An experimental model of the epithelial to mesenchymal transition and pro-fibrogenesis in urothelial cells related to bladder pain syndrome/interstitial cystitis.

BACKGROUND: Suitable in vitro models are needed to investigate urothelial epithelial to mesenchymal transition (EMT) and pro-fibrogenesis phenotype in bladder pain syndrome/interstitial cystitis (BPS/IC). This study is to establish a novel experimental BPS/IC cell model and explore how different concentrations of tumor necrosis factor (TNF)-α influence the EMT and pro-fibrogenesis phenotype of urothelial cells. METHODS: SV-HUC-1 urothelial cells were cultured with 2, 10, or 50 ng/mL TNF-α to mimic chronic inflammatory stimulation. The EMT and pro-fibrogenesis phenotype, including production of collagen I and pro-fibrosis cytokines, were estimated after 72 h of culture. RESULTS: The bladder urothelial cells of BPS/IC exhibited upregulated vimentin, TNF-α and TNF receptor, downregulated E-cadherin, and increased collagen I. Higher concentrations of TNF-α (10 and 50 ng/mL) produced an obvious mesenchymal morphology, enhanced invasion and migratory capacity, increased expression of vimentin, and decreased expression of E-cadherin. Collagen I was increased in cells treated with 2 and 10 ng/mL TNF-α after 72 h. Secretion of interleukin (IL)-6 and IL-8 was promoted with 10 and 50 ng/mL TNF-α, while that of IL-1ß or transforming growth factor-ß was unaffected. Slug and Smad2 were upregulated by TNF-α after 72 h. The Smad pathway was activated most strongly with 10 ng/mL TNF-α and Slug pathway activation was positively correlated with the concentration of TNF-α. CONCLUSIONS: Sustained 10 ng/mL TNF-α stimulation induced the EMT and pro-fibrogenesis phenotype resembling BPS/IC in SV-HUC-1 cells. Minor inflammatory stimulation induced the pro-fibrogenesis phenotype while severe inflammatory stimulation was more likely to produce significant EMT changes. Different degrees of activation of the Slug and Smad pathways may underlie this phenomenon.

The Effects of Neurokinin-1 Receptor Antagonist in an Experimental Autoimmune Cystitis Model Resembling Bladder Pain Syndrome/Interstitial Cystitis.

To identify the effects of the neurokinin-1 receptor (NK1R) antagonist aprepitant in treating pelvic pain, micturition symptoms, and bladder inflammation in mice with experimental autoimmune cystitis (EAC) similar to bladder pain syndrome/interstitial cystitis (BPS/IC). Female C57BL/6 mice were divided into the following three groups: normal control, EAC, and EAC plus aprepitant. EAC was induced in mice by duplicate immunization with bladder homogenate. In the EAC model group, EAC mice were given PBS by gavage once a day during the fourth week. In the EAC plus aprepitant group, aprepitant was administered instead of PBS in the same way. After 4 weeks, pelvic pain threshold and urination habits of mice were analyzed, as well as the bladder weight to body weight ratio, and histologic assessment of the expression of IL-1ß, TNF-α, intercellular adhesion molecule 1 (ICAM-1), and NK1R in bladder tissue. EAC mice mimicked the phenotype and pathophysiologic lesions of BPS/IC well. Compared to PBS-treated EAC mice, the mice treated with aprepitant exhibited higher pain threshold values, less number of total urine spots or small urine spots, lower bladder weight to body weight ratio, and reduced bladder inflammation with less mast cell infiltration and decreased expressions of IL-1ß, TNF-α, and ICAM-1 in bladder tissue. There was no difference in NK1R expression in bladders treated with or without aprepitant. The NK1R antagonist aprepitant relieved pelvic pain, urinary symptoms, and bladder inflammation in EAC mice. This indicated that NK1R may be a novel therapeutic target in BPS/IC treatment.

Establishment of a Novel Autoimmune Experimental Model of Bladder Pain Syndrome/Interstitial Cystitis in C57BL/6 Mice.

The aim of this study is to identify whether vaccinating twice with bladder homogenate can establish a new model of experimental autoimmune cystitis (EAC) in C57BL/6 strain mice. C57BL/6 mice were vaccinated with bladder homogenate in complete Freund's adjuvant (CFA) and boost immunized with bladder homogenate in incomplete Freund's adjuvant (IFA) after 2 weeks were used as the EAC model. Mice immunized with phosphate-buffered saline (PBS) in CFA or IFA were used as the control. Micturition habits and suprapubic-pelvic pain threshold were measured 4 weeks after primary immunization. Bladder to body weight ratios and expression of inflammatory cytokines and neurokinin 1 receptor (NK1R) were then examined. Histologic and immunohistochemical examination of the bladder was carried out, and IL-1ß, IFN-γ, and TNF-α production by the kidneys, liver, and lungs was also tested. Double-immunized mice were extensively sensitive to pressure applied on the pelvic area (P < 0.001). Compared to single-immunized mice or controls, double-immunized mice showed more micturition frequency, lower urine output per micturition, higher bladder to body weight ratio, and significant elevation in the expression of inflammatory cytokines, including IL-1ß, IL-4, IL-6, IL-10, IFN-γ, and TNF-α (all P < 0.05). NK1R gene expression was significantly increased in double-immunized mice compared to the other three groups (P < 0.001). A nonspecific immune response occurred in the liver but was much weaker than bladder inflammation. Our dual immunization EAC model in C57BL/6 mice can effectively mimic the symptoms and pathophysiologic characteristics of BPS/IC and thus can be widely used to investigate the pathogenesis and therapeutic strategies of BPS/IC.