Oxidative Stress Contributes to Overactive Bladder in the Transgenic Sickle Cell Mouse

Introduction: Lower urinary tract symptoms (LUTS) such as nocturia, urinary frequency, and urgency have been clinically reported in children and adults with sickle cell disease (SCD; Portocarrero et al J Urol 2012; Anele et al Neurourol Urodyn 2015), but little is known about the pathophysiology of micturition dysfunction in SCD patients. Transgenic sickle cell mice have been employed to better understand SCD pathophysiology. Recently, we reported that Berkeley SCD mice display underactive bladder and reduced detrusor smooth muscle contractions (Claudino et al Plos One, 2015). The homozygous Townes transgenic sickle cell mouse is another model for SCD, but urinary bladder dysfunction has never been studied in this model. Considering that SCD is associated with elevated ROS production, we hypothesized that increased ROS levels lead to alterations in the urinary bladder function of Townes SCD mice. Thus, the aim of this study was to evaluate the micturition and detrusor smooth muscle contractions in Townes transgenic sickle cell mice.

Methods: Townes transgenic sickle cell mice and C57BL/6 mice (control) aged 3 to 4 months old were used. Cystometry was performed to evaluate the urinary function in vivo. In separate protocols, the urinary bladder was removed and placed in Krebs solution. Two longitudinal detrusor smooth muscle strips with intact urothelium were obtained from each bladder. The strips were mounted in 4-ml organ baths containing Krebs-Henseleit solution at 37°C continuously bubbled with a mixture of 95% O₂ and 5% CO₂ (pH 7.4). Cumulative concentration-response curves to muscarinic receptor agonist carbachol (CCh; 0.01-100 μM), chloride potassium (KCl; 1-300mM) and frequency-response curves for electrical field stimulation (EFS; 1-32Hz) were obtained in strips of detrusor smooth muscle from both control and SCD mice.mRNA expression for gp91^phox and SOD-1 in bladder were also evaluated.

Results: Micturition in SCD mice was irregular, and characterized by significant (P < 0.05) increases in the frequency of voiding contractions and nonvoiding contractions (n=5). Voiding pressure, basal pressure, capacity and threshold pressure were not changed in SCD mice. Carbachol (0.01 - 100 µM) induced concentration-dependent detrusor smooth muscle contractions in both control and SCD mice, but maximal contractile responses were significantly lower (P < 0.05) in SCD compared to control mice (1.40 ± 0.4 and 2.9 ± 0.2 mN/mg, respectively; n=5). Likewise, EFS-induced neurogenic detrusor contractions in SCD mice were 29% lower (P < 0.05) compared to the control (n=10). Contractile responses induced by KCl (1 - 300 mM) did not differ between control and SCD group (n=5). The mRNA expression for the NADPH subunit gp91^phox in bladder was about 2-fold higher (P<0.05) in SCD mice compared to the control group, whereas no changes in the mRNA expression for SOD-1 were observed among groups (n=6). Infiltration by inflammatory cells (mononuclear cells) was seen in the submucosa and muscular area of bladder from SCD mice (n=4).

Conclusion: Our study shows that the overactive bladder seen in SCD mouse is associated with upregulation of NADPH oxidase subunit gp91phox. Interestingly, in the contrast, the Berkeley SCD mouse exhibits an atonic detrusor smooth muscle and underactive bladder (Claudino et al Plos One 2015). One may speculate therefore that the overactive bladder in Townes SCD may progress to underactive bladder at latter stages. To confirm this hypothesis, we will evaluate the urinary bladder function in older SCD mice.

Financial Support: FAPESP