Description: Analysis of MafB –/– mice has suggested that the MAFB transcription factor was essential to islet α- and β-cell formation during development, although the postnatal physiological impact could not be studied here because these mutants died due to problems in neural development. Pancreas-wide mutant mice were generated to compare the postnatal significance of MafB ( MafB panc ) and MafA/B ( MafAB panc ) with deficiencies associated with the related β-cell-enriched MafA mutant (MafA panc ). Insulin + cell production and β-cell activity were merely delayed in MafB panc islets until MafA was comprehensively expressed in this cell population. We propose that MafA compensates for the absence of MafB in MafB panc mice, which is supported by the death of MafAB panc mice soon after birth from hyperglycemia. However, glucose-induced glucagon secretion was compromised in adult MafB panc islet α-cells. Based upon these results, we conclude that MafB is only essential to islet α-cell activity and not β-cell. Interestingly, a notable difference between mice and humans is that MAFB is coexpressed with MAFA in adult human islet β-cells. Here, we show that nonhuman primate (NHP) islet α- and β-cells also produce MAFB, implying that MAFB represents a unique signature and likely important regulator of the primate islet β-cell.

Description: Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which plays an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP-converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom-designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure that induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch-induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch-induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nanomolar level) about 10 times higher than the ATP concentration in serosal chamber (subnanomolar level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data, showing that ATP-converting enzymes ENTPD3 and alkaline phosphatase are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude, and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch.

Description: Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18–24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations.

Description: Void spot assays (VSA) and cystometry are two of the most common tests performed in mice to assess lower urinary tract function. Assay protocols and methodology vary greatly among laboratories, and little is known about reproducibility of results generated by different laboratories. We performed VSA in four mouse strains, comparing males with females and comparing results between two independent laboratories. Unique aspects of the current study include direct comparison of results of VSA performed in a similar manner in two locations and comparison of cystometry performed using two different rates of infusion in these two laboratories. Both assays were performed in male and female 129S1/SvImJ, C57BL/6J, NOD/ShiLtJ, and CAST/EiJ mice, and cystometry was performed under urethane anesthesia (10/group). Assays were performed and results analyzed as previously described. Results obtained in female mice were compared with previously reported values. Results of lower urinary tract function testing in mice vary in a consistent manner with strain and sex. Variables in husbandry, testing techniques, and analysis of results can significantly affect conclusions, particularly those obtained by cystometry. Although VSA results were remarkably similar between the two laboratories, consistent methods for performing lower urinary tract function testing in mice are required to compare results among studies with confidence.

Description: Ketamine is a popular choice for young drug abusers. Ketamine abuse causes lower urinary tract symptoms, with the underlying pathophysiology poorly understood. Disruption of urothelial barrier function has been hypothesized to be a major mechanism for ketamine cystitis, yet the direct evidence of impaired urothelial barrier function is still lacking. To address this question, 8-wk-old female C57BL/6J mice were injected intraperitoneally with 30 mg·kg –1 ·day –1 ketamine for 12 wk to induce ketamine cystitis. A spontaneous voiding spot assay showed that ketamine-treated mice had increased primary voiding spot numbers and smaller primary voiding spot sizes than control mice ( P 〈 0.05), indicating a contracted bladder and bladder overactivity. Consistently, significantly increased voiding frequency was observed in ketamine-treated mice on cystometrograms. These functional experiments indicate that ketamine induces voiding dysfunction in mice. Surprisingly, urothelial permeability in ketamine-treated mice was not changed when measured using an Ussing chamber system with isotopic urea and water. Mouse urothelial structure was also not altered, and intact umbrella cell structure was observed by both transmission and scanning electron microscopy. Furthermore, immunostaining and confocal microscopy confirmed the presence of a well-defined distribution of zonula occuldens-1 in tight junctions and uroplakin in umbrella cells. In conclusion, these data indicate that ketamine injection induces voiding dysfunction in mice but does not necessarily disrupt mouse bladder barrier function. Disruption of urothelial barrier function may not be the major mechanism in ketamine cystitis.

Description: The voiding spot assay (VSA) on filter paper is an increasingly popular method for studying lower urinary tract physiology in mice. However, the ways VSAs are performed differ significantly between laboratories, and many variables are introduced compared with the mouse’s normal housing situation. Rodents are intelligent social animals, and it is increasingly understood that social and environmental stresses have significant effects on their physiology. Surprisingly, little is known about whether change of environment during VSA affects mouse voiding and what the best methodologies are for retaining "natural" micturition patterns. It is well known that stress-related neuropeptide corticotropin-releasing factor is significantly elevated and induces dramatic voiding changes when rodents encounter stresses. Therefore we hypothesized that changes in the environmental situation could potentially alter voiding during VSA. We have examined multiple factors to test whether they affect female mouse voiding patterns during VSA, including cage type, cage floor, water availability, water bottle location, single or group housing, and different handlers. Our results indicate that mice are surprisingly sensitive to changes in cage type and floor surface, water bottle location, and single/group housing, each of which induces significant changes in voiding patterns, indicative of a stress response. In contrast, neither changing handler nor 4 h of water deprivation affected voiding patterns. Our data indicate that VSA should be performed under conditions as close as possible to the mouse’s normal housing. Optimizing VSA methodology will be useful in uncovering voiding alterations in both genetic and disease models of lower urinary dysfunctions.