Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Construction of a three-dimensional urothelium on-chip with barrier function based on urinary flow microenvironment.

The urothelium covers the inner surface of the urinary tract, forming a urinary tract barrier. Impairment of the integrity and dysfunction of the urinary tract barrier is associated with the occurrence and development of various diseases. The development of a three-dimensional model of the urothelium is critical for pathophysiological studies of this site, especially under physiological fluid shear stress stimulated by the urinary flow. In this study, a urothelium on-chip is fabricated with micromilling and replica molding techniques, which contains a microfluidic chip for cell culture and a pump-based fluid perfusion system. The mechanical properties of the human urinary tract are simulated by adjusting the concentration and degree of amino substitution of the gelatin methacrylate hydrogel. The matrix stiffness is similar to the natural urinary tract. Pulsatile flow and periodic flow are provided to simulate the fluid environment of the upper and lower urinary tracts, respectively. The results show that the physiological fluid shear stress could promote the differentiation and maturation of urothelial cells. The model could simulate the three-dimensional structure of urothelium and urinary flow microenvironment, showing morphological structure close to the natural urothelium, specific differentiation and maturation markers (uroplakin 2, cytokeratin 20), and urothelial barrier function.

Urothelium removal does not impact mucosal activity in response to muscarinic or adrenergic receptor stimulation.

The inner lining of the urinary bladder (urothelium and lamina propria, or bladder mucosa) has an important role as a tissue barrier between stored urine and the underlying smooth muscle, as well as in the modulation and regulation of bladder contractility. However, the individual influence of the apical urothelial layer on the contractile activity of this tissue is uncertain. The aim of this experiment was to identify the contractile activity of the lamina propria after removal of the urothelium. Several methods were used to mechanically disrupt the urothelium, including dabbing the tissue with a paper towel, longitudinal swipes with a cotton bud, or a longitudinal scrape with the edge of a scalpel. Hematoxylin-eosin staining was utilized to determine the level of removal of the apical urothelial cells. Spontaneous contractile activity was measured in organ baths, and responses to the agonists carbachol and isoprenaline were obtained. Three longitudinal swipes with a cotton bud was found to be the optimal method to remove the majority of the urothelium without damaging the lamina propria. Upon removal of the urothelium, the spontaneous activity of the tissue was unaltered. Similarly, responses to carbachol (1 µM) and isoprenaline (1 µM) were not affected after removal of the urothelium. The urothelium can be effectively removed without damaging the lamina propria. This apical tissue layer is not responsible for mediating the increases to spontaneous phasic activity or tonic contractions of the bladder mucosa (urothelium with lamina propria) when muscarinic or adrenergic receptors are stimulated. This research presents the lamina propria as the important cell layer mediating the overall contractile activity of the bladder wall.

Development, regeneration and tumorigenesis of the urothelium.

The urothelium of the bladder functions as a waterproof barrier between tissue and outflowing urine. Largely quiescent during homeostasis, this unique epithelium rapidly regenerates in response to bacterial or chemical injury. The specification of the proper cell types during development and injury repair is crucial for tissue function. This Review surveys the current understanding of urothelial progenitor populations in the contexts of organogenesis, regeneration and tumorigenesis. Furthermore, we discuss pathways and signaling mechanisms involved in urothelial differentiation, and consider the relevance of this knowledge to stem cell biology and tissue regeneration.

A database on differentially expressed microRNAs during rodent bladder healing.

Urinary bladder wound healing relies on multiple biological events that are finely tuned in a spatial-temporal manner. MicroRNAs are small non-coding RNA molecules with regulatory functions. We hypothesized that microRNAs are important molecules in the coordination of normal urinary bladder wound healing. We aimed at identifying microRNAs expressed during bladder wound healing using Affymetrix global array for microRNA profiling of the rodent urinary bladder during healing of a surgically created wound. Results were validated in the rat bladders by real-time PCR (RT-PCR) using three of the differentially expressed (DE) microRNAs. The model was thereafter validated in human cells, by measuring the expression of eight of the DE microRNAs upon in vitro wound-healing assays in primary urothelial cells. Our results indicated that 508 (40%) of all rodent microRNAs were expressed in the urinary bladder during wound healing. Thirteen of these microRNAs (1%) were DE (false discovery rate (FDR) < 0.05, P < 0.05, |logfold|> 0.25) in wounded compared to non-wounded bladders. Bioinformatic analyses helped us to identify target molecules for the DE microRNAs, and biological pathways involved in tissue repair. All data are made available in an open-access database for other researchers to explore.

Time-of-day dependent changes in guinea pig bladder afferent mechano-sensitivity.

The voiding of urine has a clear circadian rhythm with increased voiding during active phases and decreased voiding during inactive phases. Bladder spinal afferents play a key role in the regulation of bladder storage and voiding, but it is unknown whether they exhibit themselves a potential circadian rhythm. Therefore, this study aimed to determine the mechano- and chemo- sensitivity of three major bladder afferent classes at two opposite day-night time points. Adult female guinea pigs underwent conscious voiding monitoring and bladder ex vivo single unit extracellular afferent recordings at 0300 h and 1500 h to determine day-night modulation of bladder afferent activity. All guinea pigs voided a higher amount of urine at 1500 h compared to 0300 h. This was due to an increased number of voids at 1500 h. The mechano-sensitivity of low- and high-threshold stretch-sensitive muscular-mucosal bladder afferents to mucosal stroking and stretch was significantly higher at 1500 h compared to 0300 h. Low-threshold stretch-insensitive mucosal afferent sensitivity to stroking was significantly higher at 1500 h compared to 0300 h. Further, the chemosensitivity of mucosal afferents to N-Oleoyl Dopamine (endogenous TRPV1 agonist) was also significantly increased at 1500 h compared to 0300 h. This data indicates that bladder afferents exhibit a significant time-of-day dependent variation in mechano-sensitivity which may influence urine voiding patterns. Further studies across a 24 h period are warranted to reveal potential circadian rhythm modulation of bladder afferent activity.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day-night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day-night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals' active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day-night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals' awake, active phase.

Sodium plays an important role in the absorption of intravesical fluid.

OBJECTIVES: To investigate the role of sodium in intravesical absorption of water in the bladder and the sodium pathway in the urothelium. METHODS: Adult female Sprague-Dawley rats received either saline or a 5% glucose solution injection into their bladders. The changes in intravesical fluid volume; concentrations of sodium and chlorine and osmolality; and expression of aquaporin-2, epithelial sodium channel, and claudins were compared after 3 hours. RESULTS: Intravesical volume decreased significantly in the saline group compared to that in the 5% glucose solution group. The expression of claudin-3 and -6 was higher in the saline group than in the glucose group. There was a significant correlation between changes in the intravesical saline volume and the concentration of sodium and chlorine. Intravesical administration of amiloride did not affect changes in the fluid volume and concentration of sodium. CONCLUSIONS: The presence of sodium is important for the absorption of intravesical fluid through aquaporin-2 in the urinary bladders of rats. Claudin-3 and -6 may be associated with the transport of sodium through the bladder urothelium.

Bladder urothelium converts bacterial lipopolysaccharide information into neural signaling via an ATP-mediated pathway to enhance the micturition reflex for rapid defense.

When bacteria enter the bladder lumen, a first-stage active defensive mechanism flushes them out. Although urinary frequency induced by bacterial cystitis is a well-known defensive response against bacteria, the underlying mechanism remains unclear. In this study, using a mouse model of acute bacterial cystitis, we demonstrate that the bladder urothelium senses luminal extracellular bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 and releases the transmitter ATP. Moreover, analysis of purinergic P2X2 and P2X3 receptor-deficient mice indicated that ATP signaling plays a pivotal role in the LPS-induced activation of L6-S1 spinal neurons through the bladder afferent pathway, resulting in rapid onset of the enhanced micturition reflex. Thus, we revealed a novel defensive mechanism against bacterial infection via an epithelial-neural interaction that induces urinary frequency prior to bacterial clearance by neutrophils of the innate immune system. Our results indicate an important defense role for the bladder urothelium as a chemical-neural transducer, converting bacterial LPS information into neural signaling via an ATP-mediated pathway, with bladder urothelial cells acting as sensory receptor cells.

Roles for urothelium in normal and aberrant urinary tract development.

Congenital anomalies of the kidney and urinary tract (CAKUTs) represent the leading cause of chronic kidney disease and end-stage kidney disease in children. Increasing evidence points to critical roles for the urothelium in the developing urinary tract and in the genesis of CAKUTs. The involvement of the urothelium in patterning the urinary tract is supported by evidence that CAKUTs can arise as a result of abnormal urothelial development. Emerging evidence indicates that congenital urinary tract obstruction triggers urothelial remodelling that stabilizes the obstructed kidney and limits renal injury. Finally, the diagnostic potential of radiological findings and urinary biomarkers derived from the urothelium of patients with CAKUTs might aid their contribution to clinical care.

Does the Urothelium of Old Mice Regenerate after Chitosan Injury as Quickly as the Urothelium of Young Mice?

The aging of organisms leads to a decreased ability of tissue to regenerate after injury. The regeneration of the bladder urothelium after induced desquamation with biopolymer chitosan has been studied in young mice but not in old mice. Chitosan is a suitable inducer of urothelial desquamation because it is known to be non-toxic. We used chitosan for desquamation of urothelial cells in order to compare the dynamics of urothelial regeneration after injury between young and old mice. Our aim was to determine whether the urothelial function and structure of old mice is restored as fast as in young mice, and to evaluate the inflammatory response due to chitosan treatment. We discovered that the urothelial function restored comparably fast in both age groups and that the urothelium of young and old mice recovered within 5 days after injury, although the onset of proliferation and differentiation appeared later in old mice. Acute inflammation markers showed some differences in the inflammatory response in young versus old mice, but in both age groups, chitosan caused short-term acute inflammation. In conclusion, the restoration of urothelial function is not impaired in old mice, but the regeneration of the urothelial structure in old mice slightly lags behind the regeneration in young mice.

The five primary prostaglandins stimulate contractions and phasic activity of the urinary bladder urothelium, lamina propria and detrusor.

BACKGROUND: Inflammation is often associated with several bladder dysfunctions, including overactive bladder (OAB) and interstitial cystitis/bladder pain syndrome (IC/PBS). As such, inflammation of the bladder and the actions of inflammatory mediators may contribute to the development of urinary symptoms. This study assessed the actions of PGE2, PGF2, PGD2, TXA2, and PGI2 on urinary bladder urothelium with lamina propria (U&LP), and detrusor smooth muscle. METHODS: Studies were carried out using isolated tissue baths, where strips of porcine bladder U&LP or detrusor were exposed to varying concentrations of prostaglandin agonists (1 µM and 10 µM). RESULTS: All assessed prostaglandin agonists contracted both the U&LP and detrusor smooth muscle, with the rank order of contractile response effectiveness as: PGE2 > PGF2α > TXA2 > PGD2 > PGI2. In U&LP, treatment with PGE2 (10 µM) increased tonic contractions by 1.36 ± 0.09 g (n = 42, p < 0.001) and phasic contractions by 40.4 ± 9.6% (n = 42, p < 0.001). In response to PGF2α (10 µM), U&LP tonic contractions increased by 0.79 ± 0.06 g (n = 14, p < 0.001) and phasic activity by 13.3% ± 5.3% (n = 15, p < 0.05). In detrusor preparations, PGE2 (10 µM) increased tonic contractions by 1.32 ± 0.13 g (n = 38, p < 0.001) and PGF2α (10 µM) by 0.97 ± 0.14 g (n = 12, p < 0.001). Only 34% (n = 48) of all detrusor preparations exhibited spontaneous activity prior to the addition of any agonist at a frequency of 2.03 ± 0.12 cpm. In preparations that did not exhibit initial phasic activity, all of the prostaglandin agonists were capable of commencing phasic activity. CONCLUSIONS: The urinary bladder U&LP and detrusor respond to a variety of prostaglandin agonists, with their activation resulting in direct contractions, as well as increases to spontaneous contractile activity. This study presents the prostaglandin receptor system as a potential therapeutic target for lower urinary tract dysfunction.

Urine as a Main Effector in Urological Tissue Engineering-A Double-Edged Sword.

In order to reconstruct injured urinary tract tissues, biodegradable scaffolds with autologous seeded cells are explored in this work. However, when cells are obtained via biopsy from individuals who have damaged organs due to infection, congenital disorders, or cancer, this can result in unhealthy engineered cells and donor site morbidity. Thus, neo-organ construction through an alternative cell source might be useful. Significant advancements in the isolation and utilization of urine-derived stem cells have provided opportunities for this less invasive, limitless, and versatile source of cells to be employed in urologic tissue-engineered replacement. These cells have a high potential to differentiate into urothelial and smooth muscle cells. However, urinary tract reconstruction via tissue engineering is peculiar as it takes place in a milieu of urine that imposes certain risks on the implanted cells and scaffolds as a result of the highly cytotoxic nature of urine and its detrimental effect on both growth and differentiation of these cells. Both of these projections should be tackled thoughtfully when designing a suitable approach for repairing urinary tract defects and applying the needful precautions is vital.

On the role of the epithelium in a model of sodium exchange in renal tubules.

In this study we present a mathematical model describing the transport of sodium in a fluid circulating in a counter-current tubular architecture, which constitutes a simplified model of Henle's loop in a kidney nephron. The model explicitly takes into account the epithelial layer at the interface between the tubular lumen and the surrounding interstitium. In a specific range of parameters, we show that explicitly accounting for transport across the apical and basolateral membranes of epithelial cells, instead of assuming a single barrier, affects the axial concentration gradient, an essential determinant of the urinary concentrating capacity. We present the solution related to the stationary system, and we perform numerical simulations to understand the physiological behaviour of the system. We prove that when time grows large, our dynamic model converges towards the stationary system at an exponential rate. In order to prove rigorously this global asymptotic stability result, we study eigen-problems of an auxiliary linear operator and its dual.

TRPV4 receptor as a functional sensory molecule in bladder urothelium: Stretch-independent, tissue-specific actions and pathological implications.

The newly recognized sensory role of bladder urothelium has generated intense interest in identifying its novel sensory molecules. Sensory receptor TRPV4 may serve such function. However, specific and physiologically relevant tissue actions of TRPV4, stretch-independent responses, and underlying mechanisms are unknown and its role in human conditions has not been examined. Here we showed TRPV4 expression in guinea-pig urothelium, suburothelium, and bladder smooth muscle, with urothelial predominance. Selective TRPV4 activation without stretch evoked significant ATP release-key urothelial sensory process, from live mucosa tissue, full-thickness bladder but not smooth muscle, and sustained muscle contractions. ATP release was mediated by Ca2+-dependent, pannexin/connexin-conductive pathway involving protein tyrosine kinase, but independent from vesicular transport and chloride channels. TRPV4 activation generated greater Ca2+ rise than purinergic activation in urothelial cells. There was intrinsic TRPV4 activity without exogeneous stimulus, causing ATP release. TRPV4 contributed to 50% stretch-induced ATP release. TRPV4 activation also triggered superoxide release. TRPV4 expression was increased with aging. Human bladder mucosa presented similarities to guinea pigs. Overactive bladders exhibited greater TRPV4-induced ATP release with age dependence. These data provide the first evidence in humans for the key functional role of TRPV4 in urothelium with specific mechanisms and identify TRPV4 up-regulation in aging and overactive bladders.

Bladder Organoids and Spheroids: Potential Tools for Normal and Diseased Tissue Modelling.

Bladder-related diseases are among the most common and costly to the healthcare system, and therefore require new approaches to treatment. Organoids and spheroids are 3D cultures that mimic organ features ex vivo and offer novel approaches for diagnostic and therapeutic assessment. The aim of this article was to provide a systematic review of the literature related to bladder organoids and spheroids, applied to disease diagnosis, characterization, and treatment. PubMed and Web of Science were utilized in March 2018 to compile 191 articles satisfying search criteria related to bladder organoids or spheroids and 58 articles were included in the final review. Finally, cell types and techniques utilized for spheroid and organoid manufacture were characterized. The applications of bladder carcinoma spheroids and organoids followed three themes: cancer characterization, diagnosis, and treatment. Tumor characterization studies included a focus on extracellular matrix, microenvironment, genetics, and growth of tumor cells. Diagnostic studies explored the use of endogenous fluorophores and white light for photodiagnosis. Treatment studies investigated cancer chemotherapy, immunotherapy, oncolytic viruses, and gene therapy. Ten studies explored hypericin as a tool for diagnostics and photodynamic therapy. Additionally, two studies applied organoids to urinary tract infections.

Human umbilical cord-derived mesenchymal stem cells enhanced HK-2 cell autophagy through MicroRNA-145 by inhibiting the PI3K/AKT/mTOR signaling pathway.

Recent studies have shown that autophagy exhibits a protective role in acute kidney injury (AKI), and the accumulation of advanced oxidation protein products (AOPP) participates in the progression of kidney diseases. Our previous study indicated that AOPP induced injury in renal tubular epithelial cells (RTECs) through autophagy inhibition. Besides, we found that human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) enhanced autophagy in AOPP-treated RTECs, but the underlying mechanism remains unclear. We regulated microRNA-145 (miR-145) expression in HK-2 cells (a cell line of RTECs), or co-cultured hUC-MSCs with HK-2 cells and studied the autophagic activity in HK-2 cells to explore the underlying mechanism. Our data demonstrated that upregulated miR-145 increased LC3 II and Beclin 1 levels, decreased p62 level, three autophagy related proteins, inhibited the phosphorylation of PI3K/AKT/mTOR, and increased LC3B-positive staining and the autophagosome number. Furthermore, hUC-MSCs enhanced autophagy and inhibited phosphorylation of PI3K/AKT/mTOR in AOPP-treated HK-2 cells, which was then partially rescued using miR-145 knockdown in the hUC-MSCs co-culture system. In conclusion, our study showed that hUC-MSCs enhanced autophagy in AOPP-treated HK-2 cells mediated by miR-145 via inhibition of the PI3K/AKT/mTOR pathway, which indicated that hUC-MSCs might serve as a prospective therapy for AKI.

Ureter tissue engineering with vessel extracellular matrix and differentiated urine-derived stem cells.

OBJECTIVE: To assess the possibility of ureter tissue engineering using vessel extracellular matrix (VECM) and differentiated urine-derived stem cells (USCs) in a rabbit model. METHODS: VECM was prepared by a modified technique. USCs were isolated from human urine samples and cultured with an induction medium for the differentiation of the cells into urothelium and smooth muscle phenotypes. For contractile phenotype conversion, the induced smooth muscle cells were transfected with the miR-199a-5p plasmid. The differentiated cells were seeded onto VECM and cultured under dynamic conditions in vitro for 2 weeks. The graft was tubularized and wrapped by two layers of the omentum of a rabbit for vascularization. Then, the maturated graft was used for ureter reconstruction in vivo. RESULTS: VECM has microporous structures that allow cell infiltration and exhibit adequate biocompatibility with seeding cells. USCs were isolated and identified by flow cytometry. After induction, the urothelium phenotype gene was confirmed at mRNA and protein levels. With the combined induction by TGF-ß1 and miR-199a-5p, the differentiated cells can express the smooth muscle phenotype gene and convert to the contractile phenotype. After seeding cells onto VECM, the induced urothelium cells formed a single epithelial layer, and the induced smooth muscle cells formed a few cell layers during dynamic culture. After 3 weeks of omental maturation, tubular graft was vascularized. At 2 months post ureter reconstruction, histological evaluation showed a clearly layered structure of ureter with multilayered urothelium over the organized smooth muscle tissue. CONCLUSION: By seeding differentiated USCs onto VECM, a tissue-engineered graft could form multilayered urothelium and organized smooth muscle tissue after ureteral reconstruction in vivo. STATEMENT OF SIGNIFICANCE: Cell-based tissue engineering offers an alternative technique for urinary tract reconstruction. In this work, we describe a novel strategy for ureter tissue engineering. We modified the techniques of vessel extracellular matrix (VECM) preparation and used a dynamic culture system for seeding cells onto VECM. We found that VECM had the trait of containing VEGF and exhibited blood vessel formation potential. Urine-derived stem cells (USCs) could be differentiated into urothelial cells and functional contractile phenotype smooth muscle cells in vitro. By seeding differentiated USCs onto VECM, a tissue-engineered graft could form multilayered urothelium and organized smooth muscle tissue after ureteral reconstruction in vivo. This strategy might be applied in clinical research for the treatment of long-segment ureteral defect.

An ex vivo bladder model with detrusor smooth muscle removed to analyse biologically active mediators released from the suburothelium.

KEY POINTS: Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure-volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. ABSTRACT: The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)-denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium-derived mediators at the luminal and anti-luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine-scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure-volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate- and lipopolysaccharide-treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM-denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium-DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding.

Immunomodulatory response of layered small intestinal submucosa in a rat bladder regeneration model.

Based on the hypothesis that bioscaffold permeability is a major factor in determining the outcome of histologically complete and functional bladder regeneration, we evaluated regeneration processes of four-layer porcine small intestinal submucosa (SIS) construct; and compared results between rat bladders augmented with single-layer SIS bioscaffolds. Sprague-Dawley female rats were subjected to hemi-cystectomy followed by anastomosis of a patch of either single- or four-layer porcine SIS. Permeability was analyzed in situ using magnetic resonance imaging (MRI) at post-operative days 7 and 14. Bladder sections excised at days 7, 14, 28, and 56 post-operation Samples were assessed by H&E and Masson's trichrome stains. Urothelial differentiation was analyzed using cytokeratin AE1/AE3, and uroplakin III (UPIII). In addition, quantitative and qualitative evaluations of neutrophils, mast cells, eosinophils, and macrophages were performed using anti-myeloperoxidase, Alcian blue, Giemsa stain, and anti-CD68 staining methods, respectively. Four-layer SIS was consistently impermeable as evidenced by the absence of intravesical administered gadolinium with diethylenetriaminepentacetate (Gd-DTPA) contrast signal in peripheral regions of augmented bladders compared with single-layer SIS bioscaffold. Elevated and sustained eosinophil and neutrophil infiltrations were prominent in four-layered SIS-augmented bladders compared with single-layer SIS with comparable impermeability. Delayed but consistent urothelial regeneration and differentiation were observed in four-layer SIS-augmented bladders; and urothelial differentiation was observed at day 56 post-augmentation. In conclusion, four-layer SIS enacts an elevated inflammatory response along with extended urothelial regeneration. Four-layer SIS may activate a different but yet to be identified mechanism for inflammatory responses. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1960-1969, 2019.