Difference in electron microscopic findings among interstitial cystitis/bladder pain syndrome with distinct clinical and cystoscopic characteristics.

Urothelial dysfunction may be a key pathomechanism underlying interstitial cystitis/bladder pain syndrome (IC/BPS). We therefore examined if clinical severity is associated with the extent of urothelial damage as revealed by electron microscopic (EM) analysis of biopsy tissue. One hundred IC/BPS patients were enrolled and 24 patients with stress urinary incontinence served as controls. Clinical symptoms were evaluated by visual analog scale pain score and O'Leary-Sant Symptom score. Bladder biopsies were obtained following cystoscopic hydrodistention. The presence of Hunner's lesions and glomerulation grade after hydrodistention were recorded and patients classified as Hunner-type IC (HIC) or non-Hunner-type IC (NHIC). HIC patients exhibited more severe defects in urothelium cell layers, including greater loss of umbrella cells, umbrella cell surface uroplakin plaque, and tight junctions between adjacent umbrella cells, compared to control and NHIC groups (all p < 0.05). Both NHIC and HIC groups demonstrated more severe lamina propria inflammatory cell infiltration than controls (p = 0.011, p < 0.001, respectively). O'Leary-Sant Symptom scores were significantly higher among patients with more severe urothelial defects (p = 0.030). Thus, urothelium cell layer defects on EM are associated with greater clinical symptom severity.

How Cancer Cells Invade Bladder Epithelium and Form Tumors: The Mouse Bladder Tumor Model as a Model of Tumor Recurrence in Patients.

Non-muscle-invasive bladder cancer is the most common form of bladder cancer. The main problem in managing bladder tumors is the high recurrence after the transurethral resection of bladder tumors (TURBT). Our study aimed to examine the fate of intravesically applied cancer cells as the implantation of cancer cells after TURBT is thought to be a cause of tumor recurrence. We established an orthotopic mouse bladder tumor model with MB49-GFP cancer cells and traced them during the first three days to define their location and contacts with normal urothelial cells. Data were obtained by Western blot, immunolabeling, and light and electron microscopy. We showed that within the first two hours, applied cancer cells adhered to the traumatized epithelium by cell projections containing α3ß1 integrin on their tips. Cancer cells then migrated through the epithelium and on day 3, they reached the basal lamina or even penetrated it. In established bladder tumors, E-cadherin and desmoplakin 1/2 were shown as feasible immunohistochemical markers of tumor margins based on the immunolabeling of various junctional proteins. Altogether, these results for the first time illustrate cancer cell implantation in vivo mimicking cellular events of tumor recurrence in bladder cancer patients.

Assessment of Urothelial Cytotoxicity at Morphological and Molecular Levels: Urinary Bladder as Target Organ.

The urinary bladder is a target organ of several toxic agents. Exposure to those agents induces mild-to-severe changes, which can be evaluated by different methods. Among them, the scanning-electron microscopy (SEM) is the "gold standard" for characterizing urothelial damage since it provides high-definition images, making it possible to detect early lesions on the surface of the urinary bladder. In addition, molecular technologies allow detecting changes in genetic material and investigating the interaction between genes and environmental stress in disease causation. The urinary bladder epithelium is where the most common type of bladder cancer occurs in humans, that is, the transitional-cell carcinoma (TCC). In animal models, the TCC can be similar to the disease in humans. Techniques to evaluate urothelium in experimental models aid in the comprehension of risk factors for urothelial carcinogenesis.

Cell proliferation of rat bladder urothelium induced by nicotine is suppressed by the NADPH oxidase inhibitor, apocynin.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis in a two-stage carcinogenesis model. Nicotine also induced cytotoxicity in the bladder urothelium in a short-term study. In the present study, male rats were treated with nicotine (40 ppm) in drinking water co-administered with the NADPH oxidase inhibitor, apocynin (0, 250 or 750 mg/kg) in diet for 4 weeks. The apocynin treatment induced no clinical toxic effects. Reduction of reactive oxygen species (ROS) by apocynin was confirmed by immunohistochemistry of 8-OHdG in the bladder urothelium. Incidences of simple hyperplasia, cell proliferation and apoptosis were reduced by apocynin treatment in the bladder urothelium. However, despite reduction of cell proliferation (labeling index), apocynin did not affect the incidence of simple hyperplasia, apoptosis, or ROS generation in the kidney pelvis urothelium, in addition to 8-OHdG positivity induced by nicotine being lower. In vitro, apocynin (500 µM) reduced ROS generation, but induced cell proliferation in bladder cancer cell lines (T24 and UMUC3 cells). These data suggest that oxidative stress may play a role in the cell proliferation of the bladder urothelium induced by nicotine.

Urinary crystal formation and urothelial effects of pyroxasulfone administered to male rats.

Pyroxasulfone induced a low incidence of urinary bladder tumors in male rats in a 2-year bioassay at 1000 and 2000 ppm, with occasional urinary calculi. No increased incidence of tumors of any tissue occurred in female rats or in mice of either gender. We performed three short-term studies to evaluate early development of pyroxasulfone-induced urinary crystals and urothelial cytotoxicity with consequent regenerative proliferation. First, male rats were treated with dietary 50, 1000 or 2000 ppm pyroxasulfone for 1, 3 or 7 days. The urothelium was examined by light and scanning electron microscopy (LM, SEM) and bromodeoxyuridine labeling index (BrdU LI). In two other studies, male rats were treated with dietary 20 000 ppm pyroxasulfone for 1 week. Urine collected at various times of day was examined by SEM and energy dispersive spectroscopy (EDS) or by LM, SEM, EDS, and infrared spectroscopy (IFS). Urinary crystals were present at various time points. EDS and IFS showed some contained calcium; others contained organic matter. Cytotoxicity was detected by SEM as cellular swelling, craters, and necrosis and by LM as cellular hypertrophy. Increased cell proliferation was detected by LM (hyperplasia), SEM (piling up of round cells), and by increased BrdU LI. There was no evidence of increased apoptosis. These findings support a mode of action for pyroxasulfone-associated bladder tumors in male rats involving formation of urinary crystals leading to urothelial cytotoxicity and regenerative proliferation. This is a high dose phenomenon, therefore, pyroxasulfone is not likely to be carcinogenic to humans at exposure levels that do not cause crystals with subsequent calculi formation in the urinary tract.

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.

Ultrastructural Analysis of Urethral Polyps against the Background of Urogenital Infection.

We performed an electron microscopic study of samples of urethral polyps obtained from 90 women (mean age 52.5±4.9 years). According to PCR and culture studies, the most common infectious agent in patients with urethral polyps is U. urealyticum (100% cases). In 70% cases, this infectious agent was present as monoinfection, of these, clinically significant concentration (>106 CFU/ml) were found in 53.3% cases. In 30% cases, associations with C. trachomatis, T. vaginalis, and M. genitalium were found. We observed significant ultrastructural heterogeneity of the epithelial cells in urethral polyps, which manifested in a combination of hyperplastic and metaplastic changes and signs of cytodestruction. Detection of mycoplasma-like bodies in connective tissue mononuclear cells and viral particles in epithelial cells during ultrastructural study, including cases with negative PCR results, indicates the pathogenetic role of latent infection in the formation of urethral polyps.

Lamina propria: The connective tissue of rat urinary bladder mucosa.

To describe and illustrate the structure of the propria, the bladder of adult rats was fixed in controlled conditions of distension and examined by light and electron microscopy. The lamina propria, ~50 µm thick in the distended bladder, consists of a superficial part (the cellular component), adjacent to the urothelium, rich in nerves, capillaries, fibroblasts and thin bundles of collagen, and a deep, thicker part (the fibrous component), adjacent to the detrusor, rich in large collagen fibres and with few fibroblasts. In the cellular part there is an extensive plexus of afferent nerve fibers and a dense capillary network (with numerous pericytes), lying close to the urothelium, that is unique to the bladder. The main resident cells are fibroblasts, adhering to each other at the end of laminar extensions without forming specialized junctions. The deep part of the lamina propria is made of thick collagen fibers, interwoven and crisscrossing each other, with a few fibroblasts in the interstitial spaces between them. In summary, the superficial part of the lamina propria has most of the bladder afferent nerves, contains many fibroblasts and has a network of suburothelial capillaries. The deep part as a whole forms an ovoid balloon of woven fibrous material that is acted upon by the detrusor musculature attached to its outer surface. The lamina propria is a strong fibrous barrier between urothelium and musculature. The abundance of collagen points to the main role for its fibroblasts, that is, the production of collagen fibrils, assisting the mechanical role of the lamina propria.

Collagen hollow structure for bladder tissue engineering.

Bladder is affected by numerous pathologies which require augmentation or replacement of the organ. Currently, the gold standard is enterocystoplasty which causes many complications. Bioengineering techniques propose options to overcome these issues. The innovative and very simple tissue engineered three-dimensional spherical bladder model reported here mimics the bladder natural shape using collagen-derived scaffold. Bladder mesenchymal cells were embedded inside the scaffold and epithelial cells seeded at its surface. Therefore, the bladder mesenchymal and urothelial cells seeded in the model were subjected to tensions similar to what is found in the native tissue. Both cell types organize themselves simultaneously within a culture period of 15 days. Our spherical model was able to demonstrate characteristics of highly advanced urothelial maturity. Hematoxylin eosin staining, the uroplakins immunodetection and electron microscopy analysis showed the impressive degree of urothelial organization. In addition, collagen remodeling was observed and smooth muscle cells, expressing myosin, presented a tendency to realign parallel to the luminal surface. With properties comparable to native tissue, our three-dimensional spherical bladder model could offer the possibility to produce tissue-engineered bladder implants. This technique could be efficient for partial replacement of pathologic bladder sites.

A non-enzymatic method for dissection of mouse bladder urothelial tissue.

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.

A non-canonical autophagy-dependent role of the ATG16L1T300A variant in urothelial vesicular trafficking and uropathogenic Escherichia coli persistence.

50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs. Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type.

Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells.

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.

Electron microscopic characteristics of interstitial cystitis/bladder pain syndrome and their association with clinical condition.

BACKGROUND: Electron microscopy (EM) characteristics of the urothelium in interstitial cystitis/bladder pain syndrome (IC/BPS) and their association with clinical condition are unclear. METHODS: Ten IC/BPS patients who were admitted for hydrodistention and 5 patients with stress urinary incontinence (control patients) were enrolled. All patients provided detailed clinical histories and underwent urodynamic studies. Cystoscopic bladder biopsies were obtained and processed for transmission EM (TEM) and scanning EM (SEM). The severity of the urothelium findings was graded on a 4-point scale (0: none, 1: mild, 2: moderate, and 3: severe). The EM findings between IC/BPS and control patients were compared; the results were analyzed using the chi-square test. RESULTS: Compared with the urothelium of control patients, the urothelium of IC/BPS patients had more severe defects of the urothelial cell layers and integrity of umbrella cells in TEM (p = 0.045 and 0.01, respectively). In SEM, umbrella cell pleomorphism increased and microplicae of the cell membrane decreased in the IC/BPS group, and both were more severe than in the control group (p = 0.022 and 0.007, respectively). The patients with moderate to severe defects of umbrella cell integrity had more severe bladder pain and smaller maximal bladder capacity (MBC) (both p = 0.010). Patients with moderate to severe defects in microplicae of the cell membrane had smaller cystometric bladder capacity and MBC (p = 0.037 and 0.047, respectively). CONCLUSIONS: The results revealed significant urothelium defects in IC/BPS, especially in the umbrella cells. Defects of umbrella cells may play an important role in the pathogenesis of IC/BPS.

Age-related endolysosome dysfunction in the rat urothelium.

Lysosomal dysfunction is associated with a number of age-related pathologies that affect all organ systems. While much research has focused on neurodegenerative diseases and aging-induced changes in neurons, much less is known about the impact that aging has on lower urinary tract function. Our studies explored age-dependent changes in the content of endo-lysosomal organelles (i.e., multivesicular bodies, lysosomes, and the product of their fusion, endolysosomes) and age-induced effects on lysosomal degradation in the urothelium, the epithelial tissue that lines the inner surface of the bladder, ureters, and renal pelvis. When examined by transmission electron microscopy, the urothelium from young adult rats (~3 months), mature adult rats (~12 months), and aged rats (~26 months old) demonstrated a progressive age-related accumulation of aberrantly large endolysosomes (up to 7µm in diameter) that contained undigested content, likely indicating impaired degradation. Stereological analysis confirmed that aged endolysosomes occupied approximately 300% more volume than their younger counterparts while no age-related change was observed in multivesicular bodies or lysosomes. Consistent with diminished endolysosomal degradation, we observed that cathepsin B activity was significantly decreased in aged versus young urothelial cell lysates as well as in live cells. Further, the endolysosomal pH of aged urothelium was higher than that of young adult (pH 6.0 vs pH 4.6). Our results indicate that there is a progressive decline in urothelial endolysosomal function during aging. How this contributes to bladder dysfunction in the elderly is discussed.

Pathophysiological effect of bladder outlet obstruction on the urothelium.

Bladder outlet obstruction (BOO) and associated lower urinary tract symptoms are major urological issues that significantly affect patient's quality of life and may lead to severe complications. The prevalence of both is increasing each year, raising the burden to health system. Therefore, casual and symptomatic treatment should be of great importance. However, management of symptoms is often difficult as their pathophysiology is multifactorial and not well elucidated. Recently urothelium has gathered much attention as one of the potential causal agents. It has been suggested that in addition to its barrier function, urothelium regulates transport through blood-urine barrier and is a part of "sensory web" by which it modulates afferent input. In this review we focus on adjustment of urothelium related to BOO in terms of its ultrastructure, barrier and transport function, and impact on "sensory web."

Adaptive changes of telocytes in the urinary bladder of patients affected by neurogenic detrusor overactivity.

Urinary bladder activity involves central and autonomic nervous systems and bladder wall. Studies on the pathogenesis of voiding disorders such as the neurogenic detrusor overactivity (NDO) due to suprasacral spinal cord lesions have emphasized the importance of an abnormal handling of the afferent signals from urothelium and lamina propria (LP). In the LP (and detrusor), three types of telocytes (TC) are present and form a 3D-network. TC are stromal cells able to form the scaffold that contains and organizes the connective components, to serve as guide for tissue (re)-modelling, to produce trophic and/or regulatory molecules, to share privileged contacts with the immune cells. Specimens of full thickness bladder wall from NDO patients were collected with the aim to investigate possible changes of the three TC types using histology, immunohistochemistry and transmission electron microscopy. The results show that NDO causes several morphological TC changes without cell loss or network interruption. With the exception of those underlying the urothelium, all the TC display signs of activation (increase in Caveolin1 and caveolae, αSMA and thin filaments, Calreticulin and amount of cisternae of the rough endoplasmic reticulum, CD34, euchromatic nuclei and large nucleoli). In all the specimens, a cell infiltrate, mainly consisting in plasma cells located in the vicinity or taking contacts with the TC, is present. In conclusion, our findings show that NDO causes significant changes of all the TC. Notably, these changes can be interpreted as TC adaptability to the pathological condition likely preserving each of their peculiar functions.

3D-electron microscopic characterization of interstitial cells in the human bladder upper lamina propria.

AIMS: To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. METHODS: Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. RESULTS: 3View-SEM image stacks as large as 59 × 59 × 17 µm3 (xyz) at a resolution of 16 × 16 × 50 nm3 and high resolution (5 × 5 × 10 nm3 ) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. CONCLUSIONS: Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.

[The role of the Randalls plaques in the pathogenesis of recurrent urolitasis].

The aim of the work was to present current concept of the pathogenesis of urolithiasis. Treatment and prevention of this disease a challenging issue. The article presents basic information about Randalls plaques that are described as calcium salt deposits on the surface of the transitional cell epithelium. The cause of Randalls plaques was the subject of many studies and is still not completely clear. To date, we can state that the deposit formation starts in the pelvicalyceal system and is directly linked to recurrent urolithiasis. The discovery of Randall plaques in the 1940s transformed the conception of stone formation, but there are even more questions about the pathogenesis of urolithiasis. In that respect, we consider it important to analyze the studies on Randalls plaques.

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.

Chitosan hydrochloride has no detrimental effect on bladder urothelial cancer cells.

Bladder cancer is among the most common and aggressive human malignant carcinomas, thus targeting and removal of bladder cancer cells is still a challenge. Although it is well known that chitosan hydrochloride (CH-HCl) causes desquamation of normal urothelial cells, its effect on cancer urothelial cells has not been recognized yet. In this in vitro study, we analyzed the cytotoxicity of 0.05% CH-HCl on three urothelial models: two cancer urothelial models, i.e. invasive and papillary urothelial neoplasms, and a normal urothelial model. The cytotoxicity of CH-HCl was evaluated with viability tests, transepithelial resistance (TER) measurements, and electron microscopy. TER measurements showed that 15-minute treatment with CH-HCl caused no reduction in TER of the cancer models, whereas the TER of the normal urothelial model significantly decreased. Furthermore, after CH-HCl treatment, the viability of cancer cells was reduced by only 5%, whereas the viability of normal cells was reduced by 30%. Ultrastructural analysis revealed necrotic cell death in all cases. We have demonstrated that although CH-HCl increases the mortality of cancer urothelial cells, it increases the mortality of normal urothelial cells even more so. However, shorter 2-minute CH-HCl treatment only temporarily increases the permeability of normal urothelial model, i.e. disrupts tight junctions and reduces TER without comprising cell viability, and enables the complete recovery of the permeability barrier after 24h. Overall, our results suggest that CH-HCl cannot be used as a self-sufficient anticancer agent for urothelial bladder cancer treatment; nevertheless a possibility of its use as an enhancer of cytostatic treatment is discussed.