Voiding defects in acute radiation cystitis driven by urothelial barrier defect through loss of E-cadherin, ZO-1 and Uroplakin III.

Long term-side effects from cancer therapies are a growing health care concern as life expectancy among cancer survivors increases. Damage to the bladder is common in patients treated with radiation therapy for pelvic cancers and can result in radiation (hemorrhagic) cystitis (RC). The disease progression of RC consists of an acute and chronic phase, separated by a symptom-free period. Gaining insight in tissue changes associated with these phases is necessary to develop appropriate interventions. Using a mouse preclinical model, we have previously shown that fibrosis and vascular damage are the predominant pathological features of chronic RC. The goal of this study was to determine the pathological changes during acute RC. We identified that radiation treatment results in a temporary increase in micturition frequency and decrease in void volume 4-8 weeks after irradiation. Histologically, the micturition defect is associated with thinning of the urothelium, loss of urothelial cell-cell adhesion and tight junction proteins and decrease in uroplakin III expression. By 12 weeks, the urothelium had regenerated and micturition patterns were similar to littermate controls. No inflammation or fibrosis were detected in bladder tissues after irradiation. We conclude that functional bladder defects during acute RC are driven primarily by a urothelial defect.

Immunohistochemistry of γ-H2AX as a method of early detection of urinary bladder carcinogenicity in mice.

Phosphorylated histone H2AX (γ-H2AX) has been demonstrated as a DNA damage marker both in vitro and in vivo. We previously reported the effects of genotoxic carcinogens in the urinary bladder of rats by immunohistochemical analysis of γ-H2AX using samples from 28-day repeated-dose tests. To evaluate the application of γ-H2AX as a biomarker of carcinogenicity in the bladder, we examined species differences in γ-H2AX formation in the urinary bladder of mice. Six-week-old male B6C3F1 mice were treated orally with 12 chemicals for 4 weeks. Immunohistochemical analysis demonstrated that N-butyl-N-(4-hydroxybutyl)nitrosamine, p-cresidine and 2-acetylaminofluorene (2-AAF), classified as genotoxic bladder carcinogens, induced significant increases in γ-H2AX levels in the bladder urothelium. In contrast, genotoxic (2-nitroanisole, glycidol, N-nitrosodiethylamine and acrylamide) and non-genotoxic (dimethylarsinic acid and melamine) non-bladder carcinogens did not upregulate γ-H2AX. Importantly, 2-nitroanisole, a potent genotoxic bladder carcinogen in rats, significantly increased the proportion of γ-H2AX-positive cells in rats only, reflecting differences in carcinogenicity in the urinary bladder between rats and mice. Significant upregulation of γ-H2AX was also induced by uracil, a non-genotoxic bladder carcinogen that may be associated with cell proliferation, as demonstrated by increased Ki67 expression. 2-AAF caused γ-H2AX formation mainly in the superficial layer, together with reduced and disorganized expression of uroplakin III, unlike in rats, suggesting the mouse-specific cytotoxicity of 2-AAF in umbrella cells. These results suggest γ-H2AX is a useful biomarker reflecting species differences in carcinogenicity in the urinary bladder.

Ex vivo construction of a novel model of bioengineered bladder mucosa: A preliminary study.

OBJECTIVE: To generate and to evaluate ex vivo a novel model of bioengineered human bladder mucosa based on fibrin-agarose biomaterials. METHODS: We first established primary cultures of stromal and epithelial cells from small biopsies of the human bladder using enzymatic digestion and selective cell culture media. Then, a bioengineered substitute of the bladder lamina propria was generated using cultured stromal cells and fibrin-agarose scaffolds, and the epithelial cells were then subcultured on top to generate a complete bladder mucosa substitute. Evaluation of this substitute was carried out by cell viability and histological analyses, immunohistochemistry for key epithelial markers and transmission electron microscopy. RESULTS: The results show a well-configured stroma substitute with a single-layer epithelium on top. This substitute was equivalent to the control bladder mucosa. After 7 days of ex vivo development, the epithelial layer expressed pancytokeratin, and cytokeratins CK7, CK8 and CK13, as well as filaggrin and ZO-2, with negative expression of CK4 and uroplakin III. A reduction of the expression of CK8, filaggrin and ZO-2 was found at day 14 of development. An immature basement membrane was detected at the transition between the epithelium and the lamina propria, with the presence of epithelial hemidesmosomes, interdigitations and immature desmosomes. CONCLUSIONS: The present results suggest that this model of bioengineered human bladder mucosa shared structural and functional similarities with the native bladder mucosa, although the epithelial cells were not fully differentiated ex vivo. We hypothesize that this bladder mucosa substitute could have potential clinical usefulness after in vivo implantation.

Uroplakin II is a more sensitive immunohistochemical marker than uroplakin III in urothelial carcinoma and its variants.

OBJECTIVES: Uroplakin (UP) II and UPIII are highly specific immunohistochemical markers for urothelial differentiation. Here we studied the sensitivity of UPII and UPIII in conventional and variant urothelial carcinomas (UCs). METHODS: Immunohistochemical staining for UPII and UPIII was performed on tissue microarray slides, including 105 conventional bladder UCs (BUCs), 90 upper urinary tract UCs (UUTUCs), and 47 micropapillary, 16 plasmacytoid, 22 small cell carcinoma, and 41 sarcomatoid UC variants. RESULTS: UPII expression was significantly higher than UPIII expression in conventional BUC (44% vs 17%, P < .001) and UUTUC (67% vs 46%, P = .045). UPIII expression was significantly higher in UUTUC than in BUC (P < .001). In UC variants, UPII expression was significantly higher than UPIII expression in micropapillary (91% vs 25%, P < .001), plasmacytoid (63% vs 6%, P < .001), and sarcomatoid (29% vs 5%, P = .032) variants. Only rare cases of the small cell carcinoma variant had focal UPII and UPIII expression. Compared with conventional UC, the sarcomatoid variant had significantly lower UPII expression, whereas the micropapillary variant had significantly higher UPII expression (P < .001). CONCLUSIONS: UPII demonstrates a significantly higher sensitivity than UPIII in conventional and variant UCs. Thus, UPII is a more valuable marker than UPIII in immunohistochemical analyses for confirming the urothelial origin of carcinomas.

Aberrant differentiation of urothelial cells in patients with ureteropelvic junction obstruction.

AIM: To investigate the urothelial changes in the pathogenesis of ureteropelvic junction obstruction (UPJ-O). METHODS: A total of 12 patients of UPJ-O were respectively studied. The expression of Annexin A7, Annexin A11, EGFR, Keratin 5, uroplakin III, and SMA in the urothelium of obstructed UPJ segment and of the normal ureter below the obstructed segment were determined by immunofluorescence. Transmission electron microscopy was used to determine the morphological changes in UPJ epithelium in compared to normal ureteral epithelium. RESULTS: We found that Annexin A7, Annexin A11, EGFR, Keratin 5, and SMA were upregulated, while uroplakin III was downregulated in the urothelium of UPJ-O patients. Furthermore, ultrastructural analyses showed that intercellular spaces between urothelial cells were dilated and the number of microvilli on superficial cells was increased in UPJ-O patients. CONCLUSIONS: We propose that a disrupted urothelial barrier in UPJ-O may results in urothelial inflammatory response and truncated differentiated urothelial cells, which may play an important role in the development and pathogenesis of UPJO.

Uroplakin II outperforms uroplakin III in diagnostically challenging settings.

AIMS: We performed a head-to-head comparison of an antibody against uroplakin III (UP3) and a new uroplakin II (UP2) antibody that remains untested in diagnostically challenging settings. METHODS AND RESULTS: We immunostained high-grade bladder neck carcinomas (n = 35), high-grade upper tract urothelial carcinomas (UC) and renal carcinomas (n = 85), metastases of UC (n = 30) and a multicancer tissue microarray (n = 88) for UP3 and UP2, and scored staining intensity and proportion. UP3 showed membranous plaque-like expression, while UP2 staining showed both membranous and cytoplasmic positivity. Significantly greater intensity (P = 0.003) and proportion (P = 0.03) of staining was noted for UP2 among bladder neck lesions, with UP2 staining showing greater sensitivity (63% versus 19%) and similar specificity (95% versus 100%) for UC over prostate carcinoma (P = 0.02). Among upper tract lesions, UP2 staining showed greater intensity and proportion than UP3 (both P < 0.001), including improved sensitivity (68% versus 23%) and equal specificity (both 100%) for UC (P = 0.006). Among UC metastases, UP2 staining showed greater intensity and proportion (both P < 0.001) with higher sensitivity (73% versus 37%, respectively, P = 0.001). Of 88 additional cases tested, no non-urothelial cases stained for either UP. CONCLUSIONS: The UP2 antibody outperforms the UP3 antibody, including in diagnostically challenging settings, and is a useful addition to the armamentarium of biomarkers for UC.

Local immune response in bladder pain syndrome/interstitial cystitis ESSIC type 3C.

INTRODUCTION AND HYPOTHESIS: Bladder pain syndrome/interstitial cystitis (BPS/IC) is identified based on subjective symptoms which lead to heterogeneous patient populations. Previous studies using gene expression arrays for BPS/IC with Hunner's lesions [European Society for the Study of Interstitial Cystitis (ESSIC) type 3C], a subtype of the condition discernible by cystoscopy, have revealed characteristic immune responses and urothelial abnormalities. This current study aimed to further characterize this subtype using a gene expression panel. We hypothesized that B-cell activation with high levels of urinary antibody concentration would be found. METHODS: Cold-cup bladder biopsies, catheterized urine and blood were collected from 15 BPS/IC ESSIC type 3C patients, 11 non-inflammatory overactive bladder (OAB) patients and eight healthy controls. Gene expression in biopsies was quantified by real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry was performed on bladder tissue and urinary immunoglobulins G and A were quantified by enzyme-linked immunosorbent assay. Statistical analyses included the Kruskal-Wallis test for non-parametric data and post hoc tests identified differences between groups. RESULTS: High expression of T- and B-cell markers (CTLA4, CD20, CD79A, IGH@), low expression of urothelial markers (KRT20, UPK1B, UPK3A), focal lymphoid aggregates in the submucosa and high immunoglobulin concentration in urine were found exclusively in BPS/IC ESSIC type 3C patients. Results for OAB were in intermediate ranges between the other two groups and UPK1B even reached significantly lower expression when compared to healthy controls. CONCLUSIONS: BPS/IC ESSIC type 3C is characterized by a local adaptive immune response with elevated urinary antibody concentrations. Quantification of urinary immunoglobulin levels could be used for a non-invasive diagnosis of BPS/IC ESSIC type 3C.

Expression and localization of four uroplakins in urothelial preneoplastic lesions.

In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.