Lower urinary tract symptoms (LUTS) 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 (CBI), which may play a key role in the development of LUTS1). However, the mechanisms underlying lower urinary tract dysfunction (LUTD) caused by CBI are not completely known. On the other hands, nitric oxide (NO) is well known to be a key mediator of vascular homeostasis. NO is generated from the conversion of L -arginine to L-citrulline by endothelial nitric oxide synthase (eNOS). Recent attentions focused on the altered function of eNOS in the atherosclerosis. Under progression of atherosclerosis, eNOS produces reactive oxygen species (ROS) rather than NO, resulting in vascular oxidative stress and endothelial dysfunction. This state is referred to as the eNOS uncoupling. This switch between NO and ROS generation by eNOS is determined by the depletion of tetrahydrobiopterin (BH4), which is eNOS cofactor. BH4 is synthesized by either the de novo or salvage pathway. The de novo pathway starts with guanosine triphosphate (GTP), which is metabolized to BH4 by GTP cyclohydrolase I (GCH-1) and sepiapterin (SP) reductase (SR). The rate-limiting enzyme in the de novo pathway is GCH-1. The salvage pathway converts dihydrobiopterin (BH2) to BH4 by dihydrofolate reductase (DHFR)2). Whether pelvic arterial occlusion affects the de novo pathway and salvage pathway for BH4 synthesis in the bladder is still controversial.  It has also not been established how eNOS uncoupling contributes to LUTD associated with chronic ischemia. has not been established. We therefore used a rat model of CBI to investigate the influence of chronic ischemia on BH4 synthase in the bladder and the association between eNOS uncoupling and 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 were harvested for pharmacological and histological examinations. We used Western blotting to measure expression levels of eNOS, SR, GCH-1, DHFR and the oxidative stress marker HIF1α in bladder tissue.

Bladder capacity (Bcap) and mean voided volume (VV) were significantly less and micturition interval (MI) was significantly shorter in the CBI group than in the Control group (p<0.01 each). Bcap and VV were significantly higher, and MI was significantly longer in the CBI/BH4 group than in the CBI group (Bcap, p=0.014; VV, p<0.01; MI, p=0.014). EM staining of bladder tissue demonstrated that the percentage of collagen in the muscle layer of the CBI group was significantly increased compared with the Control group (p<0.01). EM staining also showed a significantly decreased percentage of collagen in the muscle layer of bladder in the CBI/BH4 group as compared with the CBI group (p<0.01). Western blotting using bladder tissue showed that expressions of HIF-1α (p<0.01) and DHFR (p<0.01) were significantly higher in the CBI group than in the Control group. Expression of eNOS was also significantly increased in the CBI group as compared with the Control group (p=0.043). In the CBI/BH4 group, expressions of HIF-1α (p=0.012) and DHFR (p=0.018) were significantly decreased as compared with the CBI group. No significant differences in eNOS expression were evident between the CBI and CBI/BH4 groups. On the other hand, eNOS expression was significantly higher in the CBI/BH4 group than in the Control group (p<0.01). No significant difference in SR and GCH-1 expression was found among the three groups. With immunohistochemical staining, eNOS-positive cells were located mostly on vascular endothelium and urothelium in the bladder. In the Control group, eNOS-positive cells on the vascular endothelium were markedly reduced as compared with other groups. DHFR positive cells were also located mostly on vascular endothelium and obvious increased in the CBI group.

Our results suggested chronic ischemia caused LUTD and bladder fibrosis. In this study, bilateral iliac arterial occlusion increased the expressions of eNOS and DHFR, which can regenerate BH4 from BH2 in the salvage pathway of BH4 synthesis, in the bladder of rats. On the other hands, SR and GCH-1, enzymes in the de novo pathway, were not altered in the CBI rat model. Chronic administration with BH4 prevented increased expression of HIF1α and DHFR and LUTD, and did not affect enhanced eNOS expression in the bladder of rats.

Arterial occlusive disease promotes NO bioavailability in the bladder by increasing eNOS expression and the BH4 synthesis salvage pathway activation to protect lower urinary tract function. However, DHFR could not synthesize sufficient BH4 relative to the increased eNOS in chronically ischemic bladder, resulting in eNOS uncoupling. The eNOS uncoupling may bladder fibrosis and LUTD via decreased NO and increased ROS. The protective effect of BH4 supplementation on lower urinary tract function from chronic ischemia also suggested that deficiency of BH4 relative to the increased eNOS, which leaded to eNOS uncoupling, caused LUTD. One implication of this study was that avoiding eNOS uncoupling may be therapeutically useful in preventing chronic ischemia-related LUTD.

Pinggera, G. M. 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.
An J, et al. Role of tetrahydrobiopterin in resistance to myocardial ischemia in Brown Norway and Dahl S rats. Am J Physiol Heart Circ Physiol. 2009;297(5):H1783-91.