Micronuclei and other nuclear anomalies in exfoliated urothelial cells and urinary 8-hydroxy-deoxyguanosine levels among Turkish hairdressers.

Hairdressers are constantly occupationally exposed to many chemicals have the potential to cause allergies and carcinogenic effects, act as skin and eye irritants and induce oxidative stress and DNA damage. This study aimed to evaluate occupation-induced genotoxicity based on the presence of micronucleus (MN) and other nuclear anomalies in urothelial cells and measure oxidative DNA damage based on the 8-hydroxy-2'-deoxyguanosine level in the urine of Turkish hairdressers. Originality of this study comes from that there was no study on MN and other nuclear anomalies frequencies and oxidative DNA damage in urine samples of hairdressers in the literature. The mean±standard deviation frequency (‰) of micronucleated (MNed) cells was higher in the hairdresser group (n=56) (4.81±7.87, p<0.001) than in the control group (n=56) (0.93±1.85). Nuclear buds were not observed in either group. While the frequency of basal cells was higher in the control group (446.6±106.21) than in the hairdresser group (367.78±101.51, p<0.001), the frequency of binuclear, karyolytic, pycnotic and karyorrhectic cells were higher in the hairdresser group (0.41±0.80, p<0.001; 438.02±118.27, p<0.001; 0.43±0.76, p<0.001; and 47.27±28.40, p<0.001) than in the control group (0.04±0.27, 358.57±95.71, 0.05±0.23 and 24.41±14.50). Condensed chromatins were observed only in the hairdresser group. Specific gravity adjusted 8-hydroxy-2'-deoxyguanosine level was statistically lower in the hairdresser group (908.21±403.25 ng/mL-SG) compared to the control group (1003.09±327.09 ng/mL-SG) (p=0.024). No significant correlation was found between the 8-hydroxy-2'-deoxyguanosine level and the frequency MN. The amount of formaldehyde released during Brazilian keratin treatment was higher than the American Conference of Governmental Industrial Hygienists -Threshold Limit Value (ACGIH-TLV; 0.1 ppm). Similarly, the amount of ethyl acetate released in three salons was above the recommended limit (400 ppm). These findings suggest that hairdressers have an increased risk of genotoxicity and cytotoxicity owing to occupational exposure, regardless of age, working hours, smoking and alcohol consumption.

Expression and localization of the mechanosensitive/osmosensitive ion channel TMEM63B in the mouse urinary tract.

The epithelial cells that line the kidneys and lower urinary tract are exposed to mechanical forces including shear stress and wall tension; however, the mechanosensors that detect and respond to these stimuli remain obscure. Candidates include the OSCA/TMEM63 family of ion channels, which can function as mechanosensors and osmosensors. Using Tmem63bHA-fl/HA-fl reporter mice, we assessed the localization of HA-tagged-TMEM63B within the urinary tract by immunofluorescence coupled with confocal microscopy. In the kidneys, HA-TMEM63B was expressed by proximal tubule epithelial cells, by the intercalated cells of the collecting duct, and by the epithelial cells lining the thick ascending limb of the medulla. In the urinary tract, HA-TMEM63B was expressed by the urothelium lining the renal pelvis, ureters, bladder, and urethra. HA-TMEM63B was also expressed in closely allied organs including the epithelial cells lining the seminal vesicles, vas deferens, and lateral prostate glands of male mice and the vaginal epithelium of female mice. Our studies reveal that TMEM63B is expressed by subsets of kidney and lower urinary tract epithelial cells, which we hypothesize are sites of TMEM63B mechanosensation or osmosensation, or both.

Proteomic analysis of the urothelial cancer landscape.

Urothelial bladder cancer (UC) has a wide tumor biological spectrum with challenging prognostic stratification and relevant therapy-associated morbidity. Most molecular classifications relate only indirectly to the therapeutically relevant protein level. We improve the pre-analytics of clinical samples for proteome analyses and characterize a cohort of 434 samples with 242 tumors and 192 paired normal mucosae covering the full range of UC. We evaluate sample-wise tumor specificity and rank biomarkers by target relevance. We identify robust proteomic subtypes with prognostic information independent from histopathological groups. In silico drug prediction suggests efficacy of several compounds hitherto not in clinical use. Both in silico and in vitro data indicate predictive value of the proteomic clusters for these drugs. We underline that proteomics is relevant for personalized oncology and provide abundance and tumor specificity data for a large part of the UC proteome ( www.cancerproteins.org ).

Loss of LPAR6 and CAB39L dysregulates the basal-to-luminal urothelial differentiation program, contributing to bladder carcinogenesis.

We describe a strategy that combines histologic and molecular mapping that permits interrogation of the chronology of changes associated with cancer development on a whole-organ scale. Using this approach, we present the sequence of alterations around RB1 in the development of bladder cancer. We show that RB1 is not involved in initial expansion of the preneoplastic clone. Instead, we found a set of contiguous genes that we term "forerunner" genes whose silencing is associated with the development of plaque-like field effects initiating carcinogenesis. Specifically, we identified five candidate forerunner genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) mapping near RB1. Two of these genes, LPAR6 and CAB39L, are preferentially downregulated in the luminal and basal subtypes of bladder cancer, respectively. Their loss of function dysregulates urothelial differentiation, sensitizing the urothelium to N-butyl-N-(4-hydroxybutyl)nitrosamine-induced cancers, which recapitulate the luminal and basal subtypes of human bladder cancer.

Keratin 5 basal cells are temporally regulated developmental and tissue repair progenitors in bladder urothelium.

Urothelium forms a distensible yet impermeable barrier, senses and transduces stimuli, and defends the urinary tract from mechanical, chemical, and bacterial injuries. Biochemical and genetic labeling studies support the existence of one or more progenitor populations with the capacity to rapidly regenerate the urothelium following injury, but slow turnover, a low mitotic index, and inconsistent methodologies obscure progenitor identity. The progenitor properties of basal keratin 5 urothelial cells (K5-UCs) have been previously investigated, but those studies focused on embryonic or adult bladder urothelium. Urothelium undergoes desquamation and apoptosis after birth, which requires postnatal proliferation and restoration. Therefore, we mapped the fate of bladder K5-UCs across postnatal development/maturation and following administration of cyclophosphamide to measure homeostatic and reparative progenitor capacities, respectively. In vivo studies demonstrate that basal K5-UCs are age-restricted progenitors in neonates and juveniles, but not in adult mice. Neonatal K5-UCs retain a superior progenitor capacity in vitro, forming larger and more differentiated urothelial organoids than adult K5-UCs. Accordingly, K5-UC transcriptomes are temporally distinct, with enrichment of transcripts associated with cell proliferation and differentiation in neonates. Induction of urothelial proliferation is sufficient to restore adult K5-UC progenitor capacity. Our findings advance the understanding of urothelial progenitors and support a linear model of urothelial formation and regeneration, which may have significant impact on therapeutic development or tissue engineering strategies.NEW & NOTEWORTHY Fate mapping reveals an important linear relationship, whereby bladder basal urothelial cells give rise to intermediate and superficial cells in an age-restricted manner and contribute to tissue repair. Neonatal basal cells reprise their role as superior progenitors in vitro and display distinct transcriptional signatures, which suggest progenitor function is at least partially cell intrinsic. However, the urothelium progenitor niche cannot be overlooked, since FGF7 rescues adult basal cell progenitor function.

Scoring PD-L1 Expression in Urothelial Carcinoma: An International Multi-Institutional Study on Comparison of Manual and Artificial Intelligence Measurement Model (AIM-PD-L1) Pathology Assessments.

Assessing programmed death ligand 1 (PD-L1) expression on tumor cells (TCs) using Food and Drug Administration-approved, validated immunoassays can guide the use of immune checkpoint inhibitor (ICI) therapy in cancer treatment. However, substantial interobserver variability has been reported using these immunoassays. Artificial intelligence (AI) has the potential to accurately measure biomarker expression in tissue samples, but its reliability and comparability to standard manual scoring remain to be evaluated. This multinational study sought to compare the %TC scoring of PD-L1 expression in advanced urothelial carcinoma, assessed by either an AI Measurement Model (AIM-PD-L1) or expert pathologists. The concordance among pathologists and between pathologists and AIM-PD-L1 was determined. The positivity rate of ≥ 1%TC PD-L1 was between 20-30% for 8/10 pathologists, and the degree of agreement and scoring distribution for among pathologists and between pathologists and AIM-PD-L1 was similar both scored as a continuous variable or using the pre-defined cutoff. Numerically higher score variation was observed with the 22C3 assay than with the 28-8 assay. A 2-h training module on the 28-8 assay did not significantly impact manual assessment. Cases exhibiting significantly higher variability in the assessment of PD-L1 expression (mean absolute deviation > 10) were found to have patterns of PD-L1 staining that were more challenging to interpret. An improved understanding of sources of manual scoring variability can be applied to PD-L1 expression analysis in the clinical setting. In the future, the application of AI algorithms could serve as a valuable reference guide for pathologists while scoring PD-L1.

Differential expression of WNT5A long and short isoforms in non-muscle-invasive bladder urothelial carcinoma.

Wnt ligands belong to a family of secreted glycoproteins in which binding to a range of receptors/co-receptors activates several intracellular pathways. WNT5A, a member of the Wnt family, is classified as a non-canonical Wnt whose activation triggers planar cell polarity (PCP) and Ca+2 downstream pathways. Aberrant expression of WNT5A has been shown to play both protective and harmful roles in an array of conditions, such as inflammatory disease and cancer. In the present study, using histological, immunohistochemical, and molecular methods, we investigated the expression of two isoforms of WNT5A, WNT5A-Short (WNT5A-S) and WNT5A-Long (WNT5A-L) in bladder urothelial carcinoma (UC). Three UC cell lines (RT4, J82, and T24), as well as a normal urothelial cell line, and formalin-fixed, paraffin-embedded (FFPE) transurethral resection (TUR) tissue samples from 17 patients diagnosed with UC were included in the study. WNT5A-L was the predominantly expressed isoform in urothelial cells, although WNT5A-S was also detectable. Further, although no statistically significant difference was found between the percentage of WNT5A-S transcripts in low-grade versus high-grade tumors, we did find a difference between the percentage of WNT5A-S transcripts found in non-invasion versus invasion of the lamina propria, subgroups of non-muscle-invasive tumors. In conclusion, both WNT5A-S and WNT5A-L isoforms are expressed in UC, and the percentage of their expression levels suggests that a higher proportion of WNT5A-S transcription may be associated with lamina propria invasion, a process preceding muscle invasion.

UPK3A+ umbrella cell damage mediated by TLR3-NR2F6 triggers programmed destruction of urothelium in Hunner-type interstitial cystitis/painful bladder syndrome.

Urothelial damage and barrier dysfunction emerge as the foremost mechanisms in Hunner-type interstitial cystitis/bladder pain syndrome (HIC). Although treatments aimed at urothelial regeneration and repair have been employed, their therapeutic effectiveness remains limited due to the inadequate understanding of specific cell types involved in damage and the lack of specific molecular targets within these mechanisms. Therefore, we harnessed single-cell RNA sequencing to elucidate the heterogeneity and developmental trajectory of urothelial cells within HIC bladders. Through reclustering, we identified eight distinct clusters of urothelial cells. There was a significant reduction in UPK3A+ umbrella cells and a simultaneous increase in progenitor-like pluripotent cells (PPCs) within the HIC bladder. Pseudotime analysis of the urothelial cells in the HIC bladder revealed that cells faced challenges in differentiating into UPK3A+ umbrella cells, while PPCs exhibited substantial proliferation to compensate for the loss of UPK3A+ umbrella cells. The urothelium in HIC remains unrepaired, despite the substantial proliferation of PPCs. Thus, we propose that inhibiting the pivotal signaling pathways responsible for the injury to UPK3A+ umbrella cells is paramount for restoring the urothelial barrier and alleviating lower urinary tract symptoms in HIC patients. Subsequently, we identified key molecular pathways (TLR3 and NR2F6) associated with the injury of UPK3A+ umbrella cells in HIC urothelium. Finally, we conducted in vitro and in vivo experiments to confirm the potential of the TLR3-NR2F6 axis as a promising therapeutic target for HIC. These findings hold the potential to inhibit urothelial injury, providing promising clues for early diagnosis and functional bladder self-repair strategies for HIC patients. © 2024 The Pathological Society of Great Britain and Ireland.

Insulin receptor signaling engages bladder urothelial defenses that limit urinary tract infection.

Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We report that diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression.

Allograft inflammatory factor-1 enhances inflammation and oxidative stress via the NF-κB pathway of bladder urothelium in diabetic rat model.

OBJECTIVES: To explore the role of allograft inflammatory factor-1 (AIF-1) both in diabetic rat bladder urothelium and in high-glucose-treated human urothelial cell line (SV-HUC-1). METHODS: Inflammation and oxidative stress (OS) promote diabetic cystopathy (DCP), but the mechanisms are not fully understood. The expression level of AIF-1 in diabetic rat bladder urothelium and in the SV-HUC-1 cells treated with high glucose was detected using tissue immunofluorescence, immunohistochemistry and western blot assays. AIF-1 was knocked down and NF-κB was suppressed with the specific inhibitor BAY 11-7082 in high-glucose-treated SV-HUC-1 cells. RESULTS: High-glucose condition induced AIF-1 upregulation in vivo and in vitro. The up-regulated AIF-1 induced the production of inflammatory factors IL-6 and TNF-α and elevation of ROS. Informatics analysis suggested that NF-κB pathway is implicated in DCP. Through knockdown of AIF-1, we confirmed that AIF-1 simulated NF-κB pathway by enhancing the phosphorylation of IκB (p-IκB) and promoting the translocation of NF-κB p65 from cytoplasm into nucleus. Additionally, High-glucose-induced inflammation in SV-HUC-1 cells was attenuated by the addition of NF-κB inhibitor. CONCLUSIONS: This study provides novel information to understand the molecular regulation mechanisms of AIF-1 in DCP.

ß-Adrenoceptors regulate urothelial inflammation and zonula occludens in the bladder outlet obstruction model.

BACKGROUND: The purpose of this study was to evaluate the effects of ß-adrenoceptors (ADRBs) on the urothelial inflammation and zonula occludens (ZO) in a rat PBOO model and in an in vitro model. METHODS: The PBOO model was established by ligating the bladder neck of rats. Twenty rats were divided into 4 groups: sham operation, PBOO + normal saline, PBOO + ADRB2 agonist, PBOO + ADRB3 agonist. PBOO rats were with treated with ADRBs agonists for 3 weeks. Human urothelial cells (HUCs) were subjected to ADRBs agonist treatment or hydrostatic pressure in an in vitro model. RESULTS: In the PBOO group, there was a significant increase in the expression of MCP-1, IL-6 and RANTES compared to the sham group. By contrast, there was a post-PBOO decline in the expression of ZO-1 and ZO-2 in the urothelium. ADRB2 or ADRB3 agonists exhibited downregulated inflammatory cytokine expression and increased ZO expression in the PBOO model. The regulation of inflammation and ZO by ADRB2 and ADRB3 agonists in an in vitro model was found consistent with that in the PBOO model. Moreover, RhoA and ROCK inhibitors suppressed the expression of hydrostatic pressure-induced inflammatory cytokines. Additionally, RhoA agonist reversed the inhibitory effect of ADRBs agonists on the inflammatory secretion from HUCs. CONCLUSIONS: ADRB2 and ADRB3 agonists increased ZO protein expression in HUCs in a rat PBOO model and in an in vitro model. Furthermore, ADRB2 and ADRB3 agonists inhibited the secretion of inflammatory cytokines from HUCs by regulating the RhoA/ROCK signaling pathways.

D-Mannose reduces cellular senescence and NLRP3/GasderminD/IL-1ß-driven pyroptotic uroepithelial cell shedding in the murine bladder.

Aging is a risk factor for disease via increased susceptibility to infection, decreased ability to maintain homeostasis, inefficiency in combating stress, and decreased regenerative capacity. Multiple diseases, including urinary tract infection (UTI), are more prevalent with age; however, the mechanisms underlying the impact of aging on the urinary tract mucosa and the correlation between aging and disease remain poorly understood. Here, we show that, relative to young (8-12 weeks) mice, the urothelium of aged (18-24 months) female mice accumulates large lysosomes with reduced acid phosphatase activity and decreased overall autophagic flux in the aged urothelium, indicative of compromised cellular homeostasis. Aged bladders also exhibit basal accumulation of reactive oxygen species (ROS) and a dampened redox response, implying heightened oxidative stress. Furthermore, we identify a canonical senescence-associated secretory phenotype (SASP) in the aged urothelium, along with continuous NLRP3-inflammasome- and Gasdermin-D-dependent pyroptotic cell death. Consequently, aged mice chronically exfoliate urothelial cells, further exacerbating age-related urothelial dysfunction. Upon infection with uropathogenic E. coli, aged mice harbor increased bacterial reservoirs and are more prone to spontaneous recurrent UTI. Finally, we discover that treatment with D-mannose, a natural bioactive monosaccharide, rescues autophagy flux, reverses the SASP, and mitigates ROS and NLRP3/Gasdermin/interleukin (IL)-1ß-driven pyroptotic epithelial cell shedding in aged mice. Collectively, our results demonstrate that normal aging affects bladder physiology, with aging alone increasing baseline cellular stress and susceptibility to infection, and suggest that mannose supplementation could serve as a senotherapeutic to counter age-associated urothelial dysfunction.

Sex-Dependent Differences in Blood-Urine Barrier Are Subtle but Significant in Healthy and Chronically Inflamed Mouse Bladders.

The urothelium is a vital permeability barrier that prevents the uncontrolled flow of urinary components into and out of the bladder interstitium. Our study addressed the question of possible sex-specific variations in the urothelium of healthy mice and their impact on chronic bladder inflammation. We found that healthy female bladders have a less robust barrier function than male bladders, as indicated by significant differences in transepithelial electrical resistance (TEER) values. These differences could be attributed to detected higher claudin 2 mRNA expression and a less pronounced glycocalyx in females than in males. In addition, TEER measurements showed delayed barrier recovery in chronically inflamed female bladders. We found subtle differences in the expressions of genes involved in the regulation of the actin cytoskeleton between the sexes, as well as pronounced urothelial hyperplasia in females compensating for attenuated barrier function. The identified genetic variations in glycosylation pathways may also contribute to this divergence. Our findings add to the growing body of literature on the intricate sex-specific nuances of urothelial permeability function and their implications for chronic bladder inflammation. Understanding these differences could lead to tailored diagnostic and therapeutic approaches in the treatment of bladder disorders in the future.

Distinct effects of Fgf7 and Fgf10 on the terminal differentiation of murine bladder urothelium revealed using an organoid culture system.

BACKGROUND: Dysregulation of the terminal differentiation of bladder urothelium is associated with the pathogenesis of urinary tract disorders. Fibroblast growth factor (Fgf)7 and Fgf10 stimulate urothelial proliferation; however, their roles in cellular differentiation remain unclear. In this study, we used an organoid system to investigate the roles of these Fgfs in regulating bladder urothelium differentiation and identify their distribution patterns in the mouse bladder. METHODS: Adult bladder epithelia (AdBE) isolated from adult mouse bladder tissues (AdBTs) were used to culture adult bladder organoids (AdBOs) in the presence of Fgf7 and Fgf10. The differentiation status of the cells in AdBTs, AdBEs, AdBOs, and neonatal bladder tissues (NeoBTs) was analyzed via quantitative real-time-PCR for the presence of undifferentiated cell markers (Krt5, Trp63, and Krt14) and differentiated cell markers (Krt20, Upk1a, Upk2, and Upk3a). Organoid cell proliferation was assessed by counting cell numbers using the trypan blue method. The effects of Fgf7 and Fgf10 on organoid differentiation were assessed using different doses of Fgfs, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) signaling in these processes was tested by introducing a PPARγ agonist (Rosiglitazone) and antagonist (T0070907) to the culture. The expression patterns of Fgf7 and Fgf10 were examined via in situ hybridization of AdBTs. RESULTS: AdBOs showed higher expression of undifferentiated cell markers and lower expression of differentiated cell markers than AdBTs, NeoBTs, and AdBEs, indicating the relatively immature state of AdBOs. Differentiation of AdBOs was enhanced by Rosiglitazone and Fgf7, suggesting an interplay of intracellular signals between Fgf7 and PPARγ. Co-addition of T0070907 suppressed Fgf7-mediated differentiation, demonstrating that PPARγ is activated downstream of Fgf7 to promote cellular differentiation into umbrella cells. Furthermore, we found that Fgf7 is predominantly expressed in the umbrella cells of the urothelium, whereas Fgf10 is predominantly expressed in the urothelium and stroma of AdBTs. CONCLUSIONS: We demonstrated that unlike Fgf10, Fgf7 induces cellular differentiation via PPARγ activity and has a unique tissue distribution pattern in the adult bladder. Further studies on the Fgf7-PPARγ signaling axis would provide insights into the differentiation mechanisms toward functional umbrella cells and the pathogenesis of several urinary tract diseases.

Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane.

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Urothelium-Specific Expression of Mutationally Activated Pik3ca Initiates Early Lesions of Noninvasive Bladder Cancer.

Although approximately 70% of bladder cancers are noninvasive and have high recurrence rates, early-stage disease is understudied. The lack of models to validate the contribution of molecular drivers of bladder tumorigenesis is a significant issue. Although mutations in PIK3CA are frequent in human bladder cancer, an in vivo model for understanding their contribution to bladder tumorigenesis is unavailable. Therefore, a Upk2-Cre/Pik3caH1047R mouse model expressing one or two R26-Pik3caH1047R alleles in a urothelium-specific manner was generated. Pik3caH1047R functionality was confirmed by quantifying Akt phosphorylation, and mice were characterized by assessing urothelial thickness, nuclear atypia, and expression of luminal and basal markers at 6 and 12 months of age. While at 6 months, Pik3caH1047R mice developed increased urothelial thickness and nuclear atypia, progressive disease was not observed at 12 months. Immunohistochemistry showed urothelium maintained luminal differentiation characterized by high forkhead box A1 (Foxa1) and peroxisome proliferator-activated receptor γ expression. Surprisingly, Pik3caH1047R mice subjected to low-dose carcinogen exposure [N-butyl-N-(4-hydroxybutyl)nitrosamine] exhibited no significant differences after exposure relative to mice without exposure. Furthermore, single-sample gene set enrichment analysis of invasive human tumors showed those with mutant PIK3CA did not exhibit significantly increased phosphatidylinositol 3-kinase/AKT pathway activity compared with wild-type PIK3CA tumors. Overall, these data suggest that Pik3caH1047R can elicit early tumorigenic changes in the urothelium, but progression to invasion may require additional genetic alterations.

Urothelial Oxidative Stress and ERK Activation Mediate HMGB1-Induced Bladder Pain.

Activation of intravesical protease activated receptors-4 (PAR4) results in bladder pain through the release of urothelial macrophage migration inhibitory factor (MIF) and high mobility group box-1 (HMGB1). We aimed to identify HMGB1 downstream signaling events at the bladder that mediate HMGB1-induced bladder pain in MIF-deficient mice to exclude any MIF-related effects. We studied whether oxidative stress and ERK activation are involved by examining bladder tissue in mice treated with intravesical disulfide HMGB1 for 1 h and analyzed with Western blot and immunohistochemistry. HMGB1 intravesical treatment increased urothelium 4HNE and phospho-ERK1/2 staining, suggesting that HMGB1 increased urothelial oxidative stress and ERK activation. Furthermore, we examined the functional roles of these events. We evaluated lower abdominal mechanical thresholds (an index of bladder pain) before and 24 h after intravesical PAR4 or disulfide HMGB1. Intravesical pre-treatments (10 min prior) included: N-acetylcysteine amide (NACA, reactive oxygen species scavenger) and FR180204 (FR, selective ERK1/2 inhibitor). Awake micturition parameters (voided volume; frequency) were assessed at 24 h after treatment. Bladders were collected for histology at the end of the experiment. Pre-treatment with NACA or FR significantly prevented HMGB1-induced bladder pain. No significant effects were noted on micturition volume, frequency, inflammation, or edema. Thus, HMGB1 activates downstream urothelial oxidative stress production and ERK1/2 activation to mediate bladder pain. Further dissection of HMGB1 downstream signaling pathway may lead to novel potential therapeutic strategies to treat bladder pain.

Optogenetic urothelial cell stimulation induces bladder contractions and pelvic nerve afferent firing.

Urothelial cells, which play an essential role in barrier function, are also thought to play a sensory role in bladder physiology by releasing signaling molecules in response to sensory stimuli that act upon adjacent sensory neurons. However, it is challenging to study this communication due to the overlap in receptor expression and proximity of urothelial cells to sensory neurons. To overcome this challenge, we developed a mouse model where we can directly stimulate urothelial cells using optogenetics. We crossed a uroplakin II (UPK2) cre mouse with a mouse that expresses the light-activated cation channel channelrhodopsin-2 (ChR2) in the presence of cre expression. Optogenetic stimulation of urothelial cells cultured from UPK2-ChR2 mice initiates cellular depolarization and release of ATP. Cystometry recordings demonstrated that optical stimulation of urothelial cells increases bladder pressure and pelvic nerve activity. Increases in bladder pressure persisted, albeit to a lesser extent, when the bladder was excised in an in vitro preparation. The P2X receptor antagonist PPADS significantly reduced optically evoked bladder contractions in vivo and ex vivo. Furthermore, corresponding nerve activity was also inhibited with PPADS. Our data suggest that urothelial cells can initiate robust bladder contractions via sensory nerve signaling or contractions through local signaling mechanisms. These data support a foundation of literature demonstrating communication between sensory neurons and urothelial cells. Importantly, with further use of these optogenetic tools, we hope to scrutinize this signaling mechanism, its importance for normal micturition and nociception, and how it may be altered in pathophysiological conditions.NEW & NOTEWORTHY Urothelial cells play a sensory role in bladder function. However, it has been particularly challenging to study this communication as both sensory neurons and urothelial cells express similar sensory receptors. Here we demonstrate using an optogenetic technique, that specific urothelial stimulation alone resulted in bladder contractions. This approach will have a long-lasting impact on how we study urothelial-to-sensory neuron communication and the changes that occur under disease conditions.

Mucoadhesive Emulsion Microgels for Intravesical Drug Delivery: Preparation, Retention at Urothelium, and Biodistribution Study.

The intravesical instillation procedure is a proven method in modern urology for the treatment of bladder diseases. However, the low therapeutic efficiency and painfulness of the instillation procedure are significant limitations of this method. In the present study, we propose an approach to solving this problem by using microsized mucoadhesive macromolecular carriers based on whey protein isolate with the possibility of prolonged release of drugs as a drug delivery system. The optimal water-to-oil ratio (1:3) and whey protein isolate concentration (5%) were determined to obtain emulsion microgels with sufficient loading efficiency and mucoadhesive properties. The droplet diameter of emulsion microgels varies from 2.2 to 3.8 µm. The drug release kinetics from the emulsion microgels was evaluated. The release of the model dye in saline and artificial urine in vitro was observed for 96 h and reached up to 70% of loaded cargo for samples. The effect of emulsion microgels on the morphology and viability of two cell lines was observed: L929 mouse fibroblasts (normal adherent cells) and THP-1 human monocytes (cancer suspension cells). Developed emulsion microgels (5%, 1:3 and 1:5) showed sufficient mucoadhesion to a porcine bladder urothelium ex vivo. The biodistribution of emulsion microgels (5%, 1:3 and 1:5) in mice (n = 3) after intravesical (instillation) and systemic (intravenous) administration was assessed in vivo and ex vivo using near-infrared fluorescence live imaging for real time. It was demonstrated that intravesical instillation allows approximately 10 times more efficient accumulation of emulsion microgels in the mice urinary bladder in vivo 1 h after injection compared to systemic injection. The retention of the emulsion of mucoadhesive microgels in bladders after the intravesical instillation was observed for 24 h.

The mast cell stimulator compound 48/80 causes urothelium-dependent increases in murine urinary bladder contractility.

Mast cells and degranulation of preformed inflammatory mediators contribute to lower urinary tract symptoms. This study investigated pathways by which the mast cell stimulator compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that 1) mast cell degranulation causes spontaneous urinary bladder smooth muscle contractions and 2) these contractions are caused by urothelium-derived PGE2. Urothelium-intact and -denuded urinary bladder strips were collected from mast cell-sufficient (C57Bl/6) and mast cell-deficient (B6.Cg-Kitw-sh) mice to determine if compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were used to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell-sufficient and -deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blockade of P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished compound 48/80 responses. However, only combined blockade of PGE2 (EP1), PGF2α (FP), and thromboxane A2 (TP) receptors inhibited compound 48/80-induced responses. Thus, the effects of compound 48/80 are urothelium dependent but independent of mast cells. Furthermore, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory nonneurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using compound 48/80 to determine mast cell-dependent responses in the urinary bladder.NEW & NOTEWORTHY Urothelial cells are first responders to noxious contents of the urine. Our study demonstrates that the urothelium is not only a barrier but also a modulator of urinary bladder smooth muscle phasic activity and contractility independent of immune cell recruitment in response to an inflammatory insult.