Bladder inflammatory diseases cause various urinary symptoms, such as urinary frequency and painful urination, that impair quality of life. In this study, we used a mouse model of cyclophosphamide (CYP)-induced bladder inflammation and immortalized human urothelial (TRT-HU1) cells to explore the preventive potential of nobiletin (NOB), a polymethoxylated flavone enriched in citrus fruit peel, and investigate its mechanism of action in the bladder. Prophylaxis with PMF90 (60% NOB) attenuated the development of bladder inflammation and urinary symptoms in CYP-treated mice. PMF90 also reduced the upregulation of connexin 43 (Cx43), a major component of gap junction channels, in the bladder mucosa of CYP-treated mice. Stimulation of TRT-HU1 cells with the pro-inflammatory cytokine IL-1β increased Cx43 mRNA and protein expression and enhanced gap junction coupling—responses that were prevented by pre-treatment with NOB. In urothelium-specific Cx43 knockout (uCx43KO) mice, macroscopic signs of bladder inflammation and changes in voiding behavior induced by CYP treatment were significantly attenuated when compared to controls. These findings indicate the participation of urothelial Cx43 in the development of bladder inflammation and urinary symptoms in CYP-treated mice and provide pre-clinical evidence for the preventive potential of NOB through its anti-inflammatory effects on IL-1β signaling and urothelial Cx43 expression.

Nanotechnology represents an expanding area of research and innovation in almost every field of science, including Medicine, where nanomaterial-based products have been developed for diagnostic and therapeutic applications. Because of their small, nanoscale size, these materials exhibit unique physical and chemical properties that differ from those of each component when considered in bulk. In Nanomedicine, there is an increasing interest in harnessing these unique properties to engineer nanocarriers for the delivery of therapeutic agents. Nano-based drug delivery platforms have many advantages over conventional drug administration routes as this technology allows for local and transdermal applications of therapeutics that can bypass the first-pass metabolism, improves drug efficacy through encapsulation of hydrophobic drugs, and allows for a sustained and controlled release of encapsulated agents. In Urology, nano-based drug delivery platforms have been extensively investigated and implemented for cancer treatment. However, there is also great potential for use of nanotechnology to treat non-oncologic urogenital diseases. We provide an update on research that is paving the way for clinical translation of nanotechnology in the areas of erectile dysfunction (ED), overactive bladder (OAB), interstitial cystitis/bladder pain syndrome (IC/BPS), and catheter-associated urinary tract infections (CAUTIs). Overall, preclinical and clinical studies have proven the utility of nanomaterials both as vehicles for transdermal and intravesical delivery of therapeutic agents and for urinary catheter formulation with antimicrobial agents to treat non-oncologic urogenital diseases. Although clinical translation will be dependent on overcoming regulatory challenges, it is inevitable before there is universal adoption of this technology to treat non-oncologic urogenital diseases.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day–night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day–night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals’ active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day–night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals’ awake, active phase.

Background: Distension of hollow organs is known to release adenosine triphosphate (ATP) from the lining epithelium, which triggers local responses and activates sensory nerves to convey information to the central nervous system. However, little is known regarding participation of ATP and mediators of ATP release, such as Pannexin 1 (Panx1) channels, in mechanisms of vaginal mechanosensory transduction and of changes imposed by diabetes and menopause, conditions associated with vaginal dysfunction and risk for impaired genital arousal. Aim: To investigate if intravaginal mechanical stimulation triggers vaginal ATP release and if (a) this response involves Panx1 channels and (b) this response is altered in animal models of diabetes and menopause. Methods: Diabetic Akita female mice were used as a type 1 diabetes (T1D) model and surgical castration (ovariectomy [OVX]) as a menopause model. Panx1-null mice were used to evaluate Panx1 participation in mechanosensitive vaginal ATP release. Vaginal washes were collected from anesthetized mice at baseline (non-stimulated) and at 5 minutes after intravaginal stimulation. For the OVX and Sham groups, samples were collected before surgery and at 4, 12, 22, 24, and 28 weeks after surgery. ATP levels in vaginal washes were measured using the luciferin-luciferase assay. Panx1 mRNA levels in vaginal epithelium were quantified by quantitative polymerase chain reaction. Outcomes: The main outcome measures are quantification of mechanosensitive vaginal ATP release and evaluation of impact of Panx1 deletion, OVX, and T1D on this response. Results: Intravaginal mechanical stimulation–induced vaginal ATP release was 84% lower in Panx1-null (P < .001) and 76% lower in diabetic (P < .0001) mice compared with controls and was reduced in a progressive and significant manner in OVX mice when compared with Sham. Panx1 mRNA expression in vaginal epithelium was 44% lower in diabetics than that in controls (P < .05) and 40% lower in OVX than that in the Sham (P < .05) group. Clinical Translation: Panx1 downregulation and consequent attenuation of mechanosensitive vaginal responses may be implicated in mechanisms of female genital arousal disorder, thereby providing potential targets for novel therapies to manage this condition. Strengths & Limitations: Using animal models, we demonstrated Panx1 involvement in mechanosensitive vaginal ATP release and effects of T1D and menopause on this response and on Panx1 expression. A limitation is that sex steroid hormone levels were not measured, precluding correlations and insights into mechanisms that may regulate Panx1 expression in the vaginal epithelium. Conclusions: Panx1 channel is a component of the vaginal epithelial mechanosensory transduction system that is essential for proper vaginal response to mechanical stimulation and is targeted in T1D and menopause.

We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1 (ALDH1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

Metabolomics analysis of urine before and after overactive bladder (OAB) treatment may demonstrate a unique molecular profile, allowing predictions of responses to treatment. This feasibility study aimed to correlate changes in urinary metabolome with changes in OAB symptoms after intravesical onabotulinumtoxinA (BTX-A) injections for refractory OAB. Women 18 years or older with non-neurogenic refractory OAB were recruited to complete OAB-V8 questionnaires and submit urine samples before and after 100 units intravesical BTX-A injection. Samples were submitted to CE-TOFMS metabolomics profiling. Data were expressed as percent of change from pre-treatment and were correlated with OAB-V8 score improvement. Urinary metabolite changes in the OAB-V8 groups were compared using the Kruskal–Wallis test, and associations between metabolites and OAB-V8 scores were examined using quantile regression analysis. Of 61 urinary metabolites commonly detected before and after BTX-A, there was a statistically significant decrease in adenosine and an increase in N8-acetylspermidine and guanidinoacetic acid levels associated with OAB score improvement, suggesting that intravesical BTX-A injection modifies the urinary metabolome. These urinary metabolites could provide insight into OAB pathophysiology and help identify patients who would benefit most from chemodenervation.