A uro-protective agent with restorative actions on urethral and striated muscle morphology

Abstract

Purpose: Aging increases oxidative stress, which can have delirious effects on smooth and striated muscle resulting in bladder dysfunction. Consequently, in women aged over 60 years, urinary incontinence (UI) is a prevalent health problem. Despite the prevalence and consequences, UI continues to be undertreated simply because there are few therapeutic options.

Methods: Here we investigated whether 8-aminoguanine (8-AG), a purine nucleoside phosphorylase (PNPase inhibitor), would restore urethra and external sphincter (EUS) muscle morphology in the aged rat. Aged (> 25 months) female Fischer 344 rats were randomized to oral treatment with 8-AG (6 weeks) or placebo, and the urethra and EUS were evaluated by electron microscopy and protein expression (western immunoblotting).

Results: Aging was associated with mitochondrial degeneration in smooth and striated muscle cells as compared to young rats. We also observed a significant increase in biomarkers such as PARP, a downstream activator of oxidative/nitrosative stress. Treatment of aged rats with 8-AG normalized all abnormalities to that of a younger state.

Conclusions: 8-AG, a potent inhibitor of PNPase, reverses age-related lower urinary tract morphological and biochemical changes. Our observations support the concept that 8-AG will reverse age-induced lower urinary tract disorders such as UI. These initial findings could have therapeutic implications for the prevention and treatment of age-related UI.

Keywords: 8-Aminoguanine; Aging; Lower urinary tract; Purine nucleoside phosphorylase.

FIGS. 1

a-c Depicts representative transmission electron microscopy (EM) images (n=3 each) of urethral smooth muscle in young rats (FIG. 1a), aged rats (FIG. 1b) and aged rats treated with 8-aminoguanine (8-AG; FIG. 1c). The second row depicts representative EM images (n=3 each) of external urethral sphincter (EUS, striated muscle) in young rats (FIG. 1d), aged rats (FIG. 1e) and aged rats treated with 8-aminoguanine (8-AG; FIG. 1f). The damaged smooth muscle and abnormal mitochondrial morphology in aged rats is restored to a younger state by 8-AG treatment (arrows indicate mitochondria). Scale bars are 1 μm and magnification 15,000 (Panels a-f)

FIG.2

a-f Bar graphs depict age associated changes in urinary levels of hypoxanthine (FIG. 2a; n=14 rats), urethral nitrotyrosine (FIG. 2b, n=19 rats), alpha-smooth muscle actin (FIG. 2c; n=40 rats), significant changes in urethral cleaved PARP (FIG. 2d; n=21 rats), EUS cleaved caspase 3 (FIG 2e; n=23 rats) and in EUS cleaved PARP (FIG. 2f; n=23 rats) All of these abnormalities in aged rats were restored to a younger state by treatment with 8-aminoguanine (8-AG). Values represent means and SEMs. Statistical analysis was performed with either a student’s t-test or a Welch’s test as applicable to compare aged to young, and aged with treatment to untreated aged. Significant was considered at p<0.05. * p<0.05; ** p<0.01; *** p<0.0001.