- Email: [email protected]
Pancreatitis-associated Protein in Patients With Interstitial Cystitis
Interstitial Cystitis, Chronic Pelvic Pain, and Infection Elevated Urinary Levels and Urothelial Expression of Hepatocarcinomaintestine-pancreas/Pancreatitis-associated Protein in Patients With Interstitial Cystitis Tetsuya Makino, Hidenori Kawashima, Hiroyuki Konishi, Tatsuya Nakatani, and Hiroshi Kiyama OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
To investigate the pathophysiology of interstitial cystitis (IC), we examined urinary levels and urothelial expression of human orthologue of pancreatitis-associated protein (PAP) III, namely, hepatocarcinoma-intestine-pancreas (HIP)/PAP, in patients with IC. We have previously shown that PAP III is expressed with increased frequency in the bladder urothelium in a rat cystitis model (Takahara Y, et al. J Urol. 2008;179:1603-1609). HIP/PAP levels in the urine from patients with IC were measured by enzyme-linked immunosorbent assay, and HIP/PAP expression in the bladder tissues was assessed by immunohistochemical study. The median concentration of urinary HIP/PAP was significantly higher in patients with IC than in controls (13.67 vs 1.86 pg/mL urine, respectively, P ⬍.0001, with Mann–Whitney U test). HIP/PAP immunoreactivity was observed in the urothelium of 88.2% of patients with IC (15/17) and in 5.9% of bladder tissues of controls (1/17). Urinary HIP/PAP levels in patients with IC were positively correlated with urinary frequency and bladder pain (r ⫽ 0.429, 0.443) and inversely correlated with mean voided urine volume (r ⫽ ⫺0.488). Urinary HIP/PAP levels were significantly higher in IC patients and the apparent HIP/PAP expression in the bladder urothelium was more frequently observed among IC patients. The involvement of HIP/PAP in the pathophysiology of IC is suggested. UROLOGY 75: 933–937, 2010. © 2010 Elsevier Inc.
I
nterstitial cystitis (IC) is a chronic bladder inflammatory disorder characterized by urinary frequency, urgency, and bladder pain without evidence of bacterial cystitis or other lower urinary tract diseases. Although the etiology and pathophysiology of IC are not well understood, some studies recently postulated theories to reveal the pathogenesis of IC. In the urothelium of IC patients, loss of the glycosaminoglycan layer and abnormal expression of proteoglycan core proteins were observed, suggesting that IC involves urothelial abnormalities and dysfunction.1 Decreased urothelial barrier function is thought to cause the IC symptoms.1 Some
This work was supported by grants from Ministry of Health, Labour and Welfare of Japan, MEXT Japan, and Osaka City University. From the Departments of Anatomy and Neurobiology, and Urology, Osaka City University Graduate School of Medicine, Osaka, Japan Reprint requests: Hiroshi Kiyama, Ph.D., Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, 1-4-3 Asahimachi, Abenoku, Osaka 545-8585, Japan. E-mail: [email protected] Submitted: February 27, 2009, accepted (with revisions): May 20, 2009
© 2010 Elsevier Inc. All Rights Reserved
growth factors, cytokines, and other factors that affect the proliferation of epithelial cells are assumed to elicit the pathogenesis of IC.2 A protein called regenerating gene (Reg) was originally identified in regenerating pancreatic islet cells.3 A family of related genes has been identified and described under a rather varied nomenclature such as pancreatitis-associated protein (PAP) and hepatocarcinoma-intestine-pancreas (HIP) and has been broadly categorized into 4 groups, types I-IV.4,5 In humans, 4 genes (RegI-␣, RegI-, HIP/PAP, and RegIV) have been identified6 and HIP/ PAP (also known as RegIII-␣ in humans) belongs to type III. The members of the Reg/HIP/PAP family possess a C-type (calcium-dependent) lectin-like domain, although the function of lectin is unknown.7 Recently, it was assumed that HIP/PAP expression is associated with inflammation because of the following evidence. In acute pancreatitis, HIP/PAP was substantially expressed as a secretory protein.8 In chronic and destructive intestinal inflammatory diseases such as Crohn 0090-4295/10/$34.00 doi:10.1016/j.urology.2009.05.044
933
disease and ulcerative colitis, significant expression of HIP/PAP was observed in human gut epithelial cells.9 In terms of the function of the HIP/PAP family, controversies still remain. HIP/PAP inhibited bacterial proliferation in rat acute pancreatitis10 and PAP had antiapoptosis effects.11 Recently, it was demonstrated that HIP/ PAP played multiple roles as anti-inflammatory12 and antitumorigenesis factors.13 Previously, we have demonstrated in a rat cystitis model induced by cyclophosphamide that PAP III was expressed in the urothelium of the bladder and that another member of the Reg family, PAP I, was expressed in the primary afferent neurons of dorsal root ganglia that innervate the bladder.14 Both PAP I and PAP III belong to the type III Reg and correspond to HIP/PAP in humans. Along with the progression of bladder inflammation, PAP III expression was increased in umbrella cells of the urothelium. It is therefore assumed that umbrella cells might secret PAP III in response to inflammatory stimuli, and PAP III might play an important role as an anti-inflammatory factor in cystitis.14 From this background, we hypothesize that human HIP/PAP is expressed in the bladder of patients with IC. In this study, we measured HIP/PAP levels in the urine and HIP/PAP expression in the bladder tissues of IC patients and analyzed the association between urinary HIP/PAP levels and the severity of lower urinary symptoms in IC patients.
MATERIAL AND METHODS Patients We followed our institution’s ethics guidelines and the study was approved by the ethics committee of the Osaka City University Hospital. All patients provided informed consent. Patients with IC were diagnosed based on the National Institute of Diabetes and Digestive and Kidney Diseases clinical criteria. The 24-hour urinary frequency and mean voided urine volume were assessed by a voiding diary and bladder pain on a verbal 11-point rating scale (VRS). The VRS is a common pain scale for rating a patient’s perceived pain intensity on a numeric scale from 0-10, with 0 representing no pain and 10 representing the worst pain.15 Healthy controls were hospital personnel with no history of any urinary problems such as urinary infections, urinary frequency (⬎8 voids per 24 hours), irritative voiding symptoms, or bladder pain. All controls scored 0 on the VRS.
Enzyme-Linked Immunosorbent Assay for HIP/PAP Clean-catch urinary specimens were obtained from 27 female patients with IC and 27 female controls who were age-matched with IC patients within ⫾5 years; midstream urine was collected in a sterile container in the morning and then kept at 4°C. Specimens were transported to the laboratory and centrifuged at 1500 rpm for 5 minutes. The supernatants were stored at ⫺80°C until analysis of HIP/PAP. Urinary HIP/PAP concentrations were measured by enzyme-linked immunosorbent assay (Dynabio, Marseille, France). Urine was pipetted into wells precoated with anti-HIP/PAP antibody according to the manufacturer’s instructions. After incubation for 3 hours at room temperature, the plates were rinsed with wash buffer. 934
They were then incubated with biotinylated anti-HIP/PAP antibody. The plates were rinsed, incubated with avidin-peroxidase, and developed with tetramethylbenzidine substrate. Development was stopped with sulfuric acid, and absorbance at 450 nm was determined with an automatic plate reader.
Immunohistochemistry for HIP/PAP Biopsies were performed in 17 female IC patients, using cold cup rigid biopsy forceps on the posterior bladder wall before hydrodistension and control bladder tissues were obtained from 17 female controls (in the same way as from IC patients) who were age-matched with IC patients within ⫾5 years. Bladder tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. For hematoxylin-eosin staining and immunohistochemical study, the embedded bladder tissues were sectioned into 4-m-thick slices. They were deparaffinized, treated with 0.3% hydrogen peroxide, and then incubated with the primary goat polyclonal antibody against HIP/PAP (Santa Cruz Biotechnology, CA) in phosphate-buffered saline (PBS) after blocking with 1% bovine serum albumin in PBS. The tissues were rinsed, incubated with rabbit biotinylated anti-goat IgG (Vector Laboratories, CA), and incubated with avidin-biotin horseradish peroxidase complex (Vector Laboratories) in PBS. Staining was developed with 0.2 mg/mL 3,3=-diaminobenzidine tetrahydrochloride (Vector Laboratories). To examine the specificity of the antibody, an adsorption test was carried out using recombinant HIP/PAP proteins produced by yeast.
Statistical Analysis A comparison of the urinary levels between IC patients and controls was performed by the Mann–Whitney U test, with P ⬍.05 being considered statistically significant. A logarithmic transformation was applied to the HIP/PAP level data to normalize the distribution before analysis. The results are given as the median and 25th and 75th percentiles for continuous data. With regard to immunohistochemistry, an individual who was unaware of the identification of patients or controls read all slides. The association between urinary HIP/PAP levels and 24 hours urinary frequency, mean voided urine volume, and bladder pain was analyzed by Spearman correlation coefficient by rank. All statistical tests were two-sided.
RESULTS HIP/PAP Levels in the Urine Urinary specimens were collected from 27 female IC patients (median age: 58 years, range: 31-83 years) and from 27 female controls (median age: 58 years, range: 30-81 years). No samples had bacterial urinary tract infection. The median urinary HIP/PAP concentration was significantly higher in IC patients than in controls (Fig. 1). Immunohistochemical Localization of HIP/PAP in the Bladder Bladder tissues from 17 female IC patients (median age: 58 years, range: 30-82 years) were processed for immunohistochemistry, using an antibody specific to HIP/PAP. Control bladder tissues from 17 females (median age: 59 years, range: 34-83 years) were also examined. Hematoxy lin-eosin staining in the bladder of IC patients showed thinning, denudation, and tearing of the urothelium and UROLOGY 75 (4), 2010
Figure 1. HIP/PAP levels in the urine of IC patients and controls. Urinary HIP/PAP levels were determined by ELISA. Bars represent median levels. Median Urinary HIP/PAP concentration in IC patients (median: 13.67 pg/mL, 25th and 75th percentiles: 5.08 and 46.75) was significantly higher than in controls (median: 1.86 pg/mL, 25th and 75th percentiles: 0.80 and 3.75) (Mann–Whitney U test, P ⬍.0001). *Significantly different (P ⬍.05).
granulation tissue, edema, and even hemorrhaging of the submucosa (Fig. 2A). The prominent HIP/PAP immunoreactivity was mainly observed in the urothelium corresponding to the umbrella cell layer, whereas weak staining was seen in the basal layer (Fig. 2C). The immunoreactivity was completely abolished by the adsorption test using an excess amount of the antigen protein. The apparent positive staining was identified in the urothelium of bladder tissues from 88.2% of IC patients (15/17). The intense HIP/PAP immunoreactivity was particularly found in thinning and torn urothelia. No HIP/PAP immunoreactivity was observed in the submucosa. HIP/PAP immunoreactivity was observed in the urothelium of a bladder tissue from 5.9% of control patients (1/17) (Fig. 2D). Association Between Urinary HIP/PAP Levels and Urinary Symptoms In IC patients, urinary symptoms were examined and evaluated for their association with the urinary level of HIP/PAP. Urinary HIP/PAP levels in IC patients were significantly associated with 24-hour urinary frequency (r ⫽ 0.429, P ⫽ .029) (Fig. 3A). An inverse association between urinary HIP/PAP levels and the mean voided urine volume in IC patients was also observed (r ⫽ ⫺0.488, P ⫽ .013) (Fig. 3B). Significant association between urinary HIP/PAP levels and bladder pain in IC patients was found on a VRS (r ⫽ 0.443, P ⫽ .024) (Fig. 3C).
COMMENT In this study, we have demonstrated that the urinary HIP/PAP levels in IC patients were apparently higher than those in normal controls. Increased immunoreactivity for HIP/PAP was frequently observed in the umbrella UROLOGY 75 (4), 2010
cells of the urothelium in IC patients, suggesting that expression of HIP/PAP is induced in the urothelium and HIP/PAP is released into the urine in IC patients. Intriguingly, the urinary levels of HIP/PAP are correlated with the severity of urinary symptoms, implying that the level of HIP/PAP in urine could be a candidate marker of IC. With regard to the possible function of HIP/PAP in the urothelium, HIP/PAP may have an effect as an antiinflammatory factor. Some previous reports demonstrated the functions of some members of Reg/HIP/PAP family. PAP I suppressed inflammation by inhibiting nuclear factor-kappa B activation through a JAK/STAT3-dependent mechanism,16 and HIP/PAP functioned as an antimicrobial factor by binding to the peptidoglycans of bacterial targets.17 Moreover, PAP III in injured nerves possessed a macrophage chemoattractant activity that contributed to nerve regeneration.18 Those reports suggest that HIP/PAP expressed in the urothelium in IC patients functions as an anti-inflammatory factor, which suppresses inflammatory cytokine expression and bacterial proliferation, and which plays a role in repairing the injured bladder. Therefore, exploring the functional relevance and mechanism of HIP/PAP may provide IC patients with a new therapeutic approach using HIP/PAP as an anti-inflammatory factor. The present study revealed intense HIP/PAP immunoreactivity in the umbrella cell layer in IC patients but not in those in controls, suggesting that umbrella cells in IC patients have a crucial role as an anti-inflammatory mediator. These results coincide well with those in our previous animal study that demonstrated increased PAP III expression in the umbrella cell layer of the bladder urothelium in a rat cystitis model.14 Moreover, we demonstrated that the intense HIP/PAP immunoreactivity is especially observed in abnormal and injured urothelia in bladder tissues in IC patients. Because an increase was observed in PAP III expression along with the progression of bladder inflammation in the rat cystitis model,14 it is suggested that umbrella cells in IC patients produce and release more HIP/PAP as an anti-inflammatory factor along with the progression of IC. Therefore, urinary HIP/PAP levels and HIP/PAP expression in the urothelium in IC patients may represent the extent of the bladder inflammation. HIP/PAP that reflects the progression of IC may contribute to our understanding of the pathophysiology of IC. In an attempt to identify noninvasive diagnostic urine markers for IC, frizzled 8 protein-related antiproliferative factor that inhibits the urothelial cell growth19 and growth factors such as epidermal growth factor (EGF) and heparin-binding EGF-like growth factor, which would control cell proliferation, were proposed to be possible urine biomarkers. The antiproliferative factor activity and EGF levels in the urine of IC patients were higher than those of controls, whereas heparin binding-EGF levels were decreased in IC patients.2,20 We found that in IC patients, urinary HIP/PAP levels were associated with se935
Figure 2. Localization of HIP/PAP in bladder tissues. (A) Hematoxylin-eosin staining in the bladder of a patient with IC. Thinning and denudation of the urothelium and hemorrhaging in the submucosa were observed. (B) Hematoxylin-eosin staining in a control bladder. (C) Positive HIP/PAP immunoreactivity was seen in the urothelium of a patient with IC. Dense staining in the umbrella cell layer and weak staining in the basal cell layer were observed. (D) No HIP/PAP immunoreactivity was observed in the control bladder. Scale bar indicates 50 m.
Figure 3. Associations between urinary HIP/PAP levels and urinary symptoms in 27 patients with interstitial cystitis. (A) An association between urinary HIP/PAP levels and 24-hour urinary frequency (median: 15.0, 25th and 75th percentiles: 12.3 and 16.8) was observed (Spearman correlation, r ⫽ 0.429 and P ⫽ .029). (B) An inverse association between urinary HIP/PAP levels and the mean voided urine volume (median: 140 mL, 25th and 75th percentiles: 110 and 185) was found (r ⫽ ⫺0.488, P ⫽ .013). (C) Urinary HIP/PAP levels were associated with bladder pain on a 0-10 VRS (median: 3.0, 25th and 75th percentiles: 2.0 and 5.8) (r ⫽ 0.443, P ⫽ .024).
verity of urinary symptoms such as urinary frequency, voided urine volume, and bladder pain. The hypothesis that HIP/PAP represents the progression of the cystitis 936
supports this finding. Thus, HIP/PAP is one of candidate markers for the diagnosis of IC and also for estimating the severity of urinary symptoms. UROLOGY 75 (4), 2010
CONCLUSIONS We revealed that HIP/PAP levels in the urine were significantly higher in IC patients than in controls, and HIP/PAP expression in the urothelium was frequently observed in IC patients. Because we found a significant association between urinary HIP/PAP levels and urinary symptoms in IC patients, it is concluded that HIP/PAP may be involved in the pathogenesis of IC. Acknowledgments. J.L. Iovanna, INSERM U.624, Marseille, France, provided information about PAP enzyme-linked immunosorbent assay system. References 1. Hauser PJ, Dozmorov MG, Bane BL, et al. Abnormal expression of differentiation related proteins and proteoglycan core proteins in the urothelium of patients with interstitial cystitis. J Urol. 2008; 179:764-769. 2. Erickson DR, Xie SX, Bhavanandan VP, et al. A comparison of multiple urine markers for interstitial cystitis. J Urol. 2002;167: 2461-2469. 3. Terazono K, Yamamoto H, Takasawa S, et al. A novel gene activated in regenerating islets. J Biol Chem. 1988;263:2111-2114. 4. Okamoto H. The Reg gene family and Reg proteins: with special attention to the regeneration of pancreatic beta-cells. J Hepato Biliary Pancreat Surg. 1999;6:254-262. 5. Namikawa K, Fukushima M, Murakami K, et al. Expression of Reg/PAP family members during motor nerve regeneration in rat. Biochem Biophys Res Commun. 2005;332:126-134. 6. Nata K, Liu Y, Xu L, et al. Molecular cloning, expression and chromosomal localization of a novel human REG family gene, Reg III. Gene. 2004;340:161-170. 7. Lasserre C, Christa L, Simon MT, et al. A novel gene (HIP) activated in human primary liver cancer. Cancer Res. 1992;52: 5089-5095. 8. Iovanna JL, Keim V, Nordback I, et al., for Multicentric Study Group on Acute Pancreatitis. Serum levels of pancreatitis-associated protein as indicators of the course of acute pancreatitis. Gastroenterology. 1994;106:728-734.
UROLOGY 75 (4), 2010
9. Ogawa H, Fukushima K, Naito H, et al. Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model. Inflamm Bowel Dis. 2003;9:162-170. 10. Iovanna J, Orelle B, Keim V, et al. Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis. J Biol Chem. 1991;266:24664-24669. 11. Malka D, Vasseur S, Bodeker H, et al. Tumor necrosis factor alpha triggers antiapoptotic mechanisms in rat pancreatic cells through pancreatitis-associated protein I activation. Gastroenterology. 2000; 119:816-828. 12. Gironella M, Iovanna JL, Sans M, et al. Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease. Gut. 2005;54:1244-1253. 13. Choi B, Suh Y, Kim WH, et al. Downregulation of regenerating islet-derived 3 alpha (REG3A) in primary human gastric adenocarcinomas. Exp Mol Med. 2007;39:796-804. 14. Takahara Y, Suzuki A, Maeda M, et al. Expression of pancreatitis associated proteins in urothelium and urinary afferent neurons following cyclophosphamide induced cystitis. J Urol. 2008;179: 1603-1609. 15. Morris NR, Sabapathy S, Adams L, et al. Verbal numerical scales are as reliable and sensitive as visual analog scales for rating dyspnea in young and older subjects. Respir Physiol Neurobiol. 2007; 157:360-365. 16. Folch-Puy E, Granell S, Dagorn JC, et al. Pancreatitis-associated protein I suppresses NF-kappa B activation through a JAK/STATmediated mechanism in epithelial cells. J Immunol. 2006;176:37743779. 17. Cash HL, Whitham CV, Behrendt CL, et al. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science. 2006; 313:1126-1130. 18. Namikawa K, Okamoto T, Suzuki A, et al. Pancreatitis-associated protein-III is a novel macrophage chemoattractant implicated in nerve regeneration. J Neurosci. 2006;26:7460-7467. 19. Keay SK, Szekely Z, Conrads TP, et al. An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide. Proc Natl Acad Sci USA. 2004;101:11803-11808. 20. Zhang CO, Li ZL, Kong CZ. APF, HB-EGF, and EGF biomarkers in patients with ulcerative vs. non-ulcerative interstitial cystitis. BMC Urol. 2005;5:7.
937
METHODS
RESULTS
CONCLUSIONS
To investigate the pathophysiology of interstitial cystitis (IC), we examined urinary levels and urothelial expression of human orthologue of pancreatitis-associated protein (PAP) III, namely, hepatocarcinoma-intestine-pancreas (HIP)/PAP, in patients with IC. We have previously shown that PAP III is expressed with increased frequency in the bladder urothelium in a rat cystitis model (Takahara Y, et al. J Urol. 2008;179:1603-1609). HIP/PAP levels in the urine from patients with IC were measured by enzyme-linked immunosorbent assay, and HIP/PAP expression in the bladder tissues was assessed by immunohistochemical study. The median concentration of urinary HIP/PAP was significantly higher in patients with IC than in controls (13.67 vs 1.86 pg/mL urine, respectively, P ⬍.0001, with Mann–Whitney U test). HIP/PAP immunoreactivity was observed in the urothelium of 88.2% of patients with IC (15/17) and in 5.9% of bladder tissues of controls (1/17). Urinary HIP/PAP levels in patients with IC were positively correlated with urinary frequency and bladder pain (r ⫽ 0.429, 0.443) and inversely correlated with mean voided urine volume (r ⫽ ⫺0.488). Urinary HIP/PAP levels were significantly higher in IC patients and the apparent HIP/PAP expression in the bladder urothelium was more frequently observed among IC patients. The involvement of HIP/PAP in the pathophysiology of IC is suggested. UROLOGY 75: 933–937, 2010. © 2010 Elsevier Inc.
I
nterstitial cystitis (IC) is a chronic bladder inflammatory disorder characterized by urinary frequency, urgency, and bladder pain without evidence of bacterial cystitis or other lower urinary tract diseases. Although the etiology and pathophysiology of IC are not well understood, some studies recently postulated theories to reveal the pathogenesis of IC. In the urothelium of IC patients, loss of the glycosaminoglycan layer and abnormal expression of proteoglycan core proteins were observed, suggesting that IC involves urothelial abnormalities and dysfunction.1 Decreased urothelial barrier function is thought to cause the IC symptoms.1 Some
This work was supported by grants from Ministry of Health, Labour and Welfare of Japan, MEXT Japan, and Osaka City University. From the Departments of Anatomy and Neurobiology, and Urology, Osaka City University Graduate School of Medicine, Osaka, Japan Reprint requests: Hiroshi Kiyama, Ph.D., Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, 1-4-3 Asahimachi, Abenoku, Osaka 545-8585, Japan. E-mail: [email protected] Submitted: February 27, 2009, accepted (with revisions): May 20, 2009
© 2010 Elsevier Inc. All Rights Reserved
growth factors, cytokines, and other factors that affect the proliferation of epithelial cells are assumed to elicit the pathogenesis of IC.2 A protein called regenerating gene (Reg) was originally identified in regenerating pancreatic islet cells.3 A family of related genes has been identified and described under a rather varied nomenclature such as pancreatitis-associated protein (PAP) and hepatocarcinoma-intestine-pancreas (HIP) and has been broadly categorized into 4 groups, types I-IV.4,5 In humans, 4 genes (RegI-␣, RegI-, HIP/PAP, and RegIV) have been identified6 and HIP/ PAP (also known as RegIII-␣ in humans) belongs to type III. The members of the Reg/HIP/PAP family possess a C-type (calcium-dependent) lectin-like domain, although the function of lectin is unknown.7 Recently, it was assumed that HIP/PAP expression is associated with inflammation because of the following evidence. In acute pancreatitis, HIP/PAP was substantially expressed as a secretory protein.8 In chronic and destructive intestinal inflammatory diseases such as Crohn 0090-4295/10/$34.00 doi:10.1016/j.urology.2009.05.044
933
disease and ulcerative colitis, significant expression of HIP/PAP was observed in human gut epithelial cells.9 In terms of the function of the HIP/PAP family, controversies still remain. HIP/PAP inhibited bacterial proliferation in rat acute pancreatitis10 and PAP had antiapoptosis effects.11 Recently, it was demonstrated that HIP/ PAP played multiple roles as anti-inflammatory12 and antitumorigenesis factors.13 Previously, we have demonstrated in a rat cystitis model induced by cyclophosphamide that PAP III was expressed in the urothelium of the bladder and that another member of the Reg family, PAP I, was expressed in the primary afferent neurons of dorsal root ganglia that innervate the bladder.14 Both PAP I and PAP III belong to the type III Reg and correspond to HIP/PAP in humans. Along with the progression of bladder inflammation, PAP III expression was increased in umbrella cells of the urothelium. It is therefore assumed that umbrella cells might secret PAP III in response to inflammatory stimuli, and PAP III might play an important role as an anti-inflammatory factor in cystitis.14 From this background, we hypothesize that human HIP/PAP is expressed in the bladder of patients with IC. In this study, we measured HIP/PAP levels in the urine and HIP/PAP expression in the bladder tissues of IC patients and analyzed the association between urinary HIP/PAP levels and the severity of lower urinary symptoms in IC patients.
MATERIAL AND METHODS Patients We followed our institution’s ethics guidelines and the study was approved by the ethics committee of the Osaka City University Hospital. All patients provided informed consent. Patients with IC were diagnosed based on the National Institute of Diabetes and Digestive and Kidney Diseases clinical criteria. The 24-hour urinary frequency and mean voided urine volume were assessed by a voiding diary and bladder pain on a verbal 11-point rating scale (VRS). The VRS is a common pain scale for rating a patient’s perceived pain intensity on a numeric scale from 0-10, with 0 representing no pain and 10 representing the worst pain.15 Healthy controls were hospital personnel with no history of any urinary problems such as urinary infections, urinary frequency (⬎8 voids per 24 hours), irritative voiding symptoms, or bladder pain. All controls scored 0 on the VRS.
Enzyme-Linked Immunosorbent Assay for HIP/PAP Clean-catch urinary specimens were obtained from 27 female patients with IC and 27 female controls who were age-matched with IC patients within ⫾5 years; midstream urine was collected in a sterile container in the morning and then kept at 4°C. Specimens were transported to the laboratory and centrifuged at 1500 rpm for 5 minutes. The supernatants were stored at ⫺80°C until analysis of HIP/PAP. Urinary HIP/PAP concentrations were measured by enzyme-linked immunosorbent assay (Dynabio, Marseille, France). Urine was pipetted into wells precoated with anti-HIP/PAP antibody according to the manufacturer’s instructions. After incubation for 3 hours at room temperature, the plates were rinsed with wash buffer. 934
They were then incubated with biotinylated anti-HIP/PAP antibody. The plates were rinsed, incubated with avidin-peroxidase, and developed with tetramethylbenzidine substrate. Development was stopped with sulfuric acid, and absorbance at 450 nm was determined with an automatic plate reader.
Immunohistochemistry for HIP/PAP Biopsies were performed in 17 female IC patients, using cold cup rigid biopsy forceps on the posterior bladder wall before hydrodistension and control bladder tissues were obtained from 17 female controls (in the same way as from IC patients) who were age-matched with IC patients within ⫾5 years. Bladder tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. For hematoxylin-eosin staining and immunohistochemical study, the embedded bladder tissues were sectioned into 4-m-thick slices. They were deparaffinized, treated with 0.3% hydrogen peroxide, and then incubated with the primary goat polyclonal antibody against HIP/PAP (Santa Cruz Biotechnology, CA) in phosphate-buffered saline (PBS) after blocking with 1% bovine serum albumin in PBS. The tissues were rinsed, incubated with rabbit biotinylated anti-goat IgG (Vector Laboratories, CA), and incubated with avidin-biotin horseradish peroxidase complex (Vector Laboratories) in PBS. Staining was developed with 0.2 mg/mL 3,3=-diaminobenzidine tetrahydrochloride (Vector Laboratories). To examine the specificity of the antibody, an adsorption test was carried out using recombinant HIP/PAP proteins produced by yeast.
Statistical Analysis A comparison of the urinary levels between IC patients and controls was performed by the Mann–Whitney U test, with P ⬍.05 being considered statistically significant. A logarithmic transformation was applied to the HIP/PAP level data to normalize the distribution before analysis. The results are given as the median and 25th and 75th percentiles for continuous data. With regard to immunohistochemistry, an individual who was unaware of the identification of patients or controls read all slides. The association between urinary HIP/PAP levels and 24 hours urinary frequency, mean voided urine volume, and bladder pain was analyzed by Spearman correlation coefficient by rank. All statistical tests were two-sided.
RESULTS HIP/PAP Levels in the Urine Urinary specimens were collected from 27 female IC patients (median age: 58 years, range: 31-83 years) and from 27 female controls (median age: 58 years, range: 30-81 years). No samples had bacterial urinary tract infection. The median urinary HIP/PAP concentration was significantly higher in IC patients than in controls (Fig. 1). Immunohistochemical Localization of HIP/PAP in the Bladder Bladder tissues from 17 female IC patients (median age: 58 years, range: 30-82 years) were processed for immunohistochemistry, using an antibody specific to HIP/PAP. Control bladder tissues from 17 females (median age: 59 years, range: 34-83 years) were also examined. Hematoxy lin-eosin staining in the bladder of IC patients showed thinning, denudation, and tearing of the urothelium and UROLOGY 75 (4), 2010
Figure 1. HIP/PAP levels in the urine of IC patients and controls. Urinary HIP/PAP levels were determined by ELISA. Bars represent median levels. Median Urinary HIP/PAP concentration in IC patients (median: 13.67 pg/mL, 25th and 75th percentiles: 5.08 and 46.75) was significantly higher than in controls (median: 1.86 pg/mL, 25th and 75th percentiles: 0.80 and 3.75) (Mann–Whitney U test, P ⬍.0001). *Significantly different (P ⬍.05).
granulation tissue, edema, and even hemorrhaging of the submucosa (Fig. 2A). The prominent HIP/PAP immunoreactivity was mainly observed in the urothelium corresponding to the umbrella cell layer, whereas weak staining was seen in the basal layer (Fig. 2C). The immunoreactivity was completely abolished by the adsorption test using an excess amount of the antigen protein. The apparent positive staining was identified in the urothelium of bladder tissues from 88.2% of IC patients (15/17). The intense HIP/PAP immunoreactivity was particularly found in thinning and torn urothelia. No HIP/PAP immunoreactivity was observed in the submucosa. HIP/PAP immunoreactivity was observed in the urothelium of a bladder tissue from 5.9% of control patients (1/17) (Fig. 2D). Association Between Urinary HIP/PAP Levels and Urinary Symptoms In IC patients, urinary symptoms were examined and evaluated for their association with the urinary level of HIP/PAP. Urinary HIP/PAP levels in IC patients were significantly associated with 24-hour urinary frequency (r ⫽ 0.429, P ⫽ .029) (Fig. 3A). An inverse association between urinary HIP/PAP levels and the mean voided urine volume in IC patients was also observed (r ⫽ ⫺0.488, P ⫽ .013) (Fig. 3B). Significant association between urinary HIP/PAP levels and bladder pain in IC patients was found on a VRS (r ⫽ 0.443, P ⫽ .024) (Fig. 3C).
COMMENT In this study, we have demonstrated that the urinary HIP/PAP levels in IC patients were apparently higher than those in normal controls. Increased immunoreactivity for HIP/PAP was frequently observed in the umbrella UROLOGY 75 (4), 2010
cells of the urothelium in IC patients, suggesting that expression of HIP/PAP is induced in the urothelium and HIP/PAP is released into the urine in IC patients. Intriguingly, the urinary levels of HIP/PAP are correlated with the severity of urinary symptoms, implying that the level of HIP/PAP in urine could be a candidate marker of IC. With regard to the possible function of HIP/PAP in the urothelium, HIP/PAP may have an effect as an antiinflammatory factor. Some previous reports demonstrated the functions of some members of Reg/HIP/PAP family. PAP I suppressed inflammation by inhibiting nuclear factor-kappa B activation through a JAK/STAT3-dependent mechanism,16 and HIP/PAP functioned as an antimicrobial factor by binding to the peptidoglycans of bacterial targets.17 Moreover, PAP III in injured nerves possessed a macrophage chemoattractant activity that contributed to nerve regeneration.18 Those reports suggest that HIP/PAP expressed in the urothelium in IC patients functions as an anti-inflammatory factor, which suppresses inflammatory cytokine expression and bacterial proliferation, and which plays a role in repairing the injured bladder. Therefore, exploring the functional relevance and mechanism of HIP/PAP may provide IC patients with a new therapeutic approach using HIP/PAP as an anti-inflammatory factor. The present study revealed intense HIP/PAP immunoreactivity in the umbrella cell layer in IC patients but not in those in controls, suggesting that umbrella cells in IC patients have a crucial role as an anti-inflammatory mediator. These results coincide well with those in our previous animal study that demonstrated increased PAP III expression in the umbrella cell layer of the bladder urothelium in a rat cystitis model.14 Moreover, we demonstrated that the intense HIP/PAP immunoreactivity is especially observed in abnormal and injured urothelia in bladder tissues in IC patients. Because an increase was observed in PAP III expression along with the progression of bladder inflammation in the rat cystitis model,14 it is suggested that umbrella cells in IC patients produce and release more HIP/PAP as an anti-inflammatory factor along with the progression of IC. Therefore, urinary HIP/PAP levels and HIP/PAP expression in the urothelium in IC patients may represent the extent of the bladder inflammation. HIP/PAP that reflects the progression of IC may contribute to our understanding of the pathophysiology of IC. In an attempt to identify noninvasive diagnostic urine markers for IC, frizzled 8 protein-related antiproliferative factor that inhibits the urothelial cell growth19 and growth factors such as epidermal growth factor (EGF) and heparin-binding EGF-like growth factor, which would control cell proliferation, were proposed to be possible urine biomarkers. The antiproliferative factor activity and EGF levels in the urine of IC patients were higher than those of controls, whereas heparin binding-EGF levels were decreased in IC patients.2,20 We found that in IC patients, urinary HIP/PAP levels were associated with se935
Figure 2. Localization of HIP/PAP in bladder tissues. (A) Hematoxylin-eosin staining in the bladder of a patient with IC. Thinning and denudation of the urothelium and hemorrhaging in the submucosa were observed. (B) Hematoxylin-eosin staining in a control bladder. (C) Positive HIP/PAP immunoreactivity was seen in the urothelium of a patient with IC. Dense staining in the umbrella cell layer and weak staining in the basal cell layer were observed. (D) No HIP/PAP immunoreactivity was observed in the control bladder. Scale bar indicates 50 m.
Figure 3. Associations between urinary HIP/PAP levels and urinary symptoms in 27 patients with interstitial cystitis. (A) An association between urinary HIP/PAP levels and 24-hour urinary frequency (median: 15.0, 25th and 75th percentiles: 12.3 and 16.8) was observed (Spearman correlation, r ⫽ 0.429 and P ⫽ .029). (B) An inverse association between urinary HIP/PAP levels and the mean voided urine volume (median: 140 mL, 25th and 75th percentiles: 110 and 185) was found (r ⫽ ⫺0.488, P ⫽ .013). (C) Urinary HIP/PAP levels were associated with bladder pain on a 0-10 VRS (median: 3.0, 25th and 75th percentiles: 2.0 and 5.8) (r ⫽ 0.443, P ⫽ .024).
verity of urinary symptoms such as urinary frequency, voided urine volume, and bladder pain. The hypothesis that HIP/PAP represents the progression of the cystitis 936
supports this finding. Thus, HIP/PAP is one of candidate markers for the diagnosis of IC and also for estimating the severity of urinary symptoms. UROLOGY 75 (4), 2010
CONCLUSIONS We revealed that HIP/PAP levels in the urine were significantly higher in IC patients than in controls, and HIP/PAP expression in the urothelium was frequently observed in IC patients. Because we found a significant association between urinary HIP/PAP levels and urinary symptoms in IC patients, it is concluded that HIP/PAP may be involved in the pathogenesis of IC. Acknowledgments. J.L. Iovanna, INSERM U.624, Marseille, France, provided information about PAP enzyme-linked immunosorbent assay system. References 1. Hauser PJ, Dozmorov MG, Bane BL, et al. Abnormal expression of differentiation related proteins and proteoglycan core proteins in the urothelium of patients with interstitial cystitis. J Urol. 2008; 179:764-769. 2. Erickson DR, Xie SX, Bhavanandan VP, et al. A comparison of multiple urine markers for interstitial cystitis. J Urol. 2002;167: 2461-2469. 3. Terazono K, Yamamoto H, Takasawa S, et al. A novel gene activated in regenerating islets. J Biol Chem. 1988;263:2111-2114. 4. Okamoto H. The Reg gene family and Reg proteins: with special attention to the regeneration of pancreatic beta-cells. J Hepato Biliary Pancreat Surg. 1999;6:254-262. 5. Namikawa K, Fukushima M, Murakami K, et al. Expression of Reg/PAP family members during motor nerve regeneration in rat. Biochem Biophys Res Commun. 2005;332:126-134. 6. Nata K, Liu Y, Xu L, et al. Molecular cloning, expression and chromosomal localization of a novel human REG family gene, Reg III. Gene. 2004;340:161-170. 7. Lasserre C, Christa L, Simon MT, et al. A novel gene (HIP) activated in human primary liver cancer. Cancer Res. 1992;52: 5089-5095. 8. Iovanna JL, Keim V, Nordback I, et al., for Multicentric Study Group on Acute Pancreatitis. Serum levels of pancreatitis-associated protein as indicators of the course of acute pancreatitis. Gastroenterology. 1994;106:728-734.
UROLOGY 75 (4), 2010
9. Ogawa H, Fukushima K, Naito H, et al. Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model. Inflamm Bowel Dis. 2003;9:162-170. 10. Iovanna J, Orelle B, Keim V, et al. Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis. J Biol Chem. 1991;266:24664-24669. 11. Malka D, Vasseur S, Bodeker H, et al. Tumor necrosis factor alpha triggers antiapoptotic mechanisms in rat pancreatic cells through pancreatitis-associated protein I activation. Gastroenterology. 2000; 119:816-828. 12. Gironella M, Iovanna JL, Sans M, et al. Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease. Gut. 2005;54:1244-1253. 13. Choi B, Suh Y, Kim WH, et al. Downregulation of regenerating islet-derived 3 alpha (REG3A) in primary human gastric adenocarcinomas. Exp Mol Med. 2007;39:796-804. 14. Takahara Y, Suzuki A, Maeda M, et al. Expression of pancreatitis associated proteins in urothelium and urinary afferent neurons following cyclophosphamide induced cystitis. J Urol. 2008;179: 1603-1609. 15. Morris NR, Sabapathy S, Adams L, et al. Verbal numerical scales are as reliable and sensitive as visual analog scales for rating dyspnea in young and older subjects. Respir Physiol Neurobiol. 2007; 157:360-365. 16. Folch-Puy E, Granell S, Dagorn JC, et al. Pancreatitis-associated protein I suppresses NF-kappa B activation through a JAK/STATmediated mechanism in epithelial cells. J Immunol. 2006;176:37743779. 17. Cash HL, Whitham CV, Behrendt CL, et al. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science. 2006; 313:1126-1130. 18. Namikawa K, Okamoto T, Suzuki A, et al. Pancreatitis-associated protein-III is a novel macrophage chemoattractant implicated in nerve regeneration. J Neurosci. 2006;26:7460-7467. 19. Keay SK, Szekely Z, Conrads TP, et al. An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide. Proc Natl Acad Sci USA. 2004;101:11803-11808. 20. Zhang CO, Li ZL, Kong CZ. APF, HB-EGF, and EGF biomarkers in patients with ulcerative vs. non-ulcerative interstitial cystitis. BMC Urol. 2005;5:7.
937