Aging patients with bladder dysfunction often have lower urinary tract symptoms, which are related to both overactive and underactive bladder conditions, also known as detrusor hyperreflexia with impaired contractility (1). In recent years, the importance of nitric oxide (NO), a transmitter of non-adrenergic, non-cholinergic nerves, in the control of lower urinary tract function has been documented (2). NO promotes cyclic guanosine monophosphate (cGMP) production intracellularly in smooth muscles, reduces the intracellular calcium concentration, and enhances smooth muscle relaxation (3). However, it is not known whether changes in the expression of NO-related molecules are associated with development of lower urinary tract dysfunction during aging. In this regard, to the best of our knowledge, this is the first report, which investigated the bladder and urethral activity and NO-related molecular changes in aging rats.

Female Sprague-Dawley rats (Envigo, Fredrick, MD, USA) were divided to two different age groups: (A) control (12 weeks old, n=8) and (B) aging rats (15 months old, n=6). In both  groups, a 24 h voiding assay was performed and the urodynamic parameters using awake cystometry (CMG) and urethral perfusion pressure (UPP) recordings were evaluated under urethane anesthesia. The mRNA expression levels of NO-, ischemia-, and inflammation-related markers in urethra and bladder tissues and the levels of cGMP in the urethra were also assessed. In CMG, parameters such as opening pressure (pressure at which the urethra opens and urine flow starts), intercontraction intervals, the number of non-voiding contractions (NVCs) per voiding, postvoid residual (PVR), bladder capacity, bladder compliance, and voiding efficiency (VE) were measured. NVC was defined as an increase in intravesical pressure of more than 8 cm H2O above the baseline. In UPP recordings, parameters such as baseline urethral pressure (UP), UP nadir, UP at which the urethra starts to relax (UPUR), UP reduction (the difference between UPUR and UP nadir), and high-frequency oscillation (HFO; the amplitude of the UP changes during the micturition reflex) were measured. Urethral HFO induced by bursting activity of the striated urethral sphincter muscle is known to be necessary for its pumping function to enhance efficient voiding in rats. Gene expression of NO-related markers such as nitric oxide synthase 1 (NOS1) and protein kinase G (PKG) and ischemia- and inflammation-related markers such as hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-β1) were quantified using real-time PCR. Urethral tissues were pulverized and processed for cGMP measurements using an ELISA kit. All values are expressed as mean ± standard deviation. Mann-Whitney U test was used to evaluate statistical differences between the groups. Statistical significance was set at P < 0.05.

Body weight was significantly higher in Group B than in Group A (340.5 ± 39.0 vs 286.8 ± 19.5 g, P = 0.0037). There was no significant difference in the 24 h voiding assay results. In CMG, the number of NVC per voiding cycle and PVR were significantly higher in Group B than in Group A (0.2 ± 0.1 vs. 0.1 ± 0.0 number/min, P = 0.0080; 0.1 ± 0.0 vs. 0.0 ± 0.0 mL, P = 0.0007, respectively), and VE was significantly lower in Group B than in Group A (91.4 ± 3.7 vs. 99.4 ± 1.1 %, P = 0.0007) (Fig. 1A, B). In UPP recordings, UP reduction and HFO amplitude were significantly lower in Group B than in Group A (8.6 ± 3.1 vs. 15.5 ± 2.3 cm H2O, P = 0.0079, 1.3 ± 0.3 vs. 4.0 ± 2.0, P = 0.0079, respectively) (Fig. 1C, D). In molecular studies, mRNA expression levels of HIF-1α, VEGF, and TGF-β1 in the bladder were significantly higher in Group B than in Group A (4.0 ± 1.0 vs. 1.0 ± 0.8 fold, P = 0.0003, 3.0 ± 0.4 vs. 1.0 ± 0.2 fold, P = 0.0003, 7.8 ± 2.4 vs. 1.0 ± 0.6 fold, P = 0.0003, respectively) (Fig. 2A). The mRNA expression levels of NOS1 and PKG were significantly lower in Group B than in Group A (0.6 ± 0.2 vs. 1.0 ± 0.4 fold, P = 0.0260; 0.2 ± 0.1 vs. 1.0 ± 0.2 fold, P = 0.0022, respectively) (Fig. 2B). cGMP concentrations in the urethra were significantly lower in Group B than in Group A (0.1 ± 0.0 vs. 0.2 ± 0.0 pmol/mg of tissue, P = 0.0022) (Fig. 2C).

Animal studies focusing on aging and NO in association with urinary tract dysfunction are scarce. Our results indicate the following: (a) 15-month-old rats exhibited the bladder overactive condition as evidenced by increased NVCs and the bladder underactive condition as evidenced by increased PVR and decreased VE. The urethral dysfunction was also shown by decreases in UP reduction and HFO amplitudes, (b) aging bladder tissues showed increased mRNA expression levels of HIF-1α, VEGF, and TGF-β1, and aging urethral tissues showed decreased mRNA expression levels of NOS1 and PKG, and (c) aging urethral tissues showed decreased levels of cGMP as compared to controls, suggesting that the cGMP level in the urethra is decreased with aging, leading to impaired urethral relaxation mechanisms underlying aging-related voiding and storage dysfunction.

In aging rats, reductions in mRNA expression levels of NO-related molecules and the cGMP level in the urethra, which induced functional urethral obstruction during voiding due to impaired urethral relaxation, might play an important role in bladder overactivity, as well as in incomplete emptying and bladder underactivity. Aging rats would be a useful model for studying the natural progression of age-related lower urinary tract dysfunction, especially, in relation to alterations in the NO-mediated transmitter function.

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Continence 2S2 (2022) 100462
DOI: 10.1016/j.cont.2022.100462