Differentiation of epithelial cells in the urinary tract

Cell Tissue Res (2005) 320: 259–268 DOI 10.1007/s00441-004-1005-4 REGULAR A RTICLE Rok Romih . Peter Korošec . Wilson de Mello Jr . Kristijan Jezernik Differentiation of epithelial cells in the urinary tract Received: 21 June 2004 / Accepted: 14 September 2004 / Published online: 19 March 2005 # Springer-Verlag 2005 Abstract 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 twolayered 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 W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support. R. Romih (*) . K. Jezernik Medical faculty, Institute of Cell Biology, Lipiceva 2, 1000 Ljubljana, Slovenia e-mail: [email protected] P. Korošec University Clinic of Pulmonary and Allergic Diseases, Golnik, Slovenia W. de Mello Jr Department of Anatomy, Biosciences Institute, UNESP, Campus of Botucatu, Botucatu, SP, Brazil 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. Keywords Differentiation . Urinary tract . Urothelium . Uroplakins . Cytokeratins . Mouse (Albany strain) Introduction The urinary tract is covered with various types of epithelia that differ in the expression of cell differentiation markers and in their morphology. The major part, extending from the renal pelvis to the urinary bladder is lined by the urothelium, which typically consists of three distinct cell layers: basal, intermediate and superficial. Superficial cells, called umbrella cells, are adapted to maintain a permeability barrier between urine and blood and to cycles of contractions/distensions that occur mainly in the urinary bladder. During advanced stages of differentiation, the urothelium undergoes unique membrane specialization making a rigidlooking asymmetrical unit membrane (AUM) that covers the apical surface of the umbrella cells (Hicks 1965; Koss 1969). AUM also exists in the form of numerous fusiform vesicles in the cytoplasm of superficial and some intermediate urothelial cells (Wu et al. 1990; Yu et al. 1990). Cytokeratins (CKs) are widely used differentiation markers of various epithelia (Franke et al. 1979; Sun et al. 1979; Moll et al. 1982). The epithelium covering renal papilla (inner medulla) expresses cytokeratins of the simple epithelial type (CKs 7, 8, 18, 19), whereas the urethra expresses cytokeratins typical for stratified epithelia (CKs 4, 10, 13) and the urothelium expresses a complex mixture of both types (Achtstätter et al. 1985; Moll et al. 1988; Schaafsma et al. 1989). Cytokeratin 20 (CK20) has the most interesting distribution in the urinary tract, because it is found exclusively in fully differentiated umbrella cells (Moll et al. 1992; Romih et al. 1998). Another class of urothelial differentiation markers are the uroplakins (UPs), a group of integral membrane proteins that form AUM plaques (Wu et al. 1990, 1995; Yu et al. 1990; Walz et al. 260 1995). Thus far, four major UPs (UPIa, UPIb, UPII, UPIII) have been identified and their expression has been shown to be differentiation-related, i.e. they are expressed only in urothelial cells during advanced stages of differentiation (Lin et al. 1994; Wu et al. 1994; Yu et al. 1994). We have recently shown that another important aspect of cell differentiation is the subcellular organization of certain structural proteins. In umbrella cells, cytokeratins uniquely become organized into a complex network beneath the apical plasma membrane, whereas actin filaments almost disappear from the subapical region of these cells (Romih et al. 1999; Veranič et al. 2004). The present study focuses on the differentiation of epithelial cells of the kidney and the urethra and compares their differentiation with that of the urothelium of the urinary bladder. Various parts of the urinary tract of the male and female mouse have been examined by immunohistochemical and immunoelectron-microscopical methods with a panel of antibodies against individual UPs and CK20. The results show that, between the simple epithelium and the fully differentiated urothelium of the kidney lies a junctional zone that contains urothelial cells that do not reach advanced stages of differentiation. Umbrella cells are formed in the urothelium adjacent to the renal cortex, in the ureter, urinary bladder and proximal urethra. These cells are characterized by AUM formation and by a highly ordered CK20 network. The urothelium ends sharply between the urinary bladder neck and the proximal urethra and continues as a stratified epithelium that expresses none of the urothelial cell differentiation markers. Materials and methods Tissues Male and female Albany strain mice were anaesthetized with a ketamine/xylazine/atropine (150 mg ketamine, 10 mg xylazine, and 0.1 mg atropine/kg) mixture and perfused with appropriate fixative through the left ventricle according to the method of Sprando (1990). After fixation, tissues were dissected to obtain samples from kidney, ureter, urinary bladder and urethra. For light microscopy (except for immunofluorescence), whole kidneys were processed and embedded in the paraffin and then sectioned from the ventral part in dorsal planes until the pelvis and renal papilla were reached and used for labelling. The male urethra was divided in two halves in the region of diverticulum, thus obtaining the pelvic urethra and the penile urethra. Each part was processed and embedded in paraffin and then sectioned longitudinally until the lumen was reached. The female urethra was embedded in toto and also sectioned longitudinally. For electron microscopy, kidneys were sectioned longitudinally in the dorsal plane, thus obtaining the inner ventral part of the kidney. By two transversal sections, one cranial and one caudal, and one longitudinal section running parallel to the base of pyramid, the pelvis together with renal medulla was obtained. Each urethra was sectioned longitudinally through its entire length. In the case of Lowicryl embedding, tissues were further cut into smaller pieces that were precisely labelled to keep track of their position and orientation. For immunofluorescence, dissection was carried out as for electron microscopy except that unfixed tissue in phosphate-buffered saline (PBS) was used. Antibodies Five antisera against UPs were used in these study: (1) rabbit antiserum generated against total UPs of highly purified bovine AUM (anti-AUM); (2) rabbit antiserum against a synthetic peptide corresponding to amino acid residues 139–152 of bovine UPIa (anti-UPIa); (3) rabbit antiserum against a synthetic peptide corresponding to amino acid residues 130–149 of bovine UPIb (anti-UPIb); (4) rabbit antiserum against a synthetic peptide corresponding to amino acid residues 40–58 of bovine UPII (anti-UPII); (5) a monoclonal AU1 antibody raised against UPIII (anti-UPIII; Wu et al. 1990, 1994; Liang et al. 2001; Riedel et al. 2001). All antibodies were produced and tested in the laboratory of Prof. T.-T. Sun at the Epithelial Biology Unit, School of Medicine, New York University. Monoclonal anti-CK20 (clone Ks20.8) was obtained from Dako. Immunohistochemistry of UPs on paraffin sections The animals were perfused with Bouin’s solution and, after dissection, samples were dehydrated and embedded in paraffin. Sections were mounted on APTES-precoated slides, deparaffinized in xylene and rehydrated and endogenous peroxidase activity was blocked by 3% H2O2 in methanol. After being rinsed in TRIS-buffered saline (TBS) and pre-incubated with 20% normal swine serum (Dako) in TBS for 20 min, the sections were incubated overnight at room temperature with anti-UP antibodies diluted in TBS containing 3% bovine serum albumin (BSA). Sections were then washed and incubated for 1 h with biotinylated swine anti-rabbit (for anti-AUM, UPIa, UPIb, UPII) or rabbit anti-mouse (for AU1) secondary antibodies (Dako) diluted in TBS containing 3% BSA and then with ABComplex/ HRP (Dako) prepared according to the manufacturer’s instructions. Peroxidase activity was demonstrated with 3, 3′-diaminobenzidine. Immunofluorescence of UPs and CK20 Tissues were frozen in liquid nitrogen immediately after dissection. For detection of UPs and CK20, frozen sections were fixed with absolute ethanol for 15 min at −20°C. After fixation, sections were washed in PBS and preincubated for 30 min with 1% BSA (Sigma) and 5% fetal bovine serum (Sigma) in PBS. Primary antibodies were diluted in 1% BSA in PBS and the incubations were carried overnight at room temperature. Secondary fluorescein isothiocyanate 261 (FITC)-conjugated antibodies (Sigma) were diluted 1:100 in 1% BSA in PBS and the sections were incubated for 1 h at room temperature. After being washed in PBS, the sections were mounted in DAPI-Vectashield (Vector Laboratories). Scanning electron microscopy For scanning electron microscopy, tissues were perfused with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. After dissection, samples were rinsed in 0.1 M cacodylate buffer and postfixed with 1% OsO4 for 1 h at 4°C. Samples were dehydrated, criticalpoint dried and sputter-coated with gold before being examined in a Jeol JSM 840A. Non-specific labelling was blocked in PBS buffer containing 0.1% IGSS gelatine (Amersham Pharmacia), 0.8% BSA (Sigma) and 5% fetal bovine serum (Sigma). Sections were incubated with the five different antisera against UPs, diluted in the same buffer. After being washed in washing buffer (blocking buffer without fetal bovine serum), anti-UP antibodies were detected with anti-rabbit IgG or anti-mouse IgG secondary antibodies conjugated to 5-nm colloidal gold (Auro Probe, Amersham Pharmacia), diluted in blocking buffer, for 1.5 h at room temperature. After extensive washes, silver enhancement was used to enlarge the gold particles. Ultrathin sections were counterstained with uranyl acetate and lead citrate and viewed in Philips CM100 electron microscope. Results Immunoelectron microscopy of UPs Labelling properties of anti-UPs antibodies For immunoelectron microscopy, all reactions were performed on Lowicryl HM20-embedded material. The animals were perfused with 2% paraformaldehyde and 0.05% glutaraldehyde in PBS, pH 7.2. The dissected samples were dehydrated in 30% ethanol for 30 min at 0°C, 55% ethanol for 30 min at −15°C, 70% ethanol for 30 min at −30°C, 100% ethanol for 60 min at −50°C, infiltrated at −50°C with 75% ethanol/25% HM20 for 60 min, 50% ethanol/50% HM20 for 60 min, 25% ethanol/75% HM20 for 60 min, 100% HM20 for 60 min and 100% HM20 overnight. Tissue blocks were polymerized under UV light for 48 h at −50°C and then for 24 h at 20°C in Leica EM-AFS apparatus. Ultrathin sections were cut and collected on nickel grids. Mouse urinary bladder urothelium was taken as a control tissue to evaluate the labelling properties of antibodies against UPs. On paraffin and frozen sections, all UP antibodies labelled superficial and some intermediate urothelial cells (Fig. 1a–d). Some of the intermediate cells were only weakly labelled. Immunoelectron microscopy showed strong labelling of apical plasma membrane and membranes of fusiform vesicles in superficial cells with the anti-AUM, anti-UPIa, anti-UPIb and anti-UPIII antibodies (Fig. 1e–h). On the other hand, anti-UPII showed also some unspecific reactivity in the cytoplasm and nuclei (Fig. 1g). Fig. 1 Three-layered urothelium of urinary bladder, labelled with antibodies against UPIa (a, e), UPIb (b, f), UPII (c, g) and UPIII (d, h). a–d All four UPs are expressed in some superficial and the majority of intermediate cells, whereas basal cells are negative for all UPs. e–h All UPs are localized on the membranes of fusiform vesicles and on the apical plasma membrane of umbrella cells 262 Anatomical relationships in the renal pelvis and urethra In a dorsal section of the kidney (Fig. 2a), the renal pelvis formed an expanded funnel-like cavity surrounding the renal medulla. The medulla, which had the shape of a broad pyramid with a long apex (papilla), could be divided into outer and inner zones, based on the tubular system of the kidney. Depending on the level of the dorsal section, recesses of the pelvis cavity could be seen towards the poles of the kidney. Thus, the internal part of the renal pelvis wall was adjacent to the medulla zones, principally to the papilla, whereas the external part of pelvis wall was adjacent to the cortex, vessels and connective tissue. The rodent male urethra is divided in two parts: pelvic and penile urethra. The pelvic urethra arises ventrally at the end of urinary bladder neck and passes dorsally to the point where the ampullary duct and other accessory gland ducts enter the urinary tract (Fig. 3a). It has a thin wall compared with the lumen, but a thick layer of urethral striated muscle. At the ischiadic arch, the pelvic urethra enters the urethral diverticle and continues into the corpus spongiosum as the penile urethra, opening up in the distal extremity of the penis. The female urethra is a short tube extending from the bladder neck to the external ostium of urethra (Fig. 3b). thelium prevailed over the pseudostratified epithelium. The anti-UP labelling always had a recognizable pattern: cells in contact with the basal lamina never showed any UPstaining, whereas cells with no contacts were UP-positive (Fig. 2f–i). When the outer medulla was covered with the two-layered urothelium, the upper cells had no fusiform vesicles and the anti-UP labelling was weaker than that in the umbrella cells (compare Figs. 1h, 2i). In this two-layered epithelium, the superficial cells were CK20-positive and CK20 was seen as distinct dots in the cytoplasm (Fig. 4b). Processes of the outer medulla, generally known as secondary pyramids, usually marked the point at which the three-layered urothelium started to appear instead of twolayered; the urothelium overlaying the renal cortex was always three layers thick (Fig. 2e) The apical surface of the superficial cells had the scalloped appearance of AUM, as seen in Fig. 5b, and fusiform vesicles were present in their cytoplasm. The antibodies against CK20 reacted with the majority of superficial cells in the outer band of the outer medulla. Here, CK20 was organized into a continuous network (Fig. 4c). Immunolabelling of the superficial cells with the scalloped surface and fusiform vesicles revealed strong anti-UPIa, anti-UPIb, anti-UPII and anti-UPIII reaction on the apical plasma membranes and on the membranes of the fusiform vesicles. Urothelium Simple epithelium covering the inner zone of renal pelvis medulla (papilla) One layer of cells with microvilli on the apical surface formed the epithelium that covered the papilla (Fig. 2b–e). Cells were more columnar towards the tip of the papilla and more flattened towards the base. The epithelium covering the papilla was continuous with the lining of the larger collecting tubules. All cells were UP-negative and CK20negative in this region (Figs. 2f, 4a). Transition from simple epithelium to urothelium The transition between a simple epithelium and an epithelium with urothelial differentiation was observed in the inner band of the outer medulla (Fig. 2b). These regions gradually showed more UP-positive cells on paraffin sections (Fig. 2c–e). At the base of the pyramid (inner band of the outer medulla), the simple epithelium was first replaced by pseudostratified epithelium. The cells forming the upper layer had narrow stalks that attached these cells to the basal lamina. In these areas, the cells still had microvilli, but no fusiform vesicles in their cytoplasm and no AUM on their apical plasma membrane (Figs. 2g, 5a). This epithelium also showed no UP-staining and no CK20staining. Closer to the outer band of outer medulla, some superficial cells appeared that had no contact with the basal lamina. In their cytoplasm, vesicles of a discoidal shape were noted that were weakly labelled with all antibodies against UPs (Fig. 2g, h). Gradually, the two-layered epi- The urinary tract was covered with a urothelium that had umbrella cells in the superficial layer from the outer band of the outer medulla to the urinary bladder neck, including the epithelium adjacent to the cortex and connective tissue, the ureters and the urinary bladder (Figs. 1, 5b). Most of the cells in the intermediate layer were also anti-UPs positive Fig. 2 Transition from simple epithelium to urothelium in the " mouse kidney. a The inner medulla with papilla (IM), outer medulla (OM) and cortex (C) are delineated in a dorsal section of the kidney. The renal pelvis forms a funnel-like cavity surrounding the papilla. b–e Anti-UPIa labelling of renal pelvis epithelia. b The renal pelvis epithelium covering the inner medulla is negative (thin arrows), whereas the intensity of labelling gradually increases from the inner band (iOM) to the outer band (oOM) of the epithelium covering the outer medulla (thick arrows). The urothelium adjacent to the cortex (C) and adipose tissue (AT) is strongly labelled (arrowheads). c Higher magnification showing the thin epithelium covering the inner band of the outer medulla in which individual cells are UP-positive (arrows). d In the two-layered urothelium that covers the outer band of the outer medulla, the superficial cells are UP-positive. e The urothelium becomes three-layered between the region covering the outer medulla (left) towards the region that covers the cortex (right). All umbrella cells are labelled with anti-UPIa antibody and intermediate cells are also UP-positive in the three-layered urothelium. f–i Immunolabelling of UPIII at the ultrastructural level. f In the epithelium covering the inner medulla, cells with microvilli (arrows) are UP-negative. g, h In the epithelium covering inner band of outer medulla, cells that are in contact (arrow in g) with the basal lamina (BL) are UP-negative, whereas cells without such contact are UP-positive. i In the twolayered urothelium covering the outer band of the outer medulla, superficial cells are UPIII-positive. Note the weaker labelling of discoidal vesicles (arrows) and the apical plasma membrane compared with the strong labelling of the fusiform vesicles and plasma membrane of the umbrella cells in Fig. 1 263 264 265 3 Fig. 3 Transition from urothelium to stratified epithelium at the border between the bladder neck (BN) and proximal urethra (PU). a–d Anti-UPII labelling in male (a, c) and in female mice (b, d). The transition from UP-positive urothelium to the UP-negative epithelium is sharp. Note the male sex gland ducts (D) in a and the vaginal (V) epithelium in b. e At the ultrastructural level, fusiform vesicles and the apical plasma membrane of umbrella cells are labelled with anti-UPIb antibody in the bladder neck (BN), whereas neighbouring superficial cells of stratified epithelium in proximal urethra (PU) are UP-negative In female urethra, the urothelium also ended abruptly at the end of bladder neck and was replaced by the stratified epithelium (Fig. 3b, d). After the transition from the urothelium to the stratified urothelium, no urothelium-specific differentiation markers were detected in the urethra of males or females. Discussion In the kidney, urine flows into the renal pelvis from openings at the tip of the renal papilla. It is then propelled by the peristaltic action of the ureter into the urinary bladder, where it is stored until it is excreted through the urethra during micturition. The epithelium that covers the urinary tract is therefore adapted to the constant flushing of urine, which contains waste products of metabolism. The urinary Transition from urothelium to stratified epithelium bladder, in which urine is stored for long periods, is proIn males, the urothelium ended in the neck of the urinary tected by unique urothelial cell differentiation, which estabbladder, just at the initial part of pelvic urethra, but ventral lishes an effective blood-urine permeability barrier (Ketterer to the point at which the ampullary ductus and some sex et al. 1973; Chang et al. 1994; Negrete et al. 1996). The accessory gland ducts enter urethra; these structures were primary sites of the barrier in the bladder are terminally difsurrounded by urethral striated muscle (Fig. 3a). The tran- ferentiated superficial cells, which are marked by the forsition into a stratified columnar epithelium was sharp, with mation of AUM plaques containing UPs in the apical surface superficial umbrella cells that were UP-positive and CK20- and fusiform vesicles and by the expression of CK20 (Chang positive adjoining superficial columnar cells that were UP- et al. 1994; Negrete et al. 1996; Min et al. 2003). Here, we have studied cells of the epithelia from the kidnegative and CK20-negative (Fig. 3c, e). At the point before the change into columnar epithelium, the urothelium gen- ney, ureter, bladder and urethra for their expression, localerally consisted of more than four cell layers. Urinary tract ization and organization of specific differentiation markers. epithelia that were distal to the point of transition did not Urinary tract epithelia have been classified into four regions: express any urothelial marker. Immediately after transition, (1) the inner medulla, (2) the inner and outer bands of the the apical surface of cells showed some microvilli (Fig. 5c). outer medulla, (3) the region extending from the renal cortex but the labelled vesicles were discoid in shape and smaller than the fusiform vesicles of superficial cells. Superficial cells were CK20-positive and the CK20 was organized in an ordered network, as seen in Fig. 4c. Fig. 4 Immunofluorescence of CK20 (green) in the mouse urinary tract (L lumen, white line position of basal lamina. Nuclei are stained blue with the DAPI stain. a Epithelial cells are CK20-negative on the inner medulla. b Some superficial cells are CK20-positive on the outer medulla but the label is seen as discrete dots within their cytoplasm. c The network organization of CK20 is seen in umbrella cells in the epithelium related to the cortex Fig. 5 Apical surface of the urinary tract epithelia. a Cells with microvilli (asterisk) and cells with microridges (mr) are seen on the epithelium covering the outer medulla. b The apical surface of umbrella cells from urinary bladder has a typical rigid-looking ap- pearance, which is formed by the AUM plaques (one plaque surrounded by a ridge is encircled). c Microvilli are seen on the apical surface of the epithelium of the urethra 266 to the neck of urinary bladder and (4) the urethra. The distribution of markers in the epithelia along the urinary tract is shown in Table 1. The inner medulla (renal papilla) is covered with a simple epithelium that is mainly cuboidal except for the papilla tip, at which it is columnar and the cells have microvilli on the apical surface (Silverblatt 1974; Lacy and Schmidt-Nielsen 1979; Verani and Bulger 1982). This epithelium has been proposed to contribute to final urine formation and, thus, the AUM with its barrier function is not formed here (Lacy and Schmidt-Nielsen 1979). At the base of papilla, Verani and Bulger (1982) have detected occasional plaques on cuboidal cells of the one-layered epithelium. In our study, no urothelial markers have been detected in the simple epithelium of the pyramid immersed in the renal pelvis. Some differences have been reported regarding the epithelium covering the inner band of the outer medulla. Lacy and Schmidt-Nielsen (1979) describe this epithelium as simple squamous to low cuboidal, whereas Khorshid and Moffat (1974) claim that the epithelium of the outer medulla is composed of only one, or at most, two cell layers. Silverblatt (1974) has observed that the fornix and secondary pyramid is covered with so-called intermediate cells that have features of both cell types, those with a membrane of 125-Å thickness and those with a membrane of 75-Å thickness. In this region, the majority of cells have microvilli, some of them have AUM, and cells with both AUM and microvilli have been found, but no fusiform vesicles are seen in the cytoplasm (Verani and Bulger 1982). We have noted that a pseudostratified epithelium appears at the border between the inner medulla and the outer medulla. It occurs as a two-layered epithelium, except that the upper cells are attached to the basal lamina with narrow stalks. Nearby, the two-layered epithelium also covers the inner band of the outer medulla. Our results confirm the transition between the simple epithelium and the urothelium in the outer medulla and this is supported by the detection of urothelial cell differentiation markers. We consider that the UP immunolabelling, especially at the ultrastructural level, has a great advantage over structural observations in distinguishing between the pseudostratified and two-layered epithelium. Whereas the upper cells of the pseudostratified epithelium are UP-negative, those in the two-layered epithelium are UP-positive. This indicates that the urothelium is two or more layered and is not singlelayered or pseudostratified. The superficial cells of the twolayered urothelium express all four major UPs but these cells never reach an advanced stage of differentiation characterized by the formation of umbrella cells. Such “partial” differentiation is also found in intermediate cells of normal urinary bladder, in cultured urothelial cells and in the superficial layer during early regeneration after the removal of umbrella cells (Surya et al. 1990; Romih et al. 1998, 2001). Partially differentiated urothelial cells synthesize small amounts of UPs but fail to form AUM plaques in fusiform vesicles and apical surface. Factors other than UP synthesis thus seem to be required for the advanced stage of urothelial cell differentiation. At least three factors have been proposed to contribute to the maturation of umbrella cells: mechanical forces during the contraction-expansion cycles of the bladder, the network organization of the intermediate filaments and the differentiation-dependent expression of Rab27b protein (Southgate et al. 1999; Veranič et al. 2004; Chen et al. 2003). The outer band of the outer medulla usually begins in the fornices or recesses, as observed in dorsal sections. The epithelium is mostly two-layered and its superficial cells express all the UPs tested here. Nevertheless, the UP expression in these cells is weak and the cytoplasmic vesicles are discoidal and less numerous than the fusiform vesicles in umbrella cells. UP label is also found on the apical plas- Table 1 Presence of the differentiation markers in the superficial cells in the epithelia of the urinary tract (dashed squares mixed population of some positive and some negative cells, black squares presence of a marker in all superficial cells, pseudostrat. pseudostratified) 267 ma membrane, although some microvilli on the cell surfaces lack UP label. This is in agreement with previous morphological observations that this two-layered epithelium is thinner than normal urothelium. Some superficial cells in this region are covered with microvilli, some with AUM, and some with a combination of both (Lacy and Schmidt-Nielsen 1979; Verani and Bulger 1982). More towards the cortex, the epithelium becomes three-layered and some umbrella cells appear in the superficial region. In these cells, CK20-containing keratins are organized as a continuous network. Therefore, the umbrella cells are formed here but do not constitute the entire superficial layer as yet. However, some superficial cells are found in which CK20 is not organized into a network. Some authors have described the epithelium adjacent to cortex as being two-layered to three-layered, with fewer fusiform vesicles and some microvilli (Verani and Bulger 1982). We have shown here that this epithelium is the fully developed urothelium. The umbrella cells are mature, in agreement with observations of other investigators (Silverblatt 1974; Lacy and Schmidt-Nielsen 1979). This fully mature urothelium with umbrella cells extends throughout the ureters and urinary bladder. We have shown that the epithelium in the renal pelvis gradually changes from a simple epithelium to the urothelium. The degree of cell differentiation is closely related to the osmolarity of the underlying renal tissues. The osmolarity of the interstitial fluid in the medulla decreases from the renal papilla (approximately 1,200 mOsmol/l), which is similar to urine osmolarity, towards the cortex (approximately 300 mOsmol/l), which is the same as that in the interstitial fluid of most parts of the body (Gayton and Hall 2000). We suggest that the presence of a simple epithelium without a blood-urine barrier at the inner medulla is attibutable to the absence of osmotic pressure between the papilla and surrounding urine. In the outer medulla, the osmolarity gradually decreases in relation to urine and thus it is necessary for the epithelium to form a barrier. In the renal pelvis adjacent to the cortex or connective tissue, an effective bloodurine barrier is also necessary and this demand continues in the ureter and urinary bladder. Therefore, the major part of urinary tract is covered with fully differentiated urothelium. At the distal end, the sharp transition from urothelium to stratified epithelium is located at the end of the bladder neck in males and females. This region has a strong muscular layer that blocks uncontrolled flow of urine into the urethra; thus, the distal urethra from this point to the exit from the body is flushed with urine only for short periods during micturition. Additionally, the pelvic urethra is filled with products of sex accessory glands, making the formation of a complete barrier unnecessary. We have shown here, by studying the expression, distribution and organization of urothelial cell differentiation markers, that, in the kidneys, the change from a simple epithelium to the urothelium is gradual. In the junctional zone between these two epithelia, which is positioned in the pelvic surface of the outer medulla, the expression of urothelium-specific markers is correlated with the formation of a two-layered epithelium. In cells that are in contact with basal lamina, no urothelial markers are detected. In the junctional zone, however, cells synthesize differentiation markers (UPs, CK20) but fail to organize them in the manner observed in umbrella cells. Therefore, in the urothelium of the outer medulla, differentiation does not culminate in the formation of terminally differentiated umbrella cells. On the other hand, the transition from the urothelium to a stratified epithelium in the urinary bladder neck is sharp. These studies also show that areas of epithelial transitions represent an important model for studies of cell differentiation and possibly for the role of the substratum (underlying tissues) on the differentiation pathways of epithelial cells. 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