Mouse Bladder Tissue Research Articles

Advancing mechanical testing of biological tissues and hydrogels: A buoy-based approach

Quantifying the material properties of tissues and hydrogels aids in the development of biomedical applications through better understanding of the mechanics and mechanobiological principles at play. This study introduces a mechanical testing platform designed to address challenges in measuring mm-scale tissue and hydrogel material properties. Using a floating buoy design, the platform enables horizontal submerged tensile testing with non-submersible load cells. Buoy drag testing without a sample attached resulted in signal noise (mean ± standard deviation) of −1.6E-4 ± 2.8E-2 mN for stationary recording and 1.3E-2 ± 6.7E-2 mN for maximum buoy displacement speed (1000 µm/s), suggesting the magnitude of drag forces from buoy movement are negligible in comparison to the minimum resolution of force measurement. Validation testing with latex orthodontic bands showed a ∼ 28 × reduction in signal noise with the buoy approach compared to a previously used approach, and similar force displacement recordings using the buoy approach with 2 separate hardware systems. Simultaneous imaging enabled geometrical and microstructural analysis of the sample. Murine bladder tissue was mechanically tested using two different hardware systems and testing protocols. The platform was able to accurately capture the nonlinear stress-stretch response, alongside expected strain-softening and preconditioning behavior. Stress-relaxation testing yielded results consistent with expected microstructural and viscoelastic responses of mouse bladder tissue. The versatility of the platform underscores the potential for it to integrate with various force measurement and actuator-based systems. In conclusion, this platform offers a new avenue for accurate measurement of tissue and hydrogel mechanics, facilitating mm-scale soft material research.

Transient Receptor Potential Melastatin 8 Contributes to Cystitis-Induced Neuronal Sprouting and Pain Hypersensitivity Through AKT/mTOR Signaling Pathway in Interstitial Cystitis/Bladder Pain Syndrome.

The aim of this study was to investigate the mechanism of TRPM8 in neuroproliferation and pain, as well as the relevance of the Akt/mTOR signaling pathway in mice with IC/BPS. The model of IC/BPS was established in wild and TRPM8-/- mice. The mechanical sensitivity was measured. The number of neurite segments, length of neurites, and density of neurites were all counted. IL-6 and norepinephrine levels were detected by ELISA, Western blot was used to detect protein levels of TRPM8, Akt, p-Akt, mTOR, p-mTOR. Immunofluorescence was used to detect TRPM8 expression and distribution in neurites, neurons, and sensory nerves in mouse bladder tissue. Pain threshold in the IC/BPS group was decreased, and neurite segments, length, and density were all significantly enhanced when compared to the control group. The parameters in the IC/BPS model + Menthol group were more statistically significant. Neurite number and density were lower in TRPM8 knockout-model mice than in IC/BPS model mice. The expression of TRPM8 and the ratios of p-Akt/Akt and p-mTOR/mTOR rose in the IC/BPS model group. In TRPM8 knockout-model mice, the ratios of p-Akt/Akt and p-mTOR/mTOR were not substantially different from those in the control group. TRPM8 knockout-model mice had considerably lower levels of serum IL-6 and urine norepinephrine than IC/BPS model mice. TRPM8 can induce pain hypersensitivity and sensory nerve proliferation by activating Akt/mTOR pathway and raising the expression of IL-6 and norepinephrine in IC/BPS models. These findings offer new perspectives on IC/BPS treatment.

Investigating the Impact of Estrogen Levels on Voiding Characteristics, Bladder Structure, and Related Proteins in a Mouse Model of Menopause-Induced Lower Urinary Tract Symptoms.

Lower urinary tract symptoms (LUTS) are common in postmenopausal women. These symptoms are often linked to decreased estrogen levels following menopause. This study investigated the relationship between estrogen levels, alterations in bladder tissue structure, bladder function, and the incidence of urinary frequency. An age-appropriate bilateral ovariectomized mouse model (OVX) was developed to simulate conditions of estrogen deficiency. Mice were divided into three groups: a sham-operated control group, OVX, and an estradiol-treated group. The assessments included estrogen level measurement, urination frequency, cystometry, histological analysis, immunofluorescence staining, and real-time quantitative PCR. Additionally, we quantified the expression of the mechanosensitive channel proteins Piezo1 and TRPV4 in mouse bladder tissues. Lower estrogen levels were linked to increased voiding episodes and structural changes in mouse bladder tissues, notably a significant increase in Collagen III fiber deposition. There was a detectable negative relationship between estrogen levels and the expression of Piezo1 and TRPV4, mechanosensitive proteins in mouse bladder tissues, which may influence voiding frequency and nocturia. Estrogen treatment could improve bladder function, decrease urination frequency, and reduce collagen deposition in the bladder tissues. This study explored the connection between estrogen levels and urinary frequency, potentially setting the stage for novel methods to address frequent urination symptoms in postmenopausal women.

Uropathogenic Escherichia coli infection: innate immune disorder, bladder damage, and Tailin Fang II.

Uropathogenic Escherichia coli (UPEC) activates innate immune response upon invading the urinary tract, whereas UPEC can also enter bladder epithelial cells (BECs) through interactions with fusiform vesicles on cell surfaces and subsequently escape from the vesicles into the cytoplasm to establish intracellular bacterial communities, finally evading the host immune system and leading to recurrent urinary tract infection (RUTI). Tailin Fang II (TLF-II) is a Chinese herbal formulation composed of botanicals that has been clinically proven to be effective in treating urinary tract infection (UTI). However, the underlying therapeutic mechanisms remain poorly understood. Network pharmacology analysis of TLF-II was conducted. Female Balb/C mice were transurethrally inoculated with UPEC CFT073 strain to establish the UTI mouse model. Levofloxacin was used as a positive control. Mice were randomly divided into four groups: negative control, UTI, TLF-II, and levofloxacin. Histopathological changes in bladder tissues were assessed by evaluating the bladder organ index and performing hematoxylin-eosin staining. The bacterial load in the bladder tissue and urine sample of mice was quantified. Activation of the TLR4-NF-κB pathway was investigated through immunohistochemistry and western blotting. The urinary levels of interleukin (IL)-1β and IL-6 and urine leukocyte counts were monitored. We also determined the protein expressions of markers associated with fusiform vesicles, Rab27b and Galectin-3, and levels of the phosphate transporter protein SLC20A1. Subsequently, the co-localization of Rab27b and SLC20A1 with CFT073 was examined using confocal fluorescence microscopy. Data of network pharmacology analysis suggested that TLF-II could against UTI through multiple targets and pathways associated with innate immunity and inflammation. Additionally, TLF-II significantly attenuated UPEC-induced bladder injury and reduced the bladder bacterial load. Meanwhile, TLF-II inhibited the expression of TLR4 and NF-κB on BECs and decreased the urine levels of IL-1β and IL-6 and urine leukocyte counts. TLF-II reduced SLC20A1 and Galectin-3 expressions and increased Rab27b expression. The co-localization of SLC20A1 and Rab27b with CFT073 was significantly reduced in the TLF-II group. Collectively, innate immunity and bacterial escape from fusiform vesicles play important roles in UPEC-induced bladder infections. Our findings suggest that TLF-II combats UPEC-induced bladder infections by effectively mitigating bladder inflammation and preventing bacterial escape from fusiform vesicles into the cytoplasm. The findings suggest that TLF-II is a promising option for treating UTI and reducing its recurrence.

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

BackgroundDysregulation 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.MethodsAdult 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.ResultsAdBOs 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.ConclusionsWe 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.

MP03-08 INCREASED EXPRESSION OF PRO-FIBROTIC AMPHIREGULIN IN RADIATION CYSTITIS

You have accessJournal of UrologyCME1 Apr 2023MP03-08 INCREASED EXPRESSION OF PRO-FIBROTIC AMPHIREGULIN IN RADIATION CYSTITIS Bernadette Zwaans, Sabrina Mota, Elijah Ward, Sarah Bartolone, and Michael Chancellor Bernadette ZwaansBernadette Zwaans More articles by this author , Sabrina MotaSabrina Mota More articles by this author , Elijah WardElijah Ward More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , and Michael ChancellorMichael Chancellor More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Radiation cystitis (RC) is a painful chronic bladder condition that results from radiation therapy for pelvic cancers and for which no safe and effective therapy exists. RC has a long latency; it can take years before We previously developed a pre-clinical model that closely mimics the human condition. We sought to use this model to identify and validate proteins that are altered after RT and that may drive RC. METHODS: Female C57Bl/6 mice received a single dose of 40Gy irradiation. Bladders were harvested at 1 week, 4 weeks, 3 months and 6 months after irradiation, RNA was harvested, mRNA sequencing was performed at paired-end 150bp on the Illumina NovaSeq6000 with a target of 30 million reads per sample, and bioinformatics analysis was performed. Findings of the RNA sequencing were validated using qPCR analysis in bladder tissues and human urothelial cells (SV-HUC-1). We subsequently assessed levels of our protein of interest in urine samples from prostate cancer survivors with history of external beam radiation therapy using ELISA. RESULTS: RNAseq analysis identified amphiregulin (AREG) to be significantly increased in mouse bladder tissues in response to irradiation (Figure 1). AREG expression in the bladder significantly increased over time starting at 1-week post-RT and continuing up to 6 months post-RT. AREG is a member of the Epidermal Growth Factor (EGF) family and AREG has been implicated in fibrosis in various organ systems including kidney, skin, heart, lung, and liver. These findings were validated with qPCR. Exposure of human urothelial cells to radiation also resulted in increased AREG RNA and protein expression in a dose dependent manner. In addition, AREG was detectable in the urine of prostate cancer survivors with history of pelvic radiation therapy and was significantly higher in the subset of patients with diagnosis of RC (Figure 1). CONCLUSIONS: We have identified increased AREG in bladder tissue and urothelial cells after radiation exposure. AREG has been implicated in pathological tissue fibrosis in various organ systems. As such, AREG may play an important role in mediating radiation cystitis in cancer survivors. AREG may be Further research is needed to determine if AREG could be a novel target to treat and detect bladder fibrosis. Source of Funding: Research reported in this abstract was supported by NIDDK of NIH by award K01DK114334 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e23 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bernadette Zwaans More articles by this author Sabrina Mota More articles by this author Elijah Ward More articles by this author Sarah Bartolone More articles by this author Michael Chancellor More articles by this author Expand All Advertisement PDF downloadLoading ...

MP60-12 CHIMERIC BOTULINUM NEUROTOXIN A-B HAS HIGHER NEUROMUSCULAR BLOCKADE POTENCY COMPARED TO BOTULINUM NEUROTOXIN A IN THE MOUSE BLADDER

You have accessJournal of UrologyCME1 Apr 2023MP60-12 CHIMERIC BOTULINUM NEUROTOXIN A-B HAS HIGHER NEUROMUSCULAR BLOCKADE POTENCY COMPARED TO BOTULINUM NEUROTOXIN A IN THE MOUSE BLADDER Hatim Thaker, Jie Zhang, Rosalyn Adam, Vivian Cristofaro, Maryrose Sullivan, and Min Dong Hatim ThakerHatim Thaker More articles by this author , Jie ZhangJie Zhang More articles by this author , Rosalyn AdamRosalyn Adam More articles by this author , Vivian CristofaroVivian Cristofaro More articles by this author , Maryrose SullivanMaryrose Sullivan More articles by this author , and Min DongMin Dong More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003318.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Neurogenic and idiopathic detrusor overactivity (DO) is currently treated with onabotulinumtoxin A (onaBoNT/A) as third-line therapy. However, BoNT/A requires repeated injections into the bladder and is known to have diminished treatment effect after 5 years. Novel, chimeric toxins may provide an alternative to BoNT/A in the setting of treatment failure. Chimeric BoNT/A-B is a construct that consists of the light chain (catalytic domain) of BoNT/A, paired with a receptor binding domain of BoNT/B. This receptor binding domain binds to synaptotagmin-1, recently shown to be the dominant BoNT receptor in the bladder. Here, we aim to determine the effect of a chimeric BoNT/A-B construct in mouse bladder tissues. METHODS: Gene fragments encoding the light chain and translocation domain of BoNT/A, as well as the receptor binding domain for BoNT/B, were produced via PCR and cloned into a vector to produce chimeric BoNT/A-B. To assess ex vivo smooth muscle response, wild type (WT) mouse bladders were harvested and placed in Kreb's buffer. Each bladder was cut into three longitudinal strips and mounted in tissue chambers at 37 C with continuous bubbled mixture of carbogen. Bladder strips were attached to a force transducer (Figure A) and underwent nerve-evoked contraction studies via electrical field stimulation (EFS; 1-64Hz, 30V 0.5 ms pulse width, 10 sec duration), with the addition of 0.3 or 1 nM BoNT/A-B, to generate a frequency response curve. RESULTS: Ex vivo contraction studies in the presence of 1 nM BoNT/A-B showed a reduction in 80% of nerve-evoked contractions by 100 minutes; BoNT/A had produced the same degree of reduction at only 300 minutes, reflecting the rapid onset of BoNT/A-B. At a lower dose of 0.3 nM, BoNT/A-B reduced nerve-evoked contractions by 40% at 100 minutes; BoNT/A had minimal effect (Figure B). All bladder weights were similar, and data were normalized to the cross-sectional area of the bladder strip. Carbachol dose-response curves served as controls to ensure bladder strips were viable and contractile pre- and post-exposure. CONCLUSIONS: We demonstrate chimeric BoNT/A-B has a faster onset and great potency at lower doses in smooth muscle ex vivo. This novel toxin may provide improved neuromuscular paralysis smooth muscle disorders, such as overactive and neurogenic bladder. Source of Funding: H.T. is supported by the NIH T32 Grant, AUA Urology Care Foundation Research Scholar Award, the SUFU Chemodenervation Grant, and the Office of Faculty Development at Harvard Medical School. M.D. is supported by grants from the NIH and holds the Investigator in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund. M.P.S. received support from the U.S. Department of Veterans Affairs © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e848 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hatim Thaker More articles by this author Jie Zhang More articles by this author Rosalyn Adam More articles by this author Vivian Cristofaro More articles by this author Maryrose Sullivan More articles by this author Min Dong More articles by this author Expand All Advertisement PDF downloadLoading ...

Single-cell profiling reveals various types of interstitial cells in the bladder.

Clarifying the locations, molecular markers, functions and roles of bladder interstitial cells is crucial for comprehending the pathophysiology of the bladder. This research utilized human, rat and mouse bladder single-cell sequencing, bioinformatics analysis and experimental validation. The main cell types found in human, rat and mouse bladder tissues include epithelial cells, smooth muscle cells, endothelial cells, fibroblasts, myofibroblasts, neurons and various immune cells. Our study identified two significant types of interstitial cells (PTN+ IGFBP6+ PI16 (CD364)+ CD34+ ) and myofibroblasts (STC1+ PLAT+ TNC+ ). These two types of interstitial cells are mainly located in the subepithelial lamina propria, between muscles and between muscle bundles. In the CYP (cyclophosphamide)-induced bladder injury mouse model, the interaction types and signals (MK, MIF, GDF and CXCL) of fibroblasts and myofibroblasts significantly increased compared with the normal group. However, in the aging mouse model, the signals CD34, LAMININ, GALECTIN, MK, SELPLG, ncWNT, HSPG, ICAM and ITGAL-ITGB2 of fibroblasts and myofibroblasts disappeared, but the signals PTN and SEMA3 significantly increased. Our findings identified two crucial types of interstitial cells in bladder tissue, fibroblasts and myofibroblasts, which play a significant role in normal bladder physiology, CYP-induced bladder injury and aging bladder development.

Exosomes from human urine-derived stem cells carry NRF1 to alleviate bladder fibrosis via regulating miR-301b-3p/TGFβR1 pathway.

Bladder outlet obstruction (BOO) is a common disease that always make the bladder develops from inflammation to fibrosis. This study was to investigate the effect of exosomes from human urine-derived stem cells (hUSCs) on bladder fibrosis after BOO and the underlying mechanism. The BOO mouse model was established by inserting a transurethral catheter, ligation of periurethral wire, and removal of the catheter. Mouse primary bladder smooth muscle cells (BSMCs) were isolated and treated with TGFβ1 to mimic the bladder fibrosis model in vitro. Exosomes from hUSCs (hUSC-Exos) were injected into the bladder of BOO mice and added into the culture of TGFβ1-induced BSMCs. The associated factors in mouse bladder tissues and BSMCs were detected. It was confirmed that thetreatment of hUSC-Exosalleviated mouse bladder fibrosis and down-regulated fibrotic markers (a-SMA and collagen III) in bladder tissues and TGFβ1-induced BSMCs. Overexpression of NRF1 in hUSC-Exos further improved the effects of hUSC-Exos on bladder fibrosis both in vivo and in vitro. TGFβR1 was a target of NRF1 and miR-301b-3p, and miR-301b-3p was atarget of NRF1. It was next characterized that hUSC-Exos carried NRF1 to up-regulate miR-301B-3p, thereby reducing TGFβR1level. Our results illustrated that hUSC-Exos carried NRF1 to alleviate bladder fibrosis through regulating miR-301b-3p/TGFβR1 pathway.

Detrusor Underactivity as an HCN-Mediated Failure of Resilience in Aging

Sympathetic relaxation of the bladder wall permits low pressure urine storage and allows central regulation of afferent sensitivity to volume. Impaired regulation of volume sensitivity has been linked to symptoms of underactive bladder and cystometric detrusor underactivity. Hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) are mediators of sympathomimetic-induced detrusor relaxation in young mouse bladder tissue, however in bladder strips from old female mice, HCN blockade enhanced age-diminished isoproterenol-induced relaxation. We therefore hypothesized that loss of HCN would compromise cystometric function and enhance sympathomimetic responses in old mice. Male HCN1 KO mice (20-22 mo) and their WT littermates underwent pressure-flow cystometry under urethane anesthesia to assess urinary performance at the level of the autonomic nervous system in the absence of cortical control. Following cystometry, bladders were harvested and pharmacomyography was performed on bladder strips to determine tissue-level changes in the absence of CNS input. All mice responded to continuous-fill cystometry by establishing regular filling/voiding cycles. HCN KO mice function showed discrete changes in volume sensitivity vs. WT. Bladder strip studies showed minimal response to isoproterenol regardless of HCN status, and no significant differences in response to carbachol based on HCN status. We conclude that HCN status impacts the brainstem-bladder reflexic control over urine storage/voiding, but not by regulating bladder wall tensions during filling. The absence of HCN influence on the loss of end-organ responsiveness to sympathetic control in old mice points to an increasing dependency on central control mechanisms with aging.

Sensory Receptors are Found in a Non‐sensory Organ: The Bladder

Recent research has found that “sensory” receptors (including olfactory receptors (Olfr), taste receptors (Tas), and opsins (Opn)) play functional roles throughout the body. However, the expression of these receptors has not yet been carefully examined in the bladder. In this study, we utilized customized Taqman low density gene array cards to perform quantitative PCR and identify novel sensory receptors in mouse bladder tissue. We extracted RNA from murine bladder (2 male, 2 female), and then screened for the presence of sensory receptors using custom Taqman arrays which included probesets for 43 ORs, 35 bitter TRs, 3 TRs for umami/sweet, 5 opsins, and 7 G‐proteins and/or accessory proteins associated with sensory signaling pathways, as well as a Gapdh control. Using these arrays, we found the expression of 30 sensory transcripts in all male and female bladder samples, with 10 receptors being relatively well‐expressed (Ct values<~30). These moderately expressed receptors included 2 opsins (Opn1sw, Opn3), 3 ORs (Olfr558, Olfr99, Olfr78), and 5 TRs (Tas1r3,Tas2r143, Tas2r135, Tas2r108, Tas2r126. We did not note any significant sex differences, and the top hit (Opn1sw) was the same for all four arrays. We have localized these top receptors within the bladder using two different methods: quantitative PCR on microdissected bladder tissue, and RNAscope fluorescent imaging. Although some receptors have higher relative expression in either urothelium or muscle, all of the receptors are expressed in both of these cell types. Notably, two of the ORs found in the bladder (Olfr78, Olfr558) are known to respond to bacterial metabolites, which may be relevant in the setting of cystitis/urinary tract infections (UTIs). We then screened by qRT‐PCR for a subset of five of these receptors as well as two control receptors (not detected in bladder in the initial screen) in various tissues: brain, colon, eye, heart, kidney, skeletal muscle, and tongue. We found that all five of the receptors of interest were expressed in at least one tissue in addition to bladder, with varying patterns of expression for each. Further investigation could help us better understand why these receptors are expressed in these tissues. Moreover, determining the role of these novel sensory receptors in the bladder has the potential to enhance our understanding of bladder function, and may lead to novel treatments for conditions such as cystitis/UTIs.

Plasminogen activator inhibitor-2(PAI-2) overexpression supportsbladder cancer developmentin PAI-1 knockout mice in N-butyl-N-(4-hydroxybutyl)-nitrosamine- induced bladdercancer mouse model

BackgroundAccumulating evidence suggests that plasminogen activator inhibitor-1 (PAI-1) plays an important role in bladder tumorigenesis by regulating cell cycle. However, it remains unclear whether and how inhibition of PAI-1 suppresses bladder tumorigenesis.MethodsTo elucidate the therapeutic effect of PAI-1 inhibition, we tested its tumorigenicity in PAI-1 knockout (KO) mice exposed to a known bladder carcinogen.ResultsPAI-1 deficiency did not inhibit carcinogen-induced bladder cancer in mice although carcinogen-exposed wild type mice significantly increased PAI-1 levels in bladder tissue, plasma and urine. We found that PAI-1 KO mice exposed to carcinogen tended to upregulate protein C inhibitor (PAI-3), urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA), and significantly increased PAI-2, suggesting a potential compensatory function of these molecules when PAI-1 is abrogated. Subsequent studies employing gene expression microarray using mouse bladder tissues followed by post hoc bioinformatics analysis and validation experiments by qPCR and IHC demonstrated that SERPING1 is further downregulated in PAI-1 KO mice exposed to BBN, suggesting that SERPING1 as a potential missing factor that regulate PAI-2 overexpression (compensation pathway).ConclusionsThese results indicate that serpin compensation pathway, specifically PAI-2 overexpression in this model, supports bladder cancer development when oncoprotein PAI-1 is deleted. Further investigations into PAI-1 are necessary in order to identify true potential targets for bladder cancer therapy.

Action mechanism of microRNA-1a-3p in mouse neurogenic bladder

Objective To establish a mouse model of neurogenic bladder and to investigate the changes of fibronectin 1 (FN1) and the action mechanism of microRNA (miRNA, miR)-1a-3p. Methods Mice were divided into model group and control group. The model group received subcutaneous injection of myelin oligodendrocyte glycoprotein and mycobacterium tuberculosis, and intraperitoneal injection of pertussis toxin at 48 h. The control group received subcutaneous injection of normal saline. The frequency of urination and the amount of urine output were observed. The mice were sacrificed and the bladder tissue was taken. Three bladders were selected from the model group and the control group for high-throughput sequencing. The changes of FN1 and miR-1a-3p in mouse bladder tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting. Results The sequencing results were analyzed. As the urinary system symptoms worsened, FN1 increased (4.569±0.426, t=-5.142, P<0.01), while miR-1a-3p decreased (1.623±0.312, t=-3.945, P<0.05). Compared with the control group, the expression of FN1 mRNA [2.501 (1.301, 4.251), F=99.183, P<0.01] and protein [2.937 (1.174, 4.262), F=63.834, P<0.01] in the bladder tissue of the model group was up-regulated, and that of miR-1a-3p was down-regulated [2.401 (1.250, 3.001), F=35.951, P<0.01]. The dual luciferase gene report indicated that FN1 was a direct target gene of miR-1a-3p (0.514±0.027, t=13.355, P<0.01). Conclusion The animal model of neurogenic bladder has urinary frequency, urgency, urinary retention and other symptoms, and the expression of FN1 is up-regulated, while the expression of miR-1a-3p is down-regulated. Therefore, miR-1a-3p may participate in neurogenic bladder formation through regulation of FN1. Key words: Neurogenic bladder; Fibronectin 1; MicroRNA-1a-3p

Étude de la virulence d’Acinetobacter baumannii dans un modèle d’infection expérimental du tractus urinaire

The virulence characteristics of Acinetobacter baumannii are poorly understood, especially the in vivo pathogenicity in the urinary tract.This research evaluates the degree of virulence of urinary strain of A. baumannii by visualizing the histopathological features in an experimental urinary tract infection. The infectious effects of this isolate on mice bladder and kidneys were compared to the effects of a Proteus mirabilis specie.The in vivo study was carried out by experimental induction of an ascending infection in mice and visualizing the effects on urinary organs. An ascending model of pyelonephritis was established in female mice with the test isolates. Quantitative bacterial counts and histopathological evaluations of mouse bladders and renal tissues were performed.A. baumannii was able to colonize the bladder and renal tissue of mice. Microscopic observations of the bladder urothelium and renal tissue revealed significant inflammatory changes in infected mice. Furthermore, these inflammatory effects are quite similar to those previously reported with other urinary bacteria. This induced pyelonephritis model might be considered as a promoter to understand the pathogenicity of A. baumannii.The identification of virulence factors could contribute to the development of novel therapeutic alternatives for A. baumannii infections.

Gene expression and DNA methylation regulation of arsenic in mouse bladder tissues and in human urothelial cells.

According to a report of the International Agency for Research on Cancer, arsenic and inorganic arsenic compounds are classified into Group 1 carcinogens with regard to human health. Epidemiological studies indicate that arsenic is one of the main risk factors for the development of bladder cancer. In the present study, arsenic-altered gene expression in mouse bladder tissues and in human urothelial cells was compared. In the mouse model, sodium arsenite-induced mouse urothelial hyperplasia and intracellular inclusions were present. Following DNA array analysis, four genes with differential expression were selected for quantitative real-time PCR assay. The genes were the following: Cystathionine β-synthase (CBS), adenosine A1 receptor (ADORA1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and Wnt inhibitory factor 1 (Wif1). The results indicated a significant increase in the levels of Cbs and Adora1. The analysis of the DNA CpG methylation levels of the mouse Cbs and Adora1 genes revealed no significant change. In contrast to these observations, the four genes were further analyzed in the human normal urothelial cell line SV-HUC1. The data indicated that WIF1 gene expression was decreased by sodium arsenite, whereas this was not noted for CBS, MALAT1 and ADORA1. Sodium arsenite decreased mRNA and protein expression levels of the WIF1 gene. In addition, the methylation levels of the WIF1 gene were increased. Sodium arsenite inhibited cell proliferation and promoted cell migration as demonstrated in cell functional assays. The gene status was compared in 8 human urothelial cell lines, and WIF1 mRNA expression levels were determined to be higher, whereas DNA CpG methylation levels were lower in SV-HUC1 cells compared with those noted in the other 7 bladder cancer cell lines. In summary, the data indicated that sodium arsenite decreased WIF1 gene expression and promoted cell migration. The increased methylation levels of WIF1 DNA CpG could be a potential biomarker for bladder cancer.

Sensory Receptors Are Found in a Non‐sensory Organ: The Bladder

Recent research has found that “sensory” receptors (including olfactory receptors (ORs), taste receptors (TRs), and opsins) play functional roles throughout the body. However, the expression of these receptors has not yet been carefully examined in the bladder. In this study, we utilized customized Taqman low density gene array cards to perform quantitative PCR and identify novel sensory receptors in mouse bladder tissue. We extracted RNA from murine bladder (2 male, 2 female), and then screened for the presence of sensory receptors using custom Taqman arrays which included probesets for 43 ORs, 35 bitter TRs, 3 TRs for umami/sweet, 5 opsins, and 7 G‐proteins and/or accessory proteins associated with sensory signaling pathways, as well as a Gapdh control. Using these arrays, we found the expression of 30 sensory transcripts in all male and female bladder samples, with 11 receptors being relatively well‐expressed (Ct values&lt;~30). The 11 highly expressed receptors included 2 opsins (Opn1sw, Opn3), 3 ORs (Olfr558, Olfr99, Olfr78), 5 TRs (Tas1r3, Tas2r143, Tas2r135, Tas2r108, Tas2r126), and the G protein for olfaction (Gnal). We did not note any sex differences, and the top hit (Opn1sw) was the same for all four arrays. Notably, two of the ORs found in the bladder (Olfr78, Olfr558) are known to respond to bacterial metabolites, which may be relevant in the setting of cystitis/urinary tract infections (UTIs). In the future, determining the role of these novel bladder sensory receptors has the potential to enhance our understanding of bladder function, and may lead to novel treatments for conditions such as cystitis/UTIs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The increased expression of Piezo1 and Piezo2 ion channels in human and mouse bladder carcinoma.

Piezo1/2, a mechanically activated ion channel, is believed to play an important role in bladder carcinogenesis process. Piezo1/2 expression has not been previously reported in urinary bladder carcinoma, and little is known about its significance in bladder carcinogenesis. In our study, we aimed to evaluate the Piezo1 and Piezo2 expression as developmental in mouse bladder tissue and bladder cancer tissue of mice and humans. The detection of developmental expression was performed on P0-P90 days in bladder tissue of Balb/c strain mice. Mice were divided into bladder cancer (n = 40) and control groups (n = 10). Bladder cancer in mice was created by using N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In the study, 60 human subjects were included, whose normal tissues were used as controls. After the histopathological evaluation, the expression of Piezo1/2 genes was examined by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry in tumor and normal tissues. In developmental period of the mice, Piezo1 expression increased on days 21 and 90, whereas Piezo2 expression increased on day 7 and decreased on day 90 in mouse bladder tissues. There was a significant increase in the expression of Piezo1/2 in both cancer groups compared to the control group. Piezo1 expression was significantly increased at tumor size, stage and grade. Piezo2 expression was upregulated in high grade tumors in human subjects. The developmental changes of Piezo expression on specific days demonstrate the role of these channels in bladder cancer development. The dysfunction of Piezo1/2 expression may contribute to the carcinogenesis of bladder cancer by causing proliferative changes and angiogenesis. The expression of Piezo1/2 can provide new prognostic information for disease progression.

AB295. SPR-22 Functional relevance of purinergic P2X4R in bladder smooth muscle

ObjectiveUnder physiologic conditions in animals and pathologic conditions in humans, purinergic mechanisms contribute significantly to detrusor contractions. Although the activation of P2X1 receptors (P2X1R) accounts for the largest portion of bladder smooth muscle (BSM) responses to ATP, previous reports have shown that P2X1R antagonists do not completely abolish the purinergic component of neurogenic contractions, suggesting the presence of other P2XR subtypes on BSM. P2X4R has been identified in bladder tissue, however whether this receptor is functionally relevant warrants investigation. The aim of this study was to examine the extent of P2X4R expression in BSM tissue and to investigate its pharmacological contribution to ATP-mediated detrusor contractions.MethodsP2X4R mRNA and protein expression was investigated in mouse bladder tissue (without mucosa) and in cultured BSM cells by real-time RT-PCR and western blotting respectively. In vitro isometric tension studies were performed in mouse BSM strips without mucosa. Purinergic detrusor contractions were elicited by administration of α-β-methylene-ATP (αβmATP), and the purinergic component of neurogenic contractions induced by electrical field stimulations (EFS) was isolated by pre-treatment with the muscarinic receptor antagonist atropine. The inhibitory effect of two P2X4R selective antagonists, 5-BDBD and BX430, on the αβmATP- and EFS-induced contractions was investigated in the presence of P2X1R antagonist NF449. In addition, the effect of the P2X4R positive modulator ivermectin (IVC) on αβmATP responses was investigated. Data were analyzed by a repeated measures analysis of variance.ResultsP2X4R mRNA was detected in smooth muscle from both bladder tissue and cultured BSM cells, although its expression was significantly lower than P2X1R expression. Immunoreactivity for P2X4R was detected in lysates from both mouse BSM tissue and smooth muscle cells. Functional studies indicated that although P2X1R activation is predominantly responsible for purinergic contractions in mouse detrusor, a significant portion of the contractile response to both αβmATP (22.3±7%) and EFS (27.5±4% of purinergic component of EFS) was resistant to P2X1R inhibition. This NF449-resistant component was abolished by administration of P2X4R antagonists 5-BDBD or BX430. In addition, responses to αβmATP increased significantly upon administration of IVC.ConclusionsThe expression of P2X4R in detrusor smooth muscle together with the identification of a P2X4R-sensitive component of bladder contractions suggest that the activation of this P2X receptor subtype could significantly contribute to ATP-mediated BSM responses. P2X4R may thus potentially represent a novel target for the management of detrusor dysfunctions associated with alterations in purinergic signaling.Funding Source(s)Department of Veterans Affairs, Research Service BX001790; BX002806

MP24-08 EPIGENETIC REGULATION OF DNA DAMAGE REPAIR GENE 8-OXOGUANINE DNA GLYCOSYLASE (OGG1) IN THE PYROPTOSIS PATHWAYS OF HEMORRHAGIC CYSTITIS

INTRODUCTION AND OBJECTIVES: Hemorrhagic cystitis is the severe clinical manifestation of several systemic chemotherapeutics, most notably cyclophosphamide (CPX), and other nitrogen mustard alkylating agents. 8-oxoguanine DNA glycosylase (Ogg1) is a base excision repair enzyme that edits oxidized nucleotide residues and is essential for initiation of the pyroptotic cell death pathways. We investigated the role of this enzymatic pathway in hemorrhagic cystitis to identify any targets for potential future therapies. METHODS: Several models, including an in-vitro mouse bladder fibroblast model, an in-vivo mouse model, and a cellular Ogg1 knockout model, were treated with either cyclophosphamide or acrolein to induce bladder inflammation and hemorrhagic cystitis. Nicotinamide was also added to treatment groups to limit DNA methylation. Cultured bladder fibroblasts and harvested mouse bladders tissues and cells were subjected to bisulfite sequencing and chromatin immunoprecipitation (ChIP) analysis to evaluate expression of Ogg1 and DNA methylation patterns. RESULTS: We found that Ogg1 was down-regulated with subsequent accumulationof 8-oxoguanineandactivationofNLRP3-mediated pyroptotic cell death signaling pathways when inflammation was induced with cyclophosphamide and acrolein. In the attached graphic, differences incellularadhesionandcell deathareevident in thephasecontrast images and histologic examination of mouse bladder tissues demonstrates characteristic detrusor hypertrophy in response to cyclophosphamide. In bladder fibroblasts treated with acrolein, bisulfite sequence analysis demonstratedCpGmethylation onOgg1 geneRegion III and transcription silencing. Additionally, recruitment of DNMT3b in acrolein-treated cells initiated de novo methylation in ChIP analysis. Knockout of Ogg1 in bladder fibroblasts activated signal I and II in pyroptosis. CONCLUSIONS: Epigenetic regulation of Ogg1 is essential in the pyroptosis cellular pathway in hemorrhagic cystitis. The pyroptosis signaling cascade in detrusor cells and the role of Ogg1 suggest the potential for a novel therapeutic target for prevention and treatment of this highly morbid condition.

Editorial: Therapeutic implications of circadian rhythms.

Editorial: Therapeutic implications of circadian rhythms.