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Purpose of Review Bladder pain syndrome (BPS)/interstitial cystitis (IC) can also be classified as either non-ulcerative or ulcerative, corresponding to the characteristic cystoscopic findings under hydrodistention. Promising therapeutic effects, including decreased bladder pain, have been reported from recent clinical trials using botulinum toxin A (BoNTA) for the treatment of BPS/IC. This review summarizes the current state of the literature on the underlying mechanisms of BoNTA therapy in BPS/IC as well as new forms of its application. Recent Findings BoNTA has its effect in the central nervous system in the afferent nerves as well as in the bladder wall. Besides the well-known effects of BoNTA in the nervous system, pain control as well as reduction of urinary urgency in BPS patients could be achieved by mast cell stabilization effecting histamine release as well as modulation of TRPV and PGE2 pathways, among other systems. In addition, new forms of BoNTA administration have focused on intravesical instillation of the drug in order to circumvent bladder wall injections. Hyperthermia, intravesical hydrogel, and lysosomes have been studied as new ways of BoNTA application in BPS/IC patients. From the available studies, bladder instillation of BoNTA in combination with EMDA is the most promising and effective novel approach. Summary The most promising novel application methods for BoNTA in patient with BPS/IC are bladder instillations. Future research needs to point out if bladder instillations with BoNTA with some form of bladder absorption enhancement such as hyperthermia or EMDA would be able to replace BoNTA injections in patients with BPS/IC

The Golgi complex undergoes considerable structural remodeling during differentiation of urothelial cells in vivo and in vitro. It is known that in a healthy bladder the differentiation from the basal to the superficial cell layer leads to the formation of the tightest barrier in our body, i.e., the blood–urine barrier. In this process, urothelial cells start expressing tight junctional proteins, apical membrane lipids, surface glycans, and integral membrane proteins, the uroplakins (UPs). The latter are the most abundant membrane proteins in the apical plasma membrane of differentiated superficial urothelial cells (UCs) and, in addition to well-developed tight junctions, contribute to the permeability barrier by their structural organization and by hindering endocytosis from the apical plasma membrane. By studying the transport of UPs, we were able to demonstrate their differentiation-dependent effect on the Golgi architecture. Although fragmentation of the Golgi complex is known to be associated with mitosis and apoptosis, we found that the process of Golgi fragmentation is required for delivery of certain specific urothelial differentiation cargoes to the plasma membrane as well as for cell–cell communication. In this review, we will discuss the currently known contribution of the Golgi complex to the formation of the blood–urine barrier in normal UCs and how it may be involved in the loss of the blood–urine barrier in cancer. Some open questions related to the Golgi complex in the urothelium will be highlighted.

Background Recent genome-wide studies revealed the molecular subtypes and mutational landscape of bladder cancer, which is the 10th most common cancer causing many deaths. ELF3 is one of the frequently mutated genes in bladder cancer with 14% alteration rate. It mainly functions as an epithelial transcription factor and its proper function is critical for the urothelium development. However, the impact of ELF3 mutations in bladder cancer is currently unknown. Methods and results In this study, we analysed the gene expression data available for primary bladder cancer and bladder cancer cell lines according to the mutation status of ELF3. Our results show that de-regulated genes common in cell lines and primary tissue are primarily involved in ameboidal type cell migration and cell-cell junction organization. Additionally, we identify that ELF3-mutant cases in primary samples significantly overexpress PIK3C2B and ELF3 and PIK3C2B and ELF3 are significantly co-mutated in many cancer types. Our integrative analysis with existing Hi-C data further revealed the genes proximally located to ELF3, including PIK3C2B to be upregulated in ELF3 mutant cases, potentially as a result of truncated ELF3 protein product and subsequent changes in regulatory interactions. Conclusions Our results provide important insights about how ELF3 mutation contributes to bladder tumorigenesis and uncover previously unknown dependencies.

Limited understanding of bladder cancer aetiopathology hampers progress in reducing incidence. Mutational signatures show the anti-viral apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes are responsible for the preponderance of mutations in bladder tumour genomes, but no causative viral agent has been identified. BK polyomavirus (BKPyV) is a common childhood infection that remains latent in the adult kidney, where reactivation leads to viruria. This study provides missing mechanistic evidence linking reactivated BKPyV-infection to bladder cancer risk. We used a mitotically-quiescent, functionally-differentiated model of normal human urothelium to examine BKPyV-infection. BKPyV-infection led to significantly elevated APOBEC3A and APOBEC3B protein, increased deaminase activity and greater numbers of apurinic/apyrimidinic sites in the host urothelial genome. BKPyV Large T antigen (LT-Ag) stimulated re-entry from G0 into the cell cycle through inhibition of retinoblastoma protein and activation of EZH2, E2F1 and FOXM1, with cells arresting in G2. The single-stranded DNA displacement loops formed in urothelial cells during BKPyV-infection interacted with LT-Ag to provide a substrate for APOBEC3-activity. Addition of interferon gamma (IFNγ) to infected urothelium suppressed expression of the viral genome. These results support reactivated BKPyV infections in adults as a risk factor for bladder cancer in immune-insufficient populations.

The human bladder maintains a cycle of filling, storing, and micturating throughout an individual's lifespan. The cycle relies on the ability of the bladder to expand without increasing the intravesical pressure, which is only possible with the controlled relaxation of well-complaint muscles and the congruously organized construction of the bladder wall. A competent bladder outlet, which functions in a synchronous fashion with the bladder, is also necessary for this cycle to be completed successfully without deterioration. In this paper, we aimed to review the contemporary physiological findings on bladder physiology and examine the effects of amino acids on clinical conditions affecting the bladder, with special emphasis on the available therapeutic evidence and possible future roles of the amino acids in the treatment of the bladder-related disorders.

Abstract Infections are common in patients with diabetes, but increasing antibiotic resistance hampers successful bacterial clearance and calls for alternative treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is known to influence the innate immune defense and could therefore serve as a possible target. However, the impact of high glucose on HIF-1 has received little attention and merits closer investigation. Here, we show that higher levels of proinflammatory cytokines and CAMP, encoding for the antimicrobial peptide cathelicidin, LL-37, correlate with HIF-1 in type 2 diabetic patients. Chemical activation of HIF-1 further enhanced LL-37, IL-1β, and IL-8 in human uroepithelial cells exposed to high glucose. Moreover, HIF-1 activation of transurethrally infected diabetic mice resulted in lower bacterial load. Drugs activating HIF-1 could therefore in the future potentially have a therapeutic role in clearing bacteria in diabetic patients with infections where antibiotic treatment failed. Key messages • Mohanty et al. “HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients.” • Our study highlights induction of the antimicrobial peptide, LL-37, and strengthening of the innate immunity through hypoxia-inducible factor 1 (HIF-1) in diabetes. • Our key observations are: 1. HIF-1 activation increased LL-37 expression in human urothelial cells treated with high glucose. In line with that, we demonstrated that patients with type 2 diabetes living at high altitude had increased levels of the LL-37. 2. HIF-1 activation increased IL-1β and IL-8 in human uroepithelial cells treated with high glucose concentration. 3. Pharmacological activation of HIF-1 decreased bacterial load in the urinary bladder of mice with hereditary diabetes. • We conclude that enhancing HIF-1 may along with antibiotics in the future contribute to the treatment in selected patient groups where traditional therapy is not possible.

Urothelium is a specialized multilayer epithelium that lines the urinary tract from the proximal urethra to the kidney. In addition to proliferation and differentiation during development, urothelial injury postnatally triggers a robust regenerative capacity to restore the protective barrier between the urine and tissue. Mounting evidence supports the existence of dedicated progenitor cell populations that give rise to urothelium during development and in response to injury. Understanding the cellular and molecular basis for urothelial patterning and repair will inform tissue regeneration therapies designed to ameliorate a number of structural and functional defects of the urinary tract. Here, we review the current understanding of urothelial progenitors and the signaling pathways that govern urothelial development and repair. While most published studies have focused on bladder urothelium, we also discuss literature on upper tract urothelial progenitors. Furthermore, we discuss evidence supporting existence of context-specific progenitors. This knowledge is fundamental to the development of strategies to regenerate or engineer damaged or diseased urothelium.

Purpose To engraft bladder organoids (BO) on de-epithelialized mouse colon using an epithelial replacement technique. Methods BO cultured using bladder specimens from enhanced green fluorescent protein (EGFP) transgenic mice were engrafted to replace proximal colon epithelium stripped from an approximately 1cm long target site in syngeneic wild-type recipient mice (n = 9) by exposure to ethylenediaminetetraacetic acid by infusion and flushing with phosphate buffered saline. Target sites were harvested on postoperative days 2, 7, and 28 for hematoxylin-eosin staining and immunofluorescence. Results Histology on postoperative days 7 and 28 showed BO derived EGFP + cells forming multiple layers on the luminal surface of the colon. Immunohistochemistry showed that EGFP + areas were positive for CK5 and CK14, markers for basal and immature subtype urothelium, respectively, but negative for CA2, a marker for colonic epithelium. Ki67 was detected predominantly in the basal parts of EGFP + areas on post-operative day 7 and day 28. Conclusions This is the first report of successful engraftment of BO in de-epithelialized colon with urothelial tissue reconstituted by actively-proliferating cells. This technique could be developed for augmentation cystoplasty to prevent bladder calculi formation and malignant transformation.

PURPOSE: To determine the unique contributions from elevated voiding and storage pressures in the development of fibrosis and the epithelial-to-mesenchymal transition (EMT) in urothelial cells, and how progressive BOO pressure cycling is an important mechanical cue leading to these pathological changes. MATERIALS AND METHODS: Urothelial cells isolated from control, SHAM, 2 (acute)- or 6 (chronic)-week BOO rats treated with an inflammasome inhibitor or no drug. Total RNA was isolated and RT-PCR was conducted with custom primers for pro-fibrotic and EMT genes. In separate experiments, a rat urothelial cell line was exposed to cyclic pressure regimes characteristic of acute and chronic BOO in the presence or absence of an inflammasome inhibitor. Following exposure, RT-PCR was conducted, collagen content was determined and intracellular caspase-1 activity was measured. RESULTS: Urothelial cells isolated from acute and chronic BOO rat models demonstrated expression of pro-fibrotic and EMT genes. Similarly, MYP3 rat urothelial cells subjected to pressure cycling regimes that reflect intravesical pressures in the acute or chronic BOO bladder also demonstrated increased expression of pro-fibrotic and EMT genes, along with elevated soluble collagen. Treatment with inflammasome inhibitors reduced expression of pro-fibrotic genes in the rat model and pressure cycling model but had a limited effect on EMT. CONCLUSION: These results indicate that acute and chronic BOO pressure cycling are essential in the initiation and progression of fibrosis in the bladder via the NLRP3 inflammasome, but also provide new evidence that there is also an alternative NLRP3-independent pathway leading to EMT and fibrosis.

The time-varying clearance (CL) of the PD-L1 inhibitor atezolizumab was assessed on a population of 1519 cancer patients (primarily with non-small-cell lung cancer or metastatic urothelial carcinoma) from three clinical studies. The first step was to identify the baseline covariates affecting atezolizumab CL without including time-varying components (stationary covariate model). Two time-varying models were then investigated: (1) a model allowing baseline covariates to vary over time (time-varying covariate model), (2) a model with empirical time-varying Emax CL function. The final stationary covariate model included main effects of body weight, albumin levels, tumor size, anti-drug antibodies (ADA) and gender on atezolizumab CL. Both time-varying models resulted in a clear improvement of the data fit and visual predictive checks over the stationary model. The time-varying covariate model provided the best fit of the data. In this model, the main driver for change in CL over time was variations in albumin level with an increase in serum albumin (improvement in a patient’s status) mirroring a decrease in CL. Time-varying ADAs had a small impact (9% increase in CL). None of the covariates impacted atezolizumab CL by more than ± 30% from median. The estimated maximum decrease in CL with time was 22% with the Emax model. The overall impact of covariates on atezolizumab CL did not warrant any change in atezolizumab dosing recommendations. The results support the hypothesis that variation in atezolizumab CL over time is associated with patients’ disease status, as shown with other checkpoint inhibitors.

Purpose In a previous study we have shown in a mouse model that administration of nuclear factor-kappa B (NF-κB) inhibitor thalidomide has promising therapeutic effects on early radiation cystitis (ERC) and late radiation sequelae (LRS) of the urinary bladder. The aim of this study was to evaluate in the same mice the effect of thalidomide on adherens junction (AJ) proteins in ERC and LRS. Methods Urothelial expressions of E‑cadherin and β‑catenin were assessed by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) bladder specimens over 360 days post single-dose irradiation on day 0. First, the effect of irradiation on AJ expression and then effects of thalidomide on irradiation-induced AJ alterations were assessed using three different treatment times. Results Irradiation provoked a biphasic upregulation of E‑cadherin and β‑catenin in the early phase. After a mild decrease of E‑cadherin and a pronounced decrease of β‑catenin at the end of the early phase, both increased again in the late phase. Early administration of thalidomide (day 1–15) resulted in a steeper rise in the first days, an extended and increased expression at the end of the early phase and a higher expression of β‑catenin alone at the beginning of the late phase. Conclusion Upregulation of AJ proteins is an attempt to compensate irradiation-induced impairment of urothelial barrier function. Early administration of thalidomide improves these compensatory mechanisms by inhibiting NF-κB signaling and its interfering effects.

Purpose To evaluate the pharmacokinetic properties of UGN-101, a mitomycin-containing reverse thermal gel used as primary chemoablative treatment for low-grade upper tract urothelial carcinoma (UTUC), in a subset of patients participating in a phase 3 clinical trial. Methods Pharmacokinetic parameters (Cmax, Tmax, AUC(0–6), λz, t½, and AUCinf) were evaluated in six participants (male or female, ≥ 18 years) with biopsy-proven, low-grade UTUC who received the first of 6 once-weekly instillations of UGN-101 to the renal pelvis and calyces via retrograde ureteral catheter. Plasma samples were collected prior to instillation and 30 min, 1, 2, 3, 4, 5, and 6 h post-instillation. Safety was assessed by laboratory evaluations, physical exam, and adverse event monitoring. Results The mean age of the six participants was 69 years; most were male (5/6) and Caucasian (5/6). Mean (SD) Cmax was 6.24 (4.11) ng/mL and mean Tmax was 1.79 (1.89) hours after instillation. Mean apparent t½ following instillation was 1.27 (0.63) hours. Mean total systemic exposure to mitomycin up to 6 h post-instillation was 20.30 (19.69) ng h/mL. At 6 h post-instillation, mitomycin plasma concentrations of 5/6 participants were Conclusion The reverse thermal gel formulation of UGN-101 is associated with higher concentration and extended dwell time of mitomycin in contact with the urothelium of the upper urinary tract while limiting systemic absorption of mitomycin. Registration NCT02793128; registered June 8, 2016.

Disruptions to sensory pathways in the lower urinary tract commonly occur and can give rise to lower urinary tract symptoms (LUTS). The unmet clinical need for treatment of LUTS has stimulated research into the molecular mechanisms that underlie neuronal control of the bladder and transient receptor potential (TRP) channels have emerged as key regulators of the sensory processes that regulate bladder function. TRP channels function as molecular sensors in urothelial cells and afferent nerve fibres and can be considered the origin of bladder sensations. TRP channels in the lower urinary tract contribute to the generation of normal and abnormal bladder sensations through a variety of mechanisms, and have demonstrated potential as targets for the treatment of LUTS in functional disorders of the lower urinary tract. Transient receptor potential (TRP) channels have an important role in sensory mechanisms of the lower urinary tract. Vanneste et al. discuss the involvement of TRP channels in normal and abnormal bladder sensations and their potential as therapeutic targets.

Whether pT3 urothelial carcinoma of the renal pelvis (UCRP) and urothelial carcinoma of the ureter (UCU) have the same prognosis is controversial, this study compared the prognosis of pT3 UCRP with that of pT3 UCU. We retrospectively evaluated 954 patients who underwent nephroureterectomy at our institutions between January 1983 and December 2017. All surgical specimens were reviewed by a single genitourinary pathologist. Cases of pT3 UCRP were subclassified as pT3a (urothelial carcinomas extending only to the renal medulla) and pT3b (urothelial carcinomas extending into the renal cortex and/or peripelvic adipose tissue). Fine and Gray's model was used to predict recurrence-free survival (RFS) and cancer-specific survival (CSS). A total of 493 (51.7%) had UCRP and 461 (48.3%) had UCU. Within this group, 202 patients had pT3 UCRP and 146 had pT3 UCU. The pT3 subclassification of UCRP resulted in 79 cases of pT3a and 120 of pT3b. The difference in 5-year CSS among the pT3a UCRP, pT2 UCRP, and pT2 UCU subgroups was not statistically significant (pT3a UCRP vs pT2 UCRP, HR = 0.69, p = 0.56; pT3a UCRP vs pT2 UCU, HR = 0.66, p = 0.31) However, RFS and CSS were significantly higher in the pT3a UCRP group than in the pT3b group (pT3a vs pT3b, HR = 2.59, p = 0.0038 and pT3a vs pT3b, HR = 3.10, p = 0.001). The results suggest that our proposed pT3 subclassification better predicts the prognosis of UCRP patients than does the pT3 of the current AJCC/UICC classification.

To investigate the feasibility of ureteral reconstruction using lingual mucosa graft (LMG) and evaluate the histological changes of the engrafted LMG in beagles. Twelve male beagle dogs were randomly divided into groups A, B and C (n = 4). A ventral ureteral defect was created by excising half of the ureteral wall. The length of the defect was 3 cm, 6 cm, and 10 cm in groups A, B, and C, respectively. The LMGs were harvested and employed to repair the ureteral defects in onlay fashion. Two dogs per group were sacrificed after 6 months, with additional two dogs per group sacrificed after 12 months. Intravenous urography (IVU) and macroscopic examination were performed to evaluate renal function and ureteral patency. Histological changes in the engrafted LMGs during the tissue incorporation process were assessed by histological analysis. There were no postoperative complications. Only one dog in group C developed a mild stricture near the proximal anastomosis. In the remaining 11 animals, IVU showed normal renal function and a wide ureteral caliber without stricture or fistula. The diameter of the LMG-reconstructed ureter was greater than that of the proximal and distal ureter (each p value

Rationale Ketamine is the first widely used substance with rapid-onset antidepressant action. However, there are uncertainties regarding its potential urothelial toxicity, particularly after repeated application. In the context of rising recreational ketamine use, severe side effects affecting the human urinary tract have been reported. It is assumed that ketamine interacts with bladder urothelial cells and induces apoptosis. Objectives This study aimed to assess whether single or repeated doses of esketamine used in an antidepressant indication are associated with urinary toxicity. Methods We included male and female inpatients with a current episode of depression and a diagnosis of recurrent depressive disorder, bipolar disorder or schizoaffective disorder according to ICD-10 criteria (n = 25). The esketamine treatment schedule involved a maximum of 3× weekly dosing at 0.25–0.5 mg/kg i.v. or s.c. The primary outcome was the change in urine toxicity markers (leukocytes, erythrocytes, protein and free haemoglobin). Description of demographic, clinical and laboratory data was conducted using means, standard deviations, frequencies and percentages. Changes in urinary toxicity markers over time were evaluated using linear mixed models with gender as a covariate. Results The participants received an average of 11.4 (SD 8) esketamine treatments, and an average number of 11.2 (SD 8) urine samples were analysed over the course of treatment. Neither urinary leukocyte concentration (F(20; 3.0) = 3.1; p = 0.2) nor erythrocyte concentration (F(20;2.2) = 4.1; p = 0.2) showed a significant trend towards increase during the course of esketamine treatment. Similarly, free haemoglobin and protein concentrations, which were analysed descriptively, did not display a rise during treatment. There was a significant improvement in depression ratings after esketamine treatment (p Conclusions This study is, to the best of our knowledge, the first to focus on urothelial toxicity of esketamine used in antidepressant indication and dose. The results indicate that the use of single or repeated doses of esketamine is unlikely to cause urothelial toxicity. The results are in need of confirmation as sample size was small.

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.

Stress causes symptom exacerbation in functional disorders of the urinary bladder. However, the potential mediators and underlying mechanisms of stress effects on micturition reflex function are unknown. We have characterized PACAP (Adcyap1) and PAC1 receptor (Adcyap1r1) signaling in stress-induced urinary bladder dysfunction in mice. We determined PACAP and PAC1 transcripts and protein expressions in the urinary bladder and lumbosacral dorsal root ganglia (DRG) and spinal cord in repeated variate stress (RVS) or control mouse (handling only) groups. RVS in mice significantly (p ≤ 0.01) increased serum corticosterone and urinary bladder NGF content and decreased weight gain. PACAP and PAC1 mRNA and protein were differentially regulated in lower urinary tract tissues with changes observed in lumbosacral DRG and spinal cord but not in urinary bladder. RVS exposure in mice significantly (p ≤ 0.01) increased (2.5-fold) voiding frequency as determined using conscious cystometry. Intrabladder administration of the PAC1 receptor antagonist, PACAP(6-38) (300 nM), significantly (p ≤ 0.01) increased infused volume (1.5-2.7-fold) to elicit a micturition event and increased the intercontraction interval (i.e., decreased voiding frequency) in mice exposed to RVS and in control mice, but changes were smaller in magnitude in control mice. We also evaluated the effect of PAC1 blockade at the level of the urinary bladder on pelvic sensitivity in RVS or control mouse groups using von Frey filament testing. Intrabladder administration of PACAP(6-38) (300 nM) significantly (p ≤ 0.01) reduced pelvic sensitivity following RVS. PACAP/receptor signaling in the CNS and PNS contributes to increased voiding frequency and pelvic sensitivity following RVS and may represent a potential target for therapeutic intervention.

To examine the effects of intravenous and intravesical application of vibegron, a new β3-adrenoceptor (β3-AR) agonist, on bladder function in rats with oxotremorine methiodide (oxo-M: a nonselective muscarinic receptor agonist)-induced bladder overactivity. Cystometry was performed in conscious female rats with intravesical instillation of oxo-M (200 μM). In oxo-M-treated rats, vehicle or vibegron (1 and 10 mg/kg) was cumulatively applied intravenously at 30-min intervals. In other groups of rats, oxo-M + vehicle or oxo-M + vibegron (10, 100 μM, and 1 mM) was cumulatively instilled intravesically at 60-min intervals followed by intravenous application of vibegron (10 mg/kg). Expression of β3-ARs in the bladder was also evaluated using immunohistochemical staining. Intravenous application of vibegron (10 mg/kg) significantly increased bladder capacity (1.3 times) and decreased baseline, threshold, and maximal voiding pressure compared with vehicle. Next, intravesical application of vibegron (1 mM) significantly increased threshold pressure and bladder capacity (1.2 times) compared with vehicle. Combined treatments of intravesical (1 mM) and intravenous (10 mg/kg) application of vibegron induced a significantly larger degree of increases in bladder capacity (1.4 times) compared with vehicle. In addition, β3-ARs were expressed throughout the rat bladder, mainly in the urothelium. These results suggest that vibegron excreted in urine as an unchanged compound can induce the additive inhibitory effects on bladder overactivity possibly through urothelial β3-AR activation, which inhibits the afferent limb of micturition reflex rather than the efferent function as evidenced by the increases in threshold pressure and bladder capacity without affecting bladder contractile function after intravesical vibegron application.

Purpose Non-invasive high-grade (HG) bladder cancer is a heterogeneous disease that is characterized insufficiently. First-line Bacillus Calmette-Guérin instillation fails in a substantial amount of cases and alternative bladder-preserving treatments are limited, underlining the need to promote a further molecular understanding of non-invasive HG lesions. Here, we characterized pure HG papillary urothelial bladder cancer (pure pTa HG), a potential subgroup of non-invasive HG bladder carcinomas, with regard to molecular subtype affiliation and potential for targeted therapy. Methods An immunohistochemistry panel comprising luminal (KRT20, ERBB2, ESR2, GATA3) and basal (KRT5/6, KRT14) markers as well as p53 and FGFR3 was used to analyze molecular subtype affiliations of 78 pure pTa HG/papillary pT1(a) HG samples. In 66 of these, ERBB2 fluorescence in situ hybridization was performed. Additionally, targeted sequencing (31 genes) of 19 pTa HG cases was conducted, focusing on known therapeutic targets or those described to predict response to targeted therapies noted in registered clinical trials or that are already approved. Results We found that pure pTa HG/papillary pT1(a) HG lesions were characterized by a luminal-like phenotype associated with frequent (58% of samples) moderate to high ERBB2 protein expression, rare FGFR3 alterations on genomic and protein levels, and a high frequency (89% of samples) of chromatin-modifying gene alterations. Of note, 95% of pTa HG/papillary pT1 HG cases harbored at least one potential druggable genomic alteration. Conclusions Our data should help guiding the selection of targeted therapies for investigation in future clinical trials and, additionally, may provide a basis for prospective mechanistic studies of pTa HG pathogenesis.

Background Human urothelial carcinoma (UC) has a high tendency to recur and progress to life-threatening advanced diseases. Advanced therapeutic regimens are needed to control UC development and recurrence. Methods We pursued in vitro and in vivo studies to understand the ability of a triple combination of gemcitabine, romidepsin, and cisplatin (Gem+Rom+Cis) to modulate signalling pathways, cell death, drug resistance, and tumour development. Results Our studies verified the ability of Gem+Rom+Cis to synergistically induce apoptotic cell death and reduce drug resistance in various UC cells. The ERK pathway and reactive oxygen species (ROS) played essential roles in mediating Gem+Rom+Cis-induced caspase activation, DNA oxidation and damage, glutathione reduction, and unfolded protein response. Gem+Rom+Cis preferentially induced death and reduced drug resistance in oncogenic H-Ras-expressing UC vs. counterpart cells that was associated with transcriptomic profiles related to ROS, cell death, and drug resistance. Our studies also verified the efficacy and safety of the Gem plus Rom+Cis regimen in controlling UC cell-derived xenograft tumour development and resistance. Conclusions More than 80% of UCs are associated with aberrant Ras-ERK pathway. Thus the compensatory combination of Rom with Gem and Cis should be seriously considered as an advanced regimen for treating advanced UCs, especially Ras-ERK-activated UCs.

To identify patients with metastatic urothelial cancer (mUC) unlikely to benefit from immune-checkpoint inhibitors (ICIs). We explored the predictive and prognostic values of baseline neutrophil-to-lymphocyte ratio (NLR), with cut-offs ≥ 3 and ≥ 5, and of a urothelial immune prognostic index (UIPI, based on increased NLR and LDH), on 146 patients. NLR and UIPI significantly predicted progressive disease and progression-free survival with both cut-offs (p = 0.0069, p = 0.0034, p = 0.0160, p = 0.0063; p

Primary glandular bladder tumours (bladder adenocarcinoma [BAC], urachal adenocarcinoma [UAC], urothelial carcinoma with glandular differentiation [UCg]) are rare malignancies with histological resemblance to colorectal adenocarcinoma (CORAD) in the majority of this subgroup. Definite case numbers are very low, molecular data are limited and the pathogenesis remains poorly understood. Therefore, this study was designed to complement current knowledge by in depth analysis of BAC (n = 12), UAC (n = 13), UCg (n = 11) and non-invasive glandular lesions (n = 19). In BAC, in addition to known alterations in TP53, Wnt, MAP kinase and MTOR pathway, mutations in SMAD4, ARID1A and BRAF were identified. Compared to published data on muscle invasive bladder cancer (BLCA) and CORAD, UCg exhibited frequent “urothelial” like alterations while BAC and UAC were characterised by a more “colorectal” like mutational pattern. Immunohistochemically, there was no evidence of DNA mismatch repair deficiency or PD-L1 tumour cell positivity in any sample. Depending on the used antibody 0–45% of BAC, 0–30% of UCg and 0% UAC cases exhibited PD-L1 expressing tumour associated immune cells. A single BAC (9%, 1/11) showed evidence of ARID1A protein loss, and two cases of UCg (20%, 2/10) showed loss of SMARCA1 and PBRM1, respectively. Taken together, our data suggest at least in part involvement of similar pathways driving tumourigenesis of adenocarcinomas like BAC, UAC and CORAD independent of their tissue origin. Alterations of TERT and FBXW7 in single cases of intestinal metaplasia further point towards a possible precancerous character in line with previous reports.

Urothelial carcinoma (UC) is an aggressive malignancy and has a poor prognosis in the metastatic state. Treatment of UC remains a challenge, and as a first-line regimen for advanced UC, standard platinum-based chemotherapy is unfit for many patients due to numerous comorbidities and poor performance status. Recently, five immune checkpoint inhibitors have been approved for the treatment of patients with advanced UC who were ineligible for platinum-based regimens or suffered tumor progression in post-platinum setting. However, not long ago, the U.S. Food and Drug Administration restricted the use of two common immune checkpoint blockades, atezolizumab and pembrolizumab, due to uncertain survival benefit as mono-therapy. In this scenario, we reviewed rapidly surfacing clinical trials to assess the efficacy and safety of immunotherapy targeting the PD-1 pathway for advanced UC. A comprehensive search was conducted in PubMed, EMBASE and Cochrane Library for all clinical trials where the efficacy and safety were reported. Our primary outcome was efficacy evaluated by objective response rate (ORR), 1-year overall survival (OS) rate and 1-year progression-free survival (PFS) rate, and second outcome was safety assessed by any grade and grade 3–4 treatment-related adverse events (TRAEs). We chose percentages with 95% confidence intervals (CI) as the evaluation indexes and used a random-effects model to account for heterogeneity. We included 14 clinical trials with 2674 total patients in this meta-analysis. After removing unqualified studies on the basis of sensitivity analyses, 13 studies were pooled to evaluate the overall ORR, 8 studies for the 1-year OS rate and 6 studies for the 1-year PFS rate. The pooled data of ORR, 1-year OS rate, and 1-year PFS rate were 0.20 (95% CI 0.18–0.22, I2 = 38.4%, P = 0.078), 0.50 (95% CI 0.46–0.53, I2 = 30.3%, P = 0.186), and 0.17 (95% CI 0.14–0.20, I2 = 0.0%, P = 0.668), respectively. Similarly, 13 trials were utilized to compute the pooled rate of any-grade TRAEs. The pooled estimation of any-grade was 0.65 (95% CI 0.63–0.67, I2 = 1.7%, P = 0.429). The pooled rate of grade 3–4 TRAEs subgroups with Atezolizumab, Pembrolizumab, Durvalumab, Nivolumab and Avelumab were 0.11 (95% CI 0.06–0.15, I2 = 83.5%, P = 0.000), 0.15 (95% CI 0.13–0.18, I2 = 0.0%, P = 0.971), 0.06 (95% CI 0.03–0.09, I2 = 0.0%, P = 0.566), 0.19 (95% CI 0.15–0.23, I2 = 0.0%, P = 0.480) and 0.08 (95% CI 0.05–0.11, I2 = 0.0%, P = 0.702), respectively. This study showed that the immunotherapy targeting the PD-1 pathway had durable efficacy and acceptable safety in patients with advanced UC. The comprehensive role of immune checkpoint inhibitors in comparison to other treatments needs further confirmation basing on RCTs.

In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.

Background Upper urinary tract urothelial carcinoma (UTUC) accounts for only about 5–10% of all urothelial cancers and is characterized by an aggressive and frequently rapidly fatal behavior. However, detailed knowledge of its molecular profile is still lacking. Materials and methods We identified, by chart analysis, patients who underwent radical nephroureterectomy or diagnostic biopsy for UTUC between January 2015 and August 2020 at the Santa Maria Hospital of Terni, in Italy. Eligible patients were required to have also adequate clinical informations and follow-up details. The primary objective of the study was to evaluate DNA mismatch repair (MMR) proteins and Nectin-4 immunohistochemical expression in UTUC, looking also for an eventual correlation between these molecular features. The secondary objective was to investigate genomic instability in the case of a MMR protein loss. Expression of proteins was assessed by using immunohistochemistry and microsatellite instability (MSI) performed by next generation sequencing. Nectin-4 expression was reported using an intensity scoring system (score, 0–3+), instead the expression of DNA MMR proteins was indicated as present (no loss) or not present (loss). Results Thirty four cases have been evaluated and 27 considered eligible for the study with their tumor samples analyzed. Nectin-4 was found to be expressed in 44% of cases and 18.5% of patients showed defective-MMR phenotype. We found a significant correlation between Nectin-4 expression and MSH2/MSH6 protein loss. Out of 7 patients with DNA MMR proteins loss or equivocal phenotype, 3 showed MSI. Conclusions Our pilot study suggest a possible relationship between Nectin-4 and DNA MMR protein expression in UTUC and a clinically significant correlation between defective MMR phenotype and genomic instability. Because of the possible implications of these data for innovative treatment approaches, the need for further studies in this area is warranted.

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.

SummaryLocally advanced or metastatic urothelial cancer is an aggressive form of cancer with high recurrence rates and low survival. Nectin-4 is a cell adhesion molecule commonly expressed in several tumors, including high expression in urothelial cancer. Enfortumab vedotin is an antibody–drug conjugate composed of an anti-Nectin-4 humanized monoclonal antibody linked to the microtubule disrupting agent, monomethyl auristatin E. In this phase I study (NCT03070990), Japanese patients with locally advanced/metastatic urothelial cancer treated with prior chemotherapy, or ineligible for cisplatin, were randomized 1:1 to receive 1.0 mg/kg (Arm A) or 1.25 mg/kg (Arm B) enfortumab vedotin on Days 1, 8, and 15 of each 28-day cycle. Assessing the pharmacokinetic and safety/tolerability profiles of enfortumab vedotin were primary objectives; investigator-assessed antitumor activity (RECIST v1.1) was a secondary objective. Seventeen patients (n = 9, Arm A; n = 8, Arm B) received treatment. Pharmacokinetic data suggest a dose-dependent increase in enfortumab vedotin maximum concentration and area under the concentration–time curve at Day 7. Enfortumab vedotin was well tolerated across both doses. Dysgeusia and alopecia (n = 9 each) were the most common treatment-related adverse events. Regardless of attribution, grade ≥ 3 adverse events occurring in ≥2 patients were anemia and hypertension (n = 2 each). One patient achieved a confirmed complete response (Arm A) and five achieved confirmed partial responses (n = 3, Arm A; n = 2, Arm B). Objective response and disease control rates were 35.3% and 76.5%, respectively. In Japanese patients with locally advanced/metastatic urothelial cancer, enfortumab vedotin is well tolerated with preliminary antitumor activity and a pharmacokinetic profile consistent with prior reports.

The recent genomic characterization of urothelial carcinoma by the Cancer Genome Atlas Project, made possible by the introduction of high throughput, reduced cost, and sequence analysis, has shed new insights on the biology of advanced disease. In addition, studies on imaging of advanced urothelial carcinoma have widened the knowledge on disease presentation and on pattern of metastatic spread and their correlation with the underlying biology of urothelial carcinoma. The wide range of treatments for advanced urothelial cancer, including combined chemotherapy regimens and immune checkpoint inhibitors, each result in treatment class-specific patterns of response and adverse events. Results of studies point to the need for a reliable biomarker, perhaps with imaging, that predicts prognosis and treatment response to systemic treatment, and can be used to select the most effective treatment while minimizing toxicity. This review of advanced urothelial cancer introduces the latest advances in genetic profiling, the current role of imaging, the radiographic appearance of treatment response and their toxicities, and details potential future areas of imaging research.

The Cancer Genome Atlas project introduced genomic taxonomy of basal and luminal molecular subtypes in muscle invasive bladder cancer. Fewer studies have addressed the molecular classification in non-muscle invasive bladder cancer (NMIBC). Our aim is to assess the applicability of the proposed phenotypic classification for NMIBC. Three TMAs were constructed from 193 TURBT specimens of 60 bladder cancer patients treated at one of the authors' institutions (1998-2008). Follow-up data on recurrence, grade, or stage progression was obtained. Immunohistochemistry was performed using an automated Ventana System for markers indicative of luminal (GATA3, CK20, ER, Uroplakin II, and HER2/neu) and basal (CK5/6 and CD44) phenotype. Marker expression was evaluated by 3 urologic pathologists. Using unadjusted logistic regression, we found significant association between tumor recurrence at next biopsy and CD44 expression (OR = 2.51, P = 0.03), tumor recurrence at any subsequent biopsy and ER expression (OR = 0.24, P = 0.04), and tumor grade progression at any subsequent biopsy and HER2/neu expression (OR = 0.24, P = 0.04). After adjusting for pathologic stage, we found a significant association between CK5/6 expression and tumor stage progression at either next or any subsequent biopsy (OR = 0.94, P = 0.006; and OR = 0.97, P = 0.02, respectively). Our findings suggest that individual immunohistochemical markers may be of value as prognostic factors in NMIBC.

The aim of our study was to perform comparative investigation of the tissue safety of three different endoscopic lithotripter devices including a new single-probe/dual-energy lithotripter in an in vivo animal model. The Swiss LithoClast Trilogy was compared to the Storz Calcuson and the Swiss LithoClast Vario. The safety test simulated the accidental direct contact between lithotripter probes and the urothelium, which can occur when sliding off a stone or drilling through a calculus during lithotripsy. The safety test included a smallest (1.5 mm) and largest (3.3/3.4 mm) probe diameter per device. Testing was performed in nine pigs (three animals per device). The bladder tissue was exposed to direct lithotripter probe contact at maximum power for 10 s to produce visible tissue lesions. Acute tissue trauma was evaluated using a simplified scoring model describing the expected bladder wall injuries for histological examination. After 7 days, all animals were killed, necropsied and examined post mortem. For between-group comparisons regarding microscopic histopathologic features, a Chi-square test was used. A p value

BACKGROUND: Urinary tract is subjected to a variety of disorders such as urethral stricture, which often develops as a result of scarring process. Urethral stricture can be treated by urethral dilation and urethrotomy; but in cases of long urethral strictures, substitution urethroplasty with genital skin and buccal mucosa grafts is the only option. However a number of complications such as infection as a result of hair growth in neo-urethra, and stone formation restrict the application of those grafts. Therefore, tissue engineering techniques recently emerged as an alternative approach, aiming to overcome those restrictions. The aim of this review is to provide a comprehensive coverage on the strategies employed and the translational status of urethral tissue engineering over the past years and to propose a combinatory strategy for the future of urethral tissue engineering. METHODS: Data collection was based on the key articles published in English language in years between 2006 and 2018 using the searching terms of urethral stricture and tissue engineering on PubMed database. RESULTS: Differentiation of mesenchymal stem cells into urothelial and smooth muscle cells to be used for urologic application does not offer any advantage over autologous urothelial and smooth muscle cells. Among studied scaffolds, synthetic scaffolds with proper porosity and mechanical strength is the best option to be used for urethral tissue engineering. CONCLUSION: Hypoxia-preconditioned mesenchymal stem cells in combination with autologous cells seeded on a pre-vascularized synthetic and biodegradable scaffold can be said to be the best combinatory strategy in engineering of human urethra.

Urothelial cells contribute to bladder functions, including urine storage, urine emptying, and innate immune response. Functional studies of urothelial cells usually use either freshly isolated cells or cultured cells. Most methods of isolating urothelial cells require enzymes; however, these techniques remove proteins that connect the cells and disrupt the orientation of the cells within the multilayered urothelium. In addition, PCR or immunoblot results obtained from homogenates of bladder mucosa or whole bladder do not represent pure urothelial cells. We describe a dissection process that does not require enzymes and is able to obtain pure urothelial tissues from mice and humans. This method can isolate single urothelial cells for electrophysiology in situ and can also isolate pure urothelial tissue for PCR, microarray, and immunoblot procedures. The time required to obtain urothelial tissue from one mouse bladder is 15-20 min. This method is simple and time efficient as compared with alternative methods and therefore facilitates our understanding of urothelial biology.

To evaluate the significance of the presence or absence of an “inchworm sign” on DWI for the recurrence and progression of T1 bladder cancer. We retrospectively analyzed 91 patients with pT1 urothelial carcinoma who underwent DWI prior to transurethral resection between 2007 and 2016. DWI of the dominant tumors was scrutinized for inchworm signs at b = 1000 s/mm2. The association of the presence of the inchworm sign with progression and recurrence was analyzed; progression was defined as recurrence to stage T2 or higher and/or N+, and/or M1. An inchworm sign was seen in 65 cases (71%), while it was absent in 26 cases. Among the 65, 25 (38%) had confirmed tumor recurrence, while in the remaining 26, 14 (54%) had confirmed recurrence (median time post TURB = 7.9 and 10.1 months for each). At the time of recurrence, the tumor had progressed in one (2%) inchworm-sign-positive and seven (27%) inchworm-sign-negative cases. The progression rate of inchworm-sign-negative cases was significantly higher than that of inchworm-sign-positive cases (hazard ratio = 17.2, p = 0.0017), whereas there was no significant difference in the recurrence rate between two groups. The absence of an inchworm sign and histological grade 3 were independent risk factors for progression (p

The aims of the study were to determine the effectiveness of blebbistatin (BLEB) on detrusor overactivity (DO) in an animal model induced by retinyl acetate (RA) and, because of potential urothelial permeability, to evaluate the degenerative impact of BLEB on the urothelium. Three days after RA instillation into the urinary bladder, BLEB was administered into the bladder and immediately after cystometric assessment was performed. Furthermore, Evans Blue extravasation into bladder tissue and urothelium thickness were measured. Sixty female Wistar rats were used and randomly assigned to one of four groups (n = 15 in each group): (1) control, (2) RA, (3) BLEB, and (4) RA + BLEB. RA administration induced changes in cystometric parameters reflecting DO, as previously reported. Treatment with BLEB did not significantly alter cystometric parameters in rats which did not receive RA. Administration of BLEB to rats pretreated with RA reversed changes in cystometric parameters induced by RA in basal pressure, threshold pressure, detrusor overactivity index, amplitude of nonvoiding contractions, frequency of nonvoiding contractions, voided volume, volume threshold, intercontraction interval, bladder compliance, and volume threshold to elicit nonvoiding contractions. There were no significant differences in Evans Blue extravasation into bladder tissue or urothelium thickness between the groups. The current research provides new data on the possible utility of blebbistatin in the pharmacotherapy of DO, which is an important feature of overactive bladder (OAB). Further studies in human patients with DO/OAB are warranted to confirm these preclinical results.

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.

Background Screening across a multitude of normal and malignant tissues revealed an enhanced expression of lymphocyte antigen 6 superfamily member D (LY6D) in squamous epithelium and urothelium, as well as in malignancies derived therefrom. The aim of this study was to further delineate the protein expression of LY6D in urothelial bladder cancer, with particular attention to its relationship with clinicopathological characteristics and patient outcome. Methods Immunohistochemical expression of LY6D was assessed in tissue microarrays with urothelial bladder cancer tumours from three independent patient cohorts; one with transurethral resection of the bladder (TURB) specimens of mixed tumour stages from 110 consecutive cases, one with tumours of mixed stages from 260 incident cases in a population-based cohort, and one with paired TURB specimens, resected tumours and a subset of lymph node metastases from 145 patients with muscle-invasive bladder cancer (MIBC). Chi-square and non-parametric tests were applied to examine associations of LY6D expression with clinicopathological characteristics. Kaplan-Meier and Cox regression analyses were applied to examine 5-year overall survival (OS) and recurrence free survival (RFS) in relation to LY6D expression. Results In the two cohorts with mixed stages, positive LY6D expression was denoted in 63 and 64% of the cases, respectively, and found to be significantly higher in low-grade and less invasive tumours. Negative LY6D expression was significantly associated with a reduced 5-year OS, although not independently of established prognostic factors. In the population-based cohort, LY6D expression was higher in tumours with squamous differentiation and lower in other variant histologies compared to pure urothelial tumours, and the association of LY6D expression with survival was somewhat enhanced after exclusion of the former. LY6D expression was generally lower in the MIBC cohort, and even more reduced in resected tumours compared to TURB specimens in patients who had not received neoadjuvant chemotherapy. There were no significant associations between LY6D expression and RFS, neither allover nor in relation to neoadjuvant chemotherapy. Conclusion LY6D is a marker of urothelial and squamous differentiation that may add useful diagnostic and prognostic information to better guide the clinical management of bladder cancer, given that the presence of variant histology is taken into account.

Background 5α-reductase inhibitors (5-ARIs) inhibit the pathway of converting the testosterone to dihydrotestosterone and are widely used in benign prostatic hyperplasia patients. Since androgen receptor activation may play a role in urothelial tumorigenesis, we conducted this retrospective cohort study to determine whether 5α-reductase inhibitors (5-ARIs) administration is associated with bladder cancer mortality, bladder cancer recurrence and upper tract urothelial carcinoma mortality, using the Taiwan National Health Insurance database. Methods The data of this retrospective cohort study were sourced from the Longitudinal Health Insurance Database of Taiwan, compiled by the Taiwan National Health Insurance database from 1996 to 2010. It consists of 18,530 men with bladder cancer, of whom 474 were 5-ARIs recipients and 4384 men with upper tract urothelial carcinoma, of whom 109 were 5-ARIs recipients. Propensity Score Matching on the age and geographic data was done at the ratio of 1:10. We analyzed the odds ratios (OR) and 95% confidence interval (CI) of the risk of bladder cancer death, bladder cancer recurrence rate and upper tract urothelial carcinoma related death by the 5-ARIs administration. Results Those who received 5-ARIs showed a lower risk of bladder cancer related death compared to nonusers in multivariable adjusted analysis (OR 0.835, 95% CI 0.71–0.98). However, there was no significant difference in the bladder cancer recurrence rate (OR 0.956, 95% CI 0.82–1.11) and upper tract urothelial carcinoma related mortality in multivariable adjusted analysis (OR 0.814, 95% CI 0.6–1.1). Conclusions Patients who receive 5-ARIs have lower bladder cancer related mortality compared to those who don’t. 5-ARIs may prove to be a viable strategy to improve bladder cancer outcomes.

Background Transurethral resection of the bladder tumour (TUR) is associated with a risk of bladder perforation. The underlying mechanisms and risk factors are not fully understood. The aim of this study was to determine if the bladder wall structure affects the risk of bladder perforation during TUR. Methods Fifteen patients who underwent TUR complicated by a bladder perforation (group 1) and fifteen matched controls who underwent uncomplicated TUR (group 2) were retrospectively enrolled in this morphological analysis. Surgical specimens were collected from all participating patients to describe the quality and architecture of urothelium and bladder submucosa. Immunohistochemical studies were performed with primary mouse anti-human E-cadherin, beta-catenin, type IV collagen, cytokeratin 20 and epithelial membrane antigen antibodies. The intensity of the immunohistochemical reaction was assessed using an immunoreactive score (IRS). Ultrastructural examinations were performed by transmission electron microscopy. The microscopic assessment was focused on the intensity of fibrosis in the bladder submucosa and the presence of degenerative changes in the urothelium. Results Patients’ age, sex distribution, tumour diameters, surgeon experience or cancer stage did not differ between study groups. The immunohistochemical analysis did not reveal statistically significant differences between group 1 and group 2. From a clinical point of view, ultrastructural analysis by electron microscopy showed a higher rate of severe fibrosis in group 1 (63.6% vs. 38.5%), with no differences in the rate and degree of urothelial changes. However, these differences were not statistically significant (p = 0.32). Conclusions Bladder perforation during TUR is not a result of a deficient structure of the bladder wall. Based on available evidence, the surgical technique seems to play the most important role in its prevention.

Background Bladder cancer (BC) is a common and deadly disease. Over the past decade, a number of genetic alterations have been reported in BC. Bladder urothelium expresses abundant urea transporter UT-B encoded by Slc14a1 gene at 18q12.3 locus, which plays an important role in preventing high concentrated urea-caused cell injury. Early genome-wide association studies (GWAS) showed that UT-B gene mutations are genetically linked to the urothelial bladder carcinoma (UBC). In this study, we examined whether Slc14a1 gene has been changed in UBC, which has never been reported. Case presentation A 59-year-old male was admitted to a hospital with the complaint of gross hematuria for 6 days. Ultrasonography revealed a size of 2.8 × 1.7 cm mass lesion located on the rear wall and dome of the bladder. In cystoscopic examination, papillary tumoral lesions 3.0-cm in total diameter were seen on the left wall of the bladder and 2 cm to the left ureteric orifice. Transurethral resection of bladder tumor (TURBT) was performed. Histology showed high-grade non-muscle invasive UBC. Immunostaining was negative for Syn, CK7, CK20, Villin, and positive for HER2, BRCA1, GATA3. Using a fluorescence in situ hybridization (FISH), Slc14a1 gene rearrangement was identified by a pair of break-apart DNA probes. Conclusions We for the first time report a patient diagnosed with urothelial carcinoma accompanied with split Slc14a1 gene abnormality, a crucial gene in bladder.

Background Relaxin is an endogenous protein that has been shown to have antifibrotic properties in various organ systems. There has been no characterization of relaxin’s role in the human bladder. Our objective was to characterize relaxin receptor expression in the human bladder and assess relaxin’s effect on tissue remodeling/fibrosis pathways in bladder smooth muscle cells. Methods Relaxin family peptide receptor 1 (RXFP1) and RXFP2 expression was assessed using quantitative reverse transcriptase-PCR (qRT-PCR) and immunohistochemistry (IHC) on primary bladder tissue. Primary human smooth muscle bladder cells were cultured and stimulated with various concentrations of relaxin. Western blot, qRTPCR, ELISA, and zymogram assays were used to analyze fibrosis/tissue remodeling pathway proteins. Results There was universal mRNA transcript detection and protein expression of relaxin receptors in primary bladder specimens. Immunohistochemistry demonstrated RXFP1 and RXFP2 localizing to both urothelial and smooth muscle cell layers of the bladder. 24 h of in vitro relaxin stimulation did not affect mRNA expression of selected proteins in human bladder smooth muscle cells. However, 48 h of in vitro relaxin stimulation resulted in upregulation of active (p = 0.004) and latent (p = 0.027) MMP-2 in cell lysate, and upregulation of active MMP-2 in supernatant (p = 0.04). There was a dose dependent relationship with increasing expression of MMP-2 with increasing relaxin concentration. Relaxin stimulation resulted in decreased levels of active and total TGF-β1 in supernatant and extracellular matrix (p Conclusions In the human bladder, relaxin receptors are expressed at the dome and trigone and localize to the urothelium and smooth muscle cell layers. Stimulation of human bladder SMCs with relaxin in vitro affects expression of MMP-2 and TGF-β1.

Tissue engineering allows to combine biomaterials and seeded cells to experimentally replace urinary bladder wall. The normal bladder wall however, includes branched neuronal network propagating signals which regulate urine storage and voiding. In this study we introduced a novel biocomposite built from amniotic membrane (Am) and graphene which created interface between cells and external stimuli replacing neuronal network. Graphene layers were transferred without modifying Am surface. Applied method allowed to preserve the unique bioactive characteristic of Am. Tissue engineered constructs composed from biocomposite seeded with smooth muscle cells (SMC) derived from porcine detrusor and porcine urothelial cells (UC) were used to evaluate properties of developed biomaterial. The presence of graphene layer significantly increased electrical conductivity of biocomposite. UCs and SMCs showed an organized growth pattern on graphene covered surfaces. Electrical filed stimulation (EFS) applied in vitro led additionally to increased SMCs growth and linear arrangement. 3D printed chamber equipped with 3D printed graphene based electrodes was fabricated to deliver EFS and record pressure changes caused by contracting SMCs seeded biocomposite. Observed contractile response indicated on effective SMCs stimulation mediated by graphene layer which constituted efficient cell to biomaterial interface.

To evaluate the survival benefits that lymph node dissection (LND) brought to clinically node-negative upper tract urothelial carcinoma (UTUC) patients. Non-metastatic node-negative UTUC patients were identified from the Surveillance, Epidemiology and End Results database. N0 patients were naturally divided as cN0-pNx group (clinically diagnosed as N0 without LND performed) and cNx-pN0 group (pathologically diagnosed as node-negative no matter what clinical node status they have). Of the 2731 patients included, 2240 and 491 cases were cN0-pNx and cNx-pN0, respectively. The overall survival (OS) of cNx-pN0 patients was significantly better than that of cNx-pN0 patients (p = 0.022). After propensity score matching, the survival of cNx-pN0 patients was still significantly better than cN0-pNx group. Besides, multivariate analyses showed cNx-pN0 (received LND) was an independent favorable prognostic factor for OS and CSS compared with cN0-pNx (no LND). Survival advantages of pN0 group were more significant in ≥ T2 patients and patients with tumor size ≤ 5 cm. Even in N0 patients who received adjuvant treatment, LND still brought obvious survival improvement (HRos = 0.565, p = 0.013; HRcss = 0.607, p = 0.046). LND could improve survival outcomes in patients with clinically node-negative UTUC, especially for those with muscle-invasive diseases (T2–4 stages) or smaller tumor size (≤ 5 cm). Adjuvant treatment after nephroureterectomy is incapable of replacing the therapeutic role of LND.

As part of its single technology appraisal (STA) process, the National Institute for Health and Care Excellence (NICE) invited the manufacturer (Bristol-Myers Squibb) of nivolumab (Opdivo (R)) to submit evidence of its clinical and cost effectiveness for metastatic or unresectable urothelial cancer. Kleijnen Systematic Reviews Ltd, in collaboration with Maastricht University Medical Centre+, was commissioned to act as the independent Evidence Review Group (ERG), which produced a detailed review of the evidence for the clinical and cost effectiveness of the technology, based on the company's submission to NICE. Nivolumab was compared with docetaxel, paclitaxel, best supportive care and retreatment with platinum-based chemotherapy (cisplatin plus gemcitabine, but only for patients whose disease has had an adequate response in first-line treatment). Two ongoing, phase I/II, single-arm studies for nivolumab were identified, but no studies directly compared nivolumab with any specified comparator. Evidence from directly examining the single arms of the trial data indicated little difference between the outcomes measured from the nivolumab and comparator studies. A simulated treatment comparison (STC) analysis was used in an attempt to reduce the bias induced by naive comparison, but there was no clear evidence that risk of bias was reduced. Multiple limitations in the STC were identified and remained. The effect of an analysis based on different combinations of covariates in the prediction model remains unknown. The ERG's concerns regarding the economic analysis included the use of a non-established response-based survival analysis method, which introduced additional uncertainty. The use of time-dependent hazard ratios produced overfitting and was not represented in the probabilistic sensitivity analysis. The use of a treatment stopping rule to cap treatment cost left treatment effectiveness unaltered. A relevant comparator was excluded from the base-case analysis. The revised ERG deterministic base-case incremental cost-effectiveness ratios based on the company's Appraisal Consultation Document response were 58,791 pound, 78,869 pound and 62,352 pound per quality-adjusted life-year gained versus paclitaxel, docetaxel and best supportive care, respectively. Nivolumab was dominated by cisplatin plus gemcitabine in the ERG base case. Substantial uncertainties about the relative treatment effectiveness comparing nivolumab against all comparators remained. NICE did not recommend nivolumab, within its marketing authorisation, as an option for treating locally advanced, unresectable or metastatic urothelial carcinoma in adults who have had platinum-containing therapy, and considered that nivolumab was not suitable for use within the Cancer Drugs Fund.

The majority of bladder cancers in humans are non-muscle-invasive cancers that recur frequently after standard treatment procedures. Mouse models are widely used to develop anti-tumor treatments. The purpose of our work was to establish an orthotopic mouse bladder tumor model and to explore early stages of implantation of cancerous MB49 cells in vivo using various labeling and microscopic techniques. To distinguish cancer cells from normal urothelial cells in mouse urinary bladders, we performed molecular characterization of MB49 cells before intravesical injection experiments. In this new approach we applied internalized metal nanoparticles to unequivocally discriminate cancer cells from normal cells. This method revealed that cancer cells attached to the urothelium or basal lamina within just 1 hour of intravesical injection, whereas small tumors and localized hyperplastic urothelial regions developed within two days. We found that cancer cells initially adhere to normal urothelial cells through filopodia and by focal contacts with basal lamina. This is the first in vivo characterization of intercellular contacts between cancerous and normal urothelial cells in the bladder. Our study yields new data about poorly known early events of tumorigenesis in vivo, which could be helpful for the translation into clinic.

Ureteric stents have become an indispensable tool in the armamentarium of every urologist. However, they carry their own morbidity resulting mostly from infectious or abacterial fouling and biofilm formation, and/or urothelial hyperplastic reaction. All of these may interact and lead to clinical complications. Many different stent designs and coatings have been proposed. In this study, we focused on the effect of paclitaxel-coated stents on hyperplastic proliferation of ureteral tissue, using as example anastomotic strictures after ureteroureterostomy in a rat model. Human urothelial cells (SV-HUC-1) were used to determine paclitaxel dosages in vitro. Polyurethane stents were coated with a paclitaxel containing biodegradable polymer and studied in a ureteroureterostomy rat model. 48 male 9-week-old Sprague–Dawley rats underwent either sham surgery (n = 16) or ureteroureterostomy with sutured anastomosis, and consecutive stenting with either a paclitaxel-coated or an uncoated stent (16 per group), respectively. The animals received daily intraperitoneal injections of 5-bromo-2-deoxyuridine (20 mg/ml, 100 mg/kg body weight) during the first eight postoperative days, and were sacrificed on day 28. Healing of the ureteral anastomosis and proliferation of urothelial cells was examined histologically and immunohistochemically. In vitro, a concentration of 10 ng/mm2 paclitaxel can be considered as non-toxic, while still exerting an anti-proliferative effect on urothelial cells. Histologically, typical wound healing processes were seen at the site of the ureteral anastomosis in vivo. Proliferation of urothelial cells was significantly lower in animals with paclitaxel-coated stents compared to those with uncoated stents (LI 41.27 vs. 51.58, p

PURPOSE: Transient receptor potential cation channel subfamily M member 4 (TRPM4) has been shown to play a key role in detrusor contractility under physiological conditions. In this study we investigated the potential role of TRPM4 in detrusor overactivity following spinal cord transection (SCT) in mice. METHODS: TRPM4 expression and function were evaluated in bladder tissue with or without the mucosa from spinal intact (SI) and SCT female mice (T8-T9 vertebra; 1–28 days post SCT) using PCR, western blots, immunohistochemistry and muscle strip contractility techniques. RESULTS: TRPM4 was expressed in the urothelium (UT) and detrusor smooth muscle (DSM) and was upregulated after SCT. Expression levels peaked 3–7 days post SCT in both the UT and DSM. Pharmacological block of TRPM4 with the antagonist, 9-Phenanthrol (30 µM) greatly reduced spontaneous phasic activity that developed after SCT, regardless of the presence or absence of the mucosa. CONCLUSIONS: Detrusor overactivity following spinal cord injury leads to incontinence and/or renal impairment, and represents a major health problem for which current treatments are not satisfactory. Augmented TRPM4 expression in the bladder after chronic SCT supports the hypothesis that TRPM4 channels play a role in DSM overactivity following SCT. Inhibition of TRPM4 may be beneficial for improving detrusor overactivity in SCI.

Pituitary adenylate cyclase-activating polypeptide (PACAP; AdcyaP1) and its cognate PAC1 receptor (Adcyap1r1), have tissue-specific distributions in the lower urinary tract (LUT). The afferent limb of the micturition reflex is often compromised following bladder injury, disease and inflammatory conditions. We have previously demonstrated that PACAP signaling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Thus, the present studies investigated the sensory components (e.g., urothelial cells, bladder afferent nerves) of the urinary bladder that may underlie the pathophysiology of aberrant PACAP activation. We utilized bladder-pelvic nerve preparations and urothelial sheet preparations to characterize PACAP-induced bladder afferent nerve discharge with distention and PACAP- induced Ca(2+) activity, respectively. We determined that PACAP38 (100 nM) significantly (p ≤ 0.01) increased bladder afferent nerve activity with distention that was blocked with a PAC1/VPAC2 receptor antagonist PACAP6–38 (300 nM). PACAP38 (100 nM) also increased Ca(2+) activity in urothelial cells over that observed in control preparations. Taken together, these results establish a role for PACAP signaling in bladder sensory components (e.g., urothelial cells, bladder afferent nerves) that may ultimately facilitate increased voiding frequency.

The epithelial inner layer of the lower urinary tract, i.e., the urothelium, and other parts of the mucosa are not just a passive barrier but play an active role in the sensing of stretching, neurotransmitters, paracrine mediators, hormones, and growth factors and of changes in the extracellular environment. We review the molecular and cellular mechanisms enabling the urothelium to sense such inputs and how this leads to modulation of smooth muscle contraction and relaxation. The urothelium releases various mediators including ATP, acetylcholine, prostaglandins, nitric oxide, and nerve growth factor. These may affect function and growth of smooth muscle cells and afferent nerves. However, the molecular identity of the urothelium-derived mediator directly modulating contractile and relaxant responses of isolated bladder strips has remained elusive. The morphology and function of the urothelium undergo changes with aging and in many pathophysiological conditions. Therefore, the urothelium may contribute to the therapeutic effects of established drugs to treat lower urinary tract dysfunction and may also serve as a target for novel therapeutics. However, therapeutics may also change urothelial function, and it is not always easy to determine whether such changes are part of the therapeutic response or reflect secondary alterations.