Superficial cell differentiation during embryonic and postnatal development of mouse urothelium

After drastic urothelial destruction around birth and around postnatal day 6, mouse urothelial renewal starts each time de novo. The differentiation of superficial cells during urothelial restoration was followed for the first time from embryonic day 15 to postnatal day 6 by the detection of differentiation markers: cytokeratins, uroplakins and apical membrane specialization. The differentiation markers of short-lived superficial cells were studied before and after urothelial destruction. Three distinctive types of superficial cells, typical for certain developmental period, were characterised: cells at low differentiation stage with microvilli and cilia, expressing CK7 and CK18, detected on embryonic day 15; cells at advanced differentiation stage with star-like arrangement of prominent membrane ridges, expressing CK7 and CK20, present between the two urothelial destruction events; highly differentiated cells with typically jagged apical surface, expressing CK7 and CK20, found twice during development. This cell type appears for the first time on embryonic day 18 as the terminal stage of embryonic differentiation. It was found again on postnatal day 6 as an initial stage of differentiation, leading toward terminally differentiated cells of the adult urothelium. Our work proves that apical membrane specialization is the most valuable differentiation marker of superficial cells.     

Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment

A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, syn-thesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation. Accepted: 19 August 1997     

What determines differentiation of urothelial umbrella cells?

Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization of differentiation markers, cytokeratins and uroplakins, during differentiation of umbrella cells of mouse bladder urothelium. Regenerating urothelium after destruction with cyclophosphamide was used to simulate de-novo differentiation of cells, which was followed from day 1 to day 14 after cyclophosphamide injection. Cytokeratin 7 and uroplakins co-localized in the subapical cytoplasm of superficial cells from the early stage of differentiation on. At early stages of superficial cell differentiation cytokeratin 7 was filamentary organized, and rare uroplakins were found on the membranes of relatively small cytoplasmic vesicles, which were grouped in clusters under the apical membrane. Later, cytokeratin 7 gradually reorganized into a continuous trajectorial network, and uroplakins became organized into plaques of asymmetric unit membrane, which formed fusiform vesicles. After insertion of fusiform vesicles into the apical plasma membrane, the surface acquired microridged appearance of umbrella cells. Cytokeratin 20 appeared as the last differentiation marker of umbrella cells. Cytokeratin 20 was incorporated into the pre-existing trajectorial cytokeratin network. These results indicate that differentiation of urothelial cells starts with the synthesis of differentiation-related proteins i.e., cytokeratins and uroplakins, and later with their specific organization. We consider that the umbrella cell has reached its final stage of differentiation when uroplakins form plaques of asymmetric unit membrane that are inserted into the apical plasma membrane and when cytokeratin 20 becomes included in a trajectorial cytokeratin network in the subapical area of cytoplasm.     

Differentiation-induced uroplakin III expression promotes urothelial cell death in response to uropathogenic E. coli

Uropathogenic E. coli (UPEC) expressing type 1 pili underlie most urinary tract infections (UTIs). UPEC adherence to the bladder urothelium induces a rapid apoptosis and exfoliation of terminally differentiated urothelial cells, a critical event in pathogenesis. Of the four major uroplakin proteins that are densely expressed on superficial urothelial cells, UPIa serves as the receptor for type 1-piliated UPEC, but the contributions of uroplakins to cell death are not known. We examined the role of differentiation and uroplakin expression on UPEC-induced cell death. Utilizing in vitro models of urothelial differentiation, we demonstrated induction of tissue-specific differentiation markers including uroplakins. UPEC-induced urothelial cell death was shown to increase with enhanced differentiation but required expression of uroplakin III: infection with an adenovirus encoding uroplakin III significantly increased cell death, while siRNA directed against uroplakin III abolished UPEC-induced cell death. In a murine model of UTI where superficial urothelial cells were selectively eroded to expose less differentiated cells, urothelial apoptosis was reduced, indicating a requirement for differentiation in UPEC-induced apoptosis in vivo. These data suggest that induction of uroplakin III during urothelial differentiation sensitizes cells to UPEC-induced death. Thus, uroplakin III plays a pivotal role in UTI pathogenesis.     

Rapid differentiation of superficial urothelial cells after chitosan-induced desquamation

Superficial cell desquamation followed by differentiation of newly exposed superficial cells induces regeneration of the urinary bladder epithelium, urothelium. In the present work, chitosan was evaluated as a new inducer of urothelial cell desquamation, in order to study the regeneration of mouse urothelial cells in vivo. Intravesical application of chitosan dispersion caused complete removal of only the superficial layer of cells within 20 min of treatment. Differentiation of the new superficial layer was followed by the appearance and distribution of three urothelial differentiation markers, tight junction protein ZO1, cytokeratin 20 and the maturation of the apical plasma membrane. The arrangement of ZO1 into continuous lines in individual cells of the intermediate layer was already found after 10 min of chitosan application, when desquamation had just started. The appearance of the apical membrane changed from microvillar to typically scalloped within 20 min of regeneration, while complete arrangement of the cytokeratin 20 network took 60 min. These findings provide a new perspective on the rate of the differentiation process in the urothelium and make chitosan a new and a very controllable tool for studies on urothelial regeneration.     

Temporal and spatial dimensions of postnatal growth of the mouse urinary bladder urothelium

Postnatal growth and renewal of mouse urothelium start on the day of birth. In the present study, temporal and spatial dimensions of urothelial growth were studied during the first two postnatal weeks. Quantitative analysis showed that the rate of urothelial cell proliferation is significantly higher during all 14 postnatal days than in adult mice. Three peaks of proliferative and mitotic activity were revealed: on the day of birth and postnatal day 1, on days 6 and 7, and on day 14. The high proliferation rate around the day of birth and at postnatal days 6 and 7 coincides with cell death in the urothelium. Semiquantitative analysis showed that during all 14 postnatal days, the urothelial proliferative response is mostly confined to the basal cell layer. Urothelial cells divide predominantly in parallel to the plain of the urothelium on all chosen postnatal days. Increased portions of urothelial cells, dividing perpendicularly to the urothelium were observed only on the day of birth and on postnatal day 7. Our results suggest that postnatal growth of mouse urothelium is particularly the result of an increasing number of cells in individual cell layers and not the result of an increasing number of cell layers.     

Expression and localization of four uroplakins in urothelial preneoplastic lesions

In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.     

Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support.     

Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation

Using primary explant cultures of mouse bladder, the early response of the urothelium after superficial and full-thickness injuries was investigated. In such an in vitro wound healing model, explant surfaces with a mostly desquamated urothelial superficial layer represented superficial wounds, and the exposed lamina propria at the cut edges of the explants represented full-thickness wounds. The urothelial cell ultrastructure, the expression and subcellular distribution of the tight junctional protein occludin, and differentiation-related proteins CK 20, uroplakins, and actin were followed. Since singular terminally differentiated superficial cells remained on the urothelium after superficial injury (i.e., original superficial cells), we sought to determine their role during the urothelial wound-healing process. Ultrastructural and immunocytochemical studies have revealed that restored tight junctions are the earliest cellular event during the urothelial superficial and full-thickness wound-healing process. Occludin-containing tight junctions are developed before the new superficial cells are terminally differentiated. New insights into the urothelium wound-healing process were provided by demonstrating that the original superficial cells contribute to the urothelium wound healing by developing tight junctions with de novo differentiated superficial cells and by stretching, thus providing a large urothelial surface with asymmetric unit membrane plaques.     

Differentiation-dependent localisation of tissue-type plasminogen activator in human bladder urothelium

Human bladder urothelium is able to secrete tissue-type plasminogen activator (tPA). The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder. Twenty-five samples of normal urothelium and 31 non-invasive papillary tumours from 36 patients were examined. The presence of tPA antigen was evaluated immunohistochemically. Differentiation of superficial cells was assessed by the presence of urothelial cell differentiation markers, uroplakins (UPs; immunohistochemistry) and cell's apical surface architecture (scanning electron microscopy). All tissue samples stained anti-tPA positive. In normal urothelium, the intensity of anti-tPA staining was the strongest in superficial cells, which were well-differentiated. In tumours, all cell layers stained anti-tPA positive. The intensity of anti-tPA positive reaction in the upper cell layer correlated with the percentage of anti-UP positive superficial cells. Superficial cells showed various differentiation states. The localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. Our results suggest a positive correlation between tPA secretion and cell differentiation.     

Endocytotic activity of bladder superficial urothelial cells is inversely related to their differentiation stage

The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5–15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.     

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation. The work was supported by the Ministry of Education and Sport, Government of Republic of Slovenia, Slovenia (grant no. 3311-04-831450).     

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells

The apical surface of mammalian bladder urothelium is covered by large (500-1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin-Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.     

Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development

Postnatal rat urothelium was studied from day 0 to day 14, when intense cell loss as part of tissue remodeling was expected. The morphological and biochemical characteristics of urothelial cells in the tissue and released cells were investigated by light and electron microscopy, by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay, by annexin V/propidium iodide assay, and by immunofluorescent detection of active caspases and tight-junction protein occludin. Intense apoptosis and massive desquamation were detected between postnatal days 7 and 10. During this period, active caspases and TUNEL-positive cells were found in the urothelium. Disassembled cell–cell junctions were detected between cells. The majority of desquamated cells expressed no apoptotic cell morphology, but were active caspase positive and TUNEL positive. Ann+/PI− apoptotic bodies and desquamated Ann+/PI+ cells were detected in the lumen. These results indicate that apoptosis and desquamation participate in urothelial cell loss in the rat early postnatal period, indispensable for fast urothelial remodeling during development. (J Histochem Cytochem 57:721–730, 2009)     

Urothelial umbrella cells of human ureter are heterogeneous with respect to their uroplakin composition: different degrees of urothelial maturity in ureter and bladder?

Urothelial umbrella cells are characterized by apical, rigid membrane plaques, which contain four major uroplakin proteins (UP Ia, Ib, II and III) forming UPIa/UPII and UPIb/UPIII pairs. These integral membrane proteins are thought to play an important role in maintaining the physical integrity and the permeability barrier function of the urothelium. We asked whether the four uroplakins always coexpress in the entire human lower urinary tract. We stained immunohistochemically (ABC-peroxidase method) paraffin sections of normal human ureter (n = 18) and urinary bladder (n = 10) using rabbit antibodies against UPIa, UPIb, UPII and UPIII; a recently raised mouse monoclonal antibody (MAb), AU1, and two new MAbs, AU2 and AU3, all against UPIII; and mouse MAbs against umbrella cell-associated cytokeratins CK18 and CK20. Immunoblotting showed that AU1, AU2 and AU3 antibodies all recognized the N-terminal extracellular domain of bovine UPIII. By immunohistochemistry, we found that in 15/18 cases of human ureter, but in only 2/10 cases of bladder, groups of normal-looking, CK18-positive umbrella cells lacked both UPIII and UPIb immunostaining. The UPIb/UPIII-negative cells showed either normal or reduced amounts of UPIa and UPII staining. These data were confirmed by double immunofluorescence microscopy. The distribution of the UPIb/UPIII-negative umbrella cells was not correlated with localized urothelial proliferation (Ki-67 staining) or with the distribution pattern of CK20. Similar heterogeneities were observed in bovine but not in mouse ureter. We provide the first evidence that urothelial umbrella cells are heterogeneous as some normal-looking umbrella cells can possess only one, instead of two, uroplakin pairs. This heterogeneity seems more prominent in the urothelium of human ureter than that of bladder. This finding may indicate that ureter urothelium is intrinsically different from bladder urothelium. Alternatively, a single lineage of urothelium may exhibit different phenotypes resulting from extrinsic modulations due to distinct mesenchymal influence and different degrees of pressure and stretch in bladder versus ureter. Additional studies are needed to distinguish these two possibilities and to elucidate the physiological and pathological significance of the observed urothelial and uroplakin heterogeneity.     

小鼠膀胱上皮的中间层细胞中存在Uroplakins

本文运用超薄切片、冰冻蚀刻及免疫胶体金标记等多种电镜技术并结合免疫组化、免疫荧光染色技术,直观地显示出小鼠膀胱上皮的中间层细胞存在Uroplakins,并在梭形泡膜上形成了与表层细胞类似的AUM结构,而且梭形泡的AUM结构也结合在中间纤维上。蛋白质免疫印迹反应进一步证实中间层细胞含有与表层细胞相同的Uroplakin Ⅰ和UroplakinⅢ等AUM蛋白的主要成份,从而为AUM的发生及其与细胞分化关系的研究提供了重要的实验证据。     

Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explant outgrowths of mouse bladder.

The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.     

Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases

The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.     

Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder

The localisation of actin filaments was studied in rat urothelial cells during differentiation which accompanied regeneration after cell damage induced by cyclophosphamide (CP). By immunofluorescence it was established that actin filaments equally stained along the cell circumference in basal and intermediate cells, while basolateral cell membrane expression was found in terminally differentiated superficial cells. During regeneration, after CP treatment, simple urothelial hyperplasia developed with smaller cuboidal superficial cells, in which actin filaments were equally distributed under the apical and basolateral plasma membranes. As demonstrated by immunoelectron microscopy, the apical surface of these superficial cells was covered with microvilli containing bundles of actin filaments. Within 1 week, the urothelium reverted to its normal three-layer thickness. Superficial cells became larger and flattened and the unthickened apical plasma membrane matured into a thick asymmetric unit membrane. Concomitantly actin filaments disappeared from apical areas of superficial cells while remaining abundant at basolateral areas. Our results indicate that in the urothelium subcellular distribution of actin filaments can be considered as a marker of cell differentiation. Accepted: 16 September 1999     

Restoration of the rat urothelium after cyclophosphamide treatment.

Processes leading to the recovery of a normal three-layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive loss of cells from urothelium, but the remaining cells started to express epidermal growth factor receptor (EGFR) in their plasma membranes. On day 2 after CP injection, proliferating cell nuclear antigen (PCNA) immunohistochemistry showed a rapid increase in positively stained nuclei, from which a hyperplastic urothelium developed, composed of undifferentiated cells expressing EGFR over the entire plasma membrane. Subsequently, EGFR gradually disappeared from the apical plasma membrane but remained in the basolateral membranes. After day 6, PCNA-positive nuclei in all cell layers decreased, except in basal cells. Apoptotic cells were detectable by the TUNEL assay at day 2, and increased in number in all layers of the hyperplastic urothelium until day 10, returning to the control levels by day 14. Electron microscopic evidence showed that apoptotic cells were either pinched off into the bladder lumen or phagocytosed by the neighbouring urothelial cells. Thus, the urothelium responds to the damage by intense proliferation for a week, resulting in an undifferentiated hyperplastic state. Differentiation of superficial cells then begins and damaged cells are gradually removed by apoptosis until the three-layered urothelium is fully restored by two weeks following CP treatment.