Browsing by Author "Hughes, Francis M"

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    A three dimensional nerve map of human bladder trigone.
    (Neurourology and urodynamics, 2017-04) Purves, J Todd; Spruill, Laura; Rovner, Eric; Borisko, Elyse; McCants, Alden; Mugo, Elizabeth; Wingard, Ainsley; Trusk, Thomas C; Bacro, Thierry; Hughes, Francis M
    Central efferent and afferent neural pathways to and from the human urinary bladder are well-characterized, but the location and arborization of these nerves as they traverse the serosa, muscularis, and urothelial layers are not clearly defined. The purpose of this study was to create a three dimensional map of the innervation of the human bladder trigone from the extrinsic perivesical adventitial nerve trunks to the urothelium.A male and a female human bladder were harvested from fresh frozen cadavers and fixed in formalin. The bladder neck and trigone region were serially sectioned (5 μm) and every 20th slide was stained (S100), scanned and aligned to create 3D maps.Nerve penetration into the detrusor muscle occurs with the highest frequency at the bladder neck and interureteric ridge. Nerves traveling parallel to the bladder lumen do so in the adventitia, beyond the outer border of detrusor. In females, the depth of these nerve bands is uniform at 0.7-1.7 cm below the luminal surface, the outer limits of which include the anterior vaginal wall. In the male, depth is more variable owing to detrusor hypertrophy with the minimum depth of nerves approximately 0.5 cm near the interureteric ridge and over 1 cm near the bladder neck.Myelinated neural pathways traversing in the human bladder in the region of the trigone have a discreet regional density. This 3D map of trigonal innervation may provide guidance to more precisely direct therapies for urinary incontinence or pelvic pain. Neurourol. Urodynam. 36:1015-1019, 2017. © 2016 Wiley Periodicals, Inc.
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    Bladder fibrosis during outlet obstruction is triggered through the NLRP3 inflammasome and the production of IL-1β.
    (American journal of physiology. Renal physiology, 2017-09) Hughes, Francis M; Sexton, Stephanie J; Jin, Huixia; Govada, Vihasa; Purves, J Todd
    Bladder outlet obstruction (BOO) triggers inflammation in the bladder through the NLRP3 inflammasome. BOO also activates fibrosis, which is largely responsible for the decompensation of the bladder in the chronic state. Because fibrosis can be driven by inflammation, we have explored a role for NLRP3 (and IL-1β produced by NLRP3) in the activation and progression of BOO-induced fibrosis. Female rats were divided into five groups: 1) control, 2) sham, 3) BOO + vehicle, 4) BOO + the NLRP3 inhibitor glyburide, or 5) BOO + the IL-1β receptor antagonist anakinra. Fibrosis was assessed by Masson's trichrome stain, collagen secretion via Sirius Red, and protein localization by immunofluorescence. BOO increased collagen production in the bladder, which was blocked by glyburide and anakinra, clearly implicating the NLRP3/IL-1β pathway in fibrosis. The collagen was primarily found in the lamina propria and the smooth muscle, while IL-1 receptor 1 and prolyl 4-hydroylase (an enzyme involved in the intracellular modification of collagen) both localized to the urothelium and the smooth muscle. Lysyl oxidase, the enzyme involved in the final extracellular assembly of mature collagen fibrils, was found to some extent in the lamina propria where its expression was greatly enhanced during BOO. In vitro studies demonstrated isolated urothelial cells from BOO rats secreted substantially more collagen than controls, and collagen expression in control cultures could be directly stimulated by IL-1β. In summary, NLRP3-derived-IL-1β triggers fibrosis during BOO, most likely through an autocrine loop in which IL-1β acts on urothelia to drive collagen production.
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    Calcium Pyrophosphate And Monosodium Urate Activate The NLRP3 Inflammasome Within Bladder Urothelium Via Reactive Oxygen Species And TXNIP.
    (Research and reports in urology, 2019-01) Harper, Shelby N; Leidig, Patrick D; Hughes, Francis M; Jin, Huixia; Purves, J Todd
    Objective:To investigate the in vitro activation of the NLRP3 inflammasome within bladder urothelium by stone-forming components. Further, to describe the contributions of reactive oxygen species (ROS) and thioredoxin-interacting protein (TXNIP), an important structural component of the inflammasome, to this activation. Methods:Urothelial cells were harvested and incubated overnight. For agonist studies, cells were treated with varying concentrations of calcium pyrophosphate (CPPD) and monosodium urate (MSU). For inhibitor studies, cells were treated with either N-acetylcysteine (NAC) (1 hr) or Verapamil (4 hrs) prior to incubation with either CPPD (62.5 ug/mL) or MSU (1.25 ug/mL) for 24 hrs. Untreated controls were incubated with ATP (1.25 mM) for 1 hr to maximally stimulate NLRP3 inflammasome activity (measured as caspase-1 cleavage of the fluorogenic substrate Ac-YVAD-AFC). Results are reported as a percentage of maximum ATP response. Results:CPPD and MSU activate caspase-1 in urothelial cells in a dose-dependent manner, reaching ~50% and ~25% of the ATP response, respectively. Pre-treatment with the general ROS scavenger NAC reduces this activation in a dose-dependent manner. Additionally, activation was suppressed through treatment with Verapamil, a known downregulator of TXNIP expression. Conclusion:The stone components CPPD and MSU activate NLRP3 in an ROS and TXNIP-dependent manner in bladder urothelium. These findings demonstrate the importance of ROS and TXNIP, and suggest that targeting either may be a way to decrease stone-dependent NLRP3 inflammation within the bladder.
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    Inflammation triggered by the NLRP3 inflammasome is a critical driver of diabetic bladder dysfunction.
    (Frontiers in physiology, 2022-01) Hughes, Francis M; Odom, Michael R; Cervantes, Anissa; Purves, J Todd
    Diabetes is a rapidly expanding epidemic projected to affect as many as 1 in 3 Americans by 2050. This disease is characterized by devastating complications brought about high glucose and metabolic derangement. The most common of these complications is diabetic bladder dysfunction (DBD) and estimates suggest that 50-80% of patients experience this disorder. Unfortunately, the Epidemiology of Diabetes Interventions and Complications Study suggests that strict glucose control does not decrease ones risk for incontinence, although it does decrease the risk of other complications such as retinopathy, nephropathy and neuropathy. Thus, there is a significant unmet need to better understand DBD in order to develop targeted therapies to alleviate patient suffering. Recently, the research community has come to understand that diabetes produces a systemic state of low-level inflammation known as meta-inflammation and attention has focused on a role for the sterile inflammation-inducing structure known as the NLRP3 inflammasome. In this review, we will examine the evidence that NLRP3 plays a central role in inducing DBD and driving its progression towards an underactive phenotype.
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    NLRP3/IL-1β mediates denervation during bladder outlet obstruction in rats.
    (Neurourology and urodynamics, 2018-03) Lütolf, Robin; Hughes, Francis M; Inouye, Brian M; Jin, Huixia; McMains, Jennifer C; Pak, Elena S; Hannan, Johanna L; Purves, J Todd
    Denervation of the bladder is a detrimental consequence of bladder outlet obstruction (BOO). We have previously shown that, during BOO, inflammation triggered by the NLRP3 inflammasome in the urothelia mediates physiological bladder dysfunction and downstream fibrosis in rats. The aim of this study was to assess the effect of NLRP3-mediated inflammation on bladder denervation during BOO.There were five groups of rats: (i) Control (no surgery); (ii) Sham-operated; (iii) BOO rats given vehicle; (iv) BOO rats given the NLRP3 inhibitor glyburide; and (v) BOO rats given the IL-1 receptor antagonist anakinra. BOO was constructed by ligating the urethra over a 1 mm catheter and removing the catheter. Medications were given prior to surgery and once daily for 12 days. Bladder sections were stained for PGP9.5, a pan-neuronal marker. Whole transverse sections were used to identify and count nerves while assessing cross-sectional area. For in vitro studies, pelvic ganglion neurons were isolated and treated with IL-1β. After a 48 h incubation apoptosis, neurite length and branching were assessed.In obstructed bladders, the number of nerves decreased while total area increased, indicating a loss of cell number and/or branching. The decrease in nerve density was blocked by glyburide or anakinra, clearly implicating the NLRP3 pathway in denervation. In vitro analysis demonstrated that IL-1β, a product of the inflammasome, induced apoptosis in pelvic ganglion neurons, suggesting one mechanism of BOO-induced denervation is NLRP3/IL-1β triggered apoptosis.The NLRP3/IL-1β-mediated inflammation pathway plays a significant role in denervation during BOO.
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    The Emerging Role of Inflammasomes as Central Mediators in Inflammatory Bladder Pathology.
    (Current urology, 2018-02) Inouye, Brian M; Hughes, Francis M; Sexton, Stephanie J; Purves, J Todd
    Irritative voiding symptoms (e.g. increased frequency and urgency) occur in many common pathologic conditions such as urinary tract infections and bladder outlet obstruction, and these conditions are well-established to have underlying inflammation that directly triggers these symptoms. However, it remains unclear as to how such diverse stimuli individually generate a common inflammatory process. Jürg Tschopp provided substantial insight into this conundrum when, working with extracts from THP-1 cells, he reported the existence of the inflammasome. He described it as a structure that senses multiple diverse signals from intracellular/extracellular sources and pathogens and triggers inflammation by the maturation and release of the pro-inflammatory cytokines interleukin-1β and interleukin-18. Recently, many of these sensors were found in the bladder and the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, has been shown to be a central mediator of inflammation in several urological diseases. In this review, we introduce the nucleotide-binding domain, leucine-rich-containing family, pyrin domaincontaining-3 inflammasome, highlight its emerging role in several common urologic conditions, and speculate on the potential involvement of other inflammasomes in bladder pathology.
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    The NLRP3 Inflammasome Mediates Inflammation Produced by Bladder Outlet Obstruction.
    (The Journal of urology, 2016-05) Hughes, Francis M; Hill, Hayden M; Wood, Case M; Edmondson, Andrew T; Dumas, Aliya; Foo, Wen-Chi; Oelsen, James M; Rac, Goran; Purves, J Todd
    While bladder outlet obstruction is well established to elicit an inflammatory reaction in the bladder that leads to overactive bladder and fibrosis, little is known about the mechanism by which this is initiated. NLRs (NOD-like receptors) and the structures that they form (inflammasomes) have been identified as sensors of cellular damage, including pressure induced damage, and triggers of inflammation. Recently we identified these structures in the urothelium. In this study we assessed the role of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome in bladder dysfunction resulting from bladder outlet obstruction.Bladder outlet obstruction was created in female rats by inserting a 1 mm outer diameter transurethral catheter, tying a silk ligature around the urethra and removing the catheter. Untreated and sham operated rats served as controls. Rats with bladder outlet obstruction were given vehicle (10% ethanol) or 10 mg/kg glyburide (a NLRP3 inhibitor) orally daily for 12 days. Inflammasome activity, bladder hypertrophy, inflammation and bladder function (urodynamics) were assessed.Bladder outlet obstruction increased urothelial inflammasome activity, bladder hypertrophy and inflammation, and decreased voided volume. Glyburide blocked inflammasome activation, reduced hypertrophy and prevented inflammation. The decrease in voided volume was also attenuated by glyburide mechanistically as an increase in detrusor contraction duration and voiding period.Results suggest the importance of the NLRP3 inflammasome in the induction of inflammation and bladder dysfunction secondary to bladder outlet obstruction. Arresting these processes with NLRP3 inhibitors may prove useful to treat the symptoms that they produce.
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    The potential repertoire of the innate immune system in the bladder: expression of pattern recognition receptors in the rat bladder and a rat urothelial cell line (MYP3 cells).
    (International urology and nephrology, 2015-12) Hughes, Francis M; Turner, David P; Todd Purves, J
    The urothelium is a frontline sensor of the lower urinary tract, sampling the bladder lumen and stimulating an immune response to infectious and noxious agents. Pattern recognition receptors (PRRs) recognize such agents and coordinate the innate response, often by forming inflammasomes that activate caspase-1 and the release of interleukin-1. We have shown the presence of one PRR (NLRP3) in the urothelia and its central role in the inflammatory response to cyclophosphamide. The purpose of this study was to (1) assess the likely range of the PPR response by assessing the repertoire present in the rat bladder and (2) determine the utility of the MYP3 rat urothelia cell line for in vitro studies by assessing its PPR repertoire and functional responsiveness.Immunohistochemistry was performed for seven PPRs (NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4 and AIM2) on bladder sections and MYP3 cells. For functionality, MYP3 cells were challenged with the quintessential NLRP3 activator ATP and assessed for caspase-1 activation.All PPRs examined were expressed in the bladder and localized to the urothelial layer with several also in the detrusor (none in the interstitia). MYP3 cells also expressed all PRRs with a variable intracellular location. ATP-stimulated caspase-1 activity in MYP3 cells in a dose-dependent manner was reduced by knockdown of NLRP3 expression.The results suggest that the bladder possesses the capacity to initiate an innate immune response to a wide array of uropathological agents and the MYP3 cells will provide an excellent investigational tool for this field.
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    Why Are Some People with Lower Urinary Tract Symptoms (LUTS) Depressed? New Evidence That Peripheral Inflammation in the Bladder Causes Central Inflammation and Mood Disorders.
    (International journal of molecular sciences, 2023-02) Hughes, Francis M; Odom, Michael R; Cervantes, Anissa; Livingston, Austin J; Purves, J Todd
    Anecdotal evidence has long suggested that patients with lower urinary tract symptoms (LUTS) develop mood disorders, such as depression and anxiety, at a higher rate than the general population and recent prospective studies have confirmed this link. Breakthroughs in our understanding of the diseases underlying LUTS have shown that many have a substantial inflammatory component and great strides have been made recently in our understanding of how this inflammation is triggered. Meanwhile, studies on mood disorders have found that many are associated with central neuroinflammation, most notably in the hippocampus. Excitingly, work on other diseases characterized by peripheral inflammation has shown that they can trigger central neuroinflammation and mood disorders. In this review, we discuss the current evidence tying LUTS to mood disorders, its possible bidirectionally, and inflammation as a common mechanism. We also review modern theories of inflammation and depression. Finally, we discuss exciting new animal studies that directly tie two bladder conditions characterized by extensive bladder inflammation (cyclophosphamide-induced hemorrhagic cystitis and bladder outlet obstruction) to neuroinflammation and depression. We conclude with a discussion of possible mechanisms by which peripheral inflammation is translated into central neuroinflammation with the resulting psychiatric concerns.