Uroplakin (UP) Ia, UP Ib, UP II, and UP III are known to be critical factors in normal urothelial barrier function (1). Impairment of UP II has been reported to cause mast cell infiltration in mice bladders. Current attention has focused on the association between urothelial barrier dysfunction and lower urinary tract dysfunction (LUTD). Defects in UPs lead to abnormal voiding patterns (2). Recent studies have suggested that mast cells contribute to bladder hyperactivity. Mast cells play an important role in detrusor overactivity induced by visceral hypersensitivity in rats (3). Mast cell tryptase (MCT) can regulate neuronal activity by cleaving Protease-activated Recepter 2 (PAR2), which is expressed on C-fiber. However, it has not been established whether or not urothelial barrier dysfunction causes bladder hyperactivity through PAR2 activation by MCT.
On the other hand, pelvic arterial occlusive disease, including atherosclerosis, has been suggested to cause chronic bladder ischemia, which may play a key role in the development of LUTD in both men and women. The mechanisms underlying the changes in bladder function caused by chronic ischemia have not been completely elucidated. 
This study aims to investigate the effects of arterial occlusive disease-related chronic ischemia on UPs, mast cells and PAR2 in the bladder using a rat model of chronic bladder ischemia (CBI).

Adult male Sprague-Dawley rats (16 weeks old) were divided into two groups (control and CBI; n = 10 each). The CBI group underwent balloon endothelial injury of bilateral iliac arteries and received a 2% cholesterol diet for 8 weeks to induce arterial occlusive disease-related chronic ischemia. The control group received a regular diet for 8 weeks. After monitoring urine output for 24 h, the bladders and common iliac arteries were harvested for pharmacological and histological examinations. Western blot analysis was used to measure the expression of UP Ia, UP Ib, UP II, UP III, MCT, PAR2 and HIF1α, an oxidative stress marker, in the bladder of this rat model. The bladders were processed for immunohistochemical and methylene blue staining. All values are expressed as mean ± standard deviation. Values of P < 0.05 were considered statistically significant.

There was no statistically significant difference in the wet weights of the bladders in the two groups (control vs CBI: 0.237 ± 0.054 g vs 0.241 ±0.055 ml, P = 0.888). Metabolic cage studies showed that the mean and maximum voided volumes were significantly smaller in the CBI group than in the control group (control vs. CBI, mean voided volume: 1.46 ± 0.33 ml vs 1.01 ±0.21 ml, P = 0.001; maximum voided volume: 2.62 ± 0.60 ml vs 2.01 ± 0.41 ml, P = 0.023). Mean arterial wall thickness was significantly greater in the CBI group (116.3 ± 23.8 μm) than in the control group (82.0 ± 26.3 μm; P < 0.001). Western blot analysis showed expression of HIF1α (P = 0.034), MCT (P = 0.024) and PAR2 (P = 0.021) were significantly increased, and UP II expression was significantly decreased (P = 0.011) in the CBI group as compared with the control group. However, no significant differences were found in UP Ia, UP Ib or UP III expression between the two groups. Immunohistochemical staining revealed that UP II-positive cells were located mostly on the urothelium. The percentage of UP II-positive cells was significantly lower in the CBI group (63.0 ± 0.11%) than in the control group (92.1 ± 0.1%, P < 0.001). Methylene blue staining revealed the number of mast cells were significantly higher in the CBI group than in the control group (P = 0.034).

In this study, increased levels of the oxidative stress marker and decreased voided volume in the CBI group indicate that pelvic arterial occlusive disease causes an ischemia/reperfusion injury and LUTD in a rat model of chronic bladder ischemia. Our results also demonstrate that expression of UP II in the urothelium was reduced in chronically ischemic rat bladders. One possible explanation is that chronic ischemia impairs urothelial barrier function through reduced expression of UP II in rats. Urothelial barrier dysfunction contributed to mast cell infiltration and increased PAR2 on C-fiber, resulting in PAR2 activation by mast cell tryptase. Activation of PAR2 may induce LUTD by stimulating C-fiber in a rat model of chronic bladder ischemia.

Our results suggest that chronic ischemia induced by pelvic arterial occlusive disease may cause urothelial barrier dysfunction through reduced expression of UP II. A possible explanation for this is urothelial barrier dysfunction associated with CBI induced LUTD secondary to increased expression of PAR2 and mast cell tryptase.

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