Increased oxidative stress with age negatively impacts the urinary bladder urothelium

de Rijk M1, Wolf-Johnston A2, Kullmann A2, Taiclet S2, Shiva S2, Birder L2

Research Type

Pure and Applied Science / Translational

Abstract Category

Geriatrics / Gerontology

Abstract 242
On Demand Geriatrics / Gerontology
Scientific Open Discussion Session 19
Animal Study Basic Science Biochemistry Gerontology Sensory Dysfunction
1. Department of Urology, School for Mental Health and Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands, 2. Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh PA 15261 USA

Mathijs M de Rijk



Hypothesis / aims of study
The prevalence of lower urinary tract symptoms (LUTS), characterized by problems regarding storage and/or voiding of urine, is known to drastically increase with age. The increased incidence of LUTS is often related to age-related changes in the urinary tract, surrounding structures (e.g. prostate) or the central or peripheral nervous system [1,2]. In an aging society LUTS are, therefore, highly relevant and increasingly important. When involuntary loss of urine occurs together with a decrease in mobility, as is often the case in aging, this can lead to a decline in self-reliance and is often one of the decisive factors in the decision for older adults to move to institutionalized care. 
	The inner surface of the bladder is covered with a mucosal membrane. This bladder mucosa consists of a multi-layered urothelium and underlying lamina propria. The urothelium fulfills crucial tasks in serving as a protective barrier protecting the underlying bladder tissue from the harsh chemical composition of urine, and exhibits signaling properties via release of mediators within the bladder wall that affect bladder functioning. The proper execution of these tasks is highly dependent on healthy functioning of urothelial cells [3]. 
	The exact mechanisms by which aging related processes influence urothelial health and bladder functioning are not yet fully understood. In the current study we aimed to further elucidate aging related changes in urothelial and mucosal biochemistry. We investigated biomarkers for oxidative stress in mucosal tissue samples collected from young and aged rodents, and assessed cellular respiration, mitochondrial functioning and basal ROS in urothelial cell (UTC) cultures. We hypothesized that aging increases oxidative stress which in the long term may damage multiple components of the LUT system.
Study design, materials and methods
The current study was approved by the local animal ethics committee, and institutional/national guidelines for the care and use of animals were followed. We collected mucosal tissue samples from young (3–4 mo) and aged (25–30 mo) rats. Tissue was evaluated for nitrotyrosine and cytochrome C expression by western immunoblotting. Urothelial cells were cultured for single cell imaging to analyze basal levels of reactive oxygen species and mitochondrial membrane potential. Mitochondrial bioenergetics were investigated by Seahorse assay.
Our results indicate that aging is associated with a significant increase in biomarkers that have been associated with increased oxidative stress. We observed a significant increase in nitrotyrosine (p = 0.006) and cytochrome C (p = 0.04) in bladder mucosa samples obtained from young and aged rodents (Fig. 1.A, 1.B). 
	In order to localize the observation of significant increases in ROS to the urothelium we measured DHR123 in UTC cultures. DHR123 is an indicator of ROS which passively diffuses across membranes and oxidizes to cationic rhodamine 123 in mitochondria. Basal ROS production was significantly higher in UTC cultures from aged animals compared to young animals (p = <0.001, Fig. 1.C).
	We then aimed to investigate the detrimental effects of the increased ROS production in UTC cultures from aged animals on mitochondrial health. Mitochondrial membrane potential (Ψm), an indicator of cell health, measured by TMRM intensity, was significantly more depolarized in UTC cultures isolated from aged animals compared to young animals (p = 0.01). Indicating that urothelial mitochondrial health significantly is altered as a function of age (Fig. 1.D).
	The bioenergetic profile of UTC was generated using the seahorse assay (Fig. 2.A). The oxygen consumption rate (OCR) measured at baseline (p = 0.001), maximal respiratory rate (p = 0.013), and the spare respiratory capacity (p = 0.017) all proved to be significantly decreased in aged animals when compared to young animals (Fig. 2.B).
Interpretation of results
Based on the findings reported, we argue that the aging process has a substantial impact on urothelial health and that the mechanisms outlined may contribute to the deterioration of bladder morphology and bladder functioning, which is known to be characteristic for the high prevalence of LUTS in older adults. A healthy urothelium is crucial for the maintenance of optimal barrier and signaling properties. We postulate that the observed decline of urothelial health and bioenergetic profile with advanced age may alter the capacities of the urothelium to optimally execute these barrier and signaling functions.
Concluding message
In the present work we show that aging is associated with a significant increase in oxidative stress in bladder mucosa, and that mitochondrial Ψm and mitochondrial OCR are significantly decreased in UTC cultures from aged bladders compared to that obtained from younger animals. We propose that these changes might significantly impact the barrier and signaling properties of the urothelium and potentially play an important role in the increased prevalence of LUTS in older adults. Future research should aim to further investigate the nature of the relationship between oxidative stress and bladder functioning with the potential to identify novel therapeutic targets which might improve the quality of life and self-reliance of many older adults suffering from LUTS.
Figure 1
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  1. Gibson W, Wagg A. Incontinence in the elderly,'normal'ageing, or unaddressed pathology? Nature Reviews Urology. 2017;14(7):440-448.
  2. Suskind AM. The aging overactive bladder: A review of aging-related changes from the brain to the bladder. Curr Bladder Dysfunc. 2017;12(1):42-47.
  3. Fry CH, Vahabi B. The Role of the Mucosa in Normal and Abnormal Bladder Function. Basic Clin Pharmacol Toxicol. 2016;119 Suppl 3:57-62.
Funding None Clinical Trial No Subjects Animal Species Rat Ethics Committee IACUC University of Pittsburgh
04/04/2024 11:07:32