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 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.

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.

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.