J. Todd Purves

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.

The NACHT, LRR and PYD Domains-Containing protein 3 (NLRP3) Inflammasome Mediates Inflammation and Voiding Dysfunction in a Lipopolysaccharide-Induced Rat Model of Cystitis

OBJECTIVE NOD-like receptors (NLRs) sense sterile and non-sterile signals and form inflammasomes which trigger an inflammatory response through the activation of caspase-1 and release of IL-1β. Recently we have shown the presence of several NLRs in the bladder urothelia and demonstrated the importance of NLRP3 in bladder outlet obstruction and cyclophosphamide-induced cystitis, both models of sterile inflammation. In this study we explore a role for NLRP3 in mediating the response to LPS, a key antigen of uropathogenic bacteria. METHOD In order to bypass the protective glycosaminoglycan layer lining the urothelium, LPS was directly injected into the bladder wall of Sprague-Dawley rats. Glyburide (a NLRP3 inhibitor) or vehicle was administered orally prior to and after injection. Rats were analyzed 24 h later. Inflammasome activity (caspase-1 activity, IL-1β release) and inflammation (Evans Blue extravasation, bladder weight) were assessed, as was physiological bladder function (urodynamics). RESULTS Injection of LPS stimulated inflammasome activation (caspase-1 activity) and the release of IL-1β into the urine which was prevented by glyburide. Likewise, LPS increased inflammation, (bladder weight and the extravasation of Evans blue dye), and this was reversed by glyburide. Functionally, animals injected with saline alone demonstrated decreased voiding volume as measured by urodynamics. In the presence of LPS, additional urinary dysfunction was evident with decreased voiding pressures and threshold pressures. The decrease in voiding pressure was blocked by glyburide but the decrease in threshold pressure was not, suggesting that LPS has significant effects mediated by inflammasome-dependent and -independent mechanisms. CONCLUSION Overall, the results demonstrate the potential importance of inflammasomes in bacterial cystitis as well as the ability of the bladder wall injection technique to isolate the in vivo effects of specific inflammasome ligands to the physiological changes associated with cystitis.

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)

AbstractPurpose 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.MethodsImmunohistochemistry 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.ResultsAll 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.ConclusionThe 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.

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

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 Massons 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.

Is the Economic Impact and Utilization of Imaging Studies for Pediatric Urolithiasis Across the United States Increasing

OBJECTIVE To identify longitudinal trends of economic impact and resource utilization for management of pediatric urolithiasis using national databases. METHODS We analyzed the 2006-2012 Nationwide Emergency Department Sample and Nationwide Inpatient Sample. We used ICD-9 (International Classification of Diseases) codes to identify patients (≤18 years) diagnosed with urolithiasis. Diagnostic imaging and surgeries were identified using ICD-9 and Current Procedural Technology codes. We abstracted demographic, imaging, procedure, and charge data. Weighted descriptive statistics were calculated to describe the populations demographics and economic expenditures by clinical setting and year. RESULTS In total, 45,333 inpatient admissions (68% females) and 234,559 emergency department encounters (63% females) were identified. Most patients (84%) were teenagers and the southern region of the United States was the most common geographic region for all encounters (44%). There was no significant trend in number of urolithiasis encounters over the period studied. Utilization of all imaging techniques increased; in particular, computed tomography was used in 23% of encounters in 2006 and 40% in 2012 (P < .0001). The mean charge per emergency department visit increased by 60% from

Cyclophosphamide induces an early wave of acrolein-independent apoptosis in the urothelium

3645 in 2006 to

The Emerging Role of Inflammasomes as Central Mediators in Inflammatory Bladder Pathology.

5827 in 2012 (P < .0001). The mean charge increased for inpatient admissions by 102%, from

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

16,399 in 2006 to

A three dimensional nerve map of human bladder trigone

33,205 in 2012 (P < .0001). Total charges increased 72% over the study period from

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

230 million in 2006 to