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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 · 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
![]() |
(1) |
![]() |
(2) |
![]() |
(3) |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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.
|
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.
|
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.
|
|
|
|
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Hicks, RM,
Ketterer B,
and
Warren RC.
The ultrastructure and chemistry of the luminal plasma membrane of the mammalian urinary bladder: a structure with low permeability to water and ions.
Philos Trans R Soc Lond B Biol Sci
268:
23-38,
1974[ISI].
2.
Hill, WG,
Rivers RL,
and
Zeidel ML.
Role of leaflet asymmetry in the permeability of model biological membranes to protons, solutes and gases.
J Gen Physiol
114:
404-414,
1999.
3.
Lavelle, JP,
Apodaca G,
Meyers SD,
Ruiz WG,
and
Zeidel ML.
Disruption of the guinea pig urinary bladder permeability barrier in noninfectious cystitis.
Am J Physiol Renal Physiol
274:
F205-F214,
1998
4.
Lavelle, JP,
Myers S,
Doty D,
Buffington A,
Zeidel ML,
and
Apodaca G.
Urothelial pathophysiological changes in feline interstitial cystitis: a human interstitial cystitis model.
Am J Physiol Renal Physiol
278:
F540-F553,
2000
5.
Lewis, SA,
Berg JR,
and
Kleine TJ.
Modulation of epithelial permeability by extracellular macromolecules.
Physiol Rev
75:
561-590,
1995
6.
Lewis, SA,
and
Hanrahan JW.
Physiological approaches for studying mammalian urinary bladder epithelium.
Methods Enzymol
192:
632-650,
1990[Medline].
7.
Negrete, HO,
Lavelle JP,
Berg J,
Lewis SA,
and
Zeidel ML.
Permeability properties of the intact mammalian bladder epithelium.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F886-F894,
1996
8.
Parsons, CL,
Boychuk D,
Jones S,
Hurst R,
and
Callahan H.
Bladder surface glycosaminoglycans: an epithelial permeability barrier.
J Urol
143:
139-142,
1990[ISI][Medline].
9.
Parsons, CL,
Lilly JD,
and
Stein P.
Epithelial dysfunction in nonbacterial cystitis (interstitial cystitis).
J Urol
145:
732-735,
1991[ISI][Medline].
10.
Stubbs, CD,
Ketterer B,
and
Hicks RM.
The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium.
Biochem Biophys Acta
558:
58-72,
1979[ISI][Medline].
11.
Truschel, ST,
Ruiz WG,
Shulman T,
Pilewski J,
Sun TT,
Zeidel ML,
and
Apodaca G.
Primary urothelial cultures: a model system to analyze umbrella cell barrier function.
J Biol Chem
274:
15020-15029,
1999
12.
Tzan, CJ,
Berg J,
and
Lewis SA.
Effect of protamine sulfate on the permeability properties of the mammalian urinary bladder.
J Membr Biol
133:
227-242,
1995.
13.
Wu, XR,
Manabe M,
Yu J,
and
Sun TT.
Large scale purification and immunolocalization of bovine uroplakins I, II, and III: molecular markers of urothelial differentiation.
J Biol Chem
265:
19170-19179,
1990
14.
Yu, J,
Manabe M,
Wu XR,
Xu C,
Surya B,
and
Sun TT.
Uroplakin I: a 27 kD protein associated with the asymmetric unit membrane of mammalian urothelium.
J Cell Biol
111:
1207-1216,
1990[Abstract].
15.
Zeidel, ML.
Low permeabilities of apical membranes of barrier epithelia: what makes water-tight membranes water-tight?
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F243-F245,
1996