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-1β. 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-1β 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.

Acute kidney injury (AKI) is a common and potentially lifethreatening complication after ischemia/reperfusion and exposure to nephrotoxic agents. In this study, we examined the efficacy and mechanism(s) of suramin in promoting recovery from glycerol-induced AKI, a model of rhabdomyolysis-induced AKI. After intramuscular glycerol injection (10 ml of 50% glycerol per kilogram) into male Sprague-Dawley rats, serum creatinine maximally increased at 24 to 72 h and then decreased at 120 h. Creatinine clearance (CrCl) decreased 75% at 24 to 72 h and increased at 120 h. Suramin (1 mg/kg i.v.) administered 24 h after glycerol accelerated recovery of renal function as demonstrated by increased CrCl, decreased renal kidney injury molecule-1, and improved histopathology 72 h after glycerol injection. Suramin treatment decreased interleukin-1␤ (IL-1␤) mRNA, transforming growth factor-␤ 1 (TGF-␤ 1 ), phospho-p65 of nuclear factor-B (NF-B), and cleaved caspase-3 at 48 h compared with glycerol alone. Suramin treatment also decreased glycerol-induced activation of intracellular adhesion molecule-1 (ICAM-1) and leukocyte infiltration at 72 h. Urinary/ renal neutrophil gelatinase-associated lipocalin 2 (NGAL) levels, hemeoxygenase-1 expression, and renal cell proliferation were increased by suramin compared with glycerol alone at 72 h. Mechanistically, suramin decreases early glycerol-induced proinflammatory (IL-1␤ and NF-B) and growth inhibitory (TGF-␤ 1 ) mediators, resulting in the prevention of late downstream inflammatory effects (ICAM-1 and leukocyte infiltration) and increasing compensatory nephrogenic repair. These results support the hypothesis that delayed administration of suramin is effective in abrogating apoptosis, attenuating inflammation, and enhancing nephrogenic repair after glycerol-induced AKI.

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 (Evan’s 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 Evan’s 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 neurotensin hexapapetide fragment NT(8–13) is a potent analgesic when administered directly to the central nervous system, but does not cross the blood brain barrier. A total of 43 novel derivatives of NT(8–13) were evaluated with one, ABS212 (1), being most active in four rat models of pain when administered peripherally. Compound 1 binds to human neurotensin receptors 1 and 2 with IC50s of 10.6 and 54.2 nM, respectively and tolerance to the compound in a rat pain model did not develop after 12 days of daily administration. When administered peripherally, serum levels and neurotensin receptor binding potency of 1 peaked within 5 min and returned to baseline within 90–120 min, however analgesic activity remained near maximum for >240 min. This could be due to its metabolism into an active fragment; however, all 4- and 5-mer hydrolysis products were inactive. This pharmacokinetic/pharmacodynamic dichotomy is discussed. Compound 1 is a candidate for development as a first-in-class analgesic.

Kappa-opioid agonists are particularly efficacious in the treatment of peripheral pain but suffer from central nervous system (CNS)-mediated effects that limit their development. One promising kappa-agonist is the peptidic compound CR665. Although not orally available, CR665 given i.v. exhibits high peripheral to CNS selectivity and benefits patients with visceral and neuropathic pain. In this study we have generated a series of derivatives of CR665 and screened them for oral activity in the acetic acid-induced rat writhing assay for peripheral pain. Five compounds were further screened for specificity of activation of kappa receptors as well as agonism and antagonism at mu and delta receptors, which can lead to off-target effects. All active derivatives engaged the kappa receptor with EC50s in the low nM range while agonist selectivity for kappa over mu or delta was >11,000-200,000-fold. No antagonist activity was detected. One compound was chosen for further analysis (Compound 9). An oral dose response of 9 in rats yielded an EC50 of 4.7 mg/kg, approaching a druggable level for an oral analgesic. To assess the peripheral selectivity of this compound an i.v. dose response in rats was assessed in the writhing assay and hotplate assay (an assay of CNS-mediated pain). The EC50 in the writhing assay was 0.032 mg/kg while no activity was detectable in the hotplate assay at doses as high as 30 mg/kg, indicating a peripheral selectivity of >900-fold. We propose that compound 9 is a candidate for development as an orally-available peripherally-restricted kappa agonist.