Strict glucose control and elimination of NLRP3-induced inflammation prevents diabetic bladder dysfunction in the female Akita mouse model.

PURPOSE: Diabetic bladder dysfunction (DBD) is the most common diabetic complication. Logically, regulation of blood glucose should reverse dysfunction, but the Epidemiology of Diabetes Interventions and Complications study found strict control ineffective. However, it is possible that strict control may prevent DBD if initiated before symptoms appear. We examine the effect of early glucose control on development of DBD in the female diabetic Akita mouse (Type 1) and test the potential of inhibiting/deleting NLRP3 as adjunct therapy to glucose control. MATERIALS AND METHODS: Female Akita mice were bred NLRP3+/+ or NLRP3-/-. At 6 weeks of age, diabetics received either no glucose control or insulin pellets (s.c., Linshin) designed to poorly or strictly control blood glucose. At Week 15, blood glucose (glucometer), the extravasation potential of bladder (an indirect measurement of inflammation) and bladder function (urodynamics) were assessed. RESULTS: Blood glucose of diabetics was reduced in poorly controlled and strongly reduced in strictly controlled groups. Levels were not affected by deletion of NLRP3. Evans blue dye extravasation correlated with glucose control and was eliminated in the NLRP3-/- groups. Urodynamics found markers of overactivity in diabetics which was improved in the poorly controlled group and eliminated in the strictly controlled group. In the NLRP3-/- mice, no bladder dysfunction developed, regardless of glucose control. CONCLUSIONS: Early-initiated strict glycemic control and NLRP3 elimination can effectively prevent DBD, suggesting hyperglycemia acts through NLRP3-induced inflammation to trigger DBD.

Specialized pro-resolution mediators in the bladder: effects of resolvin E1 on diabetic bladder dysfunction in the type 1 diabetic male Akita mouse model.

BACKGROUND: One of the most common, but least studied, diabetic complication is diabetic bladder dysfunction. Current therapies include glucose control and symptom-based interventions. However, efficacy of these therapies is mixed and often have undesirable side effects. Diabetes is now known to be a chronic inflammatory disease. Specialized pro-resolving mediators are a class of compounds that promote the resolution of inflammation and have been shown to be effective in treating chronic inflammatory conditions. In this study we examine the ability of resolvin E1 to improve signs of diabetic bladder dysfunction. METHODS: Male Akita mice (Type 1 diabetic) develop hyperglycemia at 4 weeks and signs of bladder underactivity by 15 weeks. Starting at 15 weeks, mice were given one or two weeks of daily resolvin E1 and compared to age-matched wild type and untreated Akita mice. RESULTS: Resolvin E1 did not affect diabetic blood glucose after one week, although there was a slight decrease after two weeks. Diabetes decreased body weight and increased bladder weights and this was not affected by resolvin E1. Evan's blue dye extravasation (an indirect index of inflammation) was dramatically suppressed after one week of resolvin E1 treatment, but, surprisingly, had returned to diabetic levels after two weeks of treatment. Using cystometry, untreated Akita mice showed signs of underactivity (increased void volumes and intercontraction intervals). One week of resolvin E1treatment restored these cystometric findings back to control levels. After two weeks of treatment, cystometric changes were changed from controls but still significantly different from untreated levels, indicating a durable treatment effect even in the presence of increased inflammation at 2 weeks. CONCLUSIONS: Resolvin E1 has a beneficial effect on diabetic bladder dysfunction in the type 1 diabetic male Akita mouse model.

Male Akita mice develop signs of bladder underactivity independent of NLRP3 as a result of a decrease in neurotransmitter release from efferent neurons.

Diabetic bladder dysfunction (DBD) is a prevalent diabetic complication that is recalcitrant to glucose control. Using the Akita mouse model (type 1) bred to be NLR family pyrin domain containing 3 (NLRP3)+/+ or NLRP3-/-, we have previously found that females (mild hyperglycemia) progress from an overactive to underactive bladder phenotype and that this progression was dependent on NLRP3-induced inflammation. Here, we examined DBD in the male Akita mouse (severe hyperglycemia) and found by urodynamics only a compensated underactive-like phenotype (increased void volume and decreased frequency but unchanged efficiency). Surprisingly, this phenotype was still present in the NLRP3-/- strain and so was not dependent on NLRP3 inflammasome-induced inflammation. To examine the cause of the compensated underactive-like phenotype, we assessed overall nerve bundle density and afferent nerve bundles (Aδ-fibers). Both were decreased in density during diabetes, but denervation was absent in the diabetic NLRP3-/- strain so it was deemed unlikely to cause the underactive-like symptoms. Changes in bladder smooth muscle contractility to cell depolarization and receptor activation were also not responsible as KCl (depolarizing agent), carbachol (muscarinic agonist), and α,ß-methylene-ATP (purinergic agonist) elicited equivalent contractions in denuded bladder strips in all groups. However, electrical field stimulation revealed a diabetes-induced decrease in contractility that was not blocked in the NLRP3-/- strain, suggesting that the bladder compensated underactive-like phenotype in the male Akita mouse is likely through a decrease in efferent neurotransmitter release.NEW & NOTEWORTHY In this study, we show that diabetic bladder dysfunction (the most common diabetic complication) manifests through different mechanisms that may be related to severity of hyperglycemia and/or sex. Male Akita mice, which have severe hyperglycemia, develop bladder underactivity as a result of a decrease in efferent neurotransmitter release that is independent of inflammation. This contrasts with females, who have milder hyperglycemia, where diabetic bladder dysfunction progresses from overactivity to underactivity in an inflammation-dependent manner.

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.

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.

Diabetes causes NLRP3-dependent barrier dysfunction in mice with detrusor overactivity but not underactivity.

Approximately half of the patients with diabetes develop diabetic bladder dysfunction (DBD). The initiation and progression of DBD is largely attributed to inflammation due to dysregulated glucose and the production of toxic metabolites that activate the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome. NLRP3 activation leads to the production and release of proinflammatory cytokines and causes urothelial pyroptosis, a form of programmed cell necrosis, which we hypothesize compromises urothelial barrier integrity. Here, we investigated how NLRP3-dependent inflammation impacts barrier function during the progression of diabetes using a type 1 diabetic female Akita mouse model that progresses from an early overactive to a late underactive detrusor phenotype at 15 and 30 wk, respectively. To determine the specific role of NLRP3, Akita mice were crossbred with mice lacking the NLRP3 gene. To determine barrier function, permeability to small molecules was assessed, ex vivo using Evans blue dye and in vivo using sulfo-NHS-biotin. Both ex vivo and in vivo permeabilities were increased in diabetic mice at 15 wk. Expression of uroplakin and tight junction components was also significantly downregulated at 15 wk. Interestingly, diabetic mice lacking the NLRP3 gene showed no evidence of barrier damage or downregulation of barrier genes and proteins. At the 30-wk time point, ex vivo and in vivo barrier damage as well as barrier component downregulation was no longer evident in diabetic mice, suggesting urothelial repair or remodeling occurs between the overactive and underactive stages of DBD. Collectively, these findings demonstrate the role of NLRP3-mediated inflammation in urothelial barrier damage associated with detrusor overactivity but not underactivity.NEW & NOTEWORTHY This is the first study to demonstrate that NLRP3-mediated inflammation is responsible for urothelial barrier damage in type 1 diabetic female Akita mice with an overactive bladder. Eliminating the NLRP3 gene in these diabetic mice prevented barrier damage as a result of diabetes. By the time female Akita mice develop an underactive phenotype, the urothelial barrier has been restored, suggesting that inflammation is a critical causative factor early in the development of diabetic bladder dysfunction.

A Multidimensional Bioinformatic Platform for the Study of Human Response to Surgery.

OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

Specialized proresolution mediators in the bladder: annexin-A1 normalizes inflammation and bladder dysfunction during bladder outlet obstruction.

Bladder outlet obstruction (BOO) is ultimately experienced by ≈90% of men, most commonly secondary to benign prostatic hyperplasia. Inflammation is a critical driver of BOO pathology in the bladder and can be divided into two critical steps: initiation and resolution. Although great strides have been made toward understanding the initiation of inflammation in the bladder [through the NLR family pyrin domain containing 3 (NLRP3) inflammasome], no studies have examined resolution. Resolution is controlled by five classes of compounds known as specialized proresolving mediators (SPMs), all of which bind to one or more of the seven different receptors. Using immunocytochemistry, we showed the presence of six of the known SPM receptors in the bladder of control and BOO rats; the seventh SPM receptor has no rodent homolog. Expression was predominantly localized to urothelia, often with some expression in smooth muscle, but little to none in interstitial cells. We next examined the therapeutic potential of the annexin-A1 resolution system, also present in control and BOO bladders. Using the peptide mimetic Ac2-26, we blocked inflammation-initiating pathways (NLRP3 activation), diminished BOO-induced inflammation (Evans blue dye extravasation), and normalized bladder dysfunction (urodynamics). Excitingly, Ac2-26 also promoted faster and more complete functional recovery after surgical deobstruction. Together, the results demonstrate that the bladder expresses a wide variety of potential proresolving pathways and that modulation of just one of these pathways can alleviate many detrimental aspects of BOO and speed recovery after deobstruction. This work establishes a precedent for future studies evaluating SPM effectiveness in resolving the many conditions associated with bladder inflammation.NEW & NOTEWORTHY To our knowledge, this is the first study of proinflammation-resolving pathways in the bladder, which is the basis of a new pharmacological genus-dubbed "resolution pharmacology" aimed at reducing inflammation without creating an immunocompromised state. Inflammation plays a causative or exacerbating role in numerous bladder maladies. We documented proresolution receptors in the rat bladder and the effectiveness of a specialized proresolving mediator, annexin-A1, in alleviating detrimental aspects of bladder outlet obstruction and speeding recovery after deobstruction.

Cyclophosphamide-induced cystitis results in NLRP3-mediated inflammation in the hippocampus and symptoms of depression in rats.

Recent breakthroughs demonstrate that peripheral diseases can trigger inflammation in the brain, causing psychosocial maladies, including depression. While few direct studies have been made, anecdotal reports associate urological disorders with mental dysfunction. Thus, we investigated if insults targeted at the bladder might elicit behavioral alterations. Moreover, the mechanism of neuroinflammation elicited by other peripheral diseases involves the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which is present in microglia in the brain and cleaves and activates proinflammatory cytokines such as IL-1ß. Thus, we further explored the importance of NLRP3 in behavioral and neuroinflammatory changes. Here, we used the well-studied cyclophosphamide (CP)-treated rat model. Importantly, CP and its metabolites do not cross the blood-brain barrier or trigger inflammation in the gut, so that any neuroinflammation is likely secondary to bladder injury. We found that CP triggered an increase in inflammasome activity (caspase-1 activity) in the hippocampus but not in the pons. Evans blue extravasation demonstrated breakdown of the blood-brain barrier in the hippocampal region and activated microglia were present in the fascia dentata. Both changes were dependent on NLRP3 activation and prevented with 2-mercaptoethane sulfonate sodium (Mesna), which masks the effects of the CP metabolite acrolein in the urine. Finally, CP-treated rats displayed depressive symptoms that were prevented by NLRP3 inhibition or treatment with Mesna or an antidepressant. Thus, we conclude that CP-induced cystitis causes NLRP3-dependent hippocampal inflammation leading to depression symptoms in rats. This study proposes the first-ever causative explanation of the previously anecdotal link between benign bladder disorders and mood disorders.

A possible mechanism underlying mood disorders associated with LUTS: Chronic bladder outlet obstruction causes NLRP3-dependent inflammation in the hippocampus and depressive behavior in rats.

AIMS: Reports link urinary dysfunction and mood disorders, such as depression, but a causative mechanism has never been postulated. Contemporary discoveries demonstrate a local inflammatory response in peripheral organs can trigger inflammation in the brain, particularly the hippocampus, mediated through the NLRP3 inflammasome. Critically, central inflammation causes depressive behavior. Since bladder outlet obstruction (BOO) evokes a local inflammatory response in the bladder, we hypothesize it will induce NLRP3-dependent inflammation in the hippocampus and depressive behavior. METHODS: There were four groups of rats: control, sham, BOO, or BOO + glyburide (an NLRP3 inhibitor). BOO was created by urethral ligation over a 1 mm catheter. Sham was tied loosely. Glyburide was provided by slow-release pellet (subcutaneous 50 mg, 21 day, replaced as needed). Rats were analyzed 12 weeks post-op for: hippocampal inflammation, microglial density, neurogenesis, and depression symptoms (open field and sucrose preference). RESULTS: BOO elicited hippocampal inflammation, accompanied by an increase in activated microglia (22%) and a decrease in neurogenesis (35%), which was blocked by glyburide. In addition, BOO rats displayed anxiety (57% decrease in exploratory behavior in the open field assay) and anhedonia (21% decrease in sucrose preference), two symptoms of depression. Like inflammation, these symptoms were diminished by glyburide to levels not statistically significantly different from controls. CONCLUSIONS: BOO, a bladder-localized event, stimulates NLRP3-dependent inflammation in the rat hippocampus after 12 weeks and this inflammation causes depressive behavior. This is the first mechanistic explanation of the link between BOO and depression and provides evidence for a distinct bladder-brain axis.

Bladder decompensation and reduction in nerve density in a rat model of chronic bladder outlet obstruction are attenuated with the NLRP3 inhibitor glyburide.

Bladder outlet obstruction (BOO) leads to progressive voiding dysfunction. Acutely, obstruction triggers inflammation that drives bladder dysfunction. Over time, inflammation leads to decreased bladder nerve density and increased fibrosis, responsible for eventual decompensation and irreversibility. We have previously shown that BOO triggers inflammation, reduced bladder nerve density and increased fibrosis via activation of the NLRP3 inflammasome in an acutely obstructed (12-day) rat model. However, as BOO progresses, the bladder may become decompensated with an increase in postvoid residual volume and decreased voiding efficiency. Currently, we have examined rat bladder function and nerve densities after chronic BOO to determine whether NLRP3 plays a role in the decompensation at this stage. Four groups were examined: control, sham-operated, BOO, or BOO+gly (glyburide; an NLRP3 inhibitor). After 42 days, bladder weight, inflammation (Evans blue), urodynamics, and nerve density were measured. BOO greatly enhanced bladder weights and inflammation, while inflammation was prevented by glyburide. Voiding pressures were increased, and flow rates decreased in BOO and BOO+gly groups, demonstrating physical obstruction. No difference in frequency or voided volume was detected. However, postvoid residual volumes were greatly increased in BOO rats while BOO+gly rats were not different than controls. Moreover, there was a dramatic decrease in voiding efficiency in the chronic BOO rats, which was prevented with glyburide treatment. Finally, a reduction in nerve density was apparent with BOO and attenuated with glyburide. Together the results suggest a critical role for NLRP3 in mediating bladder decompensation and nerve density during chronic BOO.

NLRP3/IL-1ß mediates denervation during bladder outlet obstruction in rats.

AIMS: 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. METHODS: 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-1ß. After a 48 h incubation apoptosis, neurite length and branching were assessed. RESULTS: 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-1ß, a product of the inflammasome, induced apoptosis in pelvic ganglion neurons, suggesting one mechanism of BOO-induced denervation is NLRP3/IL-1ß triggered apoptosis. CONCLUSIONS: The NLRP3/IL-1ß-mediated inflammation pathway plays a significant role in denervation during BOO.

Bladder fibrosis during outlet obstruction is triggered through the NLRP3 inflammasome and the production of IL-1ß.

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-1ß 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-1ß 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-1ß 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-1ß. In summary, NLRP3-derived-IL-1ß triggers fibrosis during BOO, most likely through an autocrine loop in which IL-1ß acts on urothelia to drive collagen production.

A three dimensional nerve map of human bladder trigone.

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Inflammasomes in the urinary tract: a disease-based review.

Inflammasomes are supramolecular structures that sense molecular patterns from pathogenic organisms or damaged cells and trigger an innate immune response, most commonly through production of the proinflammatory cytokines IL-1ß and IL-18, but also through less understood mechanisms independent of these cytokines. Great strides have been made in understanding these structures and their dysfunction in various inflammatory diseases, lending new insights into urological and renal problems. From a clinical perspective, benign urinary pathology almost universally involves the inflammatory process, and understanding how inflammasomes translate etiological conditions (diabetes, obstruction, stones, urinary tract infections, etc.) into acute and chronic inflammatory responses is critical to understanding these diseases at a molecular level. To date, inflammasome components have been found in the bladder, prostate, and kidney and have been shown to be activated in response to several infectious and noninfectious insults. In this review, we summarize what is known regarding inflammasomes in both the upper and lower urinary tract and describe several common disease states where they potentially play critical roles.

The NLRP3 Inflammasome Mediates Inflammation Produced by Bladder Outlet Obstruction.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A novel urodynamic model for lower urinary tract assessment in awake rats.

OBJECTIVES: To develop a urodynamic model incorporating external urethral sphincter (EUS) electromyography (EMG) in awake rats. MATERIALS AND METHODS: Bladder catheters and EUS EMG electrodes were implanted in female Sprague Dawley rats. Assessments were performed in awake, lightly restrained rats on postoperative day 12-14. Measurements were repeated in the same rat on day 16 under urethane anaesthesia. Urodynamics and EUS EMG were performed simultaneously. In addition, serum creatinine and bladder histology was assessed. RESULTS: No significant differences in urodynamic parameters were found between bladder catheter only vs bladder catheter and EUS EMG electrode groups. Urethane anaesthesia evoked prominent changes in both urodynamic parameters and EUS EMG. Serum creatinine was within the normal limits in all rats. Bladder weight and bladder wall thickness were significantly increased in both the bladder catheter only and the bladder catheter and EUS EMG group compared with controls. CONCLUSIONS: Our novel urodynamic model allows repetitive measurements of both bladder and EUS function at different time points in the same rat under fully awake conditions and opens promising avenues to investigate lower urinary tract dysfunction in a translational approach.

S-Nitrosoglutathione protects the spinal bladder: novel therapeutic approach to post-spinal cord injury bladder remodeling.

AIMS: Bladder and renal dysfunction are secondary events of the inflammatory processes induced by spinal cord injury (SCI). S-Nitrosoglutathione (GSNO), an endogenous nitrosylating agent is pleiotropic and has anti-inflammatory property. Hence, GSNO ameliorates inflammatory sequelae observed in bladder and renal tissues after SCI. Thus, we postulate that GSNO will improve the recovery of micturition dysfunction by quenching the bladder tissue inflammation associated with SCI. METHODS: Contusion-based mild SCI was induced in female Sprague-Dawley rats. Sham operated rats served as the controls. SCI rats were gavaged daily with GSNO (50 µg/kg) or vehicle. Bladder function was assessed by urodynamics at 2 and 14 days following SCI. Urine protein concentration and osmolality were measured. Bladder and kidney tissues were analyzed by histology and immunofluorescence for a variety of endpoints related to inflammation. RESULTS: Two days after SCI, urodynamics demonstrated a hyperreflexive bladder with overflow and no clear micturition events. By Day 14, vehicle animals regained a semblance of a voiding cycle but with no definite intercontraction intervals. GSNO-treated SCI-rats showed nearly normal cystometrograms. Vehicle-treated SCI rats had increased bladder wet weight, proteinuria, and urine osmolality at Day 14, which was reversed by GSNO treatment. In addition, the SCI-induced increase in immune cell infiltration, collagen deposition, iNOS, and ICAM-1 expression and apoptosis were attenuated by GSNO. CONCLUSIONS: These results indicate that oral administration of GSNO hastens the recovery of bladder function after mild contusion-induced SCI through dampening the inflammation sequelae. These findings also suggest that GSNO-mediated redox modulation may be a novel therapeutic target for the treatment of mild SCI-induced renal and bladder dysfunction.

Inflammasomes are important mediators of cyclophosphamide-induced bladder inflammation.

Bladder inflammation (cystitis) underlies numerous bladder pathologies and is elicited by a plethora of agents such as urinary tract infections, bladder outlet obstruction, chemotherapies, and catheters. Pattern recognition receptors [Toll-like receptors (TLRs) and Nod-like receptors (NLRs)] that recognize pathogen- and/or damage-associated molecular patterns (PAMPs and/or DAMPs, respectively) are key components of the innate immune system that coordinates the production (TLRs) and maturation (NLRs) of proinflammatory IL-1ß. Despite multiple studies of TLRs in the bladder, none have investigated NLRs beyond one small survey. We now demonstrate that NLRP3 and NLRC4, and their binding partners apoptosis-associated speck-like protein containing a COOH-terminal caspase recruitment domain (ASC) and NLR family apoptosis inhibitory protein (NAIP), are expressed in the bladder and localized predominantly to the urothelia. Activated NLRs form inflammasomes that activate caspase-1. Placement of a NLRP3- or NLRC4-activating PAMP or NLRP3-activating DAMPs into the lumen of the bladder stimulated caspase-1 activity. To investigate inflammasomes in vivo, we induced cystitis with cyclophosphamide (CP, 150 mg/kg ip) in the presence or absence of the inflammasome inhibitor glyburide. Glyburide completely blocked CP-induced activation of caspase-1 and the production of IL-1ß at 4 h. At 24 h, glyburide reduced two markers of inflammation by 30-50% and reversed much of the inflammatory morphology. Furthermore, glyburide reversed changes in bladder physiology (cystometry) induced by CP. In conclusion, NLRs/inflammasomes are present in the bladder urothelia and respond to DAMPs and PAMPs, whereas NLRP3 inhibition blocks bladder dysfunction in the CP model. The coordinated response of NLRs and TLRs in the urothelia represents a first-line innate defense that may provide an important target for pharmacological intervention.

Enzyme-induced hypoxia leads to inflammation in urothelial cells in vitro.

PURPOSE: To determine the contributions of different durations of hypoxia to NLRP3 inflammasome activation in urothelial cells and how ischemic changes in bladder tissues is an important chemical que that leads to pathological changes seen in BOO. METHODS: A rat urothelial cell line (MYP3) was exposed to either a short duration (2 h) or long duration (6 h) of enzyme-induced hypoxia. Following exposure to a short duration of hypoxia, NO and ATP concentrations were measured from supernatant media and caspase-1 levels were measured from cell lysates. In a separate experiment, cells were fixed following hypoxia exposure and immunostained for HIF-1α stabilization. RESULTS: Although short exposure of low oxygen conditions resulted in a hypoxic response in MYP3 cells, as indicated by HIF-1α stabilization and increased NO activity, NLRP3 inflammasome activation was not observed as caspase-1 activity remained unchanged. However, exposure of MYP3 cells to a longer duration of hypoxia resulted in an increase in intracellular caspase-1 activity. Furthermore, treatment with antioxidant (GSH) or TXNIP inhibitor (verapamil) attenuated the hypoxia-induced increase in caspase-1 levels indicating that hypoxia primarily drives inflammation through a ROS-mediated TXNIP/NLRP3 pathway. CONCLUSION: We conclude that hypoxia induced bladder damage requires a duration that is more likely related to elevated storage pressures/hypoxia, seen in later stages of BOO, as compared to shorter duration pressure elevation/hypoxia that is encountered in normal micturition cycles or early in the BOO pathology where storage pressures are still normal.

Male Akita diabetic mice develop underactive bladder independent of NLRP3 that can be prevented with blood glucose control.

Aim: Diabetic bladder dysfunction (DBD) is the most common diabetic complication. Patients present with overactive symptoms, underactive symptoms, or both. While strict glucose control may be expected to reverse DBD, prior studies have not been supportive. However, we hypothesize that strict control, soon after hyperglycemia appears, can prevent DBD development. Moreover, 50% of adult diabetics are poorly-controlled and it is unknown how this effects development of DBD. Thus, we investigated the effect of early glucose control (poor and strict) on DBD in male Akita diabetic mice (type 1). NLRP3-induced inflammation is critical to development of DBD in female Akita. Therefore, we also hypothesized that targeting NLRP3 may control or prevent DBD in male Akita, especially in a poorly-controlled population. Methods: Akita mice (±NLRP3) were stratified into uncontrolled, poorly-controlled and strictly-controlled diabetic groups using insulin treatment (0, 0.125 or 0.25 U/day). Mice were assessed at 15 weeks for blood glucose, HbA1c, Evans blue dye extravasation (a marker of capillary permeability/inflammation) and bladder function. Results: Blood glucose was elevated in diabetics, reduced in an insulin dose-dependent manner, and not affected by NLRP3 deletion. HbA1c levels followed a similar course but were more sensitive to insulin levels. Evans blue dye extravasation was prevented with glucose control and absent in NLRP3-/- mice. Diabetics exhibited signs of underactive bladder (increased void volume, decreased frequency) that was attenuated in the uncontrolled group but absent in the well-controlled group. Deleting NLRP3 did not affect voiding function. Conclusion: Male Akita mice develop an underactive-like bladder, independent of NLRP3, which can be prevented with glucose control.