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1.
Metabolites ; 13(6)2023 May 31.
Article in English | MEDLINE | ID: mdl-37367868

ABSTRACT

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.

2.
Antioxidants (Basel) ; 12(1)2022 Dec 30.
Article in English | MEDLINE | ID: mdl-36670953

ABSTRACT

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.

3.
Function (Oxf) ; 3(5): zqac042, 2022.
Article in English | MEDLINE | ID: mdl-38989038

ABSTRACT

The bladder undergoes large shape changes as it fills and empties and experiences complex mechanical forces. These forces become abnormal in diseases of the lower urinary tract such as overactive bladder, neurogenic bladder, and urinary retention. As the primary mechanosensors linking the actin cytoskeleton to the extracellular matrix (ECM), integrins are likely to play vital roles in maintaining bladder smooth muscle (BSM) homeostasis. In a tamoxifen-inducible smooth muscle conditional knockout of ß1-integrin, there was concomitant loss of α1- and α3-integrins from BSM and upregulation of αV- and ß3-integrins. Masson's staining showed a reduction in smooth muscle with an increase in collagenous ECM. Functionally, mice exhibited a changing pattern of urination by voiding spot assay up to 8 wk after tamoxifen. By 8 wk, there was increased frequency with reductions in voided volume, consistent with overactivity. Cystometrograms confirmed that there was a significant reduction in intercontractile interval with reduced maximal bladder pressure. Muscle strip myography revealed a loss of contraction force in response to electrical field stimulation, that was entirely due to the loss of muscarinic contractility. Quantitative western blotting showed a loss of M3 receptor and no change in P2X1. qPCR on ECM and interstitial genes revealed loss of Ntpd2, a marker of an interstitial cell subpopulation; and an upregulation of S100A4, which is often associated with fibroblasts. Collectively, the data show that the loss of appropriate mechanosensation through integrins results in cellular and extracellular remodeling, and concomitant bladder dysfunction that resembles lower urinary tract symptoms seen in older people.

4.
Urology ; 154: 344.e1-344.e7, 2021 08.
Article in English | MEDLINE | ID: mdl-34010680

ABSTRACT

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.


Subject(s)
Bacterial Load , Kidney/microbiology , Urinary Bladder/microbiology , Urinary Tract Infections/microbiology , Urinary Tract Infections/physiopathology , Urinary Tract Infections/urine , Urination , Animals , Correlation of Data , Disease Models, Animal , Female , Mice , Mice, Inbred C57BL , Urine/microbiology
5.
FASEB J ; 35(4): e21447, 2021 04.
Article in English | MEDLINE | ID: mdl-33742688

ABSTRACT

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 ß1-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.


Subject(s)
Disease Models, Animal , Urinary Bladder/pathology , Urinary Retention/pathology , Animals , Biomechanical Phenomena , Female , Gene Expression Regulation , Mice , Muscle Contraction , Muscle, Smooth/pathology , Urinary Bladder/injuries , Urinary Bladder/metabolism , Urinary Retention/metabolism , Urodynamics
6.
Urology ; 141: 188.e1-188.e6, 2020 07.
Article in English | MEDLINE | ID: mdl-32201154

ABSTRACT

OBJECTIVES: To analyze factors during early stage of urinary tract infection (UTI) that are associated with development of chronic UTI. METHODS: Mice were inoculated with Uropathogenic Escherichia coli (UPEC) 2 times 24 hours apart. At 1, 3, 7, 10, 14, 21 and 28 days post infection (dpi), urine bacterial loads and voiding behavior (voiding spot assay, VSA) were measured. At 1 and 28 dpi, 32 urine inflammatory cytokines/chemokines were measured using enzyme-linked immunosorbent assay (ELISA). Bladder and kidney cytokines/chemokines were measured on 28 dpi. Mice that had no more than 1 episode of urine bacterial load < 104 colony forming unit/ml during the entire 4 weeks were defined as susceptible to chronic UTI, otherwise, mice were considered resistant. RESULTS: At 28 dpi, 64.3% mice developed chronic UTI (susceptible group) and 35.7% mice did not (resistant group). Factors at 1 dpi that were predictive of chronic UTI included increased urine IL-2 (OR 11.9, 95%CI 1.1-130.8, P = .043) and increased urine IL-10 (OR 14.0, 95%CI 1.0-201.2, P = .052). At 28 dpi, there were several significant differences between the susceptible vs resistant groups including urine/tissue bacterial loads and certain urine/tissue cytokines/chemokines. CONCLUSIONS: Higher urine IL-2 and IL-10 at 1 dpi predicted chronic UTI infection in this model. There have been recent publications associating both of these cytokines to UTI susceptibility. Further explorations into IL-2 and IL-10 mediated pathways could shed light on the biology of recurrent and chronic UTI which are difficult to treat.


Subject(s)
Interleukin-10/urine , Interleukin-2/urine , Urinary Tract Infections/urine , Animals , Bacterial Load , Biomarkers/urine , Chemokines/urine , Chronic Disease , Disease Models, Animal , Disease Progression , Escherichia coli Infections/complications , Female , Mice , Mice, Inbred C57BL , Urinary Tract Infections/microbiology , Urination , Urine/microbiology
7.
Am J Physiol Renal Physiol ; 318(1): F160-F174, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31682171

ABSTRACT

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.


Subject(s)
Diabetes Mellitus, Type 1/complications , Obesity/complications , Urinary Bladder/physiopathology , Urologic Diseases/etiology , Animals , Body Weight/physiology , Diabetes Mellitus, Type 1/physiopathology , Disease Models, Animal , Female , Insulin Resistance/physiology , Male , Mice , Obesity/physiopathology , Urologic Diseases/physiopathology
8.
JCI Insight ; 4(16)2019 08 22.
Article in English | MEDLINE | ID: mdl-31434806

ABSTRACT

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.


Subject(s)
Receptor, Adenosine A2B/physiology , Receptors, Purinergic P2Y12/physiology , Urinary Bladder/physiology , Animals , Cells, Cultured , Female , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Muscle Contraction , Muscle, Smooth/physiology , Pregnancy , Signal Transduction , Urinary Bladder Diseases/metabolism
9.
Am J Physiol Renal Physiol ; 315(5): F1422-F1429, 2018 11 01.
Article in English | MEDLINE | ID: mdl-30156116

ABSTRACT

Investigators have for decades used mouse voiding patterns as end points for studying behavioral biology. It is only recently that mouse voiding patterns were adopted for study of lower urinary tract physiology. The spontaneous void spot assay (VSA), a popular micturition assessment tool, involves placing a mouse in an enclosure lined by filter paper and quantifying the resulting urine spot pattern. The VSA has advantages of being inexpensive and noninvasive, but some investigators challenge its ability to distinguish lower urinary tract function from behavioral voiding. A consensus group of investigators who regularly use the VSA was established by the National Institutes of Health in 2015 to address the strengths and weaknesses of the assay, determine whether it can be standardized across laboratories, and determine whether it can be used as a surrogate for evaluating urinary function. Here we leverage experience from the consensus group to review the history of the VSA and its uses, summarize experiments to optimize assay design for urinary physiology assessment, and make best practice recommendations for performing the assay and analyzing its results.


Subject(s)
Biological Assay/methods , Urinary Bladder/physiopathology , Urination Disorders/physiopathology , Urination , Urodynamics , Animals , Biological Assay/standards , Disease Models, Animal , Mice , Reproducibility of Results , Time Factors , Urination Disorders/diagnosis
10.
Am J Physiol Renal Physiol ; 315(4): F1067-F1080, 2018 10 01.
Article in English | MEDLINE | ID: mdl-29972322

ABSTRACT

Mouse urinary behavior is quantifiable and is used to pinpoint mechanisms of voiding dysfunction and evaluate potential human therapies. Approaches to evaluate mouse urinary function vary widely among laboratories, however, complicating cross-study comparisons. Here, we describe development and multi-institutional validation of a new tool for objective, consistent, and rapid analysis of mouse void spot assay (VSA) data. Void Whizzard is a freely available software plugin for FIJI (a distribution of ImageJ) that facilitates VSA image batch processing and data extraction. We describe its features, demonstrate them by evaluating how specific VSA method parameters influence voiding behavior, and establish Void Whizzard as an expedited method for VSA analysis. This study includes control and obese diabetic mice as models of urinary dysfunction to increase rigor and ensure relevance across distinct voiding patterns. In particular, we show that Void Whizzard is an effective tool for quantifying nonconcentric overlapping void spots, which commonly confound analyses. We also show that mouse genetics are consistently more influential than assay design parameters when it comes to VSA outcomes. None of the following procedural modifications to reduce overlapping spots masked these genetic-related differences: reduction of VSA testing duration, water access during the assay period, placement of a wire mesh cage bottom on top of or elevated over the filter paper, treatment of mesh with a hydrophobic spray, and size of wire mesh opening. The Void Whizzard software and rigorous validation of VSA methodological parameters described here advance the goal of standardizing mouse urinary phenotyping for comprehensive urinary phenome analyses.


Subject(s)
Diabetes Mellitus, Experimental/physiopathology , Software , Urination/physiology , Urodynamics/physiology , Animals , Goals , Male , Mice, Transgenic , Urinary Bladder/physiopathology
11.
Neurourol Urodyn ; 37(8): 2398-2405, 2018 11.
Article in English | MEDLINE | ID: mdl-29682797

ABSTRACT

AIMS: Symptoms from overactive bladder (OAB) and cystitis secondary to urinary tract infection (UTI) can be similar in post-menopausal women. Effects of ovariectomy (OVX) on voiding behavior after lipopolysaccharide (LPS) intravesical exposure (surrogate for cystitis) in mice were measured. Urothelial genes associated with micturition changes were identified. METHODS: Female C57BL6/J mice underwent OVX or sham surgeries (n = 10 for each). Voiding spot assays (VSA) were performed prior to surgery, 4 weeks post-surgery, and each time after 3 consecutive days of transurethral instillation of LPS. In another experiment, mice underwent either sham (n = 9) or OVX (n = 9) surgeries. Urothelial RNAs were collected 4 weeks post-surgery, day 1 and day 3 after LPS instillation. Mouse Gene 2.0 ST Arrays (entire 34 K transcripts) were used for microarray hybridization. A set of criteria was utilized to identify gene expression changes that mimicked voiding behavior changes. RESULTS: Three days after LPS exposure, OVX mice persisted with overactive whereas sham mice normalized voiding behavior. Nine urothelial paralleling voiding behavior changes were identified: IL6 (interleukin 6), IL6rα (Interleukin 6 receptor α), Ptgs2 (Prostaglandin-endoperoxide synthase 2 or COX-2), Ereg (epiregulin), Dusp6 (dual specificity phosphatase 6), Zfp948 (zinc finger protein 948), Zfp52 (Zinc finger protein 52), Gch1 (GTP cyclohydrolase 1), and Amd (S-adenosylmethionine decarboxylase). Three other genes, coding unknown proteins, were also identified: GM12840, GM23134, and GM26809. CONCLUSIONS: OVX mice persisted with increased voiding frequency after LPS. Urothelial genes that could mediate this voiding behavior include IL6, COX-2, and S-adenosylmethionine decarboxylase.


Subject(s)
Gene Expression/physiology , Lipopolysaccharides/pharmacology , Ovariectomy , Urinary Bladder/drug effects , Urination/genetics , Urothelium/metabolism , Animals , Behavior, Animal , Female , Gene Expression/drug effects , Mice , Mice, Inbred C57BL , Microarray Analysis , RNA/biosynthesis , RNA/genetics , Urinary Bladder, Overactive/chemically induced , Urinary Bladder, Overactive/genetics , Urination/drug effects , Urination/physiology
12.
Sci Rep ; 8(1): 1838, 2018 01 30.
Article in English | MEDLINE | ID: mdl-29382907

ABSTRACT

Purinergic signalling plays an important role in the regulation of bladder smooth muscle (BSM) contractility, and P2X4 receptor is expressed in the bladder wall, where it may act by forming heteromeric receptors with P2X1, the major purinergic force-generating muscle receptor. To test this hypothesis, we examined mouse BSM contractile properties in the absence and presence of selective P2X1 (NF449 & NF279) and P2X4 antagonists (5-BDBD). These drugs inhibited BSM purinergic contraction only partially, suggesting the possibility of a heteromeric receptor. However, carefully controlled co-immunoprecipitation experiments indicated that P2X1 and P2X4 do not form physically linked heteromers. Furthermore, immunofluorescence staining showed that P2X4 is not present in mouse BSM per se, but in an unknown cellular structure among BSM bundles. To investigate whether deletion of P2X4 could impact voiding function in vivo, P2X4 null mice were characterized. P2X4 null mice had normal bladder weight and morphology, normal voiding spot size and number by voiding spot assay, normal voiding interval, pressure and compliance by cystometrogram, and normal BSM contractility by myography. In conclusion, these data strongly suggest that P2X4 is not present in mouse BSM cells, does not affect smooth muscle contractility and that mice null for P2X4 exhibit normal voiding function.


Subject(s)
Receptors, Purinergic P2X4/metabolism , Animals , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Contraction/physiology , Muscle, Smooth/metabolism , Myocytes, Smooth Muscle/metabolism , Urinary Bladder/metabolism
13.
Am J Physiol Renal Physiol ; 313(6): F1274-F1280, 2017 Dec 01.
Article in English | MEDLINE | ID: mdl-28835420

ABSTRACT

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.


Subject(s)
Behavior, Animal/physiology , Urination/physiology , Animals , Environment , Female , Mice , Models, Animal , Stress, Psychological , Urinary Bladder/physiopathology , Urodynamics/genetics , Urodynamics/physiology
15.
Article in English | MEDLINE | ID: mdl-28553656

ABSTRACT

OBJECTIVES: To investigate the effect of changing the bladder filling rate during cystometry in younger (2-3 months) and older (13-14 months) C57BL/6J male mice. METHODS: Cystometry was performed on mice under anesthesia. Voiding cycles were established in each mouse at a pump delivery rate of 17 µl/min. After 30 min, the rate was increased sequentially to 25, 33, 41 and 49 µl/min. Each rate was maintained for 30 min. The following cystometric parameters were quantified: peak pressure amplitude, intercontractile interval (ICI), compliance, micturition pressure threshold and voiding efficiency. RESULTS: Bladder weights were significantly greater in older mice (42 mg vs. 27 mg, P < 0.01), but functional capacities were not different. The pressure amplitudes did not change as filling rate increased, nor did they differ between the 4-month and 13-month-old males. ICIs were not significantly different between young and mature mice. However, both groups exhibited a non-linear reduction in ICI with increasing filling rate, best described by a power curve (R2 > 0.93). Compliance was higher in the older mice at low filling rates (17 and 25 µl/min) but this difference diminished at higher rates. Compliance decreased with increasing flow rate in a non-linear manner, again with greater effects at low filling rates. Micturition pressure thresholds increased with increasing flow rate in a linear manner and older mice began voiding at higher pressures than younger. Both young and old mice exhibited voiding efficiencies of ~70%. CONCLUSIONS: The rate of volume delivery has complex effects on the timing of voiding and compliance. These findings argue for greater standardization of cystometry protocols and further investigation into afferent signaling to higher centers at different filling rates.

16.
Development ; 144(3): 400-408, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28049658

ABSTRACT

Urothelium is the protective lining of the urinary tract. The mechanisms underlying urothelial formation and maintenance are largely unknown. Here, we report the stage-specific roles of PRC2 epigenetic regulators in embryonic and adult urothelial progenitors. Without Eed, the obligatory subunit of PRC2, embryonic urothelial progenitors demonstrate reduced proliferation with concomitant dysregulation of genes including Cdkn2a (p16), Cdkn2b (p15) and Shh. These mutants display premature differentiation of keratin 5-positive (Krt5+) basal cells and ectopic expression of squamous-like differentiation markers. Deletion of Ezh2, the major enzymatic component of PRC2, causes upregulation of Upk3a+ superficial cells. Unexpectedly, Eed and Eed/Ezh2 double mutants exhibit delayed superficial cell differentiation. Furthermore, Eed regulates the proliferative and regenerative capacity of adult urothelial progenitors and prevents precocious differentiation. Collectively, these findings uncover the epigenetic mechanism by which PRC2 controls urothelial progenitor cell fate and the timing of differentiation, and further suggest an epigenetic basis of urothelial maintenance and regeneration.


Subject(s)
Polycomb Repressive Complex 2/physiology , Regeneration/physiology , Urinary Bladder/growth & development , Urinary Bladder/physiology , Urothelium/growth & development , Urothelium/physiology , Adult Stem Cells/cytology , Adult Stem Cells/physiology , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Epigenesis, Genetic , Female , Gene Expression Regulation, Developmental , Hedgehog Proteins/genetics , Hedgehog Proteins/physiology , Male , Mice , Mice, Knockout , Mice, Transgenic , Polycomb Repressive Complex 2/chemistry , Polycomb Repressive Complex 2/deficiency , Polycomb Repressive Complex 2/genetics , Protein Subunits , Regeneration/genetics , Urinary Bladder/embryology , Urothelium/embryology
17.
Curr Protoc Mouse Biol ; 5(2): 95-133, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-26069080

ABSTRACT

Despite the dramatic increase in human lifespan over the past century, there remains pronounced variability in "health-span," or the period of time in which one is generally healthy and free of disease. Much of the variability in health-span and lifespan is thought to be genetic in origin. Understanding the genetic mechanisms of aging and identifying ways to boost longevity is a primary goal in aging research. Here, we describe a pipeline of phenotypic assays for assessing mouse models of aging. This pipeline includes behavior/cognition testing, body composition analysis, and tests of kidney function, hematopoiesis, and immune function, as well as physical parameters. We also describe study design methods for assessing lifespan and health-span, and other important considerations when conducting aging research in the laboratory mouse. The tools and assays provided can assist researchers with understanding the correlative relationships between age-associated phenotypes and, ultimately, the role of specific genes in the aging process.


Subject(s)
Aging , Disease Models, Animal , Laboratory Animal Science/methods , Mice , Aging/physiology , Aging/psychology , Animal Structures/physiology , Animals , Behavior, Animal , Humans , Longevity , Mice/genetics , Mice/physiology , Research Design
18.
Am J Physiol Renal Physiol ; 308(12): F1369-78, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25904700

ABSTRACT

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.


Subject(s)
Urethane/analysis , Urinary Bladder/physiology , Urination/genetics , Urodynamics/genetics , Animals , Female , Male , Mice , Mice, Inbred NOD , Reproducibility of Results , Sex Factors , Urination/physiology , Urodynamics/physiology
20.
Clin J Am Soc Nephrol ; 10(3): 480-92, 2015 Mar 06.
Article in English | MEDLINE | ID: mdl-24742475

ABSTRACT

Urine differs greatly in ion and solute composition from plasma and contains harmful and noxious substances that must be stored for hours and then eliminated when it is socially convenient to do so. The urinary tract that handles this output is composed of a series of pressurizable muscular compartments separated by sphincteric structures. With neural input, these structures coordinate the delivery, collection, and, ultimately, expulsion of urine. Despite large osmotic and chemical gradients in this waste fluid, the bladder maintains a highly impermeable surface in the face of a physically demanding biomechanical environment, which mandates recurring cycles of surface area expansion and increased wall tension during filling, followed by rapid wall compression during voiding. Afferent neuronal inflow from mucosa and submucosa communicates sensory information about bladder fullness, and voiding is initiated consciously through coordinated central and spinal efferent outflow to the detrusor, trigonal internal sphincter, and external urethral sphincter after periods of relative quiescence. Provocative new findings suggest that in some cases, lower urinary tract symptoms, such as incontinence, urgency, frequency, overactivity, and pain may be viewed as a consequence of urothelial defects (either urothelial barrier breakdown or inappropriate signaling from urothelial cells to underlying sensory afferents and potentially interstitial cells). This review describes the physiologic and anatomic mechanisms by which urine is moved from the kidney to the bladder, stored, and then released. Relevant clinical examples of urinary tract dysfunction are also discussed.


Subject(s)
Ureter/physiology , Urethra/physiology , Urinary Bladder/anatomy & histology , Urinary Bladder/physiology , Urination/physiology , Urothelium/physiology , Humans , Interstitial Cells of Cajal/physiology , Muscle Contraction , Muscle, Smooth/physiology , Neural Pathways , Ureter/anatomy & histology , Urethra/anatomy & histology , Urinary Bladder/innervation
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