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.

Female Type 1 Diabetic Akita Mice Demonstrate Increased Bladder Contractility via FP Receptor Activation due to NLRP3-Mediated Inflammation.

BACKGROUND: Diabetic bladder dysfunction (DBD) is driven in part by inflammation which dysregulates prostaglandin release in the bladder. Precise inflammatory mechanisms responsible for such dysregulation have been elusive. Since prostaglandins impact bladder contractility, elucidating these mechanisms may yield potential therapeutic targets for DBD. In female Type 1 diabetic Akita mice, inflammation mediated by the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome is responsible for DBD. Here, we utilized female Akita mice crossbred with NLRP3 knock-out mice to determine how NLRP3-driven inflammation impacts prostaglandin release within the bladder and prostaglandin-mediated bladder contractions. METHODS: Akita mice were crossbred with NLRP3-⁣/- mice to yield four groups of non-diabetics and diabetics with and without the NLRP3 gene. Females were aged to 30 weeks when Akitas typically exhibit DBD. Urothelia and detrusors were stretched ex vivo to release prostaglandins. Prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) were quantified using enzyme linked immunosorbent assays (ELISA). In separate samples, ex vivo contractile force to PGE2 and PGF2α +/- the prostaglandin F (FP) receptor antagonist, AL8810, was measured. FP receptor protein expression was determined via western blotting. RESULTS: Stretch-induced PGE2 release increases in urothelia but decreases in detrusors of diabetics. However, PGE2-mediated bladder contractions are not impacted. Conversely, diabetics show no changes in PGF2α release, but PGF2α-mediated contractions increase significantly. This is likely due to signaling through the FP receptors as FP receptor antagonism prevents this increase and diabetics demonstrate a four-fold increase in FP receptor proteins. Without NLRP3-mediated inflammation, changes in prostaglandin release, contractility, and receptor expression do not occur. CONCLUSION: NLRP3-dependent inflammation dysregulates prostaglandin release and prostaglandin-mediated bladder contractions in diabetic female Akita mice via FP receptor upregulation.

Recurrent infections drive persistent bladder dysfunction and pain via sensory nerve sprouting and mast cell activity.

Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.

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 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.

Inflammation triggered by the NLRP3 inflammasome is a critical driver of diabetic bladder dysfunction.

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.

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.

Diabetic bladder dysfunction progresses from an overactive to an underactive phenotype in a type-1 diabetic mouse model (Akita female mouse) and is dependent on NLRP3.

AIMS: Diabetic bladder dysfunction (DBD) is a prevalent diabetic complication thought to progress from overactive (OAB) to underactive (UAB) bladder. Previously we found OAB at 15 weeks in the Akita mouse, a genetic model of Type 1 diabetes. The first aim of this study assesses bladder function at 30 weeks to assess progression. In addition, inflammation triggered by the NLRP3 inflammasome is implicated in DBD. In a second aim we assessed a role for NLRP3 by crossing Akita mice with NLRP3-/- mice. MAIN METHODS: Akita mice were bred with NLRP3-/- mice. The effect of diabetes was assessed by comparing nondiabetic to diabetic mice (all NLRP3+/+). The effect of diabetes in the absence of the NLRP3 inflammasome was assessed by comparing nondiabetic/NLRP3-/- to diabetic/NLRP3-/- mice. Mice were assessed at 30 weeks for blood glucose (glucometer), inflammation (Evans blue), bladder morphology (histology) and bladder function (urodynamics). KEY FINDINGS: At 30 weeks blood glucose of nondiabetics and diabetics was not affected by the presence of absence of NLRP3. Diabetic/NLRP3+/+ mice showed bladder inflammation and detrusor hypertrophy which was blocked in the diabetic/NLRP3-/- mice, clearly showing a role for NLRP3. When bladder function was examined, diabetic/NLRP3+/+ showed an increase in voiding volume and a decrease in frequency, two signs of underactive bladder. However, in the NLRP3-/- mice, diabetes was unable to effectuate these changes, demonstrating that NLRP3-induced inflammation is responsible for UAB symptoms in these mice. SIGNIFICANCE: Akita diabetic mice progress from OAB to UAB. NLRP3 is a possible target to treat DBD.

Specialized pro-resolution mediators in the bladder: Receptor expression and recovery of bladder function from cystitis.

Inflammation is a central process in most benign bladder disorders, and its control is a delicate balance between initiating factors and resolving factors. While recent discoveries have shown a central role for the NLRP3 inflammasome in initiation, the resolving pathways remain unexplored. Resolution is controlled by specialized pro-resolution mediators (SPMs) functioning through seven receptors (six in rodents). Here we demonstrate expression of all seven in humans (six in mice) through immunocytochemistry. Expression was universal in urothelia with most also expressed in smooth muscle. We next explored the therapeutic potential of three SPMs; Resolvin E1 (RvE1), Maresin 1 (MaR1), and Protectin D1 (PD1). SPMs promote epithelial wound/barrier repair and RvE1 triggered dose-dependent wound closure in urothelia in vitro (scratch assay) (EC90 = 12.5 nM). MaR1 and PD1 were equally effective at this concentration. In vivo analyses employed a cyclophosphamide (CP) model of bladder inflammation (Day 0-CP [150 mg/kg], Day 1 to 3 SPM [25 µg/kg/day], Day 4 - analysis). All three SPMs reduced bladder inflammation (Evans blue) and bladder weights to control levels. Effects of RvE1 were also examined by urodynamics. CP decreased void volume, increased frequency and decreased bladder capacity while RvE1 restored values to control levels. Finally, SPMs reduce fibrosis and RvE1 reduced urothelial expression of TGF-ß and collagen I to control values. Together these results expand the known SPMs active in the bladder tissue and provide promising therapeutic targets for controlling inflammation in a wide variety of inflammation-associated benign bladder diseases.

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.

BOO induces fibrosis and EMT in urothelial cells which can be recapitulated in vitro through elevated storage and voiding pressure cycles.

PURPOSE: To determine the unique contributions from elevated voiding and storage pressures in the development of fibrosis and the epithelial-to-mesenchymal transition (EMT) in urothelial cells, and how progressive BOO pressure cycling is an important mechanical cue leading to these pathological changes. MATERIALS AND METHODS: Urothelial cells isolated from control, SHAM, 2 (acute)- or 6 (chronic)-week BOO rats treated with an inflammasome inhibitor or no drug. Total RNA was isolated and RT-PCR was conducted with custom primers for pro-fibrotic and EMT genes. In separate experiments, a rat urothelial cell line was exposed to cyclic pressure regimes characteristic of acute and chronic BOO in the presence or absence of an inflammasome inhibitor. Following exposure, RT-PCR was conducted, collagen content was determined and intracellular caspase-1 activity was measured. RESULTS: Urothelial cells isolated from acute and chronic BOO rat models demonstrated expression of pro-fibrotic and EMT genes. Similarly, MYP3 rat urothelial cells subjected to pressure cycling regimes that reflect intravesical pressures in the acute or chronic BOO bladder also demonstrated increased expression of pro-fibrotic and EMT genes, along with elevated soluble collagen. Treatment with inflammasome inhibitors reduced expression of pro-fibrotic genes in the rat model and pressure cycling model but had a limited effect on EMT. CONCLUSION: These results indicate that acute and chronic BOO pressure cycling are essential in the initiation and progression of fibrosis in the bladder via the NLRP3 inflammasome, but also provide new evidence that there is also an alternative NLRP3-independent pathway leading to EMT and fibrosis.

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.

A highly polarized TH2 bladder response to infection promotes epithelial repair at the expense of preventing new infections.

Urinary tract infections (UTIs) typically evoke prompt and vigorous innate bladder immune responses, including extensive exfoliation of the epithelium. To explain the basis for the extraordinarily high recurrence rates of UTIs, we examined adaptive immune responses in mouse bladders. We found that, following each bladder infection, a highly T helper type 2 (TH2)-skewed immune response directed at bladder re-epithelialization is observed, with limited capacity to clear infection. This response is initiated by a distinct subset of CD301b+OX40L+ dendritic cells, which migrate into the bladder epithelium after infection before trafficking to lymph nodes to preferentially activate TH2 cells. The bladder epithelial repair response is cumulative and aberrant as, after multiple infections, the epithelium was markedly thickened and bladder capacity was reduced relative to controls. Thus, recurrence of UTIs and associated bladder dysfunction are the outcome of the preferential focus of the adaptive immune response on epithelial repair at the expense of bacterial clearance.

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.

Calcium Pyrophosphate And Monosodium Urate Activate The NLRP3 Inflammasome Within Bladder Urothelium Via Reactive Oxygen Species And TXNIP.

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.

NLRP3 Promotes Diabetic Bladder Dysfunction and Changes in Symptom-Specific Bladder Innervation.

The NLRP3 inflammasome senses diabetic metabolites and initiates inflammation implicated in diabetic complications and neurodegeneration. No studies have investigated NLRP3 in diabetic bladder dysfunction (DBD), despite a high clinical prevalence. In vitro, we found that numerous diabetic metabolites activate NLRP3 in primary urothelial cells. In vivo, we demonstrate NLRP3 is activated in urothelia from a genetic type 1 diabetic mouse (Akita) by week 15. We then bred an NLRP3-/- genotype into these mice and found this blocked bladder inflammation and cystometric markers of DBD. Analysis of bladder innervation established an NLRP3-dependent decrease in overall nerve density and Aδ-fibers in the bladder wall along with an increase in C-fiber populations in the urothelia, which potentially explains the decreased sense of bladder fullness reported by patients and overactivity detected early in DBD. Together, the results demonstrate the role of NLRP3 in the genesis of DBD and suggest specific NLRP3-mediated neuronal changes can produce specific DBD symptoms.

Evaluation of Poly (Carbonate-Urethane) Urea (PCUU) Scaffolds for Urinary Bladder Tissue Engineering.

Although the previous success of bladder tissue engineering demonstrated the feasibility of this technology, most polyester based scaffolds used in previous studies possess inadequate mechanical properties for organs that exhibit large deformation. The present study explored the use of various biodegradable elastomers as scaffolds for bladder tissue engineering and poly (carbonate-urethane) urea (PCUU) scaffolds mimicked urinary bladder mechanics more closely than polyglycerol sebacate-polycaprolactone (PGS-PCL) and poly (ether-urethane) urea (PEUU). The PCUU scaffolds also showed cyto-compatibility as well as increased porosity with increasing stretch indicating its ability to aid in infiltration of smooth muscle cells. Moreover, a bladder outlet obstruction (BOO) rat model was used to test the safety and efficacy of the PCUU scaffolds in treating a voiding dysfunction. Bladder augmentation with PCUU scaffolds led to enhanced survival of the rats and an increase in the bladder capacity and voiding volume over a 3 week period, indicating that the high-pressure bladder symptom common to BOO was alleviated. The histological analysis of the explanted scaffold demonstrated smooth muscle cell and connective tissue infiltration. The knowledge gained in the present study should contribute towards future improvement of bladder tissue engineering technology to ultimately aide in the treatment of bladder disorders.

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.

Diabetic bladder dysfunction is associated with bladder inflammation triggered through hyperglycemia, not polyuria.

PURPOSE: Diabetes is a grave and progressive condition characterized by debilitating complications. Diabetic bladder dysfunction (DBD) is a very common complication with no specific treatments currently available. Unlike other tissues affected by this disease, the bladder is subjected to two independent insults; 1) polyuria, created by the osmotic effects of glucose in the urine, and 2) hyperglycemia itself. Based on our understanding of inflammation as a major contributor to the underlying organ damage in several other diabetic complications, its presence in the bladder during DBD and the contribution of polyuria and hyperglycemia to its development were assessed. METHODS: Awake, restrained cystometry was performed on wild type C57BL/6 mice and diabetic (Akita) mice on a C57BL/6 background at 15 weeks of age. A subgroup of the Akita mice were treated with phlorizin, an inhibitor of sodium-glucose linked transporter types 1 and 2 that prevents glucose reabsorption in the kidney. All groups were assessed for serum glucose, 4-hour voiding totals, and inflammation in the bladder (Evans blue assay). RESULTS: Akita mice develop cystometrically-defined DBD by 15 weeks of age, as evidenced by an increase in urinary frequency, a decrease in voiding volume, and an increase in post-voiding residual volume. Phlorizin effectively normalized serum glucose in these animals while increasing the urine output. Inflammation in the bladder was present in the diabetic animals at this time point, but not detectable in animals receiving phlorizin. CONCLUSION: Inflammation in the bladder of diabetic mice correlates with the development of DBD and is triggered by hyperglycemia, not polyuria.