The role of TSG-6 and uroplakin III in bladder pain syndrome/ interstitial cystitis in rats and humans.

OBJECTIVES: We investigated the relationship between the expression of tumor necrosis factor-inducible gene 6 (TSG-6) with inflammation and integrity of the bladder epithelium in the bladder tissues of patients with bladder pain syndrome/interstitial cystitis (BPS/IC) and the mechanism of action using a rat model of BPS/IC. MATERIALS AND METHODS: Expression of TSG-6 and uroplakin III was determined by immuno- histochemistry of bladder biopsy samples from control human subjects and patients with verified BPS/IC. Our rat model of BPS/IC was employed to measure the perfusion of bladders with hyaluronidase, and assessment of the effect of TSG-6 administration on disease progression. Treatment effects were assessed by measurement of metabolic characteristics, RT-PCR of TGR-6 and interleukin-6, bladder histomorphology, and immunohistochemistry of TGR-6 and uroplakin III. RESULTS: The bladders of patients with BPS/IC had lower expression of uroplakin III and higher expression of TSG-6 than controls. Rats treated with hyaluronidase for 1 week developed the typical signs and symptoms of BPS/IC, and rats treated with hyaluronidase for 4 weeks had more serious disease. Administration of TSG-6 reversed the effects of hyaluronidase and protected against disease progression. CONCLUSION: Our results indicate that TSG-6 plays an important role in maintaining the integrity of the bladder epithelial barrier.

Intravesical hyaluronidase causes chronic cystitis in a rat model: a potential model of bladder pain syndrome/interstitial cystitis.

OBJECTIVES: To determine whether a potential rat model of bladder pain syndrome could be developed through long-term intermittent intravesical hyaluronidase. METHODS: A total of 64 female Sprague-Dawley rats were divided into a control group, a low-dose hyaluronidase (1 mg/mL) group, a high-dose hyaluronidase (4 mg/mL) group and a hyaluronic acid-treated group. Hyaluronidase was given intravesically three times a week for 1 month. Hyaluronic acid (0.5 mL, 0.8 mg/mL) was introduced intravesically to hyaluronidase-treated rats' bladders. Histological changes, cystometry, nociceptive behaviors, and messenger ribonucleic acid levels of inflammatory factors were evaluated and compared between groups. RESULTS: All hyaluronidase-treated rats showed chronic inflammation and fibrosis, increased and activated mast cells, thinned bladder epithelium with abnormal expressions of uroplakin III and zonula occluden-1, and increased levels of interleukin-6 and intercellular adhesion molecule-1 messenger ribonucleic acid. However, the inflammatory score and levels of interleukin-6 and intercellular adhesion molecule-1 were more significant in the high-dose hyaluronidase group than in the low-dose hyaluronidase group (P < 0.01). Furthermore, hyaluronidase-treated rats showed markedly decreased intercontraction intervals, bladder capacity and increased sensitivity to pain compared with controls (P < 0.01). Hyaluronic acid treatment significantly decreased the inflammatory level, number of mast cells, sensitivity to pain, levels of interleukin-6 and intercellular adhesion molecule-1, and increased intercontraction intervals and bladder capacity (P < 0.01). CONCLUSIONS: Long-term intermittent intravesical hyaluronidase could develop a severe chronic cystitis with diffused fibrosis accompanied by altered histology and bladder function. This chronic cystitis rat model can resemble the clinical and histopathological features of human bladder pain syndrome, and might be a potential valuable model for investigation of this troublesome disease.

Intercellular transfer of P-glycoprotein from the drug resistant human bladder cancer cell line BIU-87 does not require cell-to-cell contact.

PURPOSE: The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. MATERIALS AND METHODS: Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. RESULTS: The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. CONCLUSIONS: Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process.

Expression and functional role of Cdx2 in intestinal metaplasia of cystitis glandularis.

PURPOSE: Cdx2 is an essential transcription factor in intestinal epithelial cell differentiation and proliferation. However, to our knowledge the expression and role of Cdx2 in the development of intestinal cystitis glandularis, a metaplastic lesion induced by chronic inflammation, remained to be explored. MATERIALS AND METHODS: Real-time polymerase chain reaction was used to examine Cdx2, LI-cadherin and villin expression in typical and intestinal cystitis glandularis, and normal bladder tissue. Cdx2 cDNA was subcloned to the retroviral vector pLNCX2 for subsequent transfection into human bladder urothelium cells and rat bladder urothelium. Cdx2 mRNA and protein levels, and cell morphology and proliferation were assessed after transfection using real-time polymerase chain reaction, phase contrast microscopy, transmission electron microscopy and MTT assay, respectively. RESULTS: Higher mRNA levels of Cdx2, villin and LI-cadherin were detected in intestinal cystitis glandularis compared to normal bladder and typical cystitis glandularis. Only Cdx2 groups attained statistical significance (p <0.001). Retroviral over expression of Cdx2 resulted in increased mRNA and protein expression of Cdx2 as well as villin and LI-cadherin levels, and increased cell proliferation. A distinct change in cellular morphology, in which cells resembled intestinal-like cells, was also observed in vitro and in vivo. CONCLUSIONS: Cdx2 may have a critical role in regulating intestinal metaplasia in cystitis glandularis. Further studies are planned to assess the potential of using Cdx2 as a marker and therapeutic target for cystitis glandularis.

Interleukin-6 levels in female rats with protamine sulfate-induced chronic cystitis treated with hyaluronic acid.

OBJECTIVES: To measure interleukin-6 levels in a protamine sulfate-induced chronic cystitis rat model treated with hyaluronic acid, and to study the correlation among interleukin-6, bladder inflammatory degree and voiding frequency. METHODS: A chronic cystitis model was created in female rats by using long-term intermittent intravesical protamine sulfate (0.5 mL, 30 mg/mL). Then, hyaluronic acid (0.5 mL, 0.8 mg/mL) was also instilled intravesically in the rats. Interleukin-6 levels were analyzed with immunohistochemistry, real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was carried out to examine bladder inflammatory degree based on a four-point scoring system (from 0 - none to 3 - severe). Voiding patterns were investigated by cystometrography. RESULTS: According to cystometrography, protamine sulfate-induced rats had significantly shorter intercontraction intervals and less bladder capacity (P < 0.001). The bladder tissue of the rats showed severe chronic inflammation. Immunohistochemistry, reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay showed significantly higher expression of interleukin-6 (P < 0.001). After intravesical administration of hyaluronic acid, both intercontraction intervals and bladder capacity increased significantly (P < 0.001), whereas both bladder inflammatory degree and interleukin-6 levels decreased significantly (P < 0.001). Furthermore, there was a strong correlation between interleukin-6 levels and inflammatory degree (r = 0.727, P < 0.001), and also between interleukin-6 levels and voiding frequency (r = -0.761, P < 0.001). CONCLUSIONS: Intravesical administration of hyaluronic acid decreases interleukin-6 levels, as well as the severity of bladder inflammation and voiding frequency in a rat model of chronic cystitis. Interleukin-6 levels closely correlate with the inflammatory degree and voiding frequency. Thus, they can be regarded as an assessment measure of therapeutic impact.

Intravesical hyaluronic acid and alkalinized lidocaine for the treatment of severe painful bladder syndrome/interstitial cystitis.

INTRODUCTION AND HYPOTHESIS: Intravesical instillation of hyaluronic acid (HA) may restore the integrity of glycosaminoglycan layer in patients with painful bladder syndrome/interstitial cystitis (PBS/IC), and the benefit may be improved with addition of alkalinized lidocaine (AL). METHODS: 48 women with severe PBS/IC who failed oral medications were enrolled and divided into one trial and two control groups. The trial group received intravesical 40 mg HA, 10 ml of 2 % lidocaine and 5 ml of 8.4 % sodium bicarbonate on a weekly basis for 8 weeks and then monthly for 4 months with a subsequent follow-up of 24 weeks, while the two control groups received 40 mg HA and mixture of 10 ml of 2 % lidocaine and 5 ml of 8.4%sodium bicarbonate respectively following the same procedure. Response to therapy was evaluated by Global Response Assessment, voids per day, Visual Analogue Scale for pain, frequency and urgency, O'leary-Sant Interstitial Cystitis Symptom Index and Problem Index, cystoscopy and bladder capacity. RESULTS: Overall 45 patients finished this study protocol. The HA + AL group and the AL group showed significant improvement at week 2 (P < 0.01), while the HA group began to show effect at week 4 (P < 0.01). There was no improvement in the AL group at week 24 and these patients quitted the study without follow up. Contrarily, the HA + AL and HA group kept on improving till the end of the study without significant difference between the two groups. CONCLUSIONS: Intravesical instillation of HA and AL may provide both immediate and sustained relief of symptoms in severe PBS/IC in this preliminary study.