Tetrahydrobiopterin (BH4) is an essential cofactor in nitric oxide (NO) biosynthesis. NO is a key mediator of vascular homeostasis, and NO bioavailability is reduced early in vascular disease states such as hypercholesterolemia, diabetes and hypertension, as well as throughout the progression of atherosclerosis. This is a result of both decreased NO synthesis and increased NO consumption by reactive oxygen species (ROS). Decreased levels of BH4 lead to an uncoupling of NO synthase (NOS), with a shift towards production of ROS. BH4 supplementation promotes NO production and other antioxidant effects known to improve endothelium-dependent vasomotion in a number of pathological states, including coronary artery disease, arterial hypertension and ischemic reperfusion1). On the other hand, recent studies have indicated that lower urinary tract symptoms (LUTS), including nocturia, urinary incontinence and urgency, are common in both men and women, with age-related increases seen in both sexes. Epidemiologic studies have suggested that aging-associated changes in pelvic vasculature, such as atherosclerosis, eventually result in chronic bladder ischemia, which may play a key role in the development of LUTS2). However, whether BH4 supplementation affects lower urinary tract dysfunction (LUTD) in arterial occlusive disease-induced chronic bladder ischemia has not been established. We therefore used a previously described rat model of chronic bladder ischemia3) to investigate the effects of BH4 on chronic ischemia-related LUTD.

Adult Sprague-Dawley male rats (16 weeks old) were divided into control, chronic bladder ischemia (CBI) and CBI with BH4 treatment (CBI/BH4) groups. The control group received a regular diet for 8 weeks (n=10). The CBI group underwent balloon endothelial injury of bilateral iliac arteries, and received a 2% cholesterol diet for 8 weeks after the procedure to induce arterial occlusion-related chronic bladder ischemia (n=10). The CBI/BH4 group underwent the same procedure as the CBI group, and also received the 2% cholesterol diet for 8 weeks. In addition, rats in the CBI/BH4 group were orally administered BH4 at 10 mg/kg/day once daily using zondes for 8 weeks (n=10). After cystometrogram (CMG) recording in conscious animals, rats from each group were euthanized, and the bladders and common iliac arteries were harvested for pharmacological and histological examinations. We used western blotting to measure HIF1α, an oxidative stress marker, in the bladder.

Body weight and bladder wet weight did not differ significantly among the three groups. In the CBI group, iliac arteries showed obvious arterial wall thickening with neointimal formation. Mean arterial wall thickness was significantly greater in the CBI group than in the control group (Figure 1). In the organ bath study, contractile responses of muscle strips to KCl, electrical field stimulation, carbachol (Cch) and ATP were significantly lower in the CBI group than in controls. In the CMG, bladder capacity and voided volume were significantly lower and micturition interval was significantly shorter in the CBI group than in the control group.  (Table 1). Western blot analysis shows that expression of HIF1α was significantly increased in the CBI group as compared with the control group (P<0.05).
The CBI/BH4 group showed suppressed arterial wall thickening compared with the CBI group. A significant difference between CBI and CBI/BH4 groups was evident with respect to mean arterial wall thickness (Figure 1). Furthermore, significant improvements in muscle strip contractility and cystometric parameters were seen in the CBI/BH4 group compared with the CBI group (Table 1). Western blotting showed no significant difference in HIF1α expression between control and CBI/BH4 groups.

Our results suggested BH4 deficiency as one cause of LUTD-associated chronic ischemia. Chronic treatment with BH4 might prevent increases in ROS and neointimal formation, resulting in improvement of bladder hyperactivity.

The implication of this study is that BH4 may be therapeutically useful in preventing atherosclerosis and chronic ischemia-related LUTD.

Fukuda Y, Teragawa H, Matsuda K, et al. Tetrahydrobiopterin restores endothelial function of coronary arteries in patients with hypercholesterolaemia. Heart. 2002;87:264-9.
Pinggera, G. M., Mitterberger, M., Steiner, E. et al. Association of lower urinary tract symptoms and chronic ischaemia of the lower urinary tract in elderly women and men: assessment using colour Doppler ultrasonography. BJU Int. 2008;102:470-474.
Nomiya M, Yamaguchi O, Andersson KE, et al. The effect of atherosclerosis-induced chronic bladder ischemia on bladder function in the rat. Neurourol Urodyn. 2012;31: 195-200.