- Email: [email protected]
Bladder Pain Syndrome With and Without Hunner's Lesion
ARTICLE IN PRESS
Female Urology, Urodynamics, Incontinence, and Pelvic Floor Reconstructive Surgery Urothelial Functional Protein and Sensory Receptors in Patients With Interstitial Cystitis/Bladder Pain Syndrome With and Without Hunner’s Lesion Jia-Fong Jhang, Yung-Hsiang Hsu, and Hann-Chorng Kuo OBJECTIVE METHODS
RESULTS
CONCLUSION
To investigate the urothelium function and sensory receptors difference between interstitial cystitis/ bladder pain syndrome (IC/BPS) patients with or without Hunner’s lesion. Fourteen female IC/BPS patients with Hunner’s lesion (Hunner IC) and 14 age-matched IC/ BPS patients without Hunner’s lesions (non-Hunner IC) were enrolled. Bladder mucosa biopsies were obtained. Bladder inflammation, eosinophil infiltration, and urothelial denudation were graded on a 4-point scale after staining with hematoxylin and eosin. Adhesive protein E-cadherin, tryptase, and zonula occuldens-1 in the bladder tissues were assessed with immunofluorescence staining. Urothelial muscarinic receptors M2, M3, endothelial nitric oxide synthase (eNOS), and purinergic receptor P2X3 were evaluated by Western blotting. Hunner IC patients had a significantly higher mean visual analog scale pain score and smaller cystometric bladder capacity than non-Hunner IC patients. The Hunner IC bladder specimens showed more severe or moderate eosinophilic infiltration and urothelial denudation than the nonHunner IC bladder specimens did. The E-cadherin expression was significantly lower, and eNOS expression was significantly higher in the Hunner IC bladder samples than in the non-Hunner IC samples. The other functional proteins or sensory receptors did not differ between groups. Bladder inflammation and urothelial cell adhesion defects were more severe in the Hunner IC than that in the non-Hunner IC patients. eNOS was significantly higher in the Hunner IC than in the non-Hunner IC bladder samples, suggesting that eNOS expression difference may implicate different pathogenesis in 2 types of IC. UROLOGY ■■: ■■–■■, 2016. © 2016 Elsevier Inc.
M
ost clinicians and researchers have reached a consensus to use the term interstitial cystitis/bladder pain syndrome (IC/BPS) to diagnose patients with chronic pelvic pain or discomfort accompanied by at least 1 urinary symptom.1 In the European Society for the Study of Interstitial Cystitis (ESSIC) guidelines, IC/BPS patients are classified into different groups according to the findings of cystoscopy with hydrodistention and the histopathological features of bladder biopsies.1 One of the most Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan; and the Department of Pathology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan Address correspondence to: Hann-Chorng Kuo, M.D., Department of Urology, Buddhist Tzu Chi General Hospital, 707 Chung-Yang Road, Section 3, Hualien 970, Taiwan. E-mail: [email protected] Submitted: June 15, 2016, accepted (with revisions): August 22, 2016
© 2016 Elsevier Inc. All rights reserved.
often mentioned cystoscopic findings in IC/BPS is Hunner’s lesion, and it presents as a circumscriptive, reddened mucosal area with small vessels radiating toward a central scar.2 Patients with Hunner’s lesions are classified into type 3 IC/ BPS and have many more clinical symptoms and signs compared to patients without Hunner’s lesions. IC/BPS patients with Hunner’s lesion (Hunner IC) complain of sharp and stabbing bladder pain whereas IC/BPS patients without Hunner’s lesions (non-Hunner IC) describe more aching and cramping in the bladder.3 Patients with Hunner IC are also older, void more frequently, and have smaller mean bladder capacity than non-Hunner IC patients.4 However, the interstitial cystitis symptom and problem indices (ICSI and ICPI) scores do not differ between the 2 IC groups.3 The differences in the pathogenesis of Hunner IC and non-Hunner IC attract widespread interest among http://dx.doi.org/10.1016/j.urology.2016.08.029 0090-4295
1
ARTICLE IN PRESS urologists. Hunner’s lesion is considered an inflammatory lesion, presenting with ruptured mucosa and submucosa provoked by bladder distention.1 However, bladder inflammation in patients with non-Hunner IC may not exist.1 In a recent histopathological study, bladder specimens with Hunner IC had more plasma cells and neutrophil infiltration.5 The level of nitric oxide (NO) in bladder samples with Hunner IC was higher than that in the nonHunner IC samples, and mast cell infiltration was greater.6 Recently, urothelial dysfunction has been considered as a possible pathogenetic mechanism of IC/BPS.7 Our previous study also revealed higher levels of cell apoptosis, active mast cells, and abnormal E-cadherin expression in the urothelium of IC/BPS bladder specimens. However, the urothelial differences between the 2 types of IC/BPS remain unknown.8 The aim of the current study was to investigate the differences in functional proteins and sensory receptor expression in the urothelium of Hunner IC and non-Hunner IC bladders.
METHODS IC/BPS patients who were admitted to the Urology Department of Hualien Tzu Chi General Hospital from 2009 to 2015 were prospectively enrolled in the study. The diagnosis of IC/BPS was based on the ESSIC criteria (chronic pelvic pain, pressure, or discomfort perceived to be related to the urinary bladder accompanied by at least 1 other urinary symptom such as persistent urge to void or urinary frequency).1 All patients underwent comprehensive medical history reviews after admission to the hospital, including questionnaires for ICPI and ICSI. IC/BPS patients with concurrent urological problems such as acute or chronic bacterial cystitis, upper tract urolithiasis, stress urinary incontinence, or neurogenic voiding dysfunction were excluded. The patients who had undergone urology procedures in the recent 6 months such as intravesical instillation of any agent or intravesical injection were also excluded. The institutional review board and ethics committee of the Buddhist Tzu Chi General Hospital (IRB number 10161) approved the study. Each patient was informed of the study rationale and procedures and written informed consent was obtained from the patients before the bladder procedures. All patients underwent video urodynamic studies to confirm the diagnosis and rule out the coexistence of other lower urinary tract diseases, and the cystometric bladder capacity (CBC) was recorded. Potassium chloride sensitivity tests were also performed, and only the patients with positive results were recruited into the study. The IC/ BPS patients underwent cystoscopic hydrodistention under general anesthesia at an intravesical pressure of 80 cm of water. The maximal bladder capacity (MBC) and grading of glomerulation hemorrhage after pressure release were recorded. The IC/BPS patients were classified into Hunner IC and non-Hunner IC according to the cystoscopic finding of Hunner’s lesion before the bladder distention. Fourteen patients with Hunner IC were identified from our pa2
tients, and 14 age- and sex-matched non-Hunner IC patients were selected from among 232 patients for further investigation. Random cold-cup biopsies of the posterior bladder wall in patients with non-Hunner IC were obtained after cystoscopic hydrodistention. The bladder biopsies of Hunner IC were obtained at sites just around the Hunner lesion. Four biopsies were obtained from each patient. Endoscopic electrocauterization of the biopsy sites was also performed to prevent bleeding. Each specimen was 2 mm in diameter and contained mucosal and submucosal tissues, but no detrusor muscle. One specimen of each patient was sent to the pathology department for histopathological examination after staining with hematoxylin and eosin. A single pathologist who was blinded to the patient symptoms reviewed the bladder histopathology and graded the bladder inflammation, eosinophil infiltration, and urothelial denudation on a 4-point scale (0: none, 1: mild, 2: moderate, and 3: severe). The remaining bladder specimens were sent to our laboratory for immunochemical staining of E-cadherin, zonula occludens protein 1 (ZO-1), tryptase, and terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL). Western blotting was used to investigate the expression of muscarinic receptors M2, M3, purinergic receptor P2X3, adrenergic receptor β3 (β3AR), and endothelial nitric oxide synthase (eNOS) in the bladder specimens. In addition, bladder specimens were also obtained from 10 female patients with pure stress urinary incontinence, and were considered as normal controls. Immunochemical Staining and Quantification The entire bladder specimens were used for immunochemical staining in all groups. The bladder specimens were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 1 hour. Next, they were rinsed with ice-cold PBS containing 15% sucrose. Biopsy specimens were embedded in optimum cutting temperature medium and stored at −80°C. Four sections per specimen were cut using a cryostat at a thickness of 8 μm and collected on new Silane III-coated slides (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). Sections were postfixed in acetone at −20°C and blocked with rabbit serum. The sections were incubated overnight at 4°C with primary antibodies to antihuman E-cadherin (BD Biosciences, San Jose, CA), ZO-1 (Invitrogen, Burlington, ON, Canada), and tryptase (Chemicon, Temecula, CA). After rinsing the sections with 0.1% Tween-20 in PBS, goat antirabbit-IgG conjugated fluorescein isothiocyanate secondary antibodies (ANA Stec, Fremont, CA) were applied to the sections and incubated for 1 hour. Finally, the sections were counterstained. Negative controls included the isotype of the primary antibody. The expression of E-cadherin and ZO-1 in the urothelium was quantified using Image J software, developed by the National Institutes of Health (Bethesda, MD). The numbers of tryptase-positive mast cells were counted in 5 consecutive high-power fields (×400) in the area with the greatest infiltrate density, and the percentage was calculated as the number of tryptasepositive mast cells or total cells per unit area (4 μm2) × 100. UROLOGY ■■ (■■), 2016
ARTICLE IN PRESS Western Blotting Human bladder biopsy specimens were homogenized in liquid nitrogen, washed twice with ice-cold PBS, and then lysed for 10 minutes on ice using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Seongnam, South Korea). The extraction solution was supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and a phosphatase inhibitor cocktail (Roche Diagnostics). Target proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. M2, M3, P2X3, β3-AR, and eNOS were evaluated via Western blotting using primary antibody and a-tubulin (Cell Signaling Technology) as a loading control.
Table 1. Clinical parameters and urodynamic findings in Hunner and non-Hunner IC/BPS patients
ICSI ICPI OSS VAS MBC (mL) CBC (mL)
Statistical Analysis The differences in clinical parameters such as visual analog scale (VAS) and CBC between Hunner and non-Hunner IC were analyzed using the Mann-Whitney U test (nonparametric analysis of variance). The histopathologic grading results in Hunner and non-Hunner IC were analyzed with the chi-square test. The quantification results of immunochemical staining and Western blotting in Hunner IC, non-Hunner IC, and controls were also analyzed using the Kruskal-Wallis test. A P value <.05 was considered statistically significant. The Mann-Whitney U test was also used to make pairwise comparisons between the 3 groups (Hunner IC, non-Hunner IC, and control), and a P value <.017 was considered significant. Pearson’s correlation coefficients were calculated to determine the correlations between CBC, VAS, MBC, and the results of immunohistochemical staining. All calculations were performed using SPSS for Windows, version 16.0 (SPSS, Chicago, IL).
RESULTS Fourteen women with Hunner IC and 14 women with non-Hunner IC were enrolled in the study. All 28 patients completed the required urological testing. The mean age of the patients was 58.3 ± 7.3 years in both groups. Table 1 lists the clinical symptom scores and urodynamic parameters. The patients with Hunner IC had significantly higher VAS scores (P = .019) and smaller CBC (P = .021) than non-Hunner IC patients. The MBC during cystoscopic hydrodistention was also smaller in the patients with Hunner IC than in the patients with non-Hunner IC (P = .003). However, the ICPI and ICSI in patients with Hunner IC and non-Hunner IC did not significantly differ. No complication such as excessive bleeding or bladder perforation occurred after biopsy in any patient. Histopathological examination revealed more severe suburothelial inflammation in the Hunner IC than in the non-Hunner IC specimens. The Hunner IC bladder specimens also had more severe or moderate eosinophil infiltration (5/14 vs 0/14, P = .04) and urothelial denudation (6/14 vs 0/14, P = .016) in the suburothelium than did the non-Hunner IC bladder specimens (Table 2). Figure 1 and UROLOGY ■■ (■■), 2016
FS (mL) US (mL)
Hunner IC (n = 14)
Non-Hunner IC (n = 14)
16.0 (12.75-20.0) 16.0 (12.0-16.0) 32.0 (24.75-36.0) 8.0 (5.25-10.0) 500.0 (450.0-550.0) 138.0 (77.0-170.8) 106.0 (84.8-172.5) 110.0 (62.0-141.0)
13.0 (12.5-18.0) 16.0 (11.0-17.5) 29.0 (23.5-34.0) 2.0 (1.0-6.5) 625.0 (550.0-687.5) 306.0 (179.5-334.8) 140.0 (126.0-211.0) 211.0 (143.8-250.8)
P Value .383 .959 .442 .019 .003 .021 .118 .022
CBC, cystometric bladder capacity; FS, first sensation; ICPI, interstitial cystitis problem index; ICSI, interstitial cystitis symptom index; MBC, maximal bladder capacity; OSS, O’Leary-Sant symptom scores; US, urge sensation; VAS, visual analog scale.
Table 3 show the immunohistochemical staining and Western blotting results. The bladder specimens with Hunner IC expressed significantly more TUNEL (P = .002) and less E-cadherin (P < .001) than the normal controls. The non-Hunner IC bladder specimens expressed more tryptase (P = .012) than the normal controls. E-cadherin (P = .007), eNOS (P < .001), and M3 (P = .001) expression was lower in the non-Hunner IC than that in normal controls. Among the Hunner IC and non-Hunner IC bladder specimens, the expression of eNOS was significantly higher (P < .001) in the Hunner IC specimens, whereas the E-cadherin (P = .013) expression was significantly lower. However, the clinical parameters, including VAS, CBC, ICSI, and ICPI, did not significantly correlate with the immunohistochemical staining and Western blotting results (all P > .05).
DISCUSSION Recently, many researchers have demonstrated that IC/ BPS encompasses a heterogeneous spectrum of disorders, with different cystoscopic and histopathologic presentations.9 Although the ESSIC classifies IC/BPS patients according to cystoscopic and histopathologic findings, the clinical significance and differences in the pathogenesis of different IC/BPS subtypes are still unclear. There has been a long debate on whether Hunner IC and non-Hunner IC are different phenotypes of the same disease condition or 2 separate disease entities.10 Fall even proposed that the term “interstitial cystitis” should be used only in patients with bladder pain syndrome and Hunner’s lesion.11 However, laboratory evidence to differentiate between Hunner IC and non-Hunner IC remains limited. Our study revealed a decrease in the urothelial adhesion protein E-cadherin 3
ARTICLE IN PRESS Table 2. Histopathology results in Hunner and non-Hunner IC bladder Inflammation Hunner IC Non-Hunner IC
Eosinophil infiltration
Urothelium denudation
None
Mild
Moderate
Severe
None
Mild
Moderate
Severe
None
Mild
Moderate
Severe
0 9
10 5
3 0
1 0
7 14
2 0
2 0
3 0
0 8
9 6
2 0
3 0
IC, interstitial cystitis.
Figure 1. Immunohistochemical staining of the urothelium in Hunner IC and non-Hunner IC revealed that the urothelium of Hunner IC showed a greater defect of E-cadherin than the non-Hunner IC did. BPS, bladder pain, syndrome; IC, interstitial cystitis; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; ZO-1, zonula occludens protein 1. (Color version available online.)
and greater expression of eNOS in Hunner IC specimens than in non-Hunner IC specimens. The urothelium specimens with Hunner IC also had more severe inflammation and eosinophil infiltration than the non-Hunner IC specimens did. Although the term IC indicates inflammation in the urinary bladder, whether all IC/BPS bladders present with inflammation is still controversial. The ESSIC criteria include IC/BPS patients without inflammation in their bladder biopsy findings.1 Previous studies report that bladder biopsy characteristics are associated with symptoms in IC/ BPS patients.12,13 A recent study revealed many different histopathological features of bladders with Hunner IC and 4
non-Hunner IC, in which bladders with Hunner IC had more epithelial denudation, subepithelial inflammation, and neutrophil and eosinophil infiltration.5 Our current study shows similar histopathological findings, but the immunohistochemical staining of tryptase did not differ between Hunner IC and non-Hunner IC specimens. This result suggests that mast cell activation exists in the urothelium of non-Hunner IC, even though there is no histopathological evidence of inflammation. Our previous study also showed increased apoptosis and decreased E-cadherin in the urothelium of IC/BPS bladders.8 In the current study, we found less expression of E-cadherin in the Hunner IC than in the non-Hunner IC bladder specimens. This finding UROLOGY ■■ (■■), 2016
ARTICLE IN PRESS Table 3. Functional protein and sensory receptor expression in Hunner and non-Hunner IC/BPS Group 1 Normal Control (N = 10)
Group 2 Hunner IC (N = 14)
Group 3 Non-Hunner IC (N = 14)
33.63 (18.97-41.15)
8.64 (4.96-20.0)
23.34 (15.27-27.14)
<.001
ZO-1
5.31 (4.75-7.25)
4.25 (0.00-7.51)
3.63 (0.00-6.96)
.303
Tryptase
5.45 (2.99-8.18)
5.04 (2.32-11.9)
9.94 (5.88-17.11)
.025
TUNEL
0.39 (0.00-0.68)
3.57 (0.91-7.42)
1.86 (0.00-3.38)
.005
M2
1.17 (0.58-1.75)
0.55 (0.22-1.00)
1.09 (0.77-1.38)
.082
M3
1.43 (0.72-2.42)
0.62 (0.33-1.11)
0.60 (0.32-0.90)
.004
P2X3
1.57 (0.63-2.26)
1.08 (0.43-1.59)
1.46 (0.72-3.23)
.161
β3-AR
0.47 (0.22-0.94)
0.48 (0.34-0.64)
0.76 (0.25-0.99)
.469
eNOS
0.23 (0.16-0.40)
0.40 (0.30-0.50)
0.05 (0.02-.014)
<.001
E-cadherin
P Value
P Value Among Groups 1vs 2: <.001 1 vs 3: .007 2 vs 3: .013 1 vs 2: .484 1 vs 3: .102 2 vs 3: .944 1 vs 2: .944 1 vs 3: .012 2 vs 3: .048 1 vs 2: .002 1 vs 3: .035 2 vs 3: .127 1 vs 2: .072 1 vs 3: .806 2 vs 3: .028 1 vs 2: .027 1 vs 3: .001 2 vs 3: .619 1 vs 2: .099 1 vs 3: .675 2 vs 3: .091 1 vs 2: .832 1 vs 3: .613 2 vs 3: .143 1vs 2: .089 1 vs 3: <.001 2 vs 3: <.001
β3-AR, beta-3 adrenoceptor; BPS, BPS, bladder pain, syndrome; eNOS, endothelial nitric oxide synthase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; ZO-1, zonula occludens protein 1.
suggests a more severe urothelial cell adhesion defect in Hunner IC than in non-Hunner IC. Previous animal and human-cultured urothelial cell studies revealed increased expression of muscarinic receptors, P2X3, and decreased beta-1 adrenoceptor in IC/BPS bladders.14-16 However, the findings of our current study differed. When compared with normal controls, only the expression of M3 was decreased in both Hunner IC and non-Hunner IC (P = .027 and .001, respectively). The other sensory receptor expressions including M2, P2X3, and β3AR did not differ between normal control and IC/BPS patients. Among Hunner IC and non-Hunner IC patients, the expression of all of the sensory receptors tested did not differ. Further investigation of the pain receptors, such as transient receptor potential cation channel subfamily V member 1, in Hunner IC and non-Hunner IC, is necessary. NO is a signaling molecule that plays a key role in the pathogenesis of inflammation.17 Logadottir et al had reported increased intravesical NO production in Hunner IC than in non-Hunner IC.18 Recently, the inducible NOS was also found to be specifically increased in patients with Hunner IC, and it was suggested that it might be involved in the pathogenesis of the bladder inflammation.19 Current study further revealed decrease of eNOS in the non-Hunner IC patients than in the Hunner IC patients and normal control. In contrast, eNOS expression in urothelium of Hunner IC had a tendency to be higher than UROLOGY ■■ (■■), 2016
that in normal control (P = .089). eNOS is primarily responsible for the generation of NO in the vascular endothelium, and it increases vascular permeability, dysregulates angiogenesis, and impairs wound healing.20-22 It also regulates the expression of the proinflammatory molecules such as nuclear factor-κB and cyclooxygenase-2.23 Hunner’s lesion is characterized by an inflammatory lesion with increase of vascularity and centralized distribution of vessels. We suggest that eNOS expression difference between Hunner and non-Hunner IC might implicate pathogenesis difference in 2 types of IC. The c-Jun N-terminal kinase (JNK) pathway is activated by a variety of environmental stresses and inflammatory cytokines.24 JNK pathway could induce eNOS expression, and recently activated JNK pathway in bladder had been identified in IC/BPS patients.25 JNK inhibitors could also inhibit bladder inflammation in animal study.24 The anti-inflammatory effect of JNK inhibitor may have originated from reducing eNOS expression, and it might be effective in treating human Hunner IC in further studies. Inhibition of NOS could downregulate inflammatory cytokines and reduce inflammation.26 NOS inhibitor such as monomethyl-L-arginine and arginine peptides may be useful in decreasing inflammation in Hunner IC bladder. The main limitation of our study was the small number of patients; thus, the data could be heavily weighted by a limited number of extreme values. Bladder biopsies were taken at sites around the Hunner’s lesion in patients with 5
ARTICLE IN PRESS Hunner IC, whereas biopsies of patients with non-Hunner IC were taken from the bladder randomly, which might also be a source of bias in this study. Further studies investigating the different sensory receptors and inflammatory molecules are needed.
CONCLUSION The pathogeneses of both Hunner IC and non-Hunner IC involve inflammation. However, the inflammation in Hunner IC is more severe. Hunner IC features a more severe urothelial adhesive protein defect and greater expression of eNOS than non-Hunner IC. The different expression of eNOS between Hunner and non-Hunner IC might implicate different pathogenesis in these 2 types of IC. Reduced eNOS expression may be a possible further target in treating Hunner IC.
References 1. van de Merwe JP, Nordling J, Bouchelouche P, et al. Diagnostic criteria, classification, and nomenclature for painful bladder syndrome/ interstitial cystitis: an ESSIC proposal. Eur Urol. 2008;53:60-67. 2. Jhang JF, Hsu YH, Kuo HC. Characteristics and electrocauterization of Hunner’s lesions associated with bladder pain syndrome. Urol Sci. 2013;24:51-55. 3. Killinger KA, Boura JA, Peters KM. Pain in interstitial cystitis/ bladder pain syndrome: do characteristics differ in ulcerative and nonulcerative subtypes? Int Urogynecol J. 2013;24:1295-1301. 4. Peters KM, Killinger KA, Mounayer MH, Boura JA. Are ulcerative and nonulcerative interstitial cystitis/painful bladder syndrome 2 distinct diseases? A study of coexisting conditions. Urology. 2011;78:301308. 5. Maeda D, Akiyama Y, Morikawa T, et al. Hunner-type (classic) interstitial cystitis: a distinct inflammatory disorder characterized by pancystitis, with frequent expansion of clonal B-cells and epithelial denudation. PLoS ONE. 2015;10:e0143316. 6. Logadottir Y, Delbro D, Lindholm C, Fall M, Peeker R. Inflammation characteristics in bladder pain syndrome ESSIC type 3C/ classic interstitial cystitis. Int J Urol. 2014;Suppl 1:75-78. 7. Kanai A, de Groat W, Birder L, et al. Symposium report on urothelial dysfunction: pathophysiology and novel therapies. J Urol. 2006;175:1624-1629. 8. Shie JH, Kuo HC. Higher levels of cell apoptosis and abnormal E-cadherin expression in the urothelium are associated with inflammation in patients with interstitial cystitis/painful bladder syndrome. BJU Int. 2011;108:E136-E141. 9. Engeler DS, Baranowski AP, Dinis-Oliveira P, European Association of Urology, et al. The 2013 EAU guidelines on chronic pelvic pain: is management of chronic pelvic pain a habit, a philosophy, or a science? 10 years of development. Eur Urol. 2013;64:431-439.
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10. Yoshimura N. Editorial comment from Dr. Yoshimura to Interstitial cystitis is bladder pain syndrome with Hunner’s lesion. Int J Urol. 2014;21:83-84. 11. Fall M, Logadottir Y, Peeker R, Interstitial cystitis is bladder pain syndrome with Hunner’s lesion. Int J Urol. 2014;21:7982. 12. Matthews YL, Abele ST, Kusek JW, Nyberg LM, Interstitial Cystitis Database Study Group. Biopsy features are associated with primary symptoms in interstitial cystitis: results from the interstitial cystitis database study. Urology. 2001;57:67-81. 13. Leiby BE, Landis JR, Propert KJ, Tomaszewski JE, Interstitial Cystitis Data Base Study Group. Discovery of morphological subgroups that correlate with severity of symptoms in interstitial cystitis: a proposed biopsy classification system. J Urol. 2007;177:142148. 14. Ikeda Y, Birder L, Buffington C, Roppolo J, Kanai A. Mucosal muscarinic receptors enhance bladder activity in cats with feline interstitial cystitis. J Urol. 2009;181:1415-1422. 15. Sun Y, Chai TC. Up-regulation of P2X3 receptor during stretch of bladder urothelial cells from patients with interstitial cystitis. J Urol. 2004;171:448-452. 16. Buffington CA, Teng B, Somogyi GT. Norepinephrine content and adrenoceptor function in the bladder of cats with feline interstitial cystitis. J Urol. 2002;167:1876-1880. 17. Sharma JN, Al-Omran A, Parvathy SS. Role of nitric oxide in inflammatory diseases. Inflammopharmacology. 2007;15:252-259. 18. Logadottir YR, Ehren I, Fall M, Wiklund NP, Peeker R, Hanno PM. Intravesical nitric oxide production discriminates between classic and nonulcer interstitial cystitis. J Urol. 2004;171:1148-1150. 19. Logadottir Y, Hallsberg L, Fall M, Peeker R, Delbro D. Bladder pain syndrome/interstitial cystitis ESSIC type 3C: high expression of inducible nitric oxide synthase in inflammatory cells. Scand J Urol. 2013;47:52-56. 20. Fish JE, Marsden PA. Endothelial nitric oxide synthase: insight into cell-specific gene regulation in the vascular endothelium. Cell Mol Life Sci. 2006;63:144-1462. 21. Fukumura D, Gohongi T, Kadambi A, et al. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factorinduced angiogenesis and vascular permeability. Proc Natl Acad Sci USA. 2001;98:2604-2609. 22. Lowry JL, Brovkovych V, Zhang Y, Skidgel RA. Endothelial nitricoxide synthase activation generates an inducible nitric-oxide synthaselike output of nitric oxide in inflamed endothelium. J Biol Chem. 2013;288:4174-4193. 23. Ying L, Hofseth LJ. An emerging role for endothelial nitric oxide synthase in chronic inflammation and cancer. Cancer Res. 2007;67:1407-1410. 24. Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Genet Dev. 2002;12:14-21. 25. Zhao J, Wang L, Dong X, et al. The c-Jun N-terminal kinase (JNK) pathway is activated in human interstitial cystitis (IC) and rat protamine sulfate induced cystitis. Sci Rep. 2016;6:19670. 26. Ianaro A, O’Donnell CA, Di Rosa M, Liew FY. A nitric oxide synthase inhibitor reduces inflammation, down-regulates inflammatory cytokines and enhances interleukin-10 production in carrageenininduced oedema in mice. Immunology. 1994;82:370-375.
UROLOGY ■■ (■■), 2016
Female Urology, Urodynamics, Incontinence, and Pelvic Floor Reconstructive Surgery Urothelial Functional Protein and Sensory Receptors in Patients With Interstitial Cystitis/Bladder Pain Syndrome With and Without Hunner’s Lesion Jia-Fong Jhang, Yung-Hsiang Hsu, and Hann-Chorng Kuo OBJECTIVE METHODS
RESULTS
CONCLUSION
To investigate the urothelium function and sensory receptors difference between interstitial cystitis/ bladder pain syndrome (IC/BPS) patients with or without Hunner’s lesion. Fourteen female IC/BPS patients with Hunner’s lesion (Hunner IC) and 14 age-matched IC/ BPS patients without Hunner’s lesions (non-Hunner IC) were enrolled. Bladder mucosa biopsies were obtained. Bladder inflammation, eosinophil infiltration, and urothelial denudation were graded on a 4-point scale after staining with hematoxylin and eosin. Adhesive protein E-cadherin, tryptase, and zonula occuldens-1 in the bladder tissues were assessed with immunofluorescence staining. Urothelial muscarinic receptors M2, M3, endothelial nitric oxide synthase (eNOS), and purinergic receptor P2X3 were evaluated by Western blotting. Hunner IC patients had a significantly higher mean visual analog scale pain score and smaller cystometric bladder capacity than non-Hunner IC patients. The Hunner IC bladder specimens showed more severe or moderate eosinophilic infiltration and urothelial denudation than the nonHunner IC bladder specimens did. The E-cadherin expression was significantly lower, and eNOS expression was significantly higher in the Hunner IC bladder samples than in the non-Hunner IC samples. The other functional proteins or sensory receptors did not differ between groups. Bladder inflammation and urothelial cell adhesion defects were more severe in the Hunner IC than that in the non-Hunner IC patients. eNOS was significantly higher in the Hunner IC than in the non-Hunner IC bladder samples, suggesting that eNOS expression difference may implicate different pathogenesis in 2 types of IC. UROLOGY ■■: ■■–■■, 2016. © 2016 Elsevier Inc.
M
ost clinicians and researchers have reached a consensus to use the term interstitial cystitis/bladder pain syndrome (IC/BPS) to diagnose patients with chronic pelvic pain or discomfort accompanied by at least 1 urinary symptom.1 In the European Society for the Study of Interstitial Cystitis (ESSIC) guidelines, IC/BPS patients are classified into different groups according to the findings of cystoscopy with hydrodistention and the histopathological features of bladder biopsies.1 One of the most Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan; and the Department of Pathology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan Address correspondence to: Hann-Chorng Kuo, M.D., Department of Urology, Buddhist Tzu Chi General Hospital, 707 Chung-Yang Road, Section 3, Hualien 970, Taiwan. E-mail: [email protected] Submitted: June 15, 2016, accepted (with revisions): August 22, 2016
© 2016 Elsevier Inc. All rights reserved.
often mentioned cystoscopic findings in IC/BPS is Hunner’s lesion, and it presents as a circumscriptive, reddened mucosal area with small vessels radiating toward a central scar.2 Patients with Hunner’s lesions are classified into type 3 IC/ BPS and have many more clinical symptoms and signs compared to patients without Hunner’s lesions. IC/BPS patients with Hunner’s lesion (Hunner IC) complain of sharp and stabbing bladder pain whereas IC/BPS patients without Hunner’s lesions (non-Hunner IC) describe more aching and cramping in the bladder.3 Patients with Hunner IC are also older, void more frequently, and have smaller mean bladder capacity than non-Hunner IC patients.4 However, the interstitial cystitis symptom and problem indices (ICSI and ICPI) scores do not differ between the 2 IC groups.3 The differences in the pathogenesis of Hunner IC and non-Hunner IC attract widespread interest among http://dx.doi.org/10.1016/j.urology.2016.08.029 0090-4295
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ARTICLE IN PRESS urologists. Hunner’s lesion is considered an inflammatory lesion, presenting with ruptured mucosa and submucosa provoked by bladder distention.1 However, bladder inflammation in patients with non-Hunner IC may not exist.1 In a recent histopathological study, bladder specimens with Hunner IC had more plasma cells and neutrophil infiltration.5 The level of nitric oxide (NO) in bladder samples with Hunner IC was higher than that in the nonHunner IC samples, and mast cell infiltration was greater.6 Recently, urothelial dysfunction has been considered as a possible pathogenetic mechanism of IC/BPS.7 Our previous study also revealed higher levels of cell apoptosis, active mast cells, and abnormal E-cadherin expression in the urothelium of IC/BPS bladder specimens. However, the urothelial differences between the 2 types of IC/BPS remain unknown.8 The aim of the current study was to investigate the differences in functional proteins and sensory receptor expression in the urothelium of Hunner IC and non-Hunner IC bladders.
METHODS IC/BPS patients who were admitted to the Urology Department of Hualien Tzu Chi General Hospital from 2009 to 2015 were prospectively enrolled in the study. The diagnosis of IC/BPS was based on the ESSIC criteria (chronic pelvic pain, pressure, or discomfort perceived to be related to the urinary bladder accompanied by at least 1 other urinary symptom such as persistent urge to void or urinary frequency).1 All patients underwent comprehensive medical history reviews after admission to the hospital, including questionnaires for ICPI and ICSI. IC/BPS patients with concurrent urological problems such as acute or chronic bacterial cystitis, upper tract urolithiasis, stress urinary incontinence, or neurogenic voiding dysfunction were excluded. The patients who had undergone urology procedures in the recent 6 months such as intravesical instillation of any agent or intravesical injection were also excluded. The institutional review board and ethics committee of the Buddhist Tzu Chi General Hospital (IRB number 10161) approved the study. Each patient was informed of the study rationale and procedures and written informed consent was obtained from the patients before the bladder procedures. All patients underwent video urodynamic studies to confirm the diagnosis and rule out the coexistence of other lower urinary tract diseases, and the cystometric bladder capacity (CBC) was recorded. Potassium chloride sensitivity tests were also performed, and only the patients with positive results were recruited into the study. The IC/ BPS patients underwent cystoscopic hydrodistention under general anesthesia at an intravesical pressure of 80 cm of water. The maximal bladder capacity (MBC) and grading of glomerulation hemorrhage after pressure release were recorded. The IC/BPS patients were classified into Hunner IC and non-Hunner IC according to the cystoscopic finding of Hunner’s lesion before the bladder distention. Fourteen patients with Hunner IC were identified from our pa2
tients, and 14 age- and sex-matched non-Hunner IC patients were selected from among 232 patients for further investigation. Random cold-cup biopsies of the posterior bladder wall in patients with non-Hunner IC were obtained after cystoscopic hydrodistention. The bladder biopsies of Hunner IC were obtained at sites just around the Hunner lesion. Four biopsies were obtained from each patient. Endoscopic electrocauterization of the biopsy sites was also performed to prevent bleeding. Each specimen was 2 mm in diameter and contained mucosal and submucosal tissues, but no detrusor muscle. One specimen of each patient was sent to the pathology department for histopathological examination after staining with hematoxylin and eosin. A single pathologist who was blinded to the patient symptoms reviewed the bladder histopathology and graded the bladder inflammation, eosinophil infiltration, and urothelial denudation on a 4-point scale (0: none, 1: mild, 2: moderate, and 3: severe). The remaining bladder specimens were sent to our laboratory for immunochemical staining of E-cadherin, zonula occludens protein 1 (ZO-1), tryptase, and terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL). Western blotting was used to investigate the expression of muscarinic receptors M2, M3, purinergic receptor P2X3, adrenergic receptor β3 (β3AR), and endothelial nitric oxide synthase (eNOS) in the bladder specimens. In addition, bladder specimens were also obtained from 10 female patients with pure stress urinary incontinence, and were considered as normal controls. Immunochemical Staining and Quantification The entire bladder specimens were used for immunochemical staining in all groups. The bladder specimens were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 1 hour. Next, they were rinsed with ice-cold PBS containing 15% sucrose. Biopsy specimens were embedded in optimum cutting temperature medium and stored at −80°C. Four sections per specimen were cut using a cryostat at a thickness of 8 μm and collected on new Silane III-coated slides (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). Sections were postfixed in acetone at −20°C and blocked with rabbit serum. The sections were incubated overnight at 4°C with primary antibodies to antihuman E-cadherin (BD Biosciences, San Jose, CA), ZO-1 (Invitrogen, Burlington, ON, Canada), and tryptase (Chemicon, Temecula, CA). After rinsing the sections with 0.1% Tween-20 in PBS, goat antirabbit-IgG conjugated fluorescein isothiocyanate secondary antibodies (ANA Stec, Fremont, CA) were applied to the sections and incubated for 1 hour. Finally, the sections were counterstained. Negative controls included the isotype of the primary antibody. The expression of E-cadherin and ZO-1 in the urothelium was quantified using Image J software, developed by the National Institutes of Health (Bethesda, MD). The numbers of tryptase-positive mast cells were counted in 5 consecutive high-power fields (×400) in the area with the greatest infiltrate density, and the percentage was calculated as the number of tryptasepositive mast cells or total cells per unit area (4 μm2) × 100. UROLOGY ■■ (■■), 2016
ARTICLE IN PRESS Western Blotting Human bladder biopsy specimens were homogenized in liquid nitrogen, washed twice with ice-cold PBS, and then lysed for 10 minutes on ice using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Seongnam, South Korea). The extraction solution was supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and a phosphatase inhibitor cocktail (Roche Diagnostics). Target proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. M2, M3, P2X3, β3-AR, and eNOS were evaluated via Western blotting using primary antibody and a-tubulin (Cell Signaling Technology) as a loading control.
Table 1. Clinical parameters and urodynamic findings in Hunner and non-Hunner IC/BPS patients
ICSI ICPI OSS VAS MBC (mL) CBC (mL)
Statistical Analysis The differences in clinical parameters such as visual analog scale (VAS) and CBC between Hunner and non-Hunner IC were analyzed using the Mann-Whitney U test (nonparametric analysis of variance). The histopathologic grading results in Hunner and non-Hunner IC were analyzed with the chi-square test. The quantification results of immunochemical staining and Western blotting in Hunner IC, non-Hunner IC, and controls were also analyzed using the Kruskal-Wallis test. A P value <.05 was considered statistically significant. The Mann-Whitney U test was also used to make pairwise comparisons between the 3 groups (Hunner IC, non-Hunner IC, and control), and a P value <.017 was considered significant. Pearson’s correlation coefficients were calculated to determine the correlations between CBC, VAS, MBC, and the results of immunohistochemical staining. All calculations were performed using SPSS for Windows, version 16.0 (SPSS, Chicago, IL).
RESULTS Fourteen women with Hunner IC and 14 women with non-Hunner IC were enrolled in the study. All 28 patients completed the required urological testing. The mean age of the patients was 58.3 ± 7.3 years in both groups. Table 1 lists the clinical symptom scores and urodynamic parameters. The patients with Hunner IC had significantly higher VAS scores (P = .019) and smaller CBC (P = .021) than non-Hunner IC patients. The MBC during cystoscopic hydrodistention was also smaller in the patients with Hunner IC than in the patients with non-Hunner IC (P = .003). However, the ICPI and ICSI in patients with Hunner IC and non-Hunner IC did not significantly differ. No complication such as excessive bleeding or bladder perforation occurred after biopsy in any patient. Histopathological examination revealed more severe suburothelial inflammation in the Hunner IC than in the non-Hunner IC specimens. The Hunner IC bladder specimens also had more severe or moderate eosinophil infiltration (5/14 vs 0/14, P = .04) and urothelial denudation (6/14 vs 0/14, P = .016) in the suburothelium than did the non-Hunner IC bladder specimens (Table 2). Figure 1 and UROLOGY ■■ (■■), 2016
FS (mL) US (mL)
Hunner IC (n = 14)
Non-Hunner IC (n = 14)
16.0 (12.75-20.0) 16.0 (12.0-16.0) 32.0 (24.75-36.0) 8.0 (5.25-10.0) 500.0 (450.0-550.0) 138.0 (77.0-170.8) 106.0 (84.8-172.5) 110.0 (62.0-141.0)
13.0 (12.5-18.0) 16.0 (11.0-17.5) 29.0 (23.5-34.0) 2.0 (1.0-6.5) 625.0 (550.0-687.5) 306.0 (179.5-334.8) 140.0 (126.0-211.0) 211.0 (143.8-250.8)
P Value .383 .959 .442 .019 .003 .021 .118 .022
CBC, cystometric bladder capacity; FS, first sensation; ICPI, interstitial cystitis problem index; ICSI, interstitial cystitis symptom index; MBC, maximal bladder capacity; OSS, O’Leary-Sant symptom scores; US, urge sensation; VAS, visual analog scale.
Table 3 show the immunohistochemical staining and Western blotting results. The bladder specimens with Hunner IC expressed significantly more TUNEL (P = .002) and less E-cadherin (P < .001) than the normal controls. The non-Hunner IC bladder specimens expressed more tryptase (P = .012) than the normal controls. E-cadherin (P = .007), eNOS (P < .001), and M3 (P = .001) expression was lower in the non-Hunner IC than that in normal controls. Among the Hunner IC and non-Hunner IC bladder specimens, the expression of eNOS was significantly higher (P < .001) in the Hunner IC specimens, whereas the E-cadherin (P = .013) expression was significantly lower. However, the clinical parameters, including VAS, CBC, ICSI, and ICPI, did not significantly correlate with the immunohistochemical staining and Western blotting results (all P > .05).
DISCUSSION Recently, many researchers have demonstrated that IC/ BPS encompasses a heterogeneous spectrum of disorders, with different cystoscopic and histopathologic presentations.9 Although the ESSIC classifies IC/BPS patients according to cystoscopic and histopathologic findings, the clinical significance and differences in the pathogenesis of different IC/BPS subtypes are still unclear. There has been a long debate on whether Hunner IC and non-Hunner IC are different phenotypes of the same disease condition or 2 separate disease entities.10 Fall even proposed that the term “interstitial cystitis” should be used only in patients with bladder pain syndrome and Hunner’s lesion.11 However, laboratory evidence to differentiate between Hunner IC and non-Hunner IC remains limited. Our study revealed a decrease in the urothelial adhesion protein E-cadherin 3
ARTICLE IN PRESS Table 2. Histopathology results in Hunner and non-Hunner IC bladder Inflammation Hunner IC Non-Hunner IC
Eosinophil infiltration
Urothelium denudation
None
Mild
Moderate
Severe
None
Mild
Moderate
Severe
None
Mild
Moderate
Severe
0 9
10 5
3 0
1 0
7 14
2 0
2 0
3 0
0 8
9 6
2 0
3 0
IC, interstitial cystitis.
Figure 1. Immunohistochemical staining of the urothelium in Hunner IC and non-Hunner IC revealed that the urothelium of Hunner IC showed a greater defect of E-cadherin than the non-Hunner IC did. BPS, bladder pain, syndrome; IC, interstitial cystitis; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; ZO-1, zonula occludens protein 1. (Color version available online.)
and greater expression of eNOS in Hunner IC specimens than in non-Hunner IC specimens. The urothelium specimens with Hunner IC also had more severe inflammation and eosinophil infiltration than the non-Hunner IC specimens did. Although the term IC indicates inflammation in the urinary bladder, whether all IC/BPS bladders present with inflammation is still controversial. The ESSIC criteria include IC/BPS patients without inflammation in their bladder biopsy findings.1 Previous studies report that bladder biopsy characteristics are associated with symptoms in IC/ BPS patients.12,13 A recent study revealed many different histopathological features of bladders with Hunner IC and 4
non-Hunner IC, in which bladders with Hunner IC had more epithelial denudation, subepithelial inflammation, and neutrophil and eosinophil infiltration.5 Our current study shows similar histopathological findings, but the immunohistochemical staining of tryptase did not differ between Hunner IC and non-Hunner IC specimens. This result suggests that mast cell activation exists in the urothelium of non-Hunner IC, even though there is no histopathological evidence of inflammation. Our previous study also showed increased apoptosis and decreased E-cadherin in the urothelium of IC/BPS bladders.8 In the current study, we found less expression of E-cadherin in the Hunner IC than in the non-Hunner IC bladder specimens. This finding UROLOGY ■■ (■■), 2016
ARTICLE IN PRESS Table 3. Functional protein and sensory receptor expression in Hunner and non-Hunner IC/BPS Group 1 Normal Control (N = 10)
Group 2 Hunner IC (N = 14)
Group 3 Non-Hunner IC (N = 14)
33.63 (18.97-41.15)
8.64 (4.96-20.0)
23.34 (15.27-27.14)
<.001
ZO-1
5.31 (4.75-7.25)
4.25 (0.00-7.51)
3.63 (0.00-6.96)
.303
Tryptase
5.45 (2.99-8.18)
5.04 (2.32-11.9)
9.94 (5.88-17.11)
.025
TUNEL
0.39 (0.00-0.68)
3.57 (0.91-7.42)
1.86 (0.00-3.38)
.005
M2
1.17 (0.58-1.75)
0.55 (0.22-1.00)
1.09 (0.77-1.38)
.082
M3
1.43 (0.72-2.42)
0.62 (0.33-1.11)
0.60 (0.32-0.90)
.004
P2X3
1.57 (0.63-2.26)
1.08 (0.43-1.59)
1.46 (0.72-3.23)
.161
β3-AR
0.47 (0.22-0.94)
0.48 (0.34-0.64)
0.76 (0.25-0.99)
.469
eNOS
0.23 (0.16-0.40)
0.40 (0.30-0.50)
0.05 (0.02-.014)
<.001
E-cadherin
P Value
P Value Among Groups 1vs 2: <.001 1 vs 3: .007 2 vs 3: .013 1 vs 2: .484 1 vs 3: .102 2 vs 3: .944 1 vs 2: .944 1 vs 3: .012 2 vs 3: .048 1 vs 2: .002 1 vs 3: .035 2 vs 3: .127 1 vs 2: .072 1 vs 3: .806 2 vs 3: .028 1 vs 2: .027 1 vs 3: .001 2 vs 3: .619 1 vs 2: .099 1 vs 3: .675 2 vs 3: .091 1 vs 2: .832 1 vs 3: .613 2 vs 3: .143 1vs 2: .089 1 vs 3: <.001 2 vs 3: <.001
β3-AR, beta-3 adrenoceptor; BPS, BPS, bladder pain, syndrome; eNOS, endothelial nitric oxide synthase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; ZO-1, zonula occludens protein 1.
suggests a more severe urothelial cell adhesion defect in Hunner IC than in non-Hunner IC. Previous animal and human-cultured urothelial cell studies revealed increased expression of muscarinic receptors, P2X3, and decreased beta-1 adrenoceptor in IC/BPS bladders.14-16 However, the findings of our current study differed. When compared with normal controls, only the expression of M3 was decreased in both Hunner IC and non-Hunner IC (P = .027 and .001, respectively). The other sensory receptor expressions including M2, P2X3, and β3AR did not differ between normal control and IC/BPS patients. Among Hunner IC and non-Hunner IC patients, the expression of all of the sensory receptors tested did not differ. Further investigation of the pain receptors, such as transient receptor potential cation channel subfamily V member 1, in Hunner IC and non-Hunner IC, is necessary. NO is a signaling molecule that plays a key role in the pathogenesis of inflammation.17 Logadottir et al had reported increased intravesical NO production in Hunner IC than in non-Hunner IC.18 Recently, the inducible NOS was also found to be specifically increased in patients with Hunner IC, and it was suggested that it might be involved in the pathogenesis of the bladder inflammation.19 Current study further revealed decrease of eNOS in the non-Hunner IC patients than in the Hunner IC patients and normal control. In contrast, eNOS expression in urothelium of Hunner IC had a tendency to be higher than UROLOGY ■■ (■■), 2016
that in normal control (P = .089). eNOS is primarily responsible for the generation of NO in the vascular endothelium, and it increases vascular permeability, dysregulates angiogenesis, and impairs wound healing.20-22 It also regulates the expression of the proinflammatory molecules such as nuclear factor-κB and cyclooxygenase-2.23 Hunner’s lesion is characterized by an inflammatory lesion with increase of vascularity and centralized distribution of vessels. We suggest that eNOS expression difference between Hunner and non-Hunner IC might implicate pathogenesis difference in 2 types of IC. The c-Jun N-terminal kinase (JNK) pathway is activated by a variety of environmental stresses and inflammatory cytokines.24 JNK pathway could induce eNOS expression, and recently activated JNK pathway in bladder had been identified in IC/BPS patients.25 JNK inhibitors could also inhibit bladder inflammation in animal study.24 The anti-inflammatory effect of JNK inhibitor may have originated from reducing eNOS expression, and it might be effective in treating human Hunner IC in further studies. Inhibition of NOS could downregulate inflammatory cytokines and reduce inflammation.26 NOS inhibitor such as monomethyl-L-arginine and arginine peptides may be useful in decreasing inflammation in Hunner IC bladder. The main limitation of our study was the small number of patients; thus, the data could be heavily weighted by a limited number of extreme values. Bladder biopsies were taken at sites around the Hunner’s lesion in patients with 5
ARTICLE IN PRESS Hunner IC, whereas biopsies of patients with non-Hunner IC were taken from the bladder randomly, which might also be a source of bias in this study. Further studies investigating the different sensory receptors and inflammatory molecules are needed.
CONCLUSION The pathogeneses of both Hunner IC and non-Hunner IC involve inflammation. However, the inflammation in Hunner IC is more severe. Hunner IC features a more severe urothelial adhesive protein defect and greater expression of eNOS than non-Hunner IC. The different expression of eNOS between Hunner and non-Hunner IC might implicate different pathogenesis in these 2 types of IC. Reduced eNOS expression may be a possible further target in treating Hunner IC.
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