Protective Effect of a Rho-kinase Inhibitor on Bladder Dysfunction in a Rat Model of Chronic Bladder Ischemia.

OBJECTIVE: To investigate the effect of fasudil, a Rho-kinase inhibitor, on chronic ischemia-related bladder dysfunction. MATERIALS AND METHODS: Male Sprague-Dawley rats (16 weeks old) were divided into control, chronic bladder ischemia (CBI), and CBI with fasudil treatment (CBI-Fa) groups. The CBI and CBI-Fa groups underwent balloon endothelial injury of bilateral iliac arteries and received a 2% cholesterol diet for 8 weeks after the procedure to induce CBI. The CBI-Fa group was given oral fasudil (30 mg/kg/day) using zonde for 8 weeks after the procedure. The control group received a regular diet for 8 weeks. After cystometry in a conscious state, rats from each group were euthanized, and the bladders and common iliac arteries were harvested for pharmacologic and histologic examination. RESULTS: Mean wall thickness of the common iliac arteries was significantly greater in the CBI group than in controls. Contractile responses of muscle strips were significantly lower in CBI group rats than in controls. In the CBI group, micturition interval was significantly shorter, and bladder capacity was significantly lower compared with those in controls. In the CBI-Fa group, arterial wall thickening was significantly suppressed compared with the CBI group. Significant improvements in muscle strip contractility and cystometric parameters were seen in the CBI-Fa group compared with the CBI group. CONCLUSION: Our results suggest that chronic treatment with fasudil could prevent neointimal formation in arteries and bladder dysfunction in this rat model. Fasudil may be therapeutically useful in protecting bladder function in chronically ischemic bladders.

Uni-axial stretch induces actin stress fiber reorganization and activates c-Jun NH2 terminal kinase via RhoA and Rho kinase in human bladder smooth muscle cells.

BACKGROUND: Excessive mechanical overload may be involved in bladder wall remodelling. Since the activity of Rho kinase is known to be upregulated in the obstructed bladder, we investigate the roles of the RhoA/Rho kinase pathway in mechanical overloaded bladder smooth muscle cells. METHODS: Human bladder smooth muscle cells were stimulated on silicon culture plates by 15 % elongated uni-axial cyclic stretch at 1 Hz. The activity of c-Jun NH2-terminal kinase was measured by western blotting and actin stress fibers were observed by stained with phallotoxin conjugated with Alexa-Fluor 594. RESULTS: The activity of c-Jun NH2-terminal kinase 1 peaked at 30 min (4.7-fold increase vs. before stretch) and this activity was partially abrogated by the RhoA inhibitor, C3 exoenzoyme or by the Rho kinase inhibitor, Y-27632. Stretch induced the strong formation of actin stress fibers and these fibers re-orientated in a direction that was perpendicular to the stretch direction. The average angle of the fibers from the perpendicular to the direction of stretch was significantly different between before, and 4 h after, stretch. Actin stress fibers reorganization was also suppressed by the C3 exoenzyme or Y-27632. CONCLUSIONS: Bladder smooth muscle cells appear to have elaborate mechanisms for sensing mechanical stress and for adapting to mechanical stress overload by cytoskeletal remodeling and by activating cell growth signals such as c-Jun NH2-terminal kinase via RhoA/Rho kinase pathways.

On the origin of spontaneous activity in the bladder.

OBJECTIVES: To characterise separately the pharmacological profiles of spontaneous contractions from the mucosa and detrusor layers of the bladder wall and to describe the relationship in mucosa between adenosine triphosphate (ATP) release and spontaneous contractions. MATERIALS AND METHODS: Spontaneous contractions were measured (36 °C) from isolated mucosa or detrusor preparations, and intact (mucosa + detrusor) preparations from guinea-pig bladders. Potential modulators were added to the superfusate. The percentage of smooth muscle was measured in haematoxylin and eosin stained sections. ATP release was measured in superfusate samples from a fixed point above the preparation using a luciferin-luciferase assay. RESULTS: The magnitude of spontaneous contractions was in the order intact >mucosa >detrusor. The percentage of smooth muscle was least in mucosa and greatest in detrusor preparations. The pharmacological profiles of spontaneous contractions were different in mucosa and detrusor in response to P2X or P2Y receptor agonists, adenosine and capsaicin. The intact preparations showed responses intermediate to those from mucosa and detrusor preparations. Low extracellular pH generated large changes in detrusor, but not mucosa preparations. The mucosa preparations released ATP in a cyclical manner, followed by variations in spontaneous contractions. ATP release was greater in mucosa compared with detrusor, augmented by carbachol and reversed by the M2 -selective antagonist, methoctramine. CONCLUSIONS: The different pharmacological profiles of bladder mucosa and detrusor, implies different pathways for contractile activation. Also, the intermediate responses from intact preparations implies functional interaction. The temporal relationship between cyclical variation of ATP release and amplitude of spontaneous contractions is consistent with ATP release controlling spontaneous activity. Carbachol-mediated ATP release was independent of active contractile force.

Possible effect of lysophosphatidic acid on cell proliferation and involvement of lysophosphatidic acid and lysophosphatidic acid receptors in mechanical stretch-induced mitogen-activated protein kinase.

OBJECTIVES: To determine whether lysophosphatidic acid activates the mitogen-activated protein kinase and increases DNA synthesis in human bladder smooth muscle cells, and to examine the involvement of lysophosphatidic acid and lysophosphatidic acid receptor in mechanical stretch-induced mitogen-activated protein kinase activation in cultured human bladder smooth muscle cells. METHODS: TaqMan reverse transcription polymerase chain reaction was used to determine the mRNA expression levels of six lysophosphatidic acid receptor subtypes. Mitogen-activated protein kinase activity enhanced by either lysophosphatidic acid or mechanical stretch was measured by western blotting. The effect of lysophosphatidic acid on DNA synthesis was assessed by 5-bromo-2'-deoxy-uridine incorporation assay. RESULTS: Lysophosphatidic acid 1 subtype mRNA was predominantly expressed (96%). Lysophosphatidic acid activated the mitogen-activated protein kinase in a concentration-dependent manner. C-jun NH2 -terminal kinase showed the highest activity among the three subsets of the mitogen-activated protein kinase family members (c-jun NH2 -terminal kinase, extracellular signal-regulated kinases, p38). Lysophosphatidic acid also increased incorporation of 5-bromo-2'-deoxy-uridine. These responses were suppressed by Ki16425 (lysophosphatidic acid receptor antagonist). Mechanical stretch mainly induced c-jun NH2 -terminal kinase activation. This activation was partially inhibited by Ki16425. CONCLUSIONS: Lysophosphatidic acid might activate the c-jun NH2 -terminal kinase component of the mitogen-activated protein kinase family and DNA synthesis through lysophosphatidic acid receptors (presumably, through lysophosphatidic acid 1) in human bladder smooth muscle cells. The present study also implicates the involvement of lysophosphatidic acid and lysophosphatidic acid receptors in mechanical stretch-induced c-jun NH2 -terminal kinase activation. Lysophosphatidic acid receptor can be partially activated by mechanical stretching through lysophosphatidic acid-dependent or independent mechanism.

External beam radiotherapy for focal lymphoepithelioma-like carcinoma in the urinary bladder: a case report and literature review.

Lymphoepithelioma is a malignant epithelial tumor in the nasopharynx characterized by prominent lymphoid infiltration. Carcinomas that resemble lymphoepitheliomas have been called lymphoepithelioma-like carcinomas and have been reported in other organs. A tumor in the bladder is categorized by the percentage of the total area occupied by the lymphoepithelioma-like carcinoma pattern, with the prognosis dependent on the percentage. We present an 81-year-old man with stage 3 chronic obstructive pulmonary disease and a history of aortic aneurysm repair. The computed tomography scans indicated thickening and irregularity of the bladder wall, with left external iliac lymph node metastasis. His diagnosis was bladder cancer, and the clinical stage was evaluated as T3N1M0. Transurethral resection of the bladder tumor was performed, and the pathological specimen showed that the tumor was composed of undifferentiated malignant cells with sheets and nests arranged in a syncytial pattern, as well as an urothelial carcinoma lesion. A prominent lymphoid reaction accompanied the tumor. The pathological diagnosis was focal-type lymphoepithelioma-like carcinoma containing a component of urothelial carcinoma G3>G2. His general condition was such that he could not tolerate radical cystectomy or systemic chemotherapy. External beam radiotherapy (total 60 Gy) was given to the bladder, including the lymph node metastatic lesion. No cancer recurrence was detected by regular follow-up computed tomography and cystoscopy. He eventually died of other causes 48 months later. Although treatment for focal lymphoepithelioma-like carcinoma generally requires multifocal therapies, in the present case, the bladder became tumor free. We also summarize previously reported lymphoepithelioma-like carcinoma cases treated with radiotherapy.

Pelvic arterial occlusive disease affects the RhoA/Rho-kinase pathway in bladder smooth muscle.

PURPOSE: We investigated the effect of pelvic arterial occlusive disease on the RhoA/Rho-kinase pathway in a rat model of chronic bladder ischemia. MATERIALS AND METHODS: Male adult Sprague Dawley® rats at age 16 weeks were divided into arterial endothelial injury and control groups. The injury group underwent balloon endothelial injury of the bilateral iliac arteries and received a 2% cholesterol diet to induce pelvic arterial occlusive disease. The control group received a regular diet. At 8 weeks cystometrograms were performed. Bladder tissue was harvested for pharmacological studies and Western blot. RESULTS: Cystometrograms showed significantly lower bladder capacity in the arterial endothelial injury group than in controls. Organ bath studies revealed significantly decreased phasic contractions induced by carbachol in bladder strips from the injury group than from controls. In controls bladder strip tonic contractions induced by carbachol were significantly decreased compared with phasic contractions. However, no significant difference was observed between phasic and tonic contractions in the injury group. The Rho-kinase inhibitor Y-27632 produced a concentration dependent decrease in tonic contractions, which was more pronounced in the injury group. Western blot showed significantly increased RhoA and Rho-kinase ß expression in the injury group. CONCLUSIONS: Our results suggest that pelvic arterial occlusive disease can affect the RhoA/Rho-kinase pathway in the bladder. This pathway might possibly be involved in the maintenance of tonic contraction and contribute to the bladder hyperactivity caused by pelvic arterial occlusive disease.

Current techniques to improve outcomes for early return of urinary continence following robot-assisted radical prostatectomy.

Although open retropubic radical prostatectomy has been the most commonly used surgical technique for patients with localized prostate cancer for decades, robot-assisted radical prostatectomy (RARP) has recently become an alternative option and widely used in Japan as well as around the world. RARP has been shown to have higher postoperative continent rates than retropubic and laparoscopic radical prostatectomy; however, urinary incontinence has remained one of the most significant causes for concern among patients who seek surgical treatment for prostate cancer, even after the introduction of RARP. The literature has shown that certain technical modifications to improve urinary continence are advocated as potential aids to reduce the risk of urinary incontinence after RARP. These modifications might be divided into 3 categories to realize the improvement of early return of urinary continence after RARP: 1) preservation, 2) reconstruction, and 3) reinforcement of the anatomic structures in the pelvis, which will make a new supporting system after radical prostatectomy. In this review, we discuss the intraoperative techniques to improve outcomes for early return of urinary continence following RARP, and provide a critical summary of current knowledge on its outcome in the literature.

Involvement of stretch-induced Rho-kinase activation in the generation of bladder tone.

AIMS: The present study investigated the role of the Rho-kinase (ROK) pathway in the maintenance of bladder tone during the storage phase, and its effects on bladder compliance. METHODS: Muscle strips from isolated rat bladder (detrusor strips) were used to evaluate the effects of the ROK inhibitors Y-27632 and HA-1077 on resting tension, which is independent of G-protein coupled pathways. Stretch-induced ROK activation was assessed by measuring phosphorylation of MYPT1 (myosin phosphatase targeting subunit) using Western blotting. The effect of ROK inhibitors on bladder compliance during bladder filling was assessed in an in vitro whole bladder model. RESULTS: Y-27632 and HA-1077 caused concentration-dependent relaxation of detrusor strips. Stretch increased MYPT1-p[Thr853] levels by approximately 1.5-fold in normal Krebs buffer. The ROK inhibitor Y-27632 abolished the stretch-induced increase and reduced the level of MYPT1-p[Thr853] to <50% of the basal values in normal Krebs buffer. Stretch-induced phosphorylation of MYPT1 was independent of Ca(2+) originating from either extracellular or intracellular stores. When tested in the isolated whole rat bladder model, HA-1077 significantly increased bladder compliance at both 3 and 10 µM. CONCLUSIONS: This study demonstrates that the ROK pathway is constitutively active and stretch further activates the ROK pathway, which contributes to the generation of bladder tone, thereby affecting bladder compliance.

Laparoscopic nephrectomy, ex vivo angioplasty, and renal autotransplant for a renal artery aneurysm: a case report.

We describe in this report the case of a renal aneurysm in a 42-year-old woman. The aneurysm measured 27 mm in diameter, and was sited at the first bifurcation of the renal artery. We performed laparoscopic nephrectomy, ex vivo angioplasty and renal autotransplant to avoid ischemic damage to the kidney during reconstruction. The patient recovered and was discharged from the hospital without any complications. Hence, we suggest these treatments can be effectively done in patients with complex renal aneurysms.

Increased bladder activity is associated with elevated oxidative stress markers and proinflammatory cytokines in a rat model of atherosclerosis-induced chronic bladder ischemia.

AIMS: To further characterize, in a rat model, the effects of atherosclerosis-induced chronic bladder ischemia on bladder function and associated changes in oxidative stress markers and proinflammatory cytokines. METHODS: Adult Sprague-Dawley male rats were divided into three groups (arterial endothelial injury: AI, sham, naïve). The AI group (n = 14) underwent endothelial injury of the iliac arteries and received a 2% cholesterol diet. The sham group (n = 12) underwent sham operation and received a 2% cholesterol diet. The naïve group (n = 12) received a regular diet. After 8 weeks, cystometrograms (CMG) without anesthesia or restraint were performed. In bladders from each group, oxidative stress markers (8-hydroxy-2'-deoxyguanosine: 8-OHdG; malondialdehyde: MDA) and proinflammatory cytokines (IL-8 like cytokine CXCL1/CINC-1, TNF-α, IL-6) were quantified. Histological examination of the iliac arteries was also performed. RESULTS: At 8 weeks, the body and bladder wet weights were not significant different among the three groups. The micturition interval in the AI group decreased significantly compared with those in the other two groups, but maximum pressure during micturition did not change. The iliac arteries in the AI group revealed thickening of intima as well as diffuse media fibrosis at the sites of balloon injury. The levels of oxidative stress markers and proinflammatory cytokines were significantly higher in the AI than in the other groups. CONCLUSION: Oxidative stress and inflammation may be key factors in the development of bladder overactivity in atherosclerosis-induced chronic bladder ischemia.

The effect of atherosclerosis-induced chronic bladder ischemia on bladder function in the rat.

AIMS: To develop a rat model of atherosclerosis-induced chronic bladder ischemia and investigate the effect of chronic bladder ischemia on voiding behavior and bladder function. METHODS: Adult male rats were divided into three groups. The arterial injury (AI) group underwent endothelial injury of the iliac arteries and received a 2% cholesterol diet. The sham group underwent sham operation and received a 2% cholesterol diet. The control group received a regular diet. After 8 weeks, a metabolic cage study and cystometry were performed without anesthesia. Bladder blood flow was measured using a laser Doppler blood flowmeter. Histological examination of the iliac arteries and the bladder was performed. The bladder was also processed for immunohistochemical staining of oxidative stress markers. RESULTS: The metabolic cage study showed that in the AI group, voiding frequency significantly increased while voided volume significantly decreased. Cystometry showed that the frequency of reflex bladder contractions was significantly higher in the AI group. Filling-induced decrease in bladder blood flow was the greatest in the AI group. Histological study showed that in the AI group alone, atherosclerotic occlusion occurred in the iliac arteries as well as in the downstream bladder microvessels. Oxidative stress marker positive cells were more prevalent in the AI bladder than in the other bladders. CONCLUSIONS: Combined with a high-cholesterol diet, endothelial injury of iliac arteries induced arterial occlusive disease in the downstream vessels and consequent bladder ischemia in rats. This model of chronic bladder ischemia showed detrusor overactivity manifested as an increase in voiding frequency.

Effect of long-term prazosin and yohimbine administration on c-Fos expression in spinal neurons: inhibitory effect of alpha-1 and alpha-2 adrenoceptors on afferents from the lower urinary tract.

INTRODUCTION: To investigate the effects of long-term administration of the α(1)-adrenoceptor antagonist prazosin and the α(2)-adrenoceptor antagonist yohimbine on afferent inputs from the lower urinary tract by evaluating c-Fos expression in the spinal cord. MATERIALS AND METHODS: Prazosin or yohimbine was administered for 4 weeks in rats using an osmotic pump. Effects of these agents on urodynamic parameters were determined by continuous cystometry in conscious rats. After cystometry, c-Fos expression in the dorsal horn of the L6 spinal cord was measured by immunohistochemistry. RESULTS: The administration of prazosin (0.12 mg/kg/day) or yohimbine (0.10 mg/kg/day) significantly increased micturition interval and bladder capacity, but did not affect micturition pressure and residual urine volume. The numbers of c-Fos-positive neurons in the dorsal horn were significantly lower in rats that received prazosin than in controls. Yohimbine reduced the number of c-Fos-positive neurons in part of the dorsal horn. CONCLUSIONS: Long-term administration of prazosin and yohimbine at clinically recommended doses can exert inhibitory effects on afferent pathways from the lower urinary tract during the storage phase. These reductions of the afferent input result in the increased bladder capacity and increased micturition interval.

Suppression of SOCS3 increases susceptibility of renal cell carcinoma to interferon-α.

Interferon (IFN)-α is one of the most commonly used agents in immunotherapy for patients with advanced stage renal cell carcinoma. However, because of the drug resistance to IFN-α, its benefits are limited. In this study, we examined whether repression of suppressor of cytokine signaling (SOCS) proteins, which are involved in the IFN-induced signaling pathway, can overcome the IFN resistance of renal cell carcinoma. The effect of IFN-α on SOCS3 expression and cell proliferation was examined using IFN-resistant 786-O and IFN-sensitive ACHN cell lines. The effects of SOCS3-targeted siRNA on 786-O xenografts were determined by SOCS3 expression, morphological observation, and tumor volume. The SOCS3 mRNA expression level was significantly increased by IFN-α stimulation in 786-O, but not in ACHN cells. The overexpression of SOCS3 by gene transfection in ACHN cells significantly inhibited the growth-inhibitory effect of IFN-α. Suppression of SOCS3 expression in 786-O cells by siRNA activated the IFN signaling pathway through signal transducer and activator of transcription 1 phosphorylation and recovered sensitivity to IFN-α. An in vivo study indicated that co-administration of SOCS3-targeted siRNA promoted IFN-α-induced cell death and growth suppression in 786-O cell xenograft in nude mice. Morphological observation of the tumors revealed the inhibition of SOCS3-induced apoptosis, invasion of inflammatory cells and fibrosis. SOCS3 could be a key component in the resistance to IFN treatment of renal cell carcinoma. Silencing SOCS3 gene expression could be an effective strategy to enhance the antitumor effect of IFN in human renal cell carcinoma cells.

Association between antibody response against cytomegalovirus strain-specific glycoprotein H epitopes and HLA-DR.

The gH of CMV is a major target for strain-specific neutralizing antibodies. To verify whether there is a correlation between HLA-DR type and strain-specific antibodies, antibodies against CMV gH in potential donors and recipients for renal transplantation were investigated. Among 471 subjects, 404 (86%) showed reactivity to CMV gH, but no antibodies against gH were detected in 67 (14%) subjects. The positive rates were over 80% in most HLA subpopulations. Fewer subjects with HLA-DR10 and DR11 had antibodies to CMV gH than did those without HLA-DR10 and DR11. HLA-DR10 and DR11 may be associated with fewer/non-responders for strain-specific neutralizing antibodies.

Obstruction alters muscarinic receptor-coupled RhoA/Rho-kinase pathway in the urinary bladder of the rat.

AIMS: The present study investigated the effects of the bladder outlet obstruction (BOO) on the muscarinic receptor (MR)-coupled RhoA/Rho-kinase (ROK) pathway in the detrusor smooth muscle of the rat. METHODS: Detrusor muscle samples were obtained from bladders after 4 weeks of BOO and also from sham-operated control rats. Contractile responses to electrical field stimulation (EFS) and 1 microM carbachol (Cch) were determined in isolated detrusor strips. The effects of the ROK inhibitor Y-27632 on the Cch-induced phasic and sustained contractions were evaluated. Western blotting was used to determine the relative levels of RhoA expression and ROK isoform expression. RESULTS: Bladder weight increased significantly after 4 weeks of BOO. Contractile responses to EFS decreased significantly in detrusor muscle from the obstructed bladder. Cch (1 microM) induced a biphasic response consisting of an initial phasic contraction followed by a sustained contraction. Y-27632 attenuated the phasic and sustained contractions induced by Cch in both control and obstructed bladders. However, BOO caused a significant increase in contractile force during the sustained phase of the contractions induced by Cch. An inhibitory effect of Y-27632 on the sustained responses to Cch was significantly enhanced in the obstructed bladder. In accordance with the functional study, the expression of RhoA and ROK isoforms (both alpha and beta) at the protein level significantly increased in the obstructed bladder. CONCLUSIONS: These results suggest that the enhanced MR-coupled RhoA/ROK pathway contributes to the maintenance of contractile force in the obstructed bladder, as a compensatory mechanism for expelling the urine against the obstruction.

Strain-specific seroepidemiology and reinfection of cytomegalovirus.

Although there have been some reports describing the serostatus of cytomegalovirus, strain-specific antibody responses and their distribution remain unknown. In this study, ELISA using fusion proteins encompassing epitope of glycoprotein H from both AD169 and Towne strains was used to test 352 blood donors. Of these 352 donors, 207 were analyzed for strain-specific glycoprotein H antibodies. Of the 44 donors whose serum contained antibodies against both AD169 and Towne, 27 (60%) were aged 50 years or over (p = 0.0003). This may indicate serological evidence of reinfection with cytomegalovirus in the elder population. The nucleotide sequence analysis of cytomegalovirus glycoprotein H from the peripheral blood of the cytomegalovirus-positive renal transplant recipients showed that our strain-specific ELISA can reveal cytomegalovirus reinfection after transplantation.

Association of the outcome of renal transplantation with antibody response to cytomegalovirus strain-specific glycoprotein H epitopes.

BACKGROUND: Cytomegalovirus (CMV) is the most important pathogen affecting the outcome of renal transplantation. The combination of CMV-seronegative transplant recipients with CMV-seropositive transplant donors places recipients at the highest risk of CMV disease. In cases of congenital CMV infection, existing immunity only partially protected mothers from reinfection with a different genotypic strain. The effect of differences in infected CMV strains between CMV-seropositive transplant donors and CMV seropositive transplant recipients on the outcome of transplantation remains unclear. METHODS: In this prospective multicenter study, the presence of antibodies against strain-specific glycoprotein H epitopes in 84 CMV-seropositive transplant donor/CMV-seropositive transplant recipient renal transplantation cases were determined, and their relationships to acute transplant rejection, CMV infection, degree of antigenemia, and CMV disease were evaluated. RESULTS: Among the 84 donor/recipient pairs, 45 and 32 had matched and mismatched strain-specific glycoprotein H antibodies, respectively. Acute transplant rejection in the mismatched group was more frequent than it was in the matched group (63% vs. 22%; P=.005). CMV disease was also more frequently observed in the mismatched group (28% vs. 9%; P=.026). The mismatched group had a higher level of antigenemia (P=.019). CONCLUSIONS: Our results illustrate more adverse events in the cases with a CMV-seropositive transplant donor and a CMV-seropositive transplant recipient in which the glycoprotein H antibodies are mismatched, suggesting that reinfection with a different CMV strain results in more complications.