Although advancing age is a major risk factor for lower urinary tract symptoms (LUTS) including urinary incontinence, underlying biochemical changes contributing to age associated LUTS remain unknown.  HX (hypoxanthine) is a purine metabolite associated with generation of tissue damaging reactive oxygen species (ROS).  Increased oxidative damage by ROS is deleterious to cells and plays a key role in disease progression.  Elevated levels of the purine metabolite hypoxanthine (HX) may exhibit harmful effects in a number of organ systems due to the production of reactive oxygen species (ROS) when HX is further metabolized by xanthine oxidase (XO) to xanthine and then to uric acid. We hypothesize that HX, via increased ROS production, damages mitochondria leading to even more ROS production by dysfunctional mitochondria, thus triggering a cascade of oxidative stress. This hypothesis forms the basis of the present study which examines whether and how exposure of the LUT in adult rodents to uro-damaging hypoxanthine (HX) increases oxidative stress and damages all components of the LUT system, thus yielding a LUT system prone to dysfunction regardless of the proximal initiating cause. Decreasing production of uro-damaging purine metabolites could offer a geroscience-guided approach to preventing the age-associated deterioration of the LUT thereby decreasing the burden of LUT diseases.

This investigation conforms to the Guide for the Care and Use of Laboratory Animals and all studies were approved by animal care and use committees.  Adult 3-month-old female Fischer 344 (F344) rats were treated with vehicle or HX (10 mg/kg/day; 3 weeks) administered in drinking water. We have tracked the amount of water consumption/day and found no variability (demonstrating consistency) in terms of dosing. Rats were housed (24 hr after 4 hr acclimatization) in metabolic cages on an ultra-sensitive balance to record void volume and frequency and measurements were averaged for each rat.  Filling cystometry was also performed to assess additional variables, such as bladder capacity.  Leak point pressure (LPP) associated with poor bladder contractile performance was assessed. Targeted molecular approaches were used to assess biomarkers for oxidative stress and cellular damage. High resolution micro-CT imaging (Skyscan 1272, Bruker Micro-CT, Kontich, Belgium) of intact, unfixed bladders and associated post processing were used to assess possible changes in the shape and wall thickness of the bladder due to HX treatment.  Data were analyzed in GraphPad Prism 9 using Student’s t-tests (2-tailed) and 1-way ANOVA followed by appropriate post hoc tests. P<0.05 was considered significant.

Metabolic-cage studies in HX-treated rats revealed increases in voiding frequency (142±11.8%, p<0.01, n=10) and decreases in the inter-void interval (34±5.9%, p<0.05, n=10) and bladder capacity (48±13.3%, p<0.01, n=3) as compared to untreated, control rats. We also observed that HX treated rats exhibited significant changes in LPP at 50% filling (144±2.8%, p<0.05, n=3). HX exposure increased outcomes indicative of oxidative stress and pathophysiological ROS production.  These include 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); a spin-trapping reagent that traps free radicals in protein. Our findings showed significant increases in bladder protein-DMPO abducts (155±6.4%, p<0.001, n=4) with HX treatment. Protein carbonylation is an irreversible state leading to significant damage due to elevated ROS levels.  We find that HX increased levels of protein oxidation in HX treated adult bladders (184±12.7%, p<0.01, n=5) as well as pAMPK, a ‘master regulator’ of cellular energy in adult HX (201±27.7%, p<0.05, n=5) as compared to untreated controls. We also observed that HX-treatment in adult rats leads to increased caspase enzymes (236±24.2%, p<0.0001, n=10), caspases are involved in signal transduction cascades culminating in apoptosis. These findings demonstrated increased oxidative stress by HX which, over time could lead to cell damage and loss of function. High-resolution micro-CT is essential for local stress calculations and for insight into local wall remodeling.  Here, we find that HX-treatment induced bladder wall remodeling that led to a statistically significant asymmetry in wall thickness between the anterior and posterior sides of the bladder. We suggest that this is a maladaptation in response to HX treatment and will contribute to the diminished compliance which affects voiding efficiency.

These studies provide evidence that adult rats exposed to HX causes long-lasting changes in voiding behavior and multiple changes in bladder structure and function resembling alterations observed with aging. Our findings support our hypothesis that increases in the purine metabolite HX, can injure the LUT and thereby render the LUT more susceptible to a ‘triggering’ insult leading to LUTS.

The LUT is very susceptible to the impact of aging-related phenomena. While specific factors associated with aging that lead to LUT dysfunction vary among patients, substantial evidence supports a role for oxidative stress (i.e., uncontrolled increases in the production of ROS) in the pathogenesis of LUTS.  The uro-toxic purine metabolite hypoxanthine produces substantial changes in LUT form and function, mimicking many of the changes that are associated with human LUTS.  We chose to examine the effects of HX in young rats as HX is a likely factor in causing damage in older rats and therefore would be difficult to interpret the damaging effects of HX in already HX-damaged older bladders. Moreover, our findings resemble many of the biochemical, structural, and functional changes observed with aging.  These findings support the concept that uro-toxic hypoxanthine triggers a cascade of oxidative damage which injures the LUT system, resulting in a LUT system prone to dysfunction regardless of the proximal initiating cause. Taken together, these changes over time may contribute to the development of LUTS especially in the older adult.  Thus, these and other observations provide insights into mechanisms whereby biological aging may contribute to age-related dysfunction and diseases of the LUT, offering opportunities for the development of geroscience-guided approaches to LUT aging and diseases.

Continence 7S1 (2023) 100861
DOI: 10.1016/j.cont.2023.100861