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.     

Cellular basis of urothelial squamous metaplasia: roles of lineage heterogeneity and cell replacement

Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to the urothelium covering the rest of the bladder, and that lineage heterogeneity coupled with cell migration/replacement form the cellular basis for urothelial squamous metaplasia.     

Epithelial-mesenchymal interactions in prostatic development. I. morphological observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder

Tissue recombinants of embryonic urogenital sinus mesenchyme (UGM) and epithelium of the urinary bladder (urothelium, BLE) of adult rats and mice were grown for 3-30 d in male syngeneic hosts. Short-term in vivo growth indicated that prostatic morphogenesis is initiated as focal outgrowths from the basal aspect of the adult urothelium. The solid epithelial buds elongate, branch, and subsequently canalize, forming prostatic acini. After 30 d of growth in the male hosts, prostatic acini exhibit secretory activity. The marked changes in urothelial morphology induced by the UGM are accompanied by the expression of fine- structural features indicative of secretory function (rough endoplasmic reticulum, Golgi apparatus, and secretory granules). During this process, urothelial cells express prostatic histochemical markers (alkaline phosphatase, nonspecific esterase, glycosaminoglycans) and prostate-specific antigens. The expression within BLE of prostatic characteristics is associated with the loss of urothelial characteristics. These data indicate that adult urothelial cells retain a responsiveness to embryonic mesenchymal inductors. Furthermore, mesenchyme-induced changes in urothelial cytodifferentiation appear to be coupled to changes in functional activity.     

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。     

Fibroblast growth factor-10 is a mitogen for urothelial cells

Fibroblast growth factor (FGF)-10 plays an important role in regulating growth, differentiation, and repair of the urothelium. This process occurs through a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the epithelium (urothelium). In situ hybridization analysis demonstrated that (i) fibroblasts of the human lamina propria were the cell type that synthesized FGF-10 RNA and (ii) the FGF-10 gene is located at the 5p12-p13 locus of chromosome 5. Recombinant (r) preparations of human FGF-10 were found to induce proliferation of human urothelial cells in vitro and of transitional epithelium of wild-type and FGF7-null mice in vivo. Mechanistic studies with human cells indicated two modes of FGF-10 action: (i) translocation of rFGF-10 into urothelial cell nuclei and (ii) a signaling cascade that begins with the heparin-dependent phosphorylation of tyrosine residues of surface transmembrane receptors. The normal urothelial phenotype, that of quiescence, is proposed to be typified by negligible levels of FGF-10. During proliferative phases, levels of FGF-10 rise at the urothelial cell surface and/or within urothelial cell nuclei. An understanding of how FGF-10 works in conjunction with these other processes will lead to better management of many diseases of the bladder and urinary tract.     

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.     

Diffusable growth factors induce bladder smooth muscle differentiation

Bladder smooth muscle differentiation is dependent on the presence of bladder epithelium. Previously, we have shown that direct contact between the epithelium and bladder mesenchyme (BLM) is necessary for this interaction. In this study, we tested the hypothesis that bladder smooth muscle can be induced via diffusable growth factors. Fourteen-day embryonic rat bladders were separated into bladder mesenchyme (prior to smooth muscle differentiation) and epithelium by enzymatic digestion and microdissection. Six in vitro experiments were performed with either direct cellular contact or no contact (1) 14-d embryonic bladder mesenchyme (BLM) alone (control), (Contact) (2) 14-d embryonic bladders intact (control), (3) 14-d embryonic bladder mesenchyme combined with BPH-1 cells (an epithelial prostate cell line) in direct contact, (4) 14-d embryonic bladder mesenchyme with recombined bladder epithelium (BLE) in direct contact, (No Contact) (5) 14-d embryonic bladder mesenchyme with BPH-1 prostatic epithelial cells cocultured in type 1 collagen gel on the bottom of the well, and (6) 14-d embryonic bladder mesenchyme with BPH-1 epithelium cultured in a monolayer on a transwell filter. In each case the bladder tissue was cultured on Millicell-CM 0.4-microm membranes for 7 d in plastic wells using serum free medium. Growth was assessed by observing the size of the bladder organoids in histologic cross section as well as the vertical height obtained in vitro. Immunohistochemical analysis of the tissue explants was performed to assess cellular differentiation with markers for smooth muscle alpha-actin and pancytokeratin to detect epithelial cells. Control (1) bladder mesenchyme grown alone did not exhibit growth or smooth muscle and epithelial differentiation. Contact experiments (2) intact embryonic bladder, (3) embryonic bladder mesenchyme recombined with BPH-1 cells, and (4) embryonic bladder mesenchyme recombined with urothelium each exhibited excellent growth and bladder smooth muscle and epithelial differentiation. Both noncontact experiments (5) and (6) exhibited growth as well as bladder smooth muscle and epithelial differentiation but to a subjectively lesser degree than the contact experiments. Direct contact of the epithelium with bladder mesenchyme provides the optimal environment for growth and smooth muscle differentiation. Smooth muscle growth and differentiation can also occur without direct cell to cell contact and is not specific to urothelium. This data supports the hypothesis that epithelium produces diffusable growth factors that induce bladder smooth muscle.     

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.     

Mesenchymal reprogramming of adult human epithelial differentiation

The objective of this study was to determine whether neonatal rat seminal vesicle mesenchyme (rSVM) can reprogram epithelial differentiation in a fully differentiated adult human bladder epithelium. For this purpose neonatal rSVM was isolated from newborn (0-day) Sprague-Dawley rats, and normal adult human bladder epithelium (hBLE) was isolated from radical cystoprostatectomy specimens to prepare rSVM+hBLE tissue recombinants in vitro. After overnight culture the tissue recombinants were grafted beneath the renal capsule of male athymic rodent hosts and allowed to grow in vivo for 6 months. As controls, rSVM and hBLE were grafted separately and allowed to grow for the same period. Tissue recombinants and control tissue grafts were harvested, and secretions were collected for biochemical studies. Tissues were fixed both for histologic as well as immunohistochemical staining. Neonatal rSVM induced normal adult human bladder urothelium to form glandular structures resembling prostate. The induced prostatic acini were filled with secretions that expressed human prostate-specific secretory proteins. These findings demonstrate that adult human urothelial cells retain a responsiveness to neonatal prostatic mesenchymal inductors. Change in urothelial histodifferentiation was associated with change in functional activity. The ability of the neonatal rat mesenchymal tissues to induce morphologic as well as biochemical changes in normal adult human urothelium provides a basis for human tissue engineering and organ reconstruction.     

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     

Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder

Adult bladder epithelium (BLE) is induced to differentiate into glandular epithelium after association with urogenital sinus mesenchyme (UGM) and subsequent in vivo growth in syngeneic male hosts. Alteration of epithelial cytodifferentiation is associated with the expression of prostate-specific antigens, histochemical and steroid metabolic activities. These observations suggest that the inductive influence of the UGM has reprogrammed both the morphological and functional characteristics of the urothelium. In this report, differences regarding the mechanisms and effects of androgenic stimulation of prostate and bladder are exploited to determine the extent to which UGM plus BLE recombinants express a prostatelike, androgen-dependent phenotype. Results from cytosolic and autoradiographic binding studies suggest that androgen binding is induced in UGM plus BLE recombinants and that this activity is accounted for by the induced urothelial cells. In UGM plus BLE recombinants, androgen-induced [3H]thymidine or [35S]-methionine uptake analyzed by two-dimensional gel electrophoresis was qualitatively and quantitatively similar to that of prostate as opposed to bladder. These studies indicate that expression within BLE of prostatic phenotype is associated with a loss of urothelial characteristics and that androgen sensitivity is presumably a function of the inductive activities of the stroma.     

Heterospecific induction of prostatic development in tissue recombinants prepared with mouse, rat, rabbit and human tissues

Abstract. Epithelia of embryonic urogenital sinuses (UGE) or embryonic or adult urinary bladders (BLE) were associated heterospecifically with mesenchyme of the embryonic urogenital sinus (UGM). The resultant chimeric tissue recombinants prepared with mouse, rat, rabbit, and human tissues were grown for 2 to 14 weeks in male athymic nude mice. For almost all categories of permissive (UGM + UGE) or instructive (UGM + BLE) inductions, prostatic epithelial development occurred. In recombinations of mouse UGM and human fetal BLE, the bladder epithelium was induced to form glandular structures. The morphogenetic process observed was similar to that normally expressed during human prostatic development. We conclude that the mechanism of prostatic development is similar in these mammalian species.     

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.     

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.     

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.