure of the apical portion of the tissue (A, C, and E), and two sequential sections were merged to show the underlying cells (B, D, and F). In the control rat bladder cells, uroplakin III is detected at the surface of umbrella cells (green; A) and in small vesicles in intermediate cells lying ~8 µm below the umbrella cell surface (B). ZO-1 is seen at the borders of the umbrella cells, reflecting their tight junctions (A). C and D: similar sections immediately after PS treatment; sloughing of umbrella cells. E and F: obtained in tissues fixed 1 h after PS treatment. By 1 h, there is abundant uroplakin III labeling in the now-exposed intermediate cells (E). ZO-1 labeling appears between the cells at 1 h, but it is punctate and patchy (arrows, E). All images were taken at the same magnification.



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Fig. 9.   Distribution of uroplakin, ZO-1, and nuclei in urothelial cells at prolonged times after PS exposure. At 24 h (A-D) and day 5 (E and F) after PS treatment, uroplakin III labeling is seen in umbrella cells (A, D, and E) but less so in underlying cell layers (B and F). ZO-1 is present at the junctions of the apical cells and also as punctate structures (arrows, A and C) in the upper portions of the cell. Junctional complexes appear to be spotty in some areas and form parallel lines in some areas (arrowheads, D), which is consistent with immature junctional formation. At day 5, the junctions appear entirely normal and ZO-1 is not detected in small punctate structures (E).

At 24 h after PS exposure (Fig. 9, A-D), uroplakin labeling is abundant in the apical membrane of surface cells (Fig. 9A) and is expressed in a punctate manner in underlying intermediate cells (Fig. 9B). ZO-1 labeling is present in the cell borders and in the cytoplasm as well. ZO-1 labeling appears spotty along the cell borders and forms two parallel lines in some areas (Fig. 9D). Both features suggest that tight junctional formation is incomplete at 24 h after PS exposure. At day 5 after PS, uroplakin and ZO-1 labeling are entirely normal. The surface cells appear entirely normal except for their relatively small size compared with control umbrella cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Homeostasis requires that the kidney excrete and the bladder store a urine that differs markedly from the composition of plasma. Depending on the needs of the individual, in humans, urine may have osmolalities ranging from 50 to 1,200 mosmol/kgH2O, pH values between 4.5 and 10.0, and levels of ammonia, sodium, potassium, urea, and other toxins that vastly exceed the levels of these substances in plasma. Because urine is so different from plasma, the bladder must maintain large electrochemical gradients between urine and plasma for prolonged periods (15).

The bladder permeability barrier is located in the umbrella cell layer and is made up of three components: the apical membrane, the tight junctions, and a trafficking mechanism that inserts and removes apical membrane in response to filling and emptying of the bladder. The apical membrane contains specialized lipid molecules and uroplakin proteins that appear to function in concert to reduce permeability of the apical membrane to small molecules such as water, ammonia, protons, and urea (2, 7). The tight junctions sharply restrict the movement of ions, such as Na+, K+, and Cl-.

Underlying the urothelium are nociceptor fibers and the bladder musculature. When the bladder permeability barrier is breached, irritants in the urine reach these underlying structures (7). These irritants increase afferent traffic in the nociceptor fibers and lead to inflammation in the muscle layers (3, 4, 7). Increased afferent traffic and muscle irritation lead to urinary frequency and urgency, which limit the contact time that the urine has with the bladder wall (7).

By damaging the urothelium, multiple different forms of cystitis lead to temporary or more long-lasting breaches in the bladder permeability barrier. These include chemical cystitis, infectious cystitis, and interstitial cystitis. The symptoms of cystitis, frequency, urgency, and pain, appear to result from leakage of noxious urinary substituents into the layers underlying the damaged urothelium. Because millions of people suffer symptomatic cystitis that results from damage to the urothelium, understanding the mechanisms by which the urothelium repairs itself may provide clues as to how to relieve the symptoms of cystitis in these patients.

An effective model of urothelial damage requires a reproducible method for inducing selective damage to the upper layers of the urothelium, as well as measures of barrier structure and function. We chose PS because previous studies had demonstrated that this molecule permeabilizes the apical membranes of umbrella cells by interacting with specific lipids or proteins present in the apical, but not the basolateral, membrane (4, 10). We varied the concentration and timing of administration so that we could limit the damage to the umbrella cell layer. Limiting the concentration and timing of PS exposure, combined with the tendency of this molecule to interact with proteins (such as glycosaminoglycans) that are expressed most abundantly in the umbrella cells, rather than the intermediate cells below, resulted in a selective umbrella cell injury. Removal of PS ensured that the ongoing response of the urothelium represented the repair process of the epithelium, rather than continuing injury due to PS.

The histological sections demonstrate that the initial injury was confined to the urothelium. Immediately after the PS exposure, only epithelial cells are damaged. The underlying layers appear normal. As time progresses, in the absence of PS, edema and inflammation are noted in the muscularis of some specimens, suggesting that leakage of urinary substituents leads to irritation and inflammation. The electron microscopy confirms that primarily the umbrella cell layer was damaged by PS exposure.

After PS exposure, the functional data demonstrate rupture of the permeability barrier, with leakage of water and urea, as well as ions (decreased TER) that persisted for 2 days. The transmission and scanning electron microscopy demonstrate clearly that this failure of barrier function was caused by the destruction of the umbrella cells. The confocal microscopy studies showed that at the time preceding and immediately after the injury, the underlying intermediate cells have limited amounts of uroplakin and poorly developed tight junctions that have little to no ZO-1.

The loss of umbrella cells leads to what appears to be a series of steps by which intermediate cells become newly functional umbrella cells. Before loss of the umbrella cell layer, intermediate cells exhibit, at most, faint labeling for uroplakin; this labeling is sometimes not detectable. Once exposed, the intermediate cells promptly increase their expression of uroplakins over baseline, and the uroplakins accumulate in or near the apical membrane. Over the next day, tight junctions begin to develop between intermediate cells, and the cells develop the ridges characteristic of mature umbrella cells. By day 2 after injury, these ridges are more prominent, and immature tight junctions have formed. In addition, subapical vesicles have begun to accumulate, suggesting that these cells can insert apical membrane in response to stretch. These changes are reflected in near-normal water permeabilities and TER values.

By day 5, the permeability barrier has been entirely functional for several days, and the new surface epithelial cells resemble umbrella cells in all respects, except for size. By day 10, the surface cells have also reached the size of mature umbrella cells.

These results suggest that the urothelium undergoes a characteristic response to injury that restores the integrity of the permeability barrier over a period of days. This response may closely resemble the process by which senescent umbrella cells are sloughed off and replaced by underlying intermediate cells. With the availability of this model, we can now explore the signaling pathways that regulate the terminal differentiation of intermediate cells to umbrella cells. We can also explore the influence of factors in the bladder, such as urine composition and the C-type nociceptor fibers, that are involved in the recovery of barrier function.

These studies demonstrate that the urothelium undergoes a characteristic functional and structural response to injury and provide an approach to defining how this process is regulated. An understanding of how the urothelium repairs itself may lead to important insights into the care of patients with cystitis and may also shed light on the mechanisms by which bladder cancer develops.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO-1-DK-43955 and DK-48217 (to M. L. Zeidel).


    FOOTNOTES

Address for reprint requests and other correspondence: M. L. Zeidel, Laboratory of Epithelial Cell Biology, Renal-Electrolyte Div., Univ. of Pittsburgh School of Medicine, Rm. 1218, Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15213 (E-Mail: Zeidel{at}msx.dept-med.pitt.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.

February 20, 2002;10.1152/ajprenal.00307.2001

Received 3 October 2001; accepted in final form 14 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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