Inflammasomes as a Target for Treating Functional and Anatomic Obstructive Bladder Uropathy

<p class="article-intro">In this brief review we discuss the role of the inflammasomes in functionally and anatomically obstructed bladders, previous success with inhibitors of the NLRP3 inflammasome, and potential future targets for NLRP3 inhibition.</p> <p class="article-content"><div id="keypoints"> <h2>Keypoints</h2> <ul> <li>Current treatments for functional and anatomic bladder obstruction do not eliminate the recurrent insults of pressure, stretch, and hypoxia. Eventually, this leads to chronic inflammation, fibrosis, and bladder decompensation with bothersome urinary symptoms.</li> <li>The NLRP3 inflammasome is an important factor in the inflammation associated with bladder outlet obstruction and the formation of a fibrotic, denervated bladder.</li> <li>Therapeutic agents that target the NLRP3 inflammasome pathway diminish inflammation, prevent fibrosis, and preserve neurons. This translates into improved functional bladder outcomes as evidenced by urodynamic studies.</li> <li>Continued research is needed to identify inflammasome-targeted therapies that are safe and efficacious for human consumption.</li> </ul> <h2>Introduction</h2> Bladder outlet obstruction (BOO) may result from anatomic resistance, as encountered in older men with benign prostatic hyperplasia, or from functional deficits, as experienced by spinal cord injury patients with detrusor sphincter dyssynergia. In either event, the result is high bladder pressures with tension on the tissues and a state of hypoxia. These factors engage the innate immune system to produce an inflammatory state, the molecular mechanisms of which are currently being investigated.¹ In sensate patients, this inflammatory state affects the afferent nervous system to cause irritative voiding symptoms such as frequency, urgency, and urge incontinence. These overactive symptoms tend to be more bothersome to the patient than the initial obstructive symptoms, and medical therapy has a high discontinuation rate with potential for serious side effects.^{2, 3} Chronic inflammation ultimately leads to fibrosis and bladder decompensation, at which point medical or surgical treatment to remove or circumvent the obstruction is ineffective in the recovery of bladder function.^{1, 4} Overflow incontinence as the result of bladder incontractility is not an uncommon outcome after years of under-treated bladder outlet obstruction and bladder inflammation. Loss of bladder compliance leads to chronically high bladder storage pressures that can place enough stress on the upper tracts to induce renal failure. By treating inflammation at the early stages of obstruction, we propose that not only can we alleviate bothersome lower urinary tract symptoms, but also prevent progression to bladder and renal failure. <h2>Introduction to Inflammasomes and Their Role in Obstructive Uropathy</h2> The inflammasome has emerged as the primary processing unit of inflammation and a central link between inflammation and bladder pathology.^{5, 6} In 2002, Tschopp et al. introduced the inflammasome as a structure that senses and responds to a diverse array of stress signals by maturing and releasing pro-inflammatory cytokines IL-1&szlig; and IL-18 to trigger an inflammatory response.⁷ During BOO, stressed or dying cells release damage associated molecular patterns (DAMPS) which are recognized by a family of Nod-like receptors (NLRs). The NLR responds by assembling into the inflammasome, a multi-protein complex that attracts and activates caspase-1. Caspase-1, once activated, cleaves pro-IL-1&szlig; and pro- IL-18 into their active forms (IL-1&szlig; and IL- 18). These pro-inflammatory cytokines are released and the inflammatory response ensues (Fig.). Though several NLRs have been localized in the bladder, NLRP3 is the best studied.⁸ We have localized NLRP3 to the urothelium and verified the importance of the NLRP3 inflammasome in triggering the inflammation during BOO in rat models. ^{8, 9} NLRP3/IL-1&szlig;-induced inflammation produced by BOO leads to functional urodynamic changes, bladder fibrosis, and bladder denervation. Blocking the inflammatory pathway with targeted agents such as glyburide (NLRP3 inhibitor) and anakinra (IL-1R1 antagonist) reduces the functional changes, amount of fibrosis formed, and diminishes nerve loss.^9-11 Therefore, agents that target the inflammasome pathway will likely reduce the sequelae of functional and anatomic obstruction in patients with BOO.</div> <div><img src="/custom/img/files/files_datafiles_data_Zeitungen_2017_Urologik_Uro_1701_Weblinks_s26_fig.jpg" alt="" width="1419" height="1539" /></div> <div> <h2>NLRP3 as a Therapeutic Target</h2> Glyburide, a potent NLRP3 inhibitor, has been shown to prevent histologic and functional changes associated with NLRP3- inflammasome-induced inflammation in the bladder.^9-12 It is an attractive medication to study given its widespread availability as an FDA-approved oral sulfonylurea diabetes mellitus treatment, its inexpensive cost, and ease of use in animal models. However, glyburide is not FDAapproved as an anti-inflammatory and its hypoglycemic effects are likely to prohibit its use in humans at the required therapeutic dosage. Excitingly, Marchetti et al. recently reported success with the molecule 16673-34-0 in mouse cardiomyocytes. This intermediate substrate in the glyburide synthesis pathway inhibits the formation of the NLRP3 without affecting glucose metabolism.¹³<br /> Avistron (Cornwall, UK) has developed an orally available compound, MCC950. This product is a diarylsulfonylurea-containing compound known to inhibit caspase-1-dependent processing of IL-1&szlig;.¹⁴ Recently, Coll et al. demonstrated that MCC950 also inhibits NLRP3 in mice in vivo and in human cells ex vivo.15, 16 While the effect of this NLRP3-targeting molecule has not been studied in relation to bladder inflammation, its effect on other disease processes offers this as a potentially useful tool needing further studies.^{15, 17}<br /> Another NLRP3 inhibitor, &szlig;-hydroybutyrate (BHB), was recently evaluated by Youm et al. who demonstrated inhibition of NLRP3 inflammasome activation and production of active IL-1&szlig; and IL-18. Interestingly, this ketone body is elevated in a starvation state, suggesting that BHB-mediated inhibition of NLRP3 may play a critical role in the anti-inflammatory effects of caloric restriction or ketogenic diets.¹⁸ Currently, there are no pharmacologic interventions that would elevate circulating BHB as an intervention against NLRP3- mediated pro-inflammatory diseases, though this is a potential future target. <h2>IL -1R1 as a Therapeutic Target</h2> Anakinra is a recombinant form of the naturally occurring IL-1 receptor antagonist, which blockades inflammasome-dependent IL-1&szlig; signaling.¹⁹ This drug is currently FDA-approved as a second-line injectable treatment for rheumatoid arthritis. Anakinra has been utilized successfully in our lab to prevent fibrosis and neuronal changes associated with BOO in rat models.^{10, 11} Unfortunately, human utilization of this drug for anti-inflammatory effects will likely be limited by its immunosuppressive side effects, requirement for injection and the prohibitive cost of biologic therapies.^{20, 21} <h2>Caspase-1 as a Therapeutic Target</h2> InvivoGen (San Diego, CA) produces several products targeting the inflammasome. These compounds have not yet been tested in the context of bladder dysfunction, but are available for future analysis. VX-765 and Ac-YVAD-cmk are caspase-1 inhibitors that reduce the production of IL-1&szlig; and IL-18 both in vitro and in vivo in animal models.^{22, 23} Z-VAD-FMK is a pancaspase inhibitor that inhibits caspase-1 activation in cells with activated NLRP3.^{24, 25} Finally, parthenolide is an herbal NF-?B inhibitory compound that is a direct inhibitor of the protease activity of caspase-1 and is also an inhibitor of the ATPase activity of NLRP3.²⁶ <h2>Natural Products</h2> While it is tempting to focus on the synthetic innovations of the present and future, it is prudent to look to naturallyexisting products that may be developed or refined into effective treatments. Glycyrrhiza roots (licorice) have been used in traditional Chinese medicine for over 4,000 years as anti-inflammatory agents.²⁷ Isoliquirtigenin (ILG) can be isolated from Glycyrrhiza and has been shown to inhibit LPS-induced NF-?B, which is an initial priming step of inflammasome activation.^{28, 29} Recently, Honda et al. demonstrated that ILG inhibits the NLRP3 inflammasome. In fact, in mouse studies the ILG more potently inhibited the NLRP3-induced production of IL-1&szlig; and caspase-1 compared with parthenolide and glyburide (previously discussed NLRP3 inhibitors).³⁰ Shikonin is another product from the roots of Lithospermum erythrorhizon which has been used as an anti-inflammatory agent in traditional Chinese medicine. Zorman et al. recently demonstrated that shikonin inhibits the activation of the NLRP3 inflammasome by directly inhibiting caspase-1.³¹ These products portend the possibility of ILG and shikonin as therapeutic agents for the treatment of NLRP3 inflammasome-associated inflammatory pathology. <div id="fazit"> <h2>Conclusion</h2> Inflammation is a component of anatomic and functionally obstructive uropathy, and the NLRP3 inflammasome is of central importance in the inflammatory process.⁹ Agents that block the NLRP3 pathway diminish inflammation, prevent fibrosis, and preserve neurons.^9&ndash;11 Functionally, this translates into improved bladder function with decreased voiding frequency and increased voided volumes despite persistent obstruction.⁹ Naturally occurring products that inhibit this inflammatory process have been available for years. Current research should focus on improving their performance as well as on synthetic production of agents that target this pathway with the goal of reducing irritative voiding symptoms and progression to bladder decompensation in BOO.</div> </div></p> <p class="article-footer"> <a class="literatur" data-toggle="collapse" href="#collapseLiteratur" aria-expanded="false" aria-controls="collapseLiteratur" >Literatur</a> <div class="collapse" id="collapseLiteratur"> <p><strong>1</strong> Metcalfe PD et al: Bladder outlet obstruction: progression from inflammation to fibrosis. BJU Int 2010; 106: 1686- 94 <strong>2</strong> Wagg A et al: Persistence with prescribed antimuscarinic therapy for overactive bladder: a UK experience. BJU Int 2012; 110: 1767-74 <strong>3</strong> Gray SZ et al: Cumulative use of strong anticholinergics and incident dementia: a prospective cohort study. JAMA Intern Med 2015; 175: 401-7 <strong>4</strong> Jock M et al: Effect of partial bladder outlet obstruction and reversal on rabbit bladder physiology and biochemistry: duration of recovery period and severity of function. BJU Int 2014; 114: 946-54 <strong>5</strong> Purves JT, Hughes FM, Jr: Inflammasomes in the urinary tract: a disease-based review. Am. J. Physiol. Renal Physiol 2016; 311: F653-62 <strong>6</strong> Di Virgilio F: The therapeutic potential of modifying inflammasomes and NOD-like receptors. Pharmacol. Rev 2013; 65: 872- 905 <strong>7</strong> Martinon F, Burns K, Tschopp J: The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 2002; 10: 417-26 <strong>8</strong> Hughes FM, Jr., Turner DP, Todd Purves J: 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). Int. Urol. Nephrol 2015; 47: 1953-64 <strong>9</strong> Hughes FM, Jr. et al: The NLRP3 inflammasome mediates inflammation produced by bladder outlet obstruction. J. Urol 2016; 195: 1598-605 <strong>10</strong> Hughes FM, Jr., Sexton, S., Purves, JT: Fibrosis in the bladder in response to outlet obstruction is triggered through the NLRP3 inflammasome and the production of IL-1&szlig;. The Society for Pelvic Research; December 6, 2016; Charleston, SC. <strong>11</strong> L&uuml;tolf R et al: NLRP3 / IL-1&szlig; mediates denervation during bladder outlet obstruction in rats. The Society for Pelvic Research; December 6, 2016; Charleston, SC. <strong>12</strong> Lamkanfi M et al: Glyburide inhibits the Cryopyrin/ Nalp3 inflammasome. J. Cell Biol 2009; 187: 61-70 <strong>13</strong> Marchetti C et al: A novel pharmacologic inhibitor of the NLRP3 inflammasome limits myocardial injury after ischemia-reperfusion in the mouse. J. Cardiovasc. Pharmacol 2014; 63: 316-22 <strong>14</strong> Perregaux DG et al: Identification and characterization of a novel class of interleukin-1 post-translational processing inhibitors. J. Pharmacol. Exp. Ther 2001; 299: 187-97 <strong>15</strong> Coll RC et al: A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat. Med 2015; 21: 248-55 <strong>16</strong> Salla M et al: Identification, synthesis, and biological evaluation of the major human metabolite of NLRP3 inflammasome inhibitor MCC950. ACS Med. Chem. Lett 2016; 7: 1034-8 <strong>17</strong> Krishnan SM et al: Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice. Br. J. Pharmacol 2016; 173: 752-65 <strong>18</strong> Youm YH et al: The ketone metabolite beta-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease. Nat. Med 2015; 21: 263-9 <strong>19</strong> Hawkins PN, Lachmann HJ, McDermott MF: Interleukin-1-receptor antagonist in the Muckle-Wells syndrome. N. Engl. J. Med 2003; 348: 2583-4 <strong>20</strong> Menu P, Vince JE: The NLRP3 inflammasome in health and disease: the good, the bad and the ugly. Clin. Exp. Immunol 2011; 166: 1-15 <strong>21</strong> Fautrel B: Economic benefits of optimizing anchor therapy for rheumatoid arthritis. Rheumatology (Oxford). 2012; 51 Suppl 4: iv21-26 <strong>22</strong> Wannamaker W et al: (S)-1-((S)-2-{[1-(4-amino-3- chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoy l)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxotetrahydro- furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18. J. Pharmacol. Exp. Ther 2007; 321: 509-16 <strong>23</strong> Zhang Y et al: Involvement of inflammasome activation in lipopolysaccharide-induced mice depressive-like behaviors. CNS Neurosci. Ther 2014; 20: 119-24 <strong>24</strong> Slee E A et al: Benzyloxycarbonyl-Val-Ala- Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem. J 1996; 315: 21-4 <strong>25</strong> Dostert C et al: Malarial hemozoin is a Nalp3 inflammasome activating danger signal. PLoS One. 2009; 4: e6510 <strong>26</strong> Juliana C et al Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome. J. Biol. Chem 2010; 285: 9792-802 <strong>27</strong> Shibata S: A drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice. Yakugaku Zasshi. 2000; 120: 849-62 <strong>28</strong> Kim JY et al: Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-induced iNOS and COX-2 expression via the attenuation of NF-kappaB in RAW 264.7 macrophages. Eur. J. Pharmacol 2008; 584: 175-84 <strong>29</strong> Bauernfeind FG et al: Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol 2009; 183: 787-91 <strong>30</strong> Honda H et al: Isoliquiritigenin is a potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue inflammation. J. Leukoc. Biol 2014; 96: 1087-100 <strong>31</strong> Zorman J, Susjan P, Hafner-Bratkovic I: Shikonin suppresses NLRP3 and AIM2 inflammasomes by direct inhibition of caspase-1. PLoS One 2016; 11: e0159826</p> </div> </p>