Hypothesis / aims of study
The drug metformin, though approved and widely used as first line treatment for type 2 diabetes, also targets a number of biological drivers of aging, including mitochondrial function, oxidative stress, inflammation and the mTOR pathway. A number of studies have shown that older diabetics taking metformin tend to have lower rates of cancers, heart disease and dementia than their counterparts taking other oral anti-hyperglycemic medications. Targeting Aging with Metformin (TAME) is a clinical trial seeking to test the geroscience hypothesis in humans by examining the ability of metformin to slow the onset of these chronic conditions in 3000 nondiabetic older adults. Despite evidence that mitochondrial dysfunction represents an important biological hallmark of aging, its role in age-related lower urinary tract dysfunction and symptoms (LUTS) remains unexplored. There is substantive evidence that the urothelium (UT) plays a prominent role in the bladder ‘sensory web’, which impacts underlying bladder neural pathways and in turn voiding function. Our recent findings show that aging leads to significant functional defects in UT mitochondria contributing to oxidative stress which can be mitigated with metformin treatment. Because a local UT-afferent signalling pathway regulates sensory input to the central nervous system, it is likely that these changes in UT-neural signalling can influence voiding behaviour. These findings lead us to hypothesize that aging is associated with UT mitochondrial dysregulation/oxidative stress that can impair signalling in the bladder wall culminating in abnormal voiding behavior.
The aim of this study was designed to elucidate the effects of aging on UT mitochondrial dysregulation/oxidative stress, its impact on bladder function and whether metformin treatment can prevent, slow or reverse these deficits.
Study design, materials and methods
This aim was investigated in female Fisher 344 rats (n=5-7; young-3 mo; aged-25-28 mo).
• Some animals were treated for 3 weeks with Metformin (300 mg/kg/day delivered orally in drinking water).
• Bladders were collected from deeply anesthetized rats and utilized for cell culture and western blot per previously published methods (minimum of n=5 rats/group and n=5-7 cultures or tissue samples/experimental group). The volume (intensity) of each protein species was determined and normalized to total protein (Bio-Rad).
• Cultured urothelial cells (UTC) were loaded with various intracellular dyes to examine functional (intracellular calcium or mitochondrial) responses. These included: Dihydrorhodamine 123 - DHR123 (to measure reactive oxygen species - ROS) and data were quantified as fluorescence arbitrary units.
• Bladder function in awake rats was characterized by measuring voiding in metabolic cages (24 hr recordings with 4 hr acclimatization).
Aging is associated with increased urothelial senescence, oxidative stress and voiding dysfunction. In aged rat UT, we find a significant increase (22%) in basal calcium in parallel with a loss (39%) of mitofusin 2 (Mfn2), which is a protein localized to the outer mitochondrial membrane. Loss of Mfn2 can impair cellular respiration and function. We also find an increase in ROS production (both basal, 37%, and in response to inflammatory stimuli, 84%) in aged UT cells. In addition to enhanced ROS, aging UT show an increase (23%) in protein carbonylation (one of the most harmful and irreversible oxidative protein modifications). These aging-associated UT changes are mitigated in aged rats treated with metformin. We also observed that aged rats exhibit an increase in voided volume (200%) coupled with a decrease in voided frequency (34%) as compared to younger rats. Strikingly, treatment with metformin resulted in a significant decrease in void volume and increase in frequency of voids to that of a younger state.
Interpretation of results
While a number of factors likely contribute to impaired UT function with age, accumulating evidence suggests that altered cellular metabolism (i.e. accumulation of mitochondrial oxidative damage) plays a key role. Accumulation of mitochondrially-derived ROS production may be an important factor in a number of age-associated defects (cellular senescence, calcium homeostasis and protein modifications). Therapies that reduce oxidative stress- in particular metformin- are likely to restore mitochondrial balance (in part via reduction in endogenous production of ROS). This may be a viable treatment for aging-associated LUT disorders.