The Urothelium: Life in a Liquid Environment.

The urothelium, which lines the renal pelvis, ureters, urinary bladder, and proximal urethra, forms a high-resistance but adaptable barrier that surveils its mechanochemical environment and communicates changes to underlying tissues including afferent nerve fibers and the smooth muscle. The goal of this review is to summarize new insights into urothelial biology and function that have occurred in the past decade. After familiarizing the reader with key aspects of urothelial histology, we describe new insights into urothelial development and regeneration. This is followed by an extended discussion of urothelial barrier function, including information about the roles of the glycocalyx, ion and water transport, tight junctions, and the cellular and tissue shape changes and other adaptations that accompany expansion and contraction of the lower urinary tract. We also explore evidence that the urothelium can alter the water and solute composition of urine during normal physiology and in response to overdistension. We complete the review by providing an overview of our current knowledge about the urothelial environment, discussing the sensor and transducer functions of the urothelium, exploring the role of circadian rhythms in urothelial gene expression, and describing novel research tools that are likely to further advance our understanding of urothelial biology.

Krt5+ urothelial cells are developmental and tissue repair progenitors in the kidney.

Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5+) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk)+ RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk+ RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk+ RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk+ RUCs derive from embryonic and neonatal Krt5+ RUCs, whereas Krt5+ RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14. However, in response to UTO, postnatal day 14-labeled adult Krt5+ RUCs break their lineage restriction and robustly differentiate into Upk+ RUCs. Thus, Krt5+ RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk+ RUCs in a temporally restricted manner.

Mode of Surgical Injury Influences the Source of Urothelial Progenitors during Bladder Defect Repair.

The bladder urothelium functions as a urine-blood barrier and consists of basal, intermediate, and superficial cell populations. Reconstructive procedures such as augmentation cystoplasty and focal mucosal resection involve localized surgical damage to the bladder wall whereby focal segments of the urothelium and underlying submucosa are respectively removed or replaced and regeneration ensues. We demonstrate using lineage-tracing systems that urothelial regeneration following augmentation cystoplasty with acellular grafts exclusively depends on host keratin 5-expressing basal cells to repopulate all lineages of the de novo urothelium at implant sites. Conversely, repair of focal mucosal defects not only employs this mechanism, but in parallel host intermediate cell daughters expressing uroplakin 2 give rise to themselves and are also contributors to superficial cells in neotissues. These results highlight the diversity of urothelial regenerative responses to surgical injury and may lead to advancements in bladder tissue engineering approaches.

A Preclinical Study of Cell-seeded Tubularized Scaffolds Specially Secreting LL37 for Reconstruction of Long Urethral Defects.

AIM: We constructed a new artificial, long tubular acellular matrix, seeded with autologous progenitor cells transfected with the sequence to produce the antibiotic peptide LL37 and another two common seeding cells, which might be adopted for patients requiring repair of long segment of the urethra. MATERIALS AND METHODS: Autologous endothelial progenitor cells transfected by lentiviral vectors expressing antibiotic peptide LL37, as well as urothelial and smooth muscle cells from New Zealand white male rabbits, were cultured and seeded onto preconfigured acellular collagen-based tubular matrices (3 cm in length). Artificial conduits were created again in New Zealand white male rabbits and, then, evaluated by immunohistochemistry after 8 weeks. RESULTS: Cell-seeded tubularized collagen scaffolds were found to be effective in repairing long urethral defects, whereas scaffolds without cells led to poor tissue development and structures. CONCLUSION: The artificial tissue engineered tubularized scaffolds combined with genetic methods resulted in vascularized autologous grafts, which may potentially be used for urethroplasty in patients requiring repair of a long segment of the urethra.

Stage- and subunit-specific functions of polycomb repressive complex 2 in bladder urothelial formation and regeneration.

Urothelium is the protective lining of the urinary tract. The mechanisms underlying urothelial formation and maintenance are largely unknown. Here, we report the stage-specific roles of PRC2 epigenetic regulators in embryonic and adult urothelial progenitors. Without Eed, the obligatory subunit of PRC2, embryonic urothelial progenitors demonstrate reduced proliferation with concomitant dysregulation of genes including Cdkn2a (p16), Cdkn2b (p15) and Shh. These mutants display premature differentiation of keratin 5-positive (Krt5+) basal cells and ectopic expression of squamous-like differentiation markers. Deletion of Ezh2, the major enzymatic component of PRC2, causes upregulation of Upk3a+ superficial cells. Unexpectedly, Eed and Eed/Ezh2 double mutants exhibit delayed superficial cell differentiation. Furthermore, Eed regulates the proliferative and regenerative capacity of adult urothelial progenitors and prevents precocious differentiation. Collectively, these findings uncover the epigenetic mechanism by which PRC2 controls urothelial progenitor cell fate and the timing of differentiation, and further suggest an epigenetic basis of urothelial maintenance and regeneration.

Urothelial generation and regeneration in development, injury, and cancer.

Homeostatic maintenance and repair of the urothelium upon injury are required for a functional bladder in both healthy and disease conditions. Understanding the cellular and molecular mechanisms underlying the urothelial regenerative response is key to designing strategies for tissue repair and ultimately treatments for urologic diseases including urinary tract infections, voiding dysfunction, painful bladder syndrome, and bladder cancer. In this article, we review studies on urothelial ontogeny during development and regeneration following various injury modalities. Signaling pathways involved in urothelial regeneration and in urothelial carcinogenesis are also discussed. Developmental Dynamics 246:336-343, 2017. © 2016 Wiley Periodicals, Inc.

Organ-specific matrix self-assembled by mesenchymal cells improves the normal urothelial differentiation in vitro.

PURPOSE: Enterocystoplasty is the gold standard to perform bladder reconstruction. Since this technique has a high morbidity rate, several matrix scaffolds have been proposed to support the urothelial maturation. Unfortunately, epithelial cells failed to fully integrate the cell-matrix interactions and therefore appropriate signalling pathways of normal differentiation. Based on these observations, we proposed to culture bladder urothelial cells (BUC) onto a matrix self-assembled by bladder mesenchymal cells (BMC), to form a vesical model (VM). METHODS: Different serum proportions were assessed to obtain a manipulable matrix deposited by BMC. The BUC were then seeded onto the BMC's matrix to evolve in a three-dimensional culture. Haematoxylin-eosin staining, immunolabeling, scanning electron microscopy, western blot and matrix metalloproteinases analysis were performed for the VM characterization. RESULTS: We were able to obtain an original matrix made of collagen-I and presenting specific organization. Matrix remodelling was observed and led to a cellular compartmentalization. The reconstructed urothelium developed in a pseudostratified arrangement, displaying an adequate cellular polarity and apical membrane remodelling of superficial cells. Like native bladder, cytokeratin 14 immunolabeling was not observed in our VM, which indicate the conformity of the development sequence taken by BUC under the influence of the BMC's matrix. CONCLUSION: Thus, it was possible to elaborate a VM without the use of exogenous matrices. The particular characteristics of the BMC's matrix permitted the development of an urothelium that shared the phenotype of native tissue. The autologous character of our VM, and its appropriate urothelial maturation, could potentially promote a better integration after grafting.

Ureter growth and differentiation.

The mammalian ureter is a slender tube that connects the renal pelvis with the bladder. It allows the unidirectional movement of urine by means of a peristaltically active smooth muscle layer that together with fibroelastic material ensheathes a water-impermeable multilayered urothelium. The ureteric urothelium as well as the outer mesenchymal coat arise from undifferentiated precursor tissues, the distal ureteric bud and its surrounding mesenchyme, respectively. Specification, growth and differentiation of these ureteric precursor tissues are tightly linked to each other, and are highly integrated with those of the adjacent rudiments of kidney and bladder. Here, we review the current knowledge on the cellular mechanisms as well as the molecular players that guide development of the tissue architecture of the ureter and its peristalsis.

Age-related changes in afferent pathways and urothelial function in the male mouse bladder.

The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea.

Preparation and characterization of gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds.

This study aimed to develop optimal gelatin-based mucoadhesive nanocomposites as scaffolds for intravesical gene delivery to the urothelium. Hydrogels were prepared by chemically crosslinking gelatin A or B with glutaraldehyde. Physicochemical and delivery properties including hydration ratio, viscosity, size, yield, thermosensitivity, and enzymatic degradation were studied, and scanning electron microscopy (SEM) was carried out. The optimal hydrogels (H), composed of 15% gelatin A175, displayed an 81.5% yield rate, 87.1% hydration ratio, 42.9 Pa·s viscosity, and 125.8 nm particle size. The crosslinking density of the hydrogels was determined by performing pronase degradation and ninhydrin assays. In vitro lentivirus (LV) release studies involving p24 capsid protein analysis in 293T cells revealed that hydrogels containing lentivirus (H-LV) had a higher cumulative release than that observed for LV alone (3.7-, 2.3-, and 2.3-fold at days 1, 3, and 5, resp.). Lentivirus from lentivector constructed green fluorescent protein (GFP) was then entrapped in hydrogels (H-LV-GFP). H-LV-GFP showed enhanced gene delivery in AY-27 cells in vitro and to rat urothelium by intravesical instillation in vivo. Cystometrogram showed mucoadhesive H-LV reduced peak micturition and threshold pressure and increased bladder compliance. In this study, we successfully developed first optimal gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds.

Retinoid signaling in progenitors controls specification and regeneration of the urothelium.

The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.

Urothelial cell culture: stratified urothelial sheet and three-dimensional growth of urothelial structure.

Urothelial cells line the urinary tract, including the renal pelvis, ureters, bladder, superior urethra, and the central ducts of the prostate. They are highly specialized epithelial cell types possessing unique features, imparting important functional roles in the urinary system. They act as a permeability barrier and protect underlying muscle tissues from the caustic effects of urine while also expanding with bladder filling to adjust urine pressures. The multilayered urothelium is typically structured with differentiated, mature surface cells and less mature basal cells. The basal cell layer contains tissue-specific stem cells able to self-renew for the lifetime of the mammal and also produces a pool of maturing cells for tissue homeostasis. Maintaining regenerative basal cells in a culture facilitates urothelial cell growth in vitro. Additionally, epithelial-mesenchymal communication, epithelial-matrix interactions, and cytokines/growth factors are required to maintain the normal structure and function of mature urothelial cells in vitro and to induce stem cell differentiation into urothelial cells. These cultures are useful to study the biology and physiology of the urinary tract, particularly for the development of cell-based tissue engineering strategies in urology. This chapter describes methods for the isolation of urothelial cells and their maintenance in monolayer culture, and methods for the production of multilayer urothelial cell sheets and three-dimensional cocultures of urothelial and mesenchymal cells.

Analysis of 3D branching pattern: hematoxylin and eosin method.

Accurate analysis of the three-dimensional (3D) architecture of developing organs is critical to understanding how developmental defects can be linked with structural abnormalities. Here, we describe a 3D reconstruction technique of the developing kidney including the outer kidney capsule, ureteric epithelium, and developing nephrons. This 3D reconstructive process involves generating serial sections of the developing kidney, followed by histological staining. Each serial image is projected on the monitor and each tissue lineage or structure is traced. The kidney tracings are aligned and a 3D image is rendered. Each reconstructed tissue/lineage can then be subjected to quantitative analysis (e.g., surface area or volume). The reconstructed ureteric epithelium can be skeletonized to determine the branching architecture.

An EGFR-ERK-SOX9 signaling cascade links urothelial development and regeneration to cancer.

Like many carcinomas, urothelial carcinoma (UroCa) is associated with chronic injury. A better understanding of this association could inform improved strategies for preventing and treating this disease. We investigated the expression, regulation, and function of the transcriptional regulator SRY-related high-mobility group box 9 (Sox9) in urothelial development, injury repair, and cancer. In mouse bladders, Sox9 levels were high during periods of prenatal urothelial development and diminished with maturation after birth. In adult urothelial cells, Sox9 was quiescent but was rapidly induced by a variety of injuries, including exposure to the carcinogen cyclophosphamide, culture with hydrogen peroxide, and osmotic stress. Activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was required for Sox9 induction in urothelial injury and resulted from activation of the epidermal growth factor receptor (Egfr) by several Egfr ligands that were dramatically induced by injury. In UroCa cell lines, SOX9 expression was constitutively upregulated and could be suppressed by EGFR or ERK1/2 blockade. Gene knockdown showed a role for SOX9 in cell migration and invasion. Accordingly, SOX9 protein levels were preferentially induced in invasive human UroCa tissue samples (n = 84) compared with noninvasive cancers (n = 56) or benign adjacent urothelium (n = 49). These results identify a novel, potentially oncogenic signaling axis linking urothelial injury to UroCa. Inhibiting this axis is feasible through a variety of pharmacologic approaches and may have clinical utility.

Prominin-2 is a novel marker of distal tubules and collecting ducts of the human and murine kidney.

Prominin-1 (CD133) and its paralogue, prominin-2, are pentaspan membrane glycoproteins that are strongly expressed in the kidney where they have been originally cloned from. Previously, we have described the localization of prominin-1 in proximal tubules of the nephron. The spatial distribution of prominin-2, however, has not yet been documented in the kidney. We therefore examined the expression of this molecule along distinct tubular segments of the human and murine nephron using in situ hybridization and immunohistochemistry. Our findings indicated that human prominin-2 transcripts and protein were confined to distal tubules of the nephron including the thick ascending limb of Henle's loop and the distal convoluted tubule, the connecting duct and to the collecting duct system. Therein, this glycoprotein was enriched at the basolateral plasma membrane of the tubular epithelial cells with exception of the thick ascending limb where it was also found in the apical domain. This is in contrast with the exclusive apical localization of prominin-1 in epithelial cells of proximal nephron tubules. The distribution of murine prominin-2 transcripts was reminiscent of its human orthologue. In addition, a marked enrichment in the epithelium covering the papilla and in the urothelium of the renal pelvis was noted in mice. Finally, our biochemical analysis revealed that prominin-2 was released into the clinically healthy human urine as a constituent of small membrane vesicles. Collectively our data show the distribution and subcellular localization of prominin-2 within the kidney in situ and its release into the urine. Urinary detection of this protein might offer novel diagnostic approaches for studying renal diseases affecting distal segments of the nephron.

Staining patterns of keratins in the human urinary tract.

The keratins, members of the intermediate filament family, are characteristically expressed in epithelial cells. In the various types of epithelia, the keratin expression pattern is characterized by cell-type specific combinations of the keratin isotypes with a plain pattern in monolayered (simple) epithelia and more complex patterns in stratified and pseudostratified epithelia. Here we demonstrate that the transitional epithelium of the human urinary tract holds an exceptional position between the pseudostratified and stratified epithelia. We show that the simple epithelia keratins 7, 8, 18 and 19 are expressed throughout the whole epithelium as known from pseudostratified epithelia. In addition, we demonstrate expression of keratins 5, 14 and 17, otherwise present in basal cells of multilayered epithelia, and keratins 4 and 13, present in suprabasal areas of non cornified multilayered epithelia. Moreover, we report differences in expression in the various morphological parts of the urinary tract which might be related to their specific functions. Keratin 20, a typical component of the simple epithelia of the digestive tract, is present in bladder and ureter but not in the renal pelvis. Keratin 6, typical for stratified epithelia, is found only in parts of the renal pelvis. We further show that changes in keratin pattern occur during the development from embryonic to adult bladder urothelium. In contrast to adult tissue, the simple type keratins 7, 8 and 18 are not synthesized in basal embryonic cells. Further, keratin 20, present in cells facing the bladder lumen in adult urothelium, is expressed in all but the basal cells in embryonic bladder.

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.

Postnatal expression of corticotropin releasing factor (CRF) in rat urinary bladder.

Corticotropin releasing factor (CRF) is a neuropeptide expressed in micturition reflex circuitry and different roles in these reflexes have been suggested. These studies examined the expression of CRF/CRF receptors in the urinary bladder during postnatal development in the rat. Urinary bladder was harvested from rats (postnatal (P) day 0-adult) euthanized by isoflurane (4%) and thoracotomy. CRF protein expression significantly (p

Tissue engineering of urethra using human vascular endothelial growth factor gene-modified bladder urothelial cells.

Acquired or congenital abnormalities may lead to urethral damage or loss, often requiring surgical reconstruction. Urethrocutaneous fistula and strictures are common complications, due to inadequate blood supply. Thus, adequate blood supply is a key factor for successful urethral tissue reconstruction. In this study, urethral grafts were prepared by seeding rabbit bladder urothelial cells (UCs) modified with human vascular endothelial growth factor (VEGF(165)) gene in the decellularized artery matrix. A retroviral pMSCV-VEGF(165)-GFP vector was cloned by insertion of VEGF open reading frame into the vector pMSCV-GFP (murine stem cell virus [MSCV]; green fluorescent protein [GFP]). Retrovirus was generated using package cell line 293T. Rabbit UCs were expanded ex vivo and modified with either MSCV-VEGF(165)-GFP or control MSCV-GFP retrovirus. Transduction efficiency was analyzed by fluorescence-activated cell sorting. The expression of VEGF(165) was examined by immunofluorescence, reverse transcript-polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA). Decellularized rabbit artery matrix was seeded with genetically modified UCs and was subsequently cultured for 1 week prior to subcutaneous implantation into nude mice. Four weeks after implantation, the implants were harvested and analyzed by fluorescence microscopy, and by histologic and immunohistochemical staining. Ex vivo transduction efficiency of UCs was greater than 50% when concentrated retrovirus was used. The modified cells expressed both VEGF and GFP protein. Furthermore, the VEGF-modified UCs secreted VEGF in a time-dependent manner. Scanning electron microscopy and histochemical analysis of cross sections of the cultured urethral grafts showed that the seeded cells were attached and proliferated on the luminal surface of the decellularized artery matrix. In the subcutaneously implanted vessels, VEGF-modified cells significantly enhanced neovascularization and the formation of a urethral layer compared to GFP-modified cells. These results indicate that VEGF gene therapy may be a suitable approach to increase the blood supply in tissue engineering for treatment of urethral damage or loss.

Replacement of intestinal mucosa with urothelium in rat augmented bladders using intravesical photodynamic therapy with 5-aminolaevulinic acid.

PURPOSE: We evaluated the efficacy of intravesical aminolevulinic acid (delta-aminolevulinic acid hydrochloride) (Frontier Scientific, Logan, Utah) and photodynamic therapy for the removal of small intestinal mucosa in augmented bladders in a rat model. MATERIALS AND METHODS: Enterocystoplasty was performed in 70 female rats using a patch of terminal ileum. A total of 28 were used to determine the pharmacokinetics (0.3, 0.6 and 0.9 M) and dwell time (30, 60 and 90 minutes) of intravesically administered aminolevulinic acid to optimize intestinal mucosal absorption and minimize bladder mucosal absorption. The remaining augmented rats were treated with intravesical photodynamic therapy at light doses of 75, 100 and 125 J. Ileal and bladder tissues were evaluated by light microscopy. Cystometric studies to evaluate bladder volume were measured before and after photodynamic therapy. RESULTS: The concentration of 0.3 M aminolevulinic acid with a dwell time of 30 minutes resulted in an average +/- SE bowel-to-bladder concentration of 2,156 +/- 269/749 +/- 62 ng/gm (ratio 2.9:1). After photodynamic therapy histology revealed uniform ablation and replacement of the intestinal mucosa with urothelium and minimal damage to the bladder wall at all light doses. Bladder cystometry revealed no significant change in bladder capacity after photodynamic therapy. CONCLUSIONS: In the rat model intravesical aminolevulinic acid and photodynamic therapy resulted in the replacement of intestinal mucosa with urothelium, leaving the underlying muscular layer intact. This could potentially be a viable option for patients with a preexisting bladder augment.