Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis.

BACKGROUND AND PURPOSE: The aim of this study is to decipher the underlying mechanisms of CCAAT/enhancer-binding protein delta (CEBPD)-enhanced glycolysis as well as the biological significance of CEBPD and MYC coamplification in urothelial carcinoma (UC). METHODS: In vitro analyses were conducted to examine the effects of altered CEBPD or MYC expression on UC cells. The in vivo effects of CEBPD overexpression in a high-glucose environment on tumour growth were investigated in xenografted induced diabetic severe combined immunodeficiency/beige mice. Data mining was used to cross-validate the associations between CEBPD and MYC copy number and transcriptional expression, quantitative reverse transcription-polymerase chain reaction, immunohistochemistry, chromogenic in situ hybridization, and in situ hybridization targeting microRNA were performed on 635 UC patient samples and xenograft samples. UC patient survival in relation to diabetes was validated by using the National Health Insurance Research Database. RESULTS: CEBPD and MYC coamplification (29.6%) occurred at a high frequency, MYC expression promoted chromosomal instability, facilitating CEBPD copy number gain and expression. CEBPD promoted glucose uptake and lactate production by upregulating SLC2A1 and HK2, leading to mitochondrial fission, increased extracellular acidification rate and decreased oxygen consumption rate to fuel cell growth. CEBPD upregulated HK2 expression through multiple regulation pathways including MYC stabilization, suppression of FBXW7 transactivation and MYC-independent transcriptional suppression of hsa-miR-429. Clinical and xenografted experiments confirmed the growth advantage of CEBPD in relation to glucose metabolic dysregulation and the significant correlations between the expression of these genes. CONCLUSIONS: We confirmed that CEBPD has an oncogenic role in UC by activating AKT signalling and initiating metabolic reprogramming from mitochondrial oxidative phosphorylation to glycolysis to satisfy glucose addiction. These novel CEBPD- and MYC-centric multilayered positive feedback loops enhance cancer growth that could complement theranostic approaches.

Functional roles for PIEZO1 and PIEZO2 in urothelial mechanotransduction and lower urinary tract interoception.

The mechanisms that link visceral mechanosensation to the perception of internal organ status (i.e., interoception) remain elusive. In response to bladder filling, the urothelium releases ATP, which is hypothesized to stimulate voiding function by communicating the degree of bladder fullness to subjacent tissues, including afferent nerve fibers. To determine if PIEZO channels function as mechanosensors in these events, we generated conditional urothelial Piezo1-, Piezo2-, and dual Piezo1/2-knockout (KO) mice. While functional PIEZO1 channels were expressed in all urothelial cell layers, Piezo1-KO mice had a limited phenotype. Piezo2 expression was limited to a small subset of superficial umbrella cells, yet male Piezo2-KO mice exhibited incontinence (i.e., leakage) when their voiding behavior was monitored during their active dark phase. Dual Piezo1/2-KO mice had the most affected phenotype, characterized by decreased urothelial responses to mechanical stimulation, diminished ATP release, bladder hypoactivity in anesthetized Piezo1/2-KO females but not males, and urinary incontinence in both male and female Piezo1/2-KO mice during their dark phase but not inactive light one. Our studies reveal that the urothelium functions in a sex- and circadian rhythm-dependent manner to link urothelial PIEZO1/2 channel-driven mechanotransduction to normal voiding function and behavior, and in the absence of these signals, bladder dysfunction ensues.

Carcinoma urotelial del tracto urinario superior: a propósito de un caso y revisión de la literatura / Upper urinary tract urothelial carcinoma: case report and review of the literature

El carcinoma urotelial (CU) del tracto urinario superior es infrecuente y representa solo del 5%-10% de todos los CU. Estas neoplasias crecen a partir del urotelio de los cálices renales hasta el tercio distal del uréter. Se reporta el caso de un paciente masculino de 68 años de edad quien presenta enfermedad actual de 3 meses de evolución caracterizada por dolor lumbar izquierdo, tipo cólico, de leve a moderada intensidad, el cual atenúa parcialmente con el uso de AINES, asociado a hematuria visible total de predominio nocturno. El uroanálisis mostró hematuria macroscópica y la citología urinaria evidenció atipias sugerentes de carcinoma. La TAC abdomino-pélvica contrastada evidenció un defecto de llenado en relación al cáliz inferior de riñón izquierdo y plastrón ganglionar paraaórtico izquierdo. Se practicó nefroureterectomía radical izquierda evidenciando tumor de 3 x 3 x 1 cm en relación a pelvis renal extensiva a cáliz inferior invadiendo parénquima renal. El estudio histopatológico mostró un carcinoma urotelial papilar infiltrativo de alto grado con márgenes sin lesiones y ganglios linfáticos con metástasis. El paciente evoluciona satisfactoriamente durante el período postoperatorio y actualmente recibe terapia adyuvante. A pesar de ser una patología poco frecuente, puede presentarse y el urólogo debe estar en capacidad para poder enfrentarla(AU)

Upper urinary tract urothelial carcinoma (UC) is infrequent and represents only 5%-10% of all UCs. These neoplasms grow from the urothelium of renal calyces to the distal third of the ureter. A case of UC of the upper urinary tract is reported in a 68-year-old male patient with a 3-month history of left lumbar mild to moderate pain, which partially mitigates with the use of NSAIDs associated with visible total predominantly nocturnal hematuria. Macroscopic hematuria was evident and urinary cytology reported carcinoma suggestive atypias. Contrasted CT of abdomen and pelvis showed filling defect in relation to lower calyx of the left kidney and left para-aortic ganglion plastron. Radical left nephroureterectomy was performed showing a 3 x 3 x 1 cm tumor in relation to the renal pelvis extending to the lower cavity and invading renal parenchyma. Histopathology showed high grade infiltrative papillary CU with margins without lesions and lymph nodes with metastasis. Patient evolves satisfactorily in the postoperative period and is currently in adjuvant therapy. Although this pathology is rare, it can occur and the urologist must be able to face it(AU)

Additive effects of intravenous and intravesical application of vibegron, a ß3-adrenoceptor agonist, on bladder function in rats with bladder overactivity.

To examine the effects of intravenous and intravesical application of vibegron, a new ß3-adrenoceptor (ß3-AR) agonist, on bladder function in rats with oxotremorine methiodide (oxo-M: a nonselective muscarinic receptor agonist)-induced bladder overactivity. Cystometry was performed in conscious female rats with intravesical instillation of oxo-M (200 µM). In oxo-M-treated rats, vehicle or vibegron (1 and 10 mg/kg) was cumulatively applied intravenously at 30-min intervals. In other groups of rats, oxo-M + vehicle or oxo-M + vibegron (10, 100 µM, and 1 mM) was cumulatively instilled intravesically at 60-min intervals followed by intravenous application of vibegron (10 mg/kg). Expression of ß3-ARs in the bladder was also evaluated using immunohistochemical staining. Intravenous application of vibegron (10 mg/kg) significantly increased bladder capacity (1.3 times) and decreased baseline, threshold, and maximal voiding pressure compared with vehicle. Next, intravesical application of vibegron (1 mM) significantly increased threshold pressure and bladder capacity (1.2 times) compared with vehicle. Combined treatments of intravesical (1 mM) and intravenous (10 mg/kg) application of vibegron induced a significantly larger degree of increases in bladder capacity (1.4 times) compared with vehicle. In addition, ß3-ARs were expressed throughout the rat bladder, mainly in the urothelium. These results suggest that vibegron excreted in urine as an unchanged compound can induce the additive inhibitory effects on bladder overactivity possibly through urothelial ß3-AR activation, which inhibits the afferent limb of micturition reflex rather than the efferent function as evidenced by the increases in threshold pressure and bladder capacity without affecting bladder contractile function after intravesical vibegron application.

The urothelium, the urinary microbioma and men LUTS: a systematic review.

INTRODUCTION: The pathophysiology and management of male patients with lower urinary tract symptoms (LUTS) is still a matter of debate. In the past few years, the urothelium and the urinary microbiota represented important areas of research to improve the understanding and management of these patients. Aim of the present review was to summarize the available data on the urothelium and the microbiota related to male LUTS. EVIDENCE ACQUISITION: A National Center for Biotechnology Information (NCBI) PubMed search for relevant articles published between January 2000 and December 2019 was performed using the medical subjects heading "urothelium," "microbioma," "microbiota," "urobioma," "urobiota," "benign prostatic hyperplasia," "benign prostatic enlargement," "lower urinary tract symptoms," "lower urinary tract dysfunction," "men," "male," "overactive bladder," "receptors." Exclusion criteria included: animal studies and studies on muscarinic and adrenergic pathways. EVIDENCE SYNTHESIS: The urothelium has been recently evaluated in humans to evaluate new possible markers and pathways. New possible targets for the treatment of male LUTS include the neural growth factor, the cannabinoid, the vanilloid and the ATP pathways. However, studies in humans are still needed to elucidate the exact role of these pathways in the management of male patients with LUTS. The available evidence on the urinary microbioma in male is poor. Standing to the available, urinary microbioma is evident in healthy urine in males. Moreover, the urinary microbioma varies depending on the method of collection, sexually transmitted disease status, inflammation and urinary symptoms. A possible role of probiotics in the management of LUTS in women has been proposed and may have a role in male patients as well. CONCLUSIONS: The urothelium and the urinary microbiota are still poorly studied in men with LUTS. Most of the evidence and the hypothesis on the relationship between urothelium/urinary microbiota and LUTS comes from animal/in-vitro evidence while clinical trials are lacking. These pathways seem interesting even in LUTS pathogenesis in men but their possible role as a new therapeutic target is still an open debate.

Therapeutic effects of Choreito, a traditional Japanese (Kampo) medicine, on detrusor overactivity induced by acetic acid in rats.

Choreito (CRT), a traditional Japanese (Kampo) medicine, is widely used for the treatment of overactive bladder (OAB) and other lower urinary tract symptoms in Japan. This study aimed to identify the effects and therapeutic mechanism of CRT on the improvement of detrusor overactivity (DO) using an experimental rat model. Forty-five female Sprague-Dawley rats were equally divided into three groups: intravesical saline instillation with normal food (normal group), intravesical acetic acid (AA) instillation with normal food (AA group), and intravesical AA instillation with CRT (AA with CRT group). To induce a decrease in bladder capacity, instillation of 0.2% AA was used based on prior studies. Cystometric investigation was employed to clarify the effects of AA and CRT. Microcirculation was performed using a laser blood flowmeter, and the localization of hypoxia-inducible factor 1α (HIF1α) was assessed by immunohistochemistry. The bladder capacities of the normal, AA, and AA with CRT groups were 1.2 ± 0.3 mL, 0.4 ± 0.1 mL, and 0.8 ± 0.1 mL, respectively. CRT significantly attenuated AA irritation of the urinary bladder and exerted protective effects on basal pressure, micturition pressure, micturition interval, and micturition volume. Furthermore, CRT could prevent the excess blood flow and edematous change under the urothelium induced by intravesical AA instillation. No obvious changes in immunohistochemical HIF1α staining were observed among the groups. CRT attenuated DO induced by intravesical AA instillation in a rat experimental model. CRT might impart therapeutic effects on OAB via the mitigation of urothelial damage and regulation of excess blood flow.

Nicotine-induced hypoxia in rat urothelium deteriorates bladder storage functions.

AIMS: To measure the effects of nicotine on lower urinary tract symptoms (LUTS), bladder blood flow, and the urothelial markers hypoxia-inducible factor 1α (HIF1α), uroplakin III (UPIII), and aquaporin 3 (AQP3). METHODS: Ten-week-old female Sprague Dawley rats were subcutaneously injected with 2 mg/kg nicotine (n = 17) or vehicle (control, n = 18) twice daily for 13 days. Some nicotine-treated rats (n = 10) were injected daily with 1 mg/kg tadalafil for the last 6 days of nicotine treatment. One day before cystometry, the bladders of some nicotine-treated and control rats were instilled with 0.08% acetic acid. Urinary frequency and volume were measured 1 day after treatment. Blood flow in the bladder neck was measured, and the urothelia were immunochemically assayed for HIF1α, UPIII, and AQP3. RESULTS: Following acetic acid treatment, both voiding interval and micturition volume of the nicotine-treated rats were significantly lower than controls. Nicotine-treated rats had lower blood flow than controls, and the urothelial expression of HIF1α was higher than controls. Simultaneously, the expressions of UPIII and AQP3 were decreased. Tadalafil treatment increased bladder blood flow, and nicotine-treated rats had increased voiding interval and micturition volume. Further, the expression of HIF1α decreased, and both UPIII and AQP3 levels increased. CONCLUSIONS: Nicotine-treated rats stimulated by intravesicular acetic acid instillation exhibited deterioration of bladder storage functions. Changes in tissue markers in the nicotine-treated rats were consistent with hypoxia and loss of urothelial function. Restoration of blood flow reversed the nicotine effects. Nicotine may induce LUTS through reduced bladder blood flow and urothelial hypoxia.

Urinary K+ promotes irritative voiding symptoms and pain in the face of urothelial barrier dysfunction.

The internal surface of the bladder is lined by the urothelium, a stratified epithelium that forms an impermeable barrier to water and urine constituents. Abnormalities in the urothelial barrier have been described in certain forms of cystitis and were hypothesized to contribute to irritative voiding symptoms and pain by allowing the permeation of urinary K+ into suburothelial tissues, which then alters afferent signaling and smooth muscle function. Here, we examined the mechanisms underlying organ hyperactivity and pain in a model of cystitis caused by adenoviral-mediated expression of claudin-2 (Cldn2), a tight junction protein that forms paracellular pores and increases urothelial permeability. We found that in the presence of a leaky urothelium, intravesical K+ sensitizes bladder afferents and enhances their response to distension. Notably, dietary K+ restriction, a maneuver that reduces urinary K+, prevented the development of pelvic allodynia and inflammation seen in rats expressing Cldn2. Most importantly, intravesical K+ causes and is required to maintain bladder hyperactivity in rats with increased urothelial permeability. Our study demonstrates that in the face of a leaky urothelium, urinary K+ is the main determinant of afferent hyperexcitability, organ hyperactivity and pain. These findings support the notion that voiding symptoms and pain seen in forms of cystitis that coexist with urothelial barrier dysfunction could be alleviated by cutting urinary K+ levels.

Transperitoneal radical nephroureterectomy is associated with worse disease progression than retroperitoneal radical nephroureterectomy in patients with upper urinary tract urothelial carcinoma.

This study aimed to compare the oncologic outcomes between retroperitoneal radical nephroureterectomy (rRNU) and transperitoneal radical nephroureterectomy (tRNU) for the treatment of patients with upper urinary tract urothelial carcinoma (UTUC). Medical records of 743 eligible patients who underwent rRNU or tRNU between 1995 and 2015 were reviewed retrospectively. Progression-free survival (PFS), cancer-specific survival (CSS), and overall survival (OS) were compared according to the surgical approach using the Kaplan-Meier method. Predictors of PFS, CSS, and OS were analyzed with a multivariable Cox regression model. Overall, 620 (83.4%) and 123 (16.6%) patients were treated with rRNU and tRNU, respectively. Five-year CSS and OS rates were equivalent between rRNU and tRNU groups, but 5-year PFS was lower in the tRNU group than the rRNU group (P = 0.005). When patients were stratified by pathological T stage, PFS was significantly different between the two groups in favor of the rRNU group for both organ-confined disease (pTis/pTa/pT1/T2) (P = 0.022) and locally advanced disease (pT3/pT4) (P = 0.039). However, no significant differences in CSS or OS was observed when comparing the two surgical types in patients with organ-confined disease (P = 0.336 and P = 0.851) or patients with locally advanced disease (P = 0.499 and P = 0.278). tRNU was a significant predictor of PFS (hazard ratio = 1.54; P = 0.023), but not CSS or OS. Our findings indicate that the rRNU approach resulted in better PFS than the tRNU approach in patients with UTUC.

Single onabotulinumtoxinA 200U dose improved clinical symptoms but not urothelial dysfunction in neurogenic detrusor overactivity due to spinal cord injury.

OBJECTIVE: To evaluate the changes in urothelial dysfunction protein expressions in bladder after onabotulinumtoxin injection and correlate that with clinical outcomes in spinal cord injury (SCI) patients. METHODS: Twenty-six patients with neurogenic detrusor overactivity (NDO) and urinary incontinence due to suprasacral SCI were treated with onabotulinumtoxinA 200U detrusor injection. Urodynamic studies and bladder biopsies were obtained at baseline, 3, and 6 months after treatment. Biopsy tissues were investigated for E-cadherin, zonula occludens-1 (ZO-1), mast cell activity, and urothelial cell apoptosis, sensory protein expression including purinergic receptor P2X3, endothelial NOS, inducible NOS, ß3-adrenoceptors, and muscarinic receptors M2 and M3. Differences in functional protein expression between controls and SCI patients and between successful and failed treatment groups were analyzed. RESULTS: SCI patients had significantly lower E-cadherin, higher mast cell activity, increased apoptosis, decreased M3 and eNOS expressions than the controls at baseline. Of the 26 patients, 17 (65%) showed improvement in bladder capacity by >50% at 3 months; however, improvement declined by 6 months after treatment. The urothelial expression of E-cadherin and ZO-1 increased at 3 months but had declined at 6 months. The urothelial sensory protein expression did not change significantly after treatment. M3 receptor density was significantly decreased in SCI patients at baseline and patients with treatment success 3 months after injection (p = 0.01). CONCLUSION: A single injection of onabotulinumtoxinA 200U improved clinical symptoms but did not significantly alter urothelial sensory protein expression. The results imply that a single 200U onabotulinumtoxinA dose might not be adequate for urothelial dysfunction in NDO. IRB: TCGH 098-53.

Stress-induced autonomic dysregulation of mitochondrial function in the rat urothelium.

AIM: Chronic stress exacerbates the symptoms of most pain disorders including interstitial cystitis/bladder pain syndrome (IC/BPS). Abnormalities in urothelial cells (UTC) occur in this debilitating bladder condition. The sequence of events that might link stress (presumably through increased sympathetic nervous system-SNS activity) to urothelial dysfunction are unknown. Since autonomic dysregulation, mitochondrial dysfunction, and oxidative stress all occur in chronic pain, we investigated whether chronic psychological stress initiated a cascade linking these three dysfunctions. METHODS: Adult female Wistar Kyoto rats were exposed to 10 days of water avoidance stress (WAS). Bladders were then harvested for Western blot and single cell imaging in UTC cultures. RESULTS: UTC from WAS rats exhibited depolarized mitochondria membrane potential (Ψm ∼30% more depolarized compared to control), activated AMPK and altered UT mitochondria bioenergetics. Expression of the fusion protein mitofusion-2 (MFN-2) was upregulated in the mucosa, suggesting mitochondrial structural changes consistent with altered cellular metabolism. Intracellular calcium levels were elevated in cultured WAS UTC, consistent with impaired cellular function. Stimulation of cultured UTC with alpha-adrenergic (α-AR) receptor agonists increased reactive oxidative species (ROS) production, suggesting a direct action of SNS activity on UTC. Treatment of rats with guanethidine to block SNS activity prevented most of WAS-induced changes. CONCLUSIONS: Chronic stress results in persistent sympathetically mediated effects that alter UTC mitochondrial function. This may impact the urothelial barrier and signaling, which contributes to bladder dysfunction and pain. This is the first demonstration, to our knowledge, of a potential autonomic mechanism directly linking stress to mitochondrial dysfunction.

Polyploid Superficial Cells that Maintain the Urothelial Barrier Are Produced via Incomplete Cytokinesis and Endoreplication.

The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair.

NLRP3/IL-1ß mediates denervation during bladder outlet obstruction in rats.

AIMS: Denervation of the bladder is a detrimental consequence of bladder outlet obstruction (BOO). We have previously shown that, during BOO, inflammation triggered by the NLRP3 inflammasome in the urothelia mediates physiological bladder dysfunction and downstream fibrosis in rats. The aim of this study was to assess the effect of NLRP3-mediated inflammation on bladder denervation during BOO. METHODS: There were five groups of rats: (i) Control (no surgery); (ii) Sham-operated; (iii) BOO rats given vehicle; (iv) BOO rats given the NLRP3 inhibitor glyburide; and (v) BOO rats given the IL-1 receptor antagonist anakinra. BOO was constructed by ligating the urethra over a 1 mm catheter and removing the catheter. Medications were given prior to surgery and once daily for 12 days. Bladder sections were stained for PGP9.5, a pan-neuronal marker. Whole transverse sections were used to identify and count nerves while assessing cross-sectional area. For in vitro studies, pelvic ganglion neurons were isolated and treated with IL-1ß. After a 48 h incubation apoptosis, neurite length and branching were assessed. RESULTS: In obstructed bladders, the number of nerves decreased while total area increased, indicating a loss of cell number and/or branching. The decrease in nerve density was blocked by glyburide or anakinra, clearly implicating the NLRP3 pathway in denervation. In vitro analysis demonstrated that IL-1ß, a product of the inflammasome, induced apoptosis in pelvic ganglion neurons, suggesting one mechanism of BOO-induced denervation is NLRP3/IL-1ß triggered apoptosis. CONCLUSIONS: The NLRP3/IL-1ß-mediated inflammation pathway plays a significant role in denervation during BOO.

Elucidating Mechanisms of Bladder Repair after Hyaluronan Instillation in Ketamine-Induced Ulcerative Cystitis in Animal Model.

Ketamine-induced ulcerative cystitis (KIC) initially damaged the bladder mucosa and induced contracted bladder thereafter. Hyaluronan (hyaluronic acid; HA) instillation to the bladder has been used to treat KIC. The present study investigated bladder injury by urothelial defect and HA degeneration and bladder repair by urothelium proliferation and differentiation. This work was based on the hypothesis that HA treatment altered the bladder urothelial layer and the expression of hyaluronan-metabolizing enzymes and/or HA receptors in KIC. Cystometrogram study and tracing analysis of voiding behavior revealed that the ketamine-treated rats exhibited significant bladder hyperactivity with an increase in micturition frequency and a decrease in bladder capacity. The expression of inflammatory and fibrosis markers was also increased in the ketamine-treated group. Moreover, ketamine administration decreased the expression of urothelial barrier-associated protein, altered HA production, and induced abnormal urothelial differentiation, which might attribute to urothelial lining defects. However, HA instillation ameliorated bladder hyperactivity, lessened bladder mucosa damage, and decreased interstitial fibrosis. HA instillation also improved the level of HA receptors (CD44, Toll-like receptor-4, and receptor for HA-mediated motility) and HA synthases 1 to 3 and decreased the expression of hyaluronidases in the urothelial layer of bladder, resulting in enhanced mucosal regeneration. These findings suggested that HA could modulate inflammatory responses, enhance mucosal regeneration, and improve urothelial lining defects in KIC.

Inhibitory Effects of Urothelium-related Factors.

The urothelium of the bladder has long been recognized as a protective barrier between detrusor and urine. In recent years, it has become more evident that the urothelium plays a role as an active source of mediators. The urothelium can release neurotransmitters and modulators such as acetylcholine, ATP, nitric oxide, prostaglandins and neuropeptides. They exert both excitatory and inhibitory effects in modulating urinary tract motility. In addition, several studies have reported the existence of an urothelium-derived unknown inhibitory factor in the urinary bladder. By the use of a new serial cascade superfusion bioassay on guinea pig ureter, recent studies confirm that the guinea pig bladder urothelium releases a substance with inhibitory bioactivity, which was resistant to treatment with nitric oxide synthase inhibitor and cyclooxygenase inhibitor and to adenosine A1/A2 receptor blockade. Lately, a marked and quickly inactivated novel release of PGD2 from the bladder urothelium was discovered, together with localization of prostaglandin D synthase therein. PGD2 was found to have an inhibitory influence on nerve-induced contractions in guinea pig urinary bladder and on spontaneous contractions in the out-flow region. An altered release of excitatory and inhibitory factors is likely to play an important part in bladder motility disturbances, of which the prostanoids are a notable group. Due to the fact that the bladder is relaxed 99% of the time, not only excitatory mechanisms in the bladder are necessary to study, but also inhibitory mechanisms need considerable attention, which will contribute to the discovery of new targets to treat bladder motility disorders.

Urothelial Dysfunction and Chronic Inflammation in Diabetic Patients with Overactive Bladder.

OBJECTIVES: To investigate urothelial dysfunction, suburothelial inflammation, and muscarinic receptor expression in diabetic patients with overactive bladder (OAB). METHODS: A total 19 patients with OAB and diabetes mellitus (DM), 14 OAB patients without DM, and 10 healthy control subjects were enrolled in the study. Immunofluorescence staining of E-cadherin, zonula occludens-1 (ZO-1), tryptase (mast cell activation), and apoptosis tests on the bladder urothelium and suburothelium were performed. Tissue muscarinic receptor M2, M3, and purinergic receptor P2X3 levels were measured by Western blotting. Video urodynamic studies were analyzed in relation to immunofluorescence and western blotting results. RESULTS: The mean age of the controls, OAB patients without and with DM were comparable (62.9 ± 6.9, 58.4 ± 21.3, and 68.9 ± 11.3 years, respectively, P = 0.25). E-cadherin and mast cell expression in the bladder urothelium samples of the DM-OAB group and the OAB group without DM differed significantly from those of the control group (all P < 0.05). However, immunofluorescence analyses including E-cadherin, mast cells, apoptotic cell counts, and ZO-1 were comparable between OAB patients with and without DM. In addition, M3 muscarinic protein expression in the bladders of OAB patients with and without DM was significantly higher than that in the control subjects. The video urodynamic parameters did not correlate with the immunofluorescence data, M2 and M3 muscarinic receptor data, or P2X3 protein expression. CONCLUSION: Urothelial dysfunction and chronic suburothelial inflammation may contribute to the pathogeneses of OAB. However, DM does not aggravate the severity of urothelial inflammation in OAB patients.

Role of renal urothelium in the development and progression of kidney disease.

The clinical and financial impact of chronic kidney disease (CKD) is significant, while its progression and prognosis is variable and often poor. Studies using the megabladder (mgb -/- ) model of CKD show that renal urothelium plays a key role in modulating early injury responses following the development of congenital obstruction. The aim of this review is to examine the role that urothelium has in normal urinary tract development and pathogenesis. We discuss normal morphology of renal urothelium and then examine the role that uroplakins (Upks) play in its development. Histologic, biochemical, and molecular characterization of Upk1b RFP/RFP mice indicated Upk1b expression is essential for normal urinary tract development, apical plaque/asymmetric membrane unit (AUM) formation, and differentiation and functional integrity of the renal urothelium. Our studies provide the first evidence that Upk1b is directly associated with the development of congenital anomalies of the urinary tract (CAKUT), spontaneous age-dependent hydronephrosis, and dysplastic urothelia. These observations demonstrate the importance of proper urothelial differentiation in normal development and pathogenesis of the urinary tract and provide a unique working model to test the hypothesis that the complex etiology associated with CKD is dependent upon predetermined genetic susceptibilities that establish pathogenic thresholds for disease initiation and progression.

Nerve growth factor release from the urothelium increases via activation of bladder C-fiber in rats with cerebral infarction.

AIMS: There are some reports that bladder C-fibers are partially involved in detrusor overactivity in patients with brain lesions. We investigated the contribution of bladder C-fiber to decreased bladder capacity in rats with cerebral infarction. METHODS: Cerebral infarction was induced under halothane anesthesia by left middle cerebral artery occlusion with 4-0 nylon thread in female Sprague-Dawley rats. Intramural amounts of ATP and prostaglandin E2 , in vivo and in vitro ATP, NGF, and prostaglandin E2 release from the distended bladder urothelium, and changes in mRNA expressions of sensor molecules and receptors were monitored 6 h after the occlusion. Cystometry was performed in rats with or without resiniferatoxin pretreatment. RESULTS: Overexpression of sensor molecule, transient receptor potential vanilloid-type channel 1, acid-sensing ion channel 2, purinergic receptors P2X3 , and M2 /M3 muscarinic receptors was found in the bladder. These changes were accompanied by increases in ATP and NGF release from the urothelium. In contrast, when bladder C-fibers were desensitized by resiniferatoxin, no increase in NGF release from the urothelium was found either in vivo or in vitro. There was no difference in the percentage decrease in bladder capacity between cerebral infarction rats pretreated with resiniferatoxin and cerebral infarction rats without pretreatment. CONCLUSIONS: Results indicate that expression of sensor molecules in the bladder is altered by distant infarction in the brain. ATP and NGF release from the urothelium also increased. NGF release was related to activation of bladder C-fibers. Bladder C-fibers might not contribute much to decreased bladder capacity caused by cerebral infarction.

Urothelial Functional Protein and Sensory Receptors in Patients With Interstitial Cystitis/Bladder Pain Syndrome With and Without Hunner's Lesion.

OBJECTIVE: To investigate the urothelium function and sensory receptors difference between interstitial cystitis/bladder pain syndrome (IC/BPS) patients with or without Hunner's lesion. METHODS: Fourteen female IC/BPS patients with Hunner's lesion (Hunner IC) and 14 age-matched IC/BPS patients without Hunner's lesions (non-Hunner IC) were enrolled. Bladder mucosa biopsies were obtained. Bladder inflammation, eosinophil infiltration, and urothelial denudation were graded on a 4-point scale after staining with hematoxylin and eosin. Adhesive protein E-cadherin, tryptase, and zonula occuldens-1 in the bladder tissues were assessed with immunofluorescence staining. Urothelial muscarinic receptors M2, M3, endothelial nitric oxide synthase (eNOS), and purinergic receptor P2X3 were evaluated by Western blotting. RESULTS: Hunner IC patients had a significantly higher mean visual analog scale pain score and smaller cystometric bladder capacity than non-Hunner IC patients. The Hunner IC bladder specimens showed more severe or moderate eosinophilic infiltration and urothelial denudation than the non-Hunner IC bladder specimens did. The E-cadherin expression was significantly lower, and eNOS expression was significantly higher in the Hunner IC bladder samples than in the non-Hunner IC samples. The other functional proteins or sensory receptors did not differ between groups. CONCLUSION: Bladder inflammation and urothelial cell adhesion defects were more severe in the Hunner IC than that in the non-Hunner IC patients. eNOS was significantly higher in the Hunner IC than in the non-Hunner IC bladder samples, suggesting that eNOS expression difference may implicate different pathogenesis in 2 types of IC.

Functional and morphological alterations of the urinary bladder in type 2 diabetic FVB(db/db) mice.

AIMS: Diabetic bladder dysfunction (DBD) has been extensively studied in animal models of type 1 diabetes. We aimed to examine the functional and morphological alterations of the urinary bladder in a type 2 diabetes model, FVB(db/db) mice. METHODS: FVB(db/db) mice and age-matched FVB/NJ control mice were tested at either 12, 24 or 52weeks of age. Body weight, blood glucose and glycated hemoglobin (HbA1c) levels were measured. Bladder function was assessed by measurement of 24-h urination behavior and conscious cystometry. Bladder was harvested for Masson's Trichrome staining and morphometric analysis. RESULTS: The body weights of FVB(db/db) mice were twice as those of FVB/NJ control mice. The blood glucose and HbA1c levels were higher in FVB(db/db) mice at 12 and 24weeks, but not at 52weeks. A significant increase in the mean volume per void, but decrease in the voiding frequency, in FVB(db/db) mice was observed. Cystometry evaluation showed increased bladder capacity, voided volume, and peak micturition pressure in FVB(db/db) mice compared with FVB/NJ mice. Morphometric analysis revealed a significant increase in the areas of detrusor muscle and urothelium in FVB(db/db) mice. In addition, some FVB(db/db) mice, especially males at 12 and 24weeks, showed small-volume voiding during 24-h urination behavior measurement, and detrusor overactivity in the cystometry measurement. CONCLUSIONS: The FVB(db/db) mouse, displaying DBD characterized by not only increased bladder capacity, void volume, and micturition pressure, but also bladder overactivity, is a useful model to further investigate the mechanisms of type 2 diabetes-related bladder dysfunction.