Urothelial Barrier Deficits, Suburothelial Inflammation and Altered Sensory Protein Expression in Detrusor Underactivity

Author's Accepted Manuscript Urothelial Barrier Deficits, Suburothelial Inflammation and Altered Sensory Protein Expressions in Detrusor Underactivity Yuan-Hong Jiang , Hann-Chorng Kuo

PII: DOI: Reference:

S0022-5347(16)30861-8 10.1016/j.juro.2016.07.071 JURO 13887

To appear in: The Journal of Urology Accepted Date: 11 July 2016 Please cite this article as: Jiang YH, Kuo HC, Urothelial Barrier Deficits, Suburothelial Inflammation and Altered Sensory Protein Expressions in Detrusor Underactivity, The Journal of Urology® (2016), doi: 10.1016/j.juro.2016.07.071. 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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Urothelial Barrier Deficits, Suburothelial Inflammation and Altered Sensory Protein Expressions in Detrusor Underactivity

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Yuan-Hong Jiang, Hann-Chorng Kuo*

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Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan

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*Correspondence: Dr. Hann-Chorng Kuo, Department of Urology, Buddhist

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Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan

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Tel: 886-3-8561825 ext. 2117 Fax: 886-3-8560794

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E-mail: [email protected]

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Word count: abstract 249 words, text 2499 words, 2 tables, 3 figures

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Key words: detrusor underactivity, urothelial dysfunction, suburothelial

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inflammation, detrusor overactivity

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Abstract

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Purpose: The pathophysiology of detrusor underactivity remains unclear, and impaired bladder afferent function is considered one of the important etiologies. This study investigated urothelial barrier deficits, suburothelial inflammation

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and sensory proteins expressed in bladder mucosa of detrusor underactivity patients.

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Materials and Methods: Bladder mucosa biopsies were performed in 34 patients with videourodynamics proven detrusor underactivity (study group) and 10 women with stress urinary incontinence as controls. The expressions of zona occuldens-1, E-cadherin in urothelium, tryptase and apoptosis levels in suburothelium, β3 adrenoreceptor, M2, and M3 muscarinic receptors, P2X3 receptor, and inducible/ endothelial nitric oxide synthase were compared between study and control patients.

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Results: Study patients included 22 women and 12 men with a mean age of 56.3±19.7 years. Fifteen patients had a history of diabetes. Study patients had significantly lower expression of E-cadherin, and higher mast cell and apoptotic cell numbers than controls. Additionally, lower expressions of M2 and M3 muscarinic receptors, P2X3 receptors, and endothelial nitric oxide synthase, but a higher expression of β-3 adrenoreceptor, were detected in the

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(all p<0.05).

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Conclusions: Urothelial dysfunction, increased suburothelial inflammation, and altered sensory protein expressions in bladder mucosa were prominent in patients with detrusor underactivity. Impaired urothelial signaling and sensory

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study patients. In study patients, positive correlation was noted between tryptase and apoptosis levels (r=0.527), and between the expressions of M2 muscarinic receptor and P2X3 receptor (r=0.403). However, the expression of β3 adrenoreceptor was negatively correlated with that of E-cadherin (r=-0.490)

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transduction pathways appear to reflect the pathophysiology of detrusor underactivity.

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Introduction

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Detrusor underactivity (DU) or underactive bladder (UAB) is an important but under-researched issue. DU is defined as “a contraction of reduced strength or duration resulting in prolonged or incomplete emptying of the bladder” or both

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by the International Continence Society.1 Nonetheless, it has received only minimal attention. DU can be observed in many conditions including aging, bladder outlet obstruction (BOO), neurologic insults, and myogenic failure, but its pathophysiology remains unclear.2 Altered or impaired bladder afferent

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function with impaired subsequent activation of detrusor contraction has been considered an important etiology of DU.3, 4

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In the urinary bladder, urothelium serves not only a physical barrier but also as

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a sensor and transducer of sensory signaling.5 Urothelial cells express various receptors and ion channels including purinergic receptors, muscarinic

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receptors, adrenoreceptors, and transient receptor potential ion channels, which sense and respond to various intravesical stimuli. In addition, the activation of β-3 adrenoreceptors in the urothelium and suburothelium can inhibit the bladder afferent signaling and facilitate bladder storage.6 The urothelium and underlying suburothelium are in close communication and are thought to be an integrated sensory unit. Urothelial dysfunction, increased suburothelial inflammation, and apoptosis occur in many lower urinary tract

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diseases, including overactive bladder (OAB) and neurogenic detrusor overactivity (DO),7-9 and probably also in DU. BOO is also considered a contributor to the development of DU.2, 10 In BOO,

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chronic bladder ischemia and repeated ischemia/reperfusion cycles cause excessive oxidative stress, which could be responsible for the development of bladder dysfunction such as DO and DU.11 Augmented puringerigc and muscarinc receptors in urothelium through mediating the augmented/ altered bladder afferent neurotransduction are considered to be important in the

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pathophysiology of DO.12, 13 We also discovered significant urothelial dysfunction and alterations of sensory proteins including higher β-3 adrenoreceptor and lower inducible nitric oxide synthase (iNOS) expressions in the bladder mucosa of BOO patients with urodynamic DU.14 Impaired urothelial signaling and sensory transduction pathways through β-3 adrenoreceptors and the NOS pathway could advance the development of DU.

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Urothelial function and sensory proteins in the bladder mucosa could 3

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participate in the pathophysiology of DU. Evidence of this hypothesis in human subjects is rare. This study investigated urothelial barrier deficits, suburothelial inflammation, and the expression of sensory proteins in the bladder mucosa of patients with DU. The relationships between altered sensory protein

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expression and bladder dysfunction were also examined.

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MATERIALS AND METHODS Patients

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Additionally, the selected patients were ready for transurethral resection of the prostate or external urethral sphincter onabotulinumtoxin-A injection to

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improve self-voiding efficiency. Clinical investigations included transrectal sonography of the prostate for male patients and cystourethroscopy and VUDS in all patients before surgery. Exclusion criteria for analysis included active urinary tract infection, significant evidence indicative of BOO in VUDS or cystourethroscopy, interstitial cystitis, occult or overt neuropathy (including cerebrovascular accident, spinal cord injury, multiple sclerosis, and Parkinson disease), history of bladder surgery or injury, and history of pelvic floor or spine

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surgery. We also invited 10 women with genuine stress urinary incontinence that did not have any other storage or voiding dysfunction in VUDS and were ready for a pubovaginal sling procedure to serve as controls.

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From June 2012 to March 2014, we prospectively and consecutively enrolled patients who had chronic urinary retention requiring clean intermittent catheterization and had DU proven by video-urodynamic study (VUDS).

VUDS and Biopsy Procedures VUDS were conducted and the results were interpreted according to the recommendations of the International Continence Society.1, 15 VUDS parameters included maximum flow rate (Qmax), detrusor voiding pressure

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(Pdet), cystometric bladder capacity (CBC), voided volume (Vol), and post-void residual (PVR) volume. DU was defined when patients had a low voiding pressure, low flow rate, PVR volume > 300 mL, and a low voiding efficiency (Vol/CBC) (< 33%) as well as a relaxed external urethral sphincter on electromyography during voiding. During the filling phase, DO was defined as an involuntary detrusor contraction associated with urgency symptom. Cystourethroscopy was performed to exclude anatomic BOO, including bladder neck obstruction or contracture, benign prostatic obstruction in men, 4

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and urethral stricture.

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Cold cup bladder biopsies were performed concurrently in all DU patients (after the operations enhancing self-voiding efficiency) and controls (after anti-incontinence surgeries). Only bladder mucosa was taken to prevent bladder perforation. Bladder specimens were obtained from the posterior wall around 2 cm above the ureteral orifices. Any erythematous or inflammatory bladder mucosa was avoided being selected as the biopsy site. One bladder

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specimen was sent to the pathology department for histological examination to exclude the possibility of carcinoma in situ.

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This study was approved by the Institutional Review Board and Ethics

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Committee of Buddhist Tzu Chi General Hospital. Each patient was informed about the study rationale and procedures, and written informed consent was

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obtained before surgery.

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Immunofluorescence Staining and Protein Expression

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and cellular apoptosis by the terminal deoxynucleotidyl transferase dUTP nick end-labelling assay (TUNEL). The laboratory procedures were performed similarly to those in our previous study.8 The thickness of the tissue sections was 6µm. The immunofluorescence quantification was determined in 4

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Bladder tissue samples from DU patients and controls were investigated for urothelial adhesive function by E-cadherin expression, urothelial integrity by zonula occludens-1 (ZO-1) expression, mast cell activation by tryptase level,

consecutive high-power fields (X 400) in the area with the greatest density. Immunofluorescence (tryptase and TUNEL) assays were quantified by counting the number of positively stained cells per 100 cells per unit area (4 µm2) and are shown as percentages. The intensity of E-cadherin (using fluorescence microscopy) and ZO-1 (using a confocal microscope) was

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quantified using Image J processing16, which developed and was available from NIH.

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Western blotting was applied to assess the expression of sensory proteins of the bladder mucosa specimen including purinergic receptor P2X3, endothelial NOS (eNOS), iNOS, β3 adrenoceptors, and M2 and M3 muscarinic receptors. The applied first antibodies included P2X3 receptor (1:2000; GeneTex, Irvine, CA, USA), eNOS and M2 muscarinic receptors (1:1000; GeneTex, Irvine, CA, 5

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USA), iNOS (1:1500; ThermoFisher, Rockford, IL, USA), β3 adrenoceptor (1:1000; Abcam, Cambridge, UK), and M3 muscarinic receptors (1:500; GeneTex, Irvine, CA, USA), and GAPDH (1:100000; GeneTex, Irvine, CA, USA) was as the internal control. The second antibodies included donkey anti-goat (for β3 adrenoceptor) and goat anti-rabbit (for the other proteins) IgG-HRP (1:3000; Santa Cruz Biotechnology, Dallas, Texas, USA). The scanned film after gel electrophoresis was quantified using a gel documentation system (Quantity One Version 4.6.2, Bio-Rad Laboratories, Hemel Hempstead, Herts,

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UK). The laboratory procedures were performed similarly to those in our previous study.14

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Statistical Analysis

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Continuous variables were presented as means ± standard deviations, and

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categorical data were presented as numbers and percentages. Eligible DU patients were grouped according to the presence of concurrent DO or not and the presence of diabetes or not and were compared with controls. Differences

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in the results of immunofluorescence staining and Western blotting were analyzed using the Kruskal-Wallis test. In order to clarify the roles of sensory proteins in DU, correlation analysis using linear regression was performed with these values including VUDS parameters, and the sensory protein expressions.

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All calculations were done using SPSS for Windows, version 16.0 (SPSS, Chicago, IL). Differences were considered statistically significant if p values were less than 0.05.

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RESULTS

Eligible DU patients included 22 women and 12 men with a mean age of 56.3 ± 19.7 years, which was similar to that of controls (52.4 ± 10.5 years). Table 1 summarizes the demographic and VUDS data. Clinically, DU patients had a

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significantly lower Pdet, lower Qmax, and a larger PVR volume than did controls. Of the DU patients, 11 (32.4%) with concurrent DO had a significantly higher Pdet and lower bladder compliance than those without DO (both p < 0.05). Fifteen DU patients (44.1%) had the history of type II diabetes mellitus, with similar demographic and VUDS data to those without diabetes. There were no major peri- or postoperative complications such as bleeding or perforation during or after bladder biopsy procedures.

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DU patients had significantly lower expressions of M2 and M3 muscarinic receptors, P2X3 receptor, and eNOS, but a higher expression of β-3 adrenoreceptor than controls (all p<0.05) (Fig. 3). Diabetic DU patients had a higher mast cell number and higher expression of M3 muscarinic receptors

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than did non-diabetic DU patients. Within diabetic DU patients, sensory protein expressions were similar between those with and without concurrent DO. Although DU patients had a lower value of iNOS expression than controls, the difference was not significant. Sensory protein expressions in DU patients

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Immunofluorescence staining showed that DU patients had a significantly lower expression of E-cadherin but not ZO-1 in the urothelium and higher mast cell and apoptotic cell numbers in the suburothelium than did controls (Fig. 1, Table 2). In Western blotting analysis of sensory proteins of bladder mucosa,

were similar to those in patients with and without DO and between the two genders.

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Linear regression analysis showed a positive correlation between tryptase and TUNEL levels (r = 0.527) in DU patients (Fig. 2). The expression of M2 muscarinic receptors was positively correlated with that of P2X3 receptors (r = 0.403). However, the expression of β3 adrenoreceptors was negatively correlated with that of E-cadherin (r = -0.490) (all p < 0.05).

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DISCUSSION

In the present study, significant urothelial dysfunction, increased suburothelial inflammation and apoptosis, and altered sensory protein expressions in

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suburothelium in the bladder could play important roles.

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bladder mucosa were observed in DU patients. Sensory protein expressions were downregulated, which indicated that impaired bladder urothelial signaling and sensory proteins might be responsible for the pathophysiology of DU. Impaired bladder afferent function with subsequent impaired activation of detrusor contraction is one of the causes of DU,3, 4 and both the urothelium and

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UAB and OAB were thought to be opposing syndromes. In OAB, upregulation

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of bladder afferent signaling through action on purinergic, muscarinic, and vanilloid receptors pathways has been proven.12, 17-19 In contrast, the role of afferent dysfunction as a potential contributory factor in DU is increasingly recognized, including deficiency in the sensory innervation peripherally or in central reporting or signal processing.10, 20 Bladder afferent mechanisms have

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important functions in the activation of the bladder micturition reflex, which may contribute to OAB and UAB or DU by increasing and decreasing signaling respectively.

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NO pathway also participates in modulating afferent neuron activities and neurotransmission in the urinary bladder.22 Both eNOS23 and iNOS24 are significantly increased in the bladders of rats with BOO and concurrent DO. The activation of β-3 adrenoreceptors in the urothelium and suburothelium

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facilitates bladder storage and increases bladder capacity through the inhibitory action of bladder afferent signaling.6 In our recent study investigating

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BOO patients, DU subgroup had significantly higher expression of β3 adrenoreceptors and lower expression of iNOS than controls did.14 In the present study, DU patients had lower expressions of M2 and M3 muscarinic receptors, P2X3 receptors, and eNOS in addition to a non-significantly lower expression of iNOS and a higher expression of β-3 adrenoreceptor than in controls. These results highlight the function of decreased bladder urothelial-afferent signaling (affected by lower muscarinic and puringergic

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receptor expressions, and higher β-3 adrenoreceptor expression) in the pathophysiology of DU. Additionally, these signal changes could account for the clinical presentation of hyposensitivity during urine storage in patients with DU. The positive correlation between the expressions of M2 muscarinic

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Urothelium-afferent nerve interactions can influence reflex bladder contractions.21 Increased P2X3, M2, and M3 muscarinic receptors in the urothelium are thought to be important factors the pathophysiology of DO through the mediation of altered bladder afferent neurotransduction.12, 13 The

receptors and P2X3 receptors support the close interaction of bladder afferent signaling in bladders with DU.

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A new hypothesis that chronic untreated or treatment refractory OAB may progress to detrusor hyperreflexia and impaired contractility and finally UAB through the repeated cycles of ischemia and reperfusion injury has been proposed.4, 11 Urothelial dysfunction (decreased E-cadherin expression) and increased suburothelial inflammation and apoptosis were observed not only in OAB patients8 but also in DU patients in this study. The expressions of eNOS25 and iNOS26 increased in a bladder inflammation/cystitis rat model, suggesting their roles in inflammation. However, in this study, DU patients had contrary results, i.e., lower expressions of eNOS and iNOS. We hypothesize that these two syndromes may share a similar initial pathogenesis of bladder 8

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inflammation and ischemia, but UAB and DU represent a late phase of inflammatory consequence. In our study, the severity of suburothelial inflammation and sensory protein expressions between DU patients with and without DO did not differ. This was probably due to the chronic urinary retention status in our eligible DU patients, representing a relatively late and irreversible status in the disease progression.

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Multiple complex mechanisms regulate E-cadherin expression within the

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bladder urothelium.27 In a partial BOO mouse model, E-cadherin was downregulated through the activation of epithelial-mesenchymal transition-inducing transcription factors by hypoxia-inducible factor pathways.28 In a previous human BOO study, the DU subgroup of patients had significantly

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lower expression of E-cadherin than did the DO or hypersensitive subgroup, suggesting DU might represent a more severe or decompensated BOO

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impaired bladder sensation in these patients.

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status.14 In the present study, decreased expression of E-cadherin and increased expression of β3 adrenoreceptors, as well as their negative correlation in DU patients, indicates the close connection of hypoxia and

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Diabetic cystopathy is one of the common causes of DU.2 The impact of diabetes on the bladder is multifactorial, including urothelial dysfunction, neuronal impairment, changes in detrusor physiology, and systemic inflammation.29 Urothelial dysfunction with increased density of M3 muscarinic receptors in the bladder of diabetic rats was thought to be related to altered afferent function and DO.29, 30 In the present study, diabetic patients with DU

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pathophysiology.

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had more severe suburothelial inflammation and greater expression of M3 muscarinic receptors than did non-diabetic patients with DU. These findings indicate that diabetic cystopathy could have a different pathophysiology causing DU. Time-sequential evaluation of urothelial dysfunction and sensory protein expressions in diabetic cystopathy is necessary to elucidate its exact

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There are several limitations in this study. First, the case number was small,

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and controls were all female. Second, although we excluded the patients with significant neuropathy and tried to select homogeneous DU patients, the causes of DU might still be heterogeneous due to its unclear definition, possibly resulting in inconsistent study and laboratory results. Third, this study represents the observational results of this cross-sectional study, and thus,

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cannot fully elucidate the pathophysiology of DU. In the future, a well-designed study with a clear and restricted definition of DU should be conducted to clarify the exact pathological mechanisms of DU. Finally, it is difficult to separate the urothelium from suburothelium in the human bladder mucosa biopsy specimen,

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which might cause errors in the quantification of sensory protein expressions.

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Conclusion

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The results demonstrated that urothelial dysfunction, increased suburothelial inflammation, cellular apoptosis and altered sensory proteins expressions were prominent in bladders with DU. Additionally, DU had an indistinct pattern of decreased sensory proteins (including M2 and M3 muscarinic receptors,

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pathways played a major role in the development of DU.

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P2X3 receptor, and eNOS) and increased β3 adrenoreceptor in the bladder mucosa. Impaired bladder urothelial signaling and sensory transduction

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FIGURE LEGENDS

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Figure 1. Immunofluorescence staining of E-cadherin (adhesion protein),

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tryptase (mast cells), terminal deoxynucleotidyl transferase dUTP nick-end

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labeling (TUNEL) (apoptotic cells), and zonula ocludens-1 (ZO-1) (junctional

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protein) in the bladder mucosa specimens of patients with DU and controls.

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*Each target cell/protein is labeled green, and arrows indicate the presence of

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mast cells and apoptotic cells in the suburothelium. The dotted line separates

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the urothelium from the suburothelium, and “L” refers to the lumen of bladder.

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Figure 2. Correlations of the immunofluorescence staining results and the

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expressions of sensory proteins in bladder mucosa specimens of patients with

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DU. (A) Correlation of tryptase and TUNEL levels. (B) Correlation of M2

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muscarinic receptors and P2X3 receptors. (C) Correlation of β3

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adrenoreceptor and E-cadherin.

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Figure 3. Western blot analysis of sensory proteins in bladder mucosa in DU

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patients and controls.

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ACCEPTED MANUSCRIPT Table 1. Demographics and video-urodynamic study parameters in detrusor underactivity patients and controls DU

Control

P value *

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Overall

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(-)

(+)

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11

19

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(67.6%)

(32.4%)

(55.9%)

(44.1%)

(100%) 22: 12

0.026

56.3 ± 19.7

0.783

0.791

(-) Number

DM

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10: 0

8: 3

14: 9

13: 6

9: 6

Age

52.4 ±

53.0 ±

63.1 ±

59.5 ±

52.2 ±

10.5

18.6

21.1

169.0 ±

184.7 ±

132.1 ±

168.5 ±

165.3 ±

167.2 ±

54.1

96.1

58.4

95.2

80.9

88.1

266.6 ±

285.6 ±

194.2 ±

240.1 ±

275.6 ±

255.2 ±

85.1

165.1

82.2

117.6

184.5

148.1

19.6

FSF (mL)

FS (mL)

US (mL)

266.7 ±

236.1 ±

278.2 ±

306.1 ±

290.0 ±

162.1

117.9

127.0

184.5

152.0

455.4 ±

399.2 ±

469.1 ±

392.7 ±

436.4 ±

194.4

198.2

160.2

199.2

162.6

185.8

25.2 ±

2.1 ±

19.8 ±

4.7 ± 7.8

12.4 ±

8.0 ± 12.5

<0.001

3.0 ± 3.4

2.3 ± 3.3

<0.001

63.9 ± 91.3

0.001

0.001

EP

356.1±

AC C

Pdet

#

#

14.5

3.5

15.7

11.9 ±

2.0 ±

3.0 ± 2.7

(mL/sec)

6.3

3.6

Vol. (mL)

302.3 ±

67.2 ±

57.4 ±

50.1 ±

82.7±

154.3

106.3

53.7

89.9

93.2

93.3 ±

388.2 ±

341.8

419.0 ±

310.0 ±

372.7 ±

106.2

235.7

±127.0

208.7

189.0

204.9

(cmH2O)

Qmax

PVR (mL)

0.842

317.0 ±

56.0

CBC (mL)

TE D

parameters

19.8

M AN U

VUDS

SC

Sex (F: M)

0.522

0.342

16.3 1.84 ± 3.3

1

ACCEPTED MANUSCRIPT Bladder compliance

86.3 ±

81.3 ± #

135.2

38.1

41.4 ± #

35.0

46.3 ±

97.4 ±

68.0 ±

47.4

163.2

112.9

0.021

(mL/cmH2O) *: p values between control and overall DU patients. #

: p < 0.05 between DO (+) and DO (-) subgroups

$

RI PT

: p < 0.05 between DM (+) and DM (-) subgroups: there was no significant difference.

DU, detrusor underactivity; DO, detrusor overactivity; DM, diabetes mellitus; VUDS,

video-urodynamic study; FSF, first sensation of bladder filling; FS, full sensation; CBC,

cystometric bladder capacity; Pdet, detrusor voiding pressure; Qmax, maximal urinary flow

AC C

EP

TE D

M AN U

SC

rate; Vol, voided volume; PVR, post-void residual volume

2

ACCEPTED MANUSCRIPT Table 2. Immunofluorescence staining, and Western-blotting analysis data in detrusor underactivity patients and controls Control

DU

P

(N=10)

value*

Concurrent

DM

(-)

(N=34)

(+)

(-)

RI PT

DO

Overall (+)

(N=23) (N=11) (N=19) (N=15) Immunofluorescence

TUNEL

23.70 ±

15.43±

24.46 ±

8.40

20.41

14.43

5.9 ± 4.9

11.5 ±

9.2 ±

6.1

4.4

1.0 ± 1.4

ZO-1

iNOS

8.7 ±

13.4 ±

10.7 ±

$

5.1

$

5.4

5.7 6.2 ±

3.3

3.4

2.5

3.9

3.3

7.65 ±

8.39 ±

8.08 ±

7.66 ±

7.89 ±

2.70

4.67

3.95

2.68

3.41

0.54 ±

0.56 ±

0.43 ±

0.70 ±

0.55 ±

0.59

0.59

0.62

0.49

0.66

0.59

1.55 ±

0.78 ±

0.82 ±

0.52 ±

1.53 ±

0.80 ±

TE D

1.22 ±

AC C

eNOS

18.88

7.1 ±

$

$

1.03

0.35

0.35

0.39

1.83

0.34

0.46 ±

0.84 ±

0.86 ±

0.82 ±

0.90 ±

0.85 ±

0.56

0.31

0.3

0.28

0.33

0.3

1.66 ±

0.69 ±

0.59 ±

0.63 ±

0.89 ±

0.66 ±

1.17

0.53

0.54

0.43

0.99

0.53

0.34 ±

0.15 ±

0.33 ±

0.19 ±

0.23 ±

0.21 ±

0.17

0.34

0.46

0.38

0.40

0.39

0.92 ±

0.71 ±

0.78 ±

0.87 ±

0.52 ±

0.73 ±

0.77

0.66

0.54

0.64

0.53

0.61

EP

P2X3 receptor

14.14

5.5 ±

Western blotting

β-3 adrenoreceptor

21.67

6.6 ±

1.72

M3 receptor

21.02±

6.1 ±

6.37 ±

M2 receptor

16.67±

SC

Tryptase

41.30 ±

M AN U

E-cadherin

0.001

0.010

<0.001

0.098

0.004

0.048

0.033

0.006

0.007

0.455

*: p values between control and overall DU patients. #

: p < 0.05 between DO (+) and DO (-) subgroups: there was no significant difference.

$

: p < 0.05 between DM (+) and DM (-) subgroups (Tryptase p value 0.014, M3 p value 0.025)

3

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

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

eNOS = endothelial nitric oxide synthase iNOS = inducible nitric oxide synthase NOS= nitric oxide synthase OAB = overactive bladder

RI PT

BOO = bladder outlet obstruction CBC= cystometric bladder capacity DO = detrusor overactivity DU = detrusor underactivity;

SC

Pdet = detrusor voiding pressure PVR= post-void residual volume Qmax= maximal urinary flow rate TUNEL = terminal deoxynucleotidyl transferase dUTP nick-end labeling assay

AC C

EP

TE D

ZO-1 = zonula occludens-1

M AN U

UAB= underactive bladder Vol= voided volume VUDS = video urodynamic studies

1