Publications by Author: Warren G Hill

OBJECTIVES: To describe associations between voiding behavior and bacterial loads in a murine model of urinary tract infection (UTI).

METHODS: Fourteen female C57BL/6J mice were transurethrally inoculated with 108colony-forming unit uropathogenic E. coli (UPEC) UTI89 in 50 μL two times, 24 hours apart. Voiding spot assays were used to measure voiding behavior. Voiding spot assays and urine cultures were performed at various time points between 1 and 28 days postinfection (dpi). Bladder and kidney bacterial loads were measured at 28 dpi. Correlations were calculated between voiding spot assay variables and bacterial loads at different dpi. In a separate experiment, 3 female mice were infected with UPEC in the same manner for histology changes at 28-dpi in chronic UTI.

RESULTS: During the 28 days, among 14 mice, 8 developed chronic cystitis and 11 developed chronic pyelonephritis based on a priori definitions. All infected mice showed increased urinary frequency, polyuria, and decreased bladder capacity. Tissue fibrosis was also observed in the infected bladder. At 1 dpi and 28 dpi, the urinary bacterial loads were positively associated with frequency and polyuria. Bladder and kidney bacterial loads at 28 dpi were positively with frequency and polyuria.

CONCLUSIONS: Urine and tissue bacterial loads were associated with changes of voiding behavior at both 1 and 28 dpi.

Acute urinary retention (AUR) is a common urological emergency and affects a significant patient population. The inability to eliminate urine may lead to permanent damage to the bladder's structure and functioning. However, we know little about the underlying molecular sequelae to the urine retention. To closely mirror the potential high pressures that patients with AUR could experience, we catheterized anesthetized female mice via the urethra and filled the bladder by pumping saline (25 µL/min) into the bladder lumen to 50 cm or 80 cm water pressure. A water column with designated height (50 or 80 cm) was then adjusted to maintain constant pressure in the bladder lumen for 30 minutes. Functional and morphological evaluations were performed from 0 to 24 hours after AUR treatment. Mice exhibited incontinence and overactivity with diminished voiding pressure. Significant injury was confirmed which revealed bladders with disrupted urothelial barrier, edematous lamina propria, and distorted muscle bundles. Bladder smooth muscle (BSM) from pressure-treated mice have significantly diminished contraction force, suggesting that bladder voiding dysfunction can be attributed to impaired BSM contractility. Indeed, dysregulation of acetylcholine and purinergic signaling pathways were demonstrated, indicating that reduced efficacy of these pathways contributes to impaired BSM contractility. Finally, altered expression of β1-integrin and extracellular matrix mediated mechanotransduction pathways were detected, suggesting a profound remodeling process. These data demonstrated an easy to perform, quantifiable, and reproducible AUR mouse model, which mimics well the characteristics of human AUR patients, and our data generate new insights into the molecular mechanisms that occur following AUR.

Diabetic bladder dysfunction (DBD) is the most prevalent complication of diabetes mellitus (DM), affecting >50% of all patients. Currently, no specific treatment is available for this condition. In the early stages of DBD, patients typically complain of frequent urination and often have difficulty sensing when their bladders are full. Over time, bladder function deteriorates to a decompensated state in which incontinence develops. Based on studies of diabetic changes in the eye, kidney, heart, and nerves, it is now recognized that DM causes tissue damage by altering redox signaling in target organs. NADPH oxidase (NOX), whose sole function is the production of reactive oxygen species (ROS), plays a pivotal role in other well-known and bothersome diabetic complications. However, there is a substantial gap in understanding how NOX controls bladder function in health and the impact of NOX on DBD. The current review provides a thorough overview of the various NOX isoforms and their roles in bladder function and discusses the importance of further investigating the role of NOXs as a key contributor to DBD pathogenesis, either as a trigger and/or an effector and potentially as a target.

The targeted activation or inhibition of specific cell populations using chemogenetics allows the precise dissection of cellular signaling and function. Designer receptors exclusively activated by designer drugs (DREADDs) is a chemogenetic platform initially developed by mutating human muscarinic receptors to be unresponsive to endogenous acetylcholine but exclusively activated by an "inert" designer drug. Compound 21 (C21) is a new and potent DREADD agonist; however, radioligand assays from a recent report indicated its ability to bind to endogenous G protein-coupled receptors (GPCRs), including muscarinic M1-3 receptors. Whether this binding causes off-target effects is unclear. Renal innervation is important for the regulation of renal function, and the advent of chemogenetic tools provides significant opportunities for the mechanistic understanding of renal innervation and function. GPCRs such as adrenergic and muscarinic receptors play a role in renal function; thus, a careful pharmacological characterization of C21 in renal function is a prerequisite for this approach. Unexpectedly, an infusion of 1.0 mg/kg C21 in anesthetized mice caused an ∼4-fold increase in urine output and correspondingly increased the glomerular filtration rate (GFR), suggesting a C21-mediated acute diuretic effect. This acute diuresis effect was further confirmed in awake mice using voiding spot assays. The exact molecular mechanism for C21-mediated diuresis is unclear; however, we demonstrated by in vitro myography that C21 can effectively inhibit bladder smooth muscle contraction by antagonizing M3 receptors at the micromolar level, causing increased voiding size in vivo. In summary, C21 functions as a GPCR antagonist and has significant dose-dependent off-target effects in the renal system.

Lower urinary tract symptoms are extremely common in people with diabetes and obesity, but the causes are unclear. Furthermore, it has proven difficult to reliably demonstrate bladder dysfunction in diabetic mouse models, thus limiting the ability to gain mechanistic insights. Therefore, the main objective of this experimental study was to characterize diabetic bladder dysfunction in three promising polygenic mouse models of type 2 diabetes. We performed periodic assessments of glucose tolerance and micturition (void spot assay) for eight to twelve months. Males and females and high-fat diets were tested. NONcNZO10/LtJ mice did not develop bladder dysfunction over twelve months. TALLYHO/JngJ males were severely hyperglycemic from two months of age (fasted blood glucose  550 mg/dL), while females were moderately so. Although males exhibited polyuria, neither they nor the females exhibited bladder dysfunction over nine months. KK.Cg-Ay/J males and females were extremely glucose intolerant. Males exhibited polyuria, a significant increase in voiding frequency at four months (compensation), followed by a rapid drop in voiding frequency by six months (decompensation) which was accompanied by a dramatic increase in urine leakage, indicating loss of outlet control. At eight months, male bladders were dilated. Females also developed polyuria but compensated with larger voids. We conclude KK.Cg-Ay/J male mice recapitulate key symptoms noted in patients and are the best model of the three to study diabetic bladder dysfunction.

Diabetic bladder dysfunction is a frequent complication of diabetes. Although many mouse models of diabetes now exist, there has been little systematic effort to characterize them for the timing of onset and severity of bladder dysfunction. We monitored metabolic status and tested bladder function by void spot assay and limited anesthetized cystometry in both male and female mice of three models of obesity and diabetes: a type 1 diabetes model (the Akita mouse) and two type 2 diabetes models [the diet-induced obese (DIO) model and the ob/ob mouse]. Akita mice had insulin pellets implanted subcutaneously every 3 mo to mimic poorly controlled type 1 diabetes in humans. Mice were hyperglycemic by 48 days after implants. Female mice exhibited no bladder dysfunction at any age up to 20 mo and gained weight normally. In contrast, by 7 mo, male Akita mice developed a profound polyuria and failed to show normal weight gain. There were no observable signs of bladder dysfunction in either sex. DIO mice on high/low-fat diets for 16 mo exhibited mild hyperglycemia in female mice (not in male mice), mild weight gain, and no evidence of bladder dysfunction. Ob/ob mice were followed for 8 mo and became extremely obese. Male and female mice were glucose intolerant, insulin intolerant, and hyperinsulinemic at 4 mo. By 8 mo, their metabolic status had improved but was still abnormal. Urine volume increased in male mice but not in female mice. Bladder dysfunction was observed in the spotting patterns of female mice at 4 and 6 mo of age, resolving by 8 mo. We conclude there are dramatic sex-related differences in lower urinary tract function in these models. Male Akita mice may be a good model for polyuria-related bladder remodeling, whereas female ob/ob mice may better mimic storage problems related to loss of outlet control in a setting of type 2 diabetes complicated by obesity.

Abnormalities in purine availability or purinergic receptor density are commonly seen in patients with lower urinary tract symptoms (LUTS), but the underlying mechanisms relating altered receptor function to LUTS are unknown. Here we provide extensive evidence for the reciprocal interplay of multiple receptors responding to ATP, ADP (adenosine diphosphate), and adenosine, agonists that regulate bladder function significantly. ADP stimulated P2Y12 receptors, causing bladder smooth muscle (BSM) contraction, whereas adenosine signaling through potentially newly defined A2b receptors, actively inhibited BSM purinergic contractility. The modulation of adenylyl cyclase-cAMP signaling via A2b and P2Y12 interaction actively regulated bladder contractility by modulating intracellular calcium levels. KO mice lacking the receptors display diametrically opposed bladder phenotypes, with P2Y12-KO mice exhibiting an underactive bladder (UAB) phenotype with increased bladder capacity and reduced voiding frequency, whereas A2b-KO mice have an overactive bladder (OAB), with decreased capacity and increased voiding frequency. The opposing phenotypes in P2Y12-KO and A2b-KO mice not only resulted from dysregulated BSM contractility, but also from abnormal BSM cell growth. Finally, we demonstrate that i.p. administration of drugs targeting P2Y12 or A2b receptor rescues these abnormal phenotypes in both KO mice. These findings strongly indicate that P2Y12 and A2b receptors are attractive therapeutic targets for human patients with LUTS.

Caloric restriction extends healthy lifespan in multiple species1. Intermittent fasting, an alternative form of dietary restriction, is potentially more sustainable in humans, but its effectiveness remains largely unexplored2-8. Identifying the most efficacious forms of dietary restriction is key for developing interventions to improve human health and longevity9. Here we performed an extensive assessment of graded levels of caloric restriction (20% and 40%) and intermittent fasting (1 and 2 days fasting per week) on the health and survival of 960 genetically diverse female mice. We show that caloric restriction and intermittent fasting both resulted in lifespan extension in proportion to the degree of restriction. Lifespan was heritable and genetics had a larger influence on lifespan than dietary restriction. The strongest trait associations with lifespan included retention of body weight through periods of handling-an indicator of stress resilience, high lymphocyte proportion, low red blood cell distribution width and high adiposity in late life. Health effects differed between interventions and exhibited inconsistent relationships with lifespan extension. 40% caloric restriction had the strongest lifespan extension effect but led to a loss of lean mass and changes in the immune repertoire that could confer susceptibility to infections. Intermittent fasting did not extend the lifespan of mice with high pre-intervention body weight, and two-day intermittent fasting was associated with disruption of erythroid cell populations. Metabolic responses to dietary restriction, including reduced adiposity and lower fasting glucose, were not associated with increased lifespan, suggesting that dietary restriction does more than just counteract the negative effects of obesity. Our findings indicate that improving health and extending lifespan are not synonymous and raise questions about which end points are the most relevant for evaluating aging interventions in preclinical models and clinical trials.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. Among the many mediators implicated in cystitis, the overproduction of reactive oxygen species (ROS) seems to play a key role, although the main source of ROS remains unclear. This study aimed to investigate the contribution of NADPH oxidase (NOX) isoforms in ROS generation and the voiding dysfunction of cyclophosphamide (CYP, 300 mg/Kg, ip, 24 h)-induced cystitis in adult female mice, a well-recognized animal model to study IC/BPS, by using GKT137831 (5 mg/Kg, ip, three times in a 24 h period) or GSK2795039 (5 mg/Kg, ip, three times in a 24 h period) to inhibit NOX1/4 or NOX2, respectively. Our results showed that treatment with GSK2795039 improved the dysfunctional voiding behavior induced by CYP, reduced bladder edema and inflammation, and preserved the urothelial barrier integrity and tight junction occludin expression, besides inhibiting the characteristic vesical pain and bladder superoxide anion generation. In contrast, the NOX1/4 inhibitor GKT137831 had no significant protective effects. Taken together, our in vivo and ex vivo data demonstrate that NOX2 is possibly the main source of ROS observed in cystitis-induced CYP in mice. Therefore, selective inhibition of NOX2 by GSK2795039 may be a promising target for future therapies for IC/BPS.