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.

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).

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.

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.

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