Urothelial Dysfunction, Suburothelial Inflammation and Altered Sensory Protein Expression in Men with Bladder Outlet Obstruction and Various Bladder Dysfunctions: Correlation with Urodynamics

Author's Accepted Manuscript Urothelial dysfunction, suburothelial inflammation, and altered sensory protein expression in men with bladder outlet obstruction and various bladder dysfunctions: correlation with urodynamics Yuan-Hong Jiang , Cheng-Ling Lee , Hann-Chorng Kuo

PII: DOI: Reference:

S0022-5347(16)03249-3 10.1016/j.juro.2016.02.2958 JURO 13525

To appear in: The Journal of Urology Accepted Date: 23 February 2016 Please cite this article as: Jiang YH, Lee CL, Kuo HC, Urothelial dysfunction, suburothelial inflammation, and altered sensory protein expression in men with bladder outlet obstruction and various bladder dysfunctions: correlation with urodynamics, The Journal of Urology® (2016), doi: 10.1016/ j.juro.2016.02.2958. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.

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ACCEPTED MANUSCRIPT Urothelial dysfunction, suburothelial inflammation, and altered sensory protein expression in men with bladder outlet obstruction and various

Yuan-Hong Jiang, Cheng-Ling Lee, Hann-Chorng Kuo

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bladder dysfunctions: correlation with urodynamics

Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi

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University, Hualien, Taiwan

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Correspondence to: Dr. Hann-Chorng Kuo, Department of Urology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan

Tel: 886-3-8561825 ext. 2117

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Fax: 886-3-8560794 E-mail: [email protected]

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Running title: Bladder Outlet Obstruction and Urodynamics

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Word count: abstract 250 words, text 2500 words, 1 table, 2 figures

Keywords: urothelial dysfunction, bladder outlet obstruction, detrusor overactivity, detrusor underactivity

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ACCEPTED MANUSCRIPT

Abstract Purpose:

This

study

investigated

urothelial

integrity,

suburothelial

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inflammation, and expression of sensory proteins in bladder urothelium of male patients with bladder outlet obstruction and various bladder dysfunctions. Materials

and

Methods:

We

prospectively

enrolled

33

men

with

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urodynamically proven bladder outlet obstruction (study group). Bladder

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biopsies were obtained from all study patients and 10 control patients. The expressions of E-cadherin, zonula occludens-1, tryptase, and apoptosis, and TRPV1, TRPV4, β3 adrenoreceptor, M2, and M3 muscarinic receptors, P2X3 receptor, and inducible/epithelial nitric oxide synthase were compared

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between study and control patients.

Results: Study patients had significantly lower expression of E-cadherin and

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higher suburothelial mast and apoptotic cell numbers than controls did.

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Additionally, higher expressions of P2X3 and M2 muscarinic receptors and lower expression of M3 muscarinic receptor were detected in the study patients. Detrusor underactivity subgroup was characterized by significantly higher expression of β3 adrenoreceptors and lower expression of inducible nitric oxide synthase than in controls. In study patients, significantly positive correlation was noted between voided volume and E-cadherin expression (r =

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ACCEPTED MANUSCRIPT 0.372), volume at first sensation of filling and expression of β3 adrenoreceptor (r = 0.386), and detrusor pressure and expression of M2 muscarinic receptor (r

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= 0.496) in the bladder urothelium (all p < 0.05). Conclusions: Urothelial dysfunction, suburothelial inflammation, cellular apoptosis, and alterations of sensory proteins are prominent in bladder

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dysfunction secondary to bladder outlet obstruction. Impaired urothelial

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signaling and sensory transduction pathways appear to reflect the pathophysiology of bladder dysfunction and detrusor underactivity in patients

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with bladder outlet obstruction.

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ACCEPTED MANUSCRIPT INTRODUCTION The urinary bladder primarily serves two functions, storing and emptying urine.

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During storage, the bladder is relaxed to accommodate filling over time with a sufficient amount of urine to cause a significant rise in bladder pressure. During emptying, detrusor muscle contraction coordinates with the relaxation

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of the external sphincter. These dynamic changes are affected by the

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urothelium, detrusor muscles, and connective tissue of the bladder.1 The general urodynamic state of bladder outlet obstruction (BOO) during voiding is high detrusor voiding pressure (Pdet) and low flow rate, while Pdet decreases in the decompensated state.2 Concurrent detrusor overactivity (DO) during

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bladder filling in BOO is highly prevalent.3 BOO causes significant transformation of the morphology and physiology

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of the bladder.4, 5 In response to mechanical stretch stress in BOO, urothelial

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and smooth muscle cells undergo modification of gene expression and protein synthesis. This results in modification of the micturition reflex threshold, causing involuntary detrusor contractions6 and bladder hypertrophy.4 In rat studies of BOO induced DO, increased P2X3 receptor, M2, and M3 muscarinic receptors,7 increased transient receptor potential vanilloid 4 (TRPV4)8 and increased inducible nitric oxide synthase (iNOS)9 in urothelium were shown.

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ACCEPTED MANUSCRIPT Such changes of urothelial receptors mediating altered sensory transduction in suburothelium and afferent nerves are thought to play important roles in the

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pathophysiology of DO associated with BOO. Urothelial dysfunction, suburothelial inflammation, and apoptosis are characteristic of many lower urinary tract diseases,10,

11

and probably also

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BOO. Evidence shows the molecular connection between the adhesion protein

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E-cadherin and TRPV4, suggesting an association with E-cadherin and bladder sensation.12 In a recent mouse study, decreased E-cadherin expression through activation of a hypoxia-inducible factor pathway was thought to be involved in the pathogenesis of BOO.13

neurologic

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Detrusor underactivity (DU) or underactive bladder is observed in many conditions

and

myogenic

failure,

including

BOO.14

The

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pathophysiology of DU is not well understood. Altered or impaired bladder

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afferent function with subsequently impaired activation of detrusor contraction leading to DU is a significant etiology.15 Oxidative stress caused by chronic bladder ischemia and repeated ischemia/reperfusion during micturition cycles may play a role in the development of bladder dysfunction, including DO and DU, in BOO.5 The severity and duration of bladder ischemia are the important determinants

of

bladder

dysfunction.

5

Nevertheless,

the

definitive

ACCEPTED MANUSCRIPT pathophysiology of BOO developing into DO, or the decompensated status developing into DU, remains largely unknown.

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At present, the evidence for urothelium sensory proteins playing a role in bladder dysfunction caused by BOO is mainly based on animal studies. Therefore, we investigated urothelial dysfunction, suburothelial inflammation,

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and the expressions of sensory proteins in the bladder urothelium of male

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BOO patients with various types of bladder dysfunction. We also sought to clarify the relationships between altered sensory protein expression and bladder dysfunction.

Patients

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MATERIALS AND METHODS

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We prospectively and consecutively enrolled 33 men who had clinically benign

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prostate enlargement or obstruction and who were scheduled for transurethral resection of the prostate from June 2012 to January 2014. Clinical investigation included transrectal sonography of the prostate to measure the prostate volume and cystoscopy and video urodynamic studies (VUDS) before surgery. The eligible patients for analysis had no active urinary tract infection, interstitial cystitis, occult or overt neuropathy (including cerebrovascular

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ACCEPTED MANUSCRIPT accident, diabetes mellitus, multiple sclerosis, Parkinson’s disease, spinal cord injury). Furthermore, they had no history of bladder surgery or injury, previous

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transurethral surgery or prostatectomy.

VUDS and Biopsy Procedures

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VUDS were performed in accordance with the recommendations of the

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International Continence Society using C-arm fluoroscopy. VUDS parameters including maximal urinary flow rate (Qmax), Pdet, cystometric bladder capacity, voided volume (Vol), post-void residual volume (PVR), and voiding cystourethrography were determined for the diagnosis of BOO. VUDS was

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repeated at least twice to obtain a reproducible pressure flow tracing. All terminology was defined according to the recommendations of the

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International Continence Society.16 A bladder outlet obstruction index > 40 was

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considered obstructed.2 DO was defined as any involuntary phasic detrusor contraction that occurred during the filling phase, and a hypersensitive bladder (HSB) was defined as an early first sensation of bladder filling (FSF) that occurred at low bladder volume and persisted. DU was defined as a low detrusor pressure and low urine flow rate, resulting in prolonged and incomplete bladder emptying with a PVR greater than 150 mL and one-third of

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ACCEPTED MANUSCRIPT the full bladder capacity. If sphincter electromyography showed relaxed activity with an open membranous urethra during voiding, a low urine flow rate was

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considered to be due to DU. We also invited 10 men with renal tumors undergoing surgery that had no lower urinary tract symptom to serve as controls. Bladder biopsies were

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obtained from all BOO patients and controls. Cold cup bladder biopsies were

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taken from the posterior wall about 2 cm above the ureteral orifice, and only the bladder mucosa was taken to prevent bladder perforation. Bladder biopsy specimens were sent to the pathology department for hematoxylin and eosin staining to exclude the possibility of carcinoma in situ.

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This study was approved by the Institutional Review Board and Ethics Committee of Buddhist Tzu Chi General Hospital. Each patient was informed

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about the study rationale and procedures; written informed consent was

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obtained from the patients before the bladder procedures.

Immunofluorescence Staining and Quantification of Protein Expression The bladder tissue of BOO patients and controls was investigated for urothelial adhesive function by measuring E-cadherin expression. Urothelial integrity was evaluated using zonula occludens-1 (ZO-1) expression, mast cell

8

ACCEPTED MANUSCRIPT activation was examined by determining the tryptase level, and cellular apoptosis was assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (TUNEL). These laboratory procedures have been

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described in detail previously.10 Immunofluorescence (tryptase and TUNEL assays) was quantified by counting the number of positively stained cells per

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100 cells per unit area (4 µm2) and was shown as a percentage. The intensity

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of E-cadherin was quantified using Image J processing.17

Western blotting was used to assess sensory proteins including TRPV1, TRPV4, β3 adrenoceptors, inducible nitric oxide synthase (iNOS), epithelial NOS (eNOS), purinergic receptor P2X3, and muscarinic receptors including

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M2 and M3. The applied first antibody included TRPV 1, TRPV4 (Alomone Labs, Jerusalem, Israel), β3 adrenoceptor (Abcam, Cambridge, UK), iNOS

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(ThermoFisher, Rockford, IL, USA), eNOS, P2X3 receptor, and M2 and M3

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muscarinic receptors (GeneTex, Irvine, CA, USA), and GAPDH (GeneTex, Irvine, CA, USA) was as the positive control. The second antibody was goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (IgG-HRP, 1:3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Briefly, the laboratory procedures were similar to the previous study.8

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ACCEPTED MANUSCRIPT Statistical Analysis Continuous variables are presented as means ± standard deviations. Eligible

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BOO patients were grouped according to bladder dysfunction and were compared with controls. Differences in the immunofluorescence staining results and Western blotting of sensory proteins were analyzed using the

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Kruskal-Wallis test. These values were also correlated with VUDS parameters

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using linear regression. All calculations were performed using SPSS for Windows, version 16.0 (SPSS, Chicago, IL). P values of less than 0.05 were

Results

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considered significant.

The 33 men diagnosed urodynamically with BOO had a mean age of 68.5 ±

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11.1 years. The 10 male controls had a similar mean age (64.6 ± 11.5 years).

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Based on the bladder dysfunction determined by VUDS, the patients included 23 with DO/HSB and 10 with DU. No major complication such as bleeding or perforation occurred after

bladder biopsy. Table 1 summarizes the demographics, immunofluorescence, Western blotting, and VUDS data. Clinically, the patients with DU had

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ACCEPTED MANUSCRIPT decreased bladder sensation, larger bladder capacity, and lower detrusor

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contractility, with a lower Qmax rate than those in DO/HSB subgroup.

The immunofluorescence results showed that the expression of E-cadherin (but not ZO-1) in the bladder urothelium of BOO patients was

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significantly lower than in controls. DU subgroup had lower expression

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E-cadherin than DO/HSB subgroup did (p = 0.015 and 0.038, respectively) (Table 1, Figure 1). More activated mast cells and apoptotic cells were observed in the bladder mucosa of BOO patients compared with controls (p < 0.001 and 0.028, respectively).

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BOO patients had significantly higher expressions of P2X3 and M2 muscarinic receptors, and lower expression of M3 muscarinic receptors than

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controls did (all p < 0.05). Although there was no difference overall between

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BOO patients and controls, the patients in DU subgroup had significantly higher expression of β3 adrenoreceptors (p = 0.041) and lower expression of iNOS (p = 0.034) than controls did. There was no significant difference in the levels of TRPV1, TRPV4, and eNOS among the three groups. When correlating the VUDS parameters with the sensory proteins, Vol was positively (r = 0.372) correlated with the E-cadherin expression in patients

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ACCEPTED MANUSCRIPT with BOO (Figure 2). The FSF volume and β3 adrenoreceptor expression (r = 0.386) showed positive correlation, as did Pdet and M2 muscarinic receptor

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expression (r = 0.496) in the BOO bladder mucosa (all p < 0.05).

DISCUSSION

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In patients with BOO, the bladder undergoes morphologic and physiologic

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transformations of the urothelium, suburothelium, and detrusor muscle.4, 5 The urothelium is not only a barrier but also a sensor and transducer, which affects nerve activity, detrusor contraction, and eventually bladder function through evoking the release of various mediators in response to different stimuli.18 In

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the present study, urothelial dysfunction, increased suburothelial inflammation and apoptosis, and altered sensory protein expression in the bladder mucosa

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were observed in BOO patients. In addition, patients with various bladder

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dysfunctions expressed different sensory proteins in the bladder mucosa, indicating their roles in the pathophysiology of bladder dysfunction, including DO/HSB and DU.

Overdistention in BOO causing hypoxia or ischemia results in urothelial damage

(E-cadherin

repression)

through

the

activation

of

the

hypoxia-inducible factor pathways.13 In animal studies, transiently diminished

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ACCEPTED MANUSCRIPT blood flow to bladder occurred with BOO. These episodes were accompanied by the subsequent production of free radicals.19,

20

In the case of chronic

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bladder obstruction, ischemia and reperfusion cycle back and forth, resulting in further oxidative stress, energy depletion, denervation, and disturbed urothelial homeostasis. These events are postulated to involve the development of

patients

with

overactive

bladder

had

increased

suburothelial

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study,

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bladder hyperactivity that progresses to bladder underactivity.5 In a previous

inflammation, but E-cadherin expression in the bladder urothelium was not decreased.10 In the present study, patients with BOO had decreased expression of E-cadherin that was significantly lower in DU subgroup

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compared with DO/HSB subgroup. Additionally, lower voided volume was associated with lower expression of E-cadherin. This suggests a more severe

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or decompensated BOO status (i.e., DU in BOO) with more severe urothelial

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dysfunction, which also supports the hypothesis of DO progression to DU. ZO-1 is a junction protein, maintaining the highly resistant urothelial

barrier.21 Mast cells present are a part of the bladder’s natural immunity; once activated, they degranulate and release preformed inflammatory mediators such as tryptase.22 As inflammation proceeds, it induces DO via various pathways.23 Apoptosis is a process of programmed cell death in respond to

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ACCEPTED MANUSCRIPT cellular damage caused by diseases or noxious agents.24 We observed concurrent defective urothelium (decreased E-cadherin but not ZO-1),

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increased suburothelial inflammation, and apoptosis of the bladder mucosa in BOO. These findings support the role of chronic ischemia/hypoxia of the bladder and altered regulation of homeostasis.

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Urothelium-afferent nerve interactions can influence the bladder

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contraction reflex.25 Increased P2X3, M2, and M3 muscarinic receptors,7 and TRPV48 in the urothelium are thought to play important roles in the pathophysiology of BOO-induced DO by mediating bladder afferent neural transduction, as demonstrated in these previous rat studies. However, M2

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muscarinic receptors may play a larger role than M3 muscarinic receptors in the local cholinergic modulation of bladder afferent activity contributing to

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DO.26 Our study showed that BOO patients had increased expressions of

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P2X3, and M2 muscarinic receptors but lower expression of M3 muscarinic receptors in the bladder mucosa. This evidence supports the significant roles of P2X3 and M2 muscarinic receptors in bladder dysfunction caused by BOO. However, the role of M3 muscarinic receptors in human BOO bladder mucosa is still unclear. Moreover, Pdet correlated positively with M2 muscarinic receptor expression. We suggest that lowered expression of M2 muscarinic

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ACCEPTED MANUSCRIPT receptors may impairs sensory transduction pathways, which could be associated with impaired activation of detrusor contraction, resulting in DU

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related to BOO. In the bladder, β-3 adrenoreceptors are distributed in not only the detrusor but also the urothelium and suburothelium.27 Beta-3 adrenoreceptor

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activation in the urothelium and suburothelium inhibits bladder afferent neural

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transduction. In the detrusor, their activation contributes to relaxation. These two mechanisms facilitate bladder storage with increasing bladder filling,27 and β-3 agonists are effective treatments for overactive bladder.28 In the present study,

FSF

was

positively

correlated

with

the

expression

of

β-3

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adrenoreceptors, and only BOO patients with concurrent DU had higher expression of β-3 adrenoreceptors. These findings indicate that the higher

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expression of β-3 adrenoreceptors is associated with decreased bladder

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sensation, which could play a major role in detrusor contractility, especially in DU. This observation also supports the viewpoint that altered or impaired bladder sensation leads to DU.15 NOS has been identified in the urothelium, suburothelium, nerves, blood vessels, and the detrusor of the bladder, and the NO pathway plays important roles in modulating afferent activities and neurotransmission.29 Increased

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ACCEPTED MANUSCRIPT iNOS activity was identified in BOO. This activity is believed to influence the progression of pathologic changes in the bladder secondary to BOO, including DO and fibrosis of the bladder wall, with iNOS expression primarily in urothelial

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and inflammatory cells.9, 30 In the present study, DO in BOO did not result in significant changes in iNOS while DU in BOO showed significantly decreased

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iNOS activity. We hypothesized that increased iNOS activity was involved in

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the pathologic consequences of bladder dysfunction after BOO, and iNOS activity decreased with inflammation producing relatively late-phase or probably decompensated status after BOO, which contributed to the development of DU.

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BOO affected the urothelial integrity and altered the sensory receptor expression within the bladder mucosa, resulting in various bladder

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dysfunctions. In BOO, the afferent bladder sensation, which was altered and

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augmented by purinergic and muscarinic mechanisms, could be responsible for DO/HSB development. Impaired bladder sensory transduction or modulation through β-3 adrenoreceptors and the NOS pathway contributed to the development of DU. Nonetheless, the sequential changes of DO/HSB to DU in bladder dysfunction caused by BOO is still uncertain and should be clarified in future studies.

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ACCEPTED MANUSCRIPT There are several limitations to the study. First, the numbers of control and study patients were small. Second, DO/HSB subgroup represented the

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condition of preserved detrusor contractility concurrent with DO/HSB in opposition to DU. Since sensory dysfunction within DO/HSB subgroup is heterogeneous, it might result in inconsistent results of altered sensory protein

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expressions. Third, the relationship between DO/HSB and DU as bladder

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dysfunctions caused by BOO still was not elucidated. In the future, a carefully designed study should be conducted to clarify the exact pathological

CONCLUSIONS We

demonstrated

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mechanisms of bladder dysfunctions caused by BOO, particularly DU.

that

defective

urothelium,

increased

suburothelial

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inflammation, and cellular apoptosis were prominent features of bladder

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dysfunction secondary to BOO. Moreover, sensory receptor proteins expression is altered in BOO. Over-expression or under-expression of these functional or sensory proteins influences the urodynamics and contributes to the pathophysiology of bladder dysfunctions and impaired detrusor contractility in BOO.

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ACCEPTED MANUSCRIPT References 1.

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2.

Nitti VW: Pressure flow urodynamic studies: the gold standard for diagnosing bladder outlet obstruction. Rev Urol 2005; 7 Suppl 6: S14. de Nunzio C, Franco G, Rocchegiani A et al: The evolution of detrusor overactivity after

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watchful waiting, medical therapy and surgery in patients with bladder outlet obstruction. J Urol 2003; 169: 535. 4.

Mirone V, Imbimbo C, Longo N et al: The detrusor muscle: an innocent victim of bladder outlet obstruction. Eur Urol 2007; 51: 57.

Yamaguchi O, Nomiya M and Andersson KE: Functional consequences of chronic bladder ischemia. Neurourol Urodyn 2014; 33: 54.

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5.

Andersson KE: Bladder activation: afferent mechanisms. Urology 2002; 59: 43

7.

Kim JC, Yoo JS, Park EY et al: Muscarinic and purinergic receptor expression in the

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Cho KJ, Park EY, Kim HS et al: Expression of transient receptor potential vanilloid 4 and effects of ruthenium red on detrusor overactivity associated with bladder outlet obstruction in rats. World J Urol 2014; 32: 677.

Johansson R, Pandita RK, Poljakovic M et al: Activity and expression of nitric oxide synthase

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in the hypertrophied rat bladder and the effect of nitric oxide on bladder smooth muscle growth. J Urol 2002; 168: 2689. 10.

Liu HT, Shie JH, Chen SH et al: Differences in mast cell infiltration, E-cadherin, and zonula

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occludens-1 expression between patients with overactive bladder and interstitial cystitis/bladder pain syndrome. Urology 2012; 80: 225 e13. 11.

Jiang YH, Liu HT and Kuo HC: Urothelial dysfunction and chronic inflammation in patients

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with spinal cord injuries at different levels and correlation with urodynamic findings. Neurourol Urodyn 2015; 34: 757.

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Janssen DA, Hoenderop JG, Jansen KC et al: The mechanoreceptor TRPV4 is localized in

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Iguchi N, Hou A, Koul HK et al: Partial bladder outlet obstruction in mice may cause E-cadherin repression through hypoxia induced pathway. J Urol 2014; 192: 964.

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Miyazato M, Yoshimura N and Chancellor MB: The other bladder syndrome: underactive bladder. Rev Urol 2013; 15: 11.

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Chancellor MB: The overactive bladder progression to underactive bladder hypothesis. Int Urol Nephrol 2014; 46 Suppl 1: S23.

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Kaczmarek E, Gorna A and Majewski P: Techniques of image analysis for quantitative immunohistochemistry. Rocz Akad Med Bialymst 2004; 49 Suppl 1: 155. Birder L and Andersson KE: Urothelial signaling. Physiol Rev 2013; 93: 653.

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Levin RM, O'Connor LJ, Leggett RE et al: Focal hypoxia of the obstructed rabbit bladder wall

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Acharya P, Beckel J, Ruiz WG et al: Distribution of the tight junction proteins ZO-1, occludin,

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Sun HD and Chung SD: Could overactive bladder be a chronic inflammatory disorder? Incont Pelvic Floor Dysfunct 2009; 3(Suppl 1): 3.

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Kullmann FA, Artim DE, Birder LA et al: Activation of muscarinic receptors in rat bladder

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sensory pathways alters reflex bladder activity. J Neurosci 2008; 28: 1977. 26.

Matsumoto Y, Miyazato M, Furuta A et al: Differential roles of M2 and M3 muscarinic receptor subtypes in modulation of bladder afferent activity in rats. Urology 2010; 75: 862. Igawa Y, Aizawa N and Homma Y: Beta3-adrenoceptor agonists: possible role in the treatment

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Chapple C, Khullar V, Nitti VW et al: Efficacy of the beta3-adrenoceptor agonist mirabegron

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Ho MH, Bhatia NN and Khorram O: Physiologic role of nitric oxide and nitric oxide synthase

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30.

Felsen D, Dardashti K, Ostad M et al: Inducible nitric oxide synthase promotes

pathophysiological consequences of experimental bladder outlet obstruction. J Urol 2003; 169: 1569.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Immunofluorescence staining of E-cadherin (adhesion protein), tryptase (mast cells), terminal deoxynucleotidyl transferase dUTP nick-end

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labeling (TUNEL) (apoptotic cells), and zonula ocludens-1 (ZO-1) (junction protein) in the bladder urothelium of controls, patients with bladder outlet

and BOO with detrusor underactivity (DU).

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obstruction (BOO) with detrusor overactivity/hypersensitive bladder (DO/HSB)

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*Each target cell is labeled green, and arrows indicate the presence of mast cells and apoptotic cells in the suburothelium. The dotted line separates the

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urothelium from the suburothelium.

Figure 2. Correlations of video urodynamic parameters and sensory proteins

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in the bladder urothelium of patients with bladder outlet obstruction. (A)

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Correlation of voided volume (Vol) and E-cadherin expression. (B) Correlation of the volume of the first sensation of filling (FSF) and β-3 adrenoreceptor expression. (C) Correlation of voiding detrusor pressure (Pdet) and M2 muscarinic receptor expression.

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ACCEPTED MANUSCRIPT Table 1. Demographic, immunofluorescence, western blotting, and video urodynamic study parameters in bladder outlet obstruction patients and control Control

BOO Overall

DO/HSB

DU

P

P

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(n=10)

1

2

(n=33)

(n=23)

(n=10)

value

value

64.6±11.45

68.5 ±11.1

69.0±11.6

67.5±10.5

0.258

0.764

E-cadherin

27.70±10.42

15.93±13.20

18.85±13.60

9.21±9.57*

0.015

0.038

Tryptase

4.16±2.68

15.12±7.89

16.11±6.16*

12.84±10.96

0.000

0.652

TUNEL

0.85±1.31

2.64±2.57

2.47±2.41*

3.03±3.01

0.028

0.844

ZO-1

6.90±1.82

7.83±3.98

7.29±2.58

9.08±6.14

0.358

0.570

TRPV 1

0.131±0.070

0.139±0.096

0.137±0.102

0.145±0.084

0.840

0.695

TRPV 4

0.188±0.286

0.155±0.243

0.152±0.249

0.164±0.241

0.565

0.570

iNOS

0.258±0.325

0.171±0.332

0.219±0.389

0.062±0.039*

0.128

0.147

eNOS

0.094±0.088

0.104±0.096

0.119±0.107

0.071±0.058

0.885

0.254

P2X3

0.097±0.109

0.257±0.206

0.247±0.145*

0.278±0.315*

0.001

0.456

β3

0.878±0.584

1.012±0.415

0.864±0.269

1.35±0.499*

0.289

0.009

M2

0.405±0.303

0.912±1.043

1.073±1.184*

0.558±0.490

0.041

0.108

M3

1.593±0.708

0.797±0.342

0.703±0.308*

1.013±0.330*

0.000

0.024

M2/ M3

0.313±0.280

1.371±1.610

1.691±1.796*

0.634±0.685

0.001

0.012

FSF (mL)

140.2±81.8

115.2±53.98

197.8±106.9

0.042

FS (mL)

242.5±146.9

190.8±80.3

361.5±195.7

0.025

Age

M AN U

SC

IF findings

Western

AC C

EP

TE D

blotting

VUDS

1

ACCEPTED MANUSCRIPT CBC (mL)

316.0±143.5

284.8±114.6

387.7±181.3

0.036

Pdet

48.9±36.0

60.5±33.4

15.6±18.9

0.002

5.58±4.68

7.74±3.93

0.60±0.84

0.000

Vol (mL)

152.0±119.6

198.7±101.8

32.8±68.8

0.000

PVR (mL)

175.4±178.8

88.3±89.9

398.0±155.1

(cmH2O) Qmax

RI PT

(mL/s)

0.000

SC

*P value < 0.05 versus control. 1 P values between control and overall BOO patients. 2 P values between DO/HSB and DU groups within BOO. BOO, bladder outlet obstruction; DO/HSB, detrusor overactivity/

M AN U

hypersensitivity bladder; DU, detrusor underactivity; FSF, first sensation of bladder filling; FS, full sensation; CBC, cystometric bladder capacity; Pdet, detrusor voiding pressure; Qmax, maximal urinary flow rate; Vol, voided

AC C

EP

TE D

volume; PVR, post-void residual volume

2

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT BOO = bladder outlet obstruction DO = detrusor overactivity DU = detrusor underactivity; eNOS = epithelial nitric oxide synthase

RI PT

FSF= first sensation of bladder filling HSB = hypersensitivity bladder iNOS = inducible nitric oxide synthase Pdet = detrusor voiding pressure

SC

PVR= post-void residual volume Qmax= maximal urinary flow rate TRPV 4= transient receptor potential vanilloid 4 TUNEL = terminal deoxynucleotidyl transferase dUTP nick-end labeling assay

AC C

EP

TE D

M AN U

Vol= voided volume VUDS = video urodynamic studies ZO-1 = zonula occludens-1

1