430 Diabetes Volume 68, February 2019 NLRP3 Promotes Diabetic Bladder Dysfunction and Changes in Symptom-Specific Bladder Innervation Francis M. Hughes Jr.,1,2 Nathan A. Hirshman,1 Brian M. Inouye,1 Huixia Jin,1 Eloise W. Stanton,1 Chloe E. Yun,1 Leah G. Davis,1,3 Jonathan C. Routh,1,4 and J. Todd Purves1,2,4 Diabetes 2019;68:430–440 | https://doi.org/10.2337/db18-0845 The NLRP3 inflammasome senses diabetic metabolites lacking, several experimental animal models suggest pro- and initiates inflammation implicated in diabetic com- gression from OAB to decompensation (6–8). Regardless, plications and neurodegeneration. No studies have until now, the mechanism by which the metabolic dysreg- investigated NLRP3 in diabetic bladder dysfunction ulation translates into physiological dysfunction has been (DBD), despite a high clinical prevalence. In vitro, we unclear. found that numerous diabetic metabolites activate It is now appreciated that diabetes is not just a disease NLRP3 in primary urothelial cells. In vivo, we demon- of high blood glucose but also a disease of deranged strate NLRP3 is activated in urothelia from a genetic metabolism resulting in hyperglycemia and the produc- type 1 diabetic mouse (Akita) by week 15. We then bred tion of numerous metabolites such as uric acid and free an NLRP32/2 genotype into these mice and found this fatty acids (9). These metabolites trigger inflammation blocked bladder inflammation and cystometric markers that damages susceptible tissues with a resulting loss of of DBD. Analysis of bladder innervation established function (10). Recent breakthroughs in other diabetic an NLRP3-dependent decrease in overall nerve density d fi complications (nephropathy, retinopathy, and cardiomy- and A - bers in the bladder wall along with an increase fl in C-fiber populations in the urothelia, which poten- opathy) have demonstrated this in ammation results COMPLICATIONS tially explains the decreased sense of bladder fullness from activation of the nucleotide-binding oligomeriza- – reported by patients and overactivity detected early in tion domain like receptor (NLR) NLRP3, which forms fl DBD. Together, the results demonstrate the role of a supramolecular complex known as an in ammasome NLRP3 in the genesis of DBD and suggest specific (11). NLRP3-mediated neuronal changes can produce spe- NLRP3 is the best studied of the NLR family of pattern cificDBDsymptoms. receptors. In general, pattern receptors recognize mole- cules released from damaged or dying cells (or those with deranged metabolism), known as damage (or danger)– Diabetic bladder dysfunction (DBD) affects a large pro- associated molecular patterns (DAMPs), or components portion of patients with diabetes, and there is currently no of pathogens known as pathogen-associated molecular specific therapy to treat it (1,2). With ;422 million people patterns. NLRP3 is by far the best understood NLR to with diabetes worldwide in 2014 (3) and projections that recognize DAMPs and has been implicated in many as many as 1 in 3 people will be affected in the U.S. by diseases with a sterile inflammatory component, includ- 2050 (4), this represents a highly prevalent pathological ing diabetic complications. Upon recognition of DAMPs, condition without a targeted therapy. Patients with DBD via a poorly understood mechanism, NLRP3 oligomerizes may present with urinary frequency and urgency (i.e., and triggers enucleation of an adaptor protein known as overactive bladder [OAB]), detrusor underactivity and apoptosis-associated speck-like protein C. Apoptosis- bladder decompensation (5), or some combination thereof. associated speck-like protein C, in turn, interacts with Although definitive longitudinal studies in humans are procaspase-1, which is cleaved and activated through an 1Department of Surgery, Division of Urology, Duke University Medical Center, Received 1 August 2018 and accepted 6 November 2018 Durham, NC © 2018 by the American Diabetes Association. Readers may use this article as 2 Department of Bioengineering, Clemson University, Clemson, SC long as the work is properly cited, the use is educational and not for profit, and the 3 Duke Cancer Center Biostatistics, Duke University Medical Center, Durham, NC work is not altered. More information is available at http://www.diabetesjournals. 4 Department of Pediatrics, Duke University Medical Center, Durham, NC org/content/license. Corresponding author: Francis M. Hughes Jr., [email protected] diabetes.diabetesjournals.org Hughes and Associates 431 autoproteolytic process. Caspase-1, in turn, catalyzes the were used. All animals were genotyped by Transnetyx, Inc. enzymatic maturation interleukin (IL)-1b, IL-18, and (Cordova, TN) and provided to the laboratory at ;4weeks gasderminD.GasderminDformsaporeintheplasma of age. membrane, triggering a programmed necrosis called The results of genotyping were used to assign them to pyroptosis that releases IL-1b and IL-18 that act as one of the four experimental groups. Nondiabetics are proinflammatory cytokines to initiate the inflammatory “nondiab,” and diabetics are “diab.” The groups are response. Recently, we have shown that NLRP3 plays an +/+ important role in the urinary tract of the rodent (12,13). 1. NLRP3 , nondiab: homozygote wild-type (wt) NLRP3 In the rat bladder, NLRP3 is localized to the urothelium genes, homozygote wt Ins2 genes (i.e., control mice). +/+ (14,15), where it mediates sterile inflammation in sev- 2. NLRP3 , diab: homozygote wt NLRP3 genes; hetero- eral important bladder pathologies, including bladder zygote for Akita mutation at the Ins2 gene (i.e., Akita outlet obstruction and cyclophosphamide-induced hem- diabetic control). 2/2 orrhagic cystitis (15,16). Experimental models have also 3. NLRP3 , nondiab: both NLRP3 genes knocked out, implicated NLRP3 in the response to urinary tract infec- homozygote wt Ins2 genes (i.e., NLRP3 knockout con- tions (17,18). Based on the central role for NLRP3 in trol). 2/2 other diabetic complications, coupled with its importance 4. NLRP3 ,diab:bothNLRP3 genes knocked out, in sterile cystitis, we hypothesized that NLRP3-mediated heterozygote for Akita mutation at the Ins2 gene. inflammation is a crucial element in the development of This is the experimental mouse generated for this DBD. study. To address this hypothesis, we bred a genetic mouse model of type 1 diabetes (the Akita mouse) (19) with Animals were received at 5 weeks of age. Blood glucose 2/2 anondiabeticNLRP3 strain to create a novel strain becomes high in Akita mice (200–300 mg/dL) ;4 weeks of of diabetic mice lacking NLRP3. These mice were then age and remains high thereafter (19,24). Mice were grown assessed for DBD. Mechanistically, DBD results, in to 15 weeks when DBD becomes apparent (25). No changes part, from peripheral neuropathy (20,21). Therefore, in urinary dysfunction were found at previous time points we also evaluated a role for NLRP3 in the variation (25). of nerve cell populations that could explain func- tional deficits in patients with diabetes, specifically di- In Vitro Experiments +/+ minished sensation (Ad-fibers) and bladder overactivity NLRP3 , nondiab mice (i.e., control littermates) were (C-fibers). used at 7 to 8 weeks of age. Urothelial cells were isolated (26) and plated (black-walled 96-well plates) at 50,000 RESEARCH DESIGN AND METHODS cells/well in 90 mL complete media (F-12K media, 10% low- m Experimental Approach endotoxin dialyzed FBS, 10 mol/L nonessential amino acids m Our approach in this study is three-pronged: 1) in vitro [all from HyClone Laboratories, Logan, UT], 1.0 g/mL m analysis of the activation of the inflammasome in normal hydrocortisone [Sigma-Aldrich, St. Louis, MO], 10 g/mL m mouse urothelia by diabetic-associated DAMPs; 2) in vivo insulin, 5 g/mL transferrin, and 6.7 ng/mL selenium urinary function (cystometry) in diabetic mice with a ge- [Gibco, Gaithersburg, MD]). Following a 24-h incubation m netic deletion of NLRP3; and 3) quantitation of nerve (37°C,95%air/5%CO2), DAMPs (10 L) were added densities in the bladders of these mice to assess potential and incubated as indicated. One hour prior to harvest, changes in specific nerves thought to mediate specific 1.25 mmol/L ATP was added to untreated wells. In stud- DBD symptoms. ies that examined ATP doses, cells were plated for 24 h and then treated with 1 mg/mL lipopolysaccharide (LPS) Animals (Escherichia coli 055:B5; Sigma-Aldrich) in PBS or PBS All protocols adhere to the National Institutes of Health alone for 24 h before treatment with the indicated doses Guide for the Care and Use of Laboratory Animals and were of ATP for 1 h. Caspase-1 activity was then measured as approved by the Institutional Animal Care and Use Com- previously described (14). Control florescence (0 mmol/L mittee at Duke University Medical Center. Founder mice DAMP) was subtracted from all wells and results nor- from The Jackson Laboratory (Bar Harbor, ME) consisted malized to the ATP response (except for the ATP dose- of Akita (C57BL/6J-Ins2Akita/J mice; stock number response studies). 2/2 003548) (22) and NLRP3 (B6.129S6-Nlrp3tm1Bhk/J (stock number 021302) mice (23). Although the strain Histological Preparation 2/2 of origin for the NLRP3 mice (129S6/SvEvTac) is Bladders were formalin-fixed and paraffin-embedded in different from the Akita background (C57BL/6J), these a transverse orientation. Sections (5 mm) from the lower mice have been backcrossed to C57BL/6J for .11 gen- third of the bladder were stained with anti-NLRP3 erations (https://www.jax.org/). Mice were bred by the (1:100) (catalog number LS-C334192; LifeSpan BioScien- Breeding Core Facility at Duke University through an ces, Inc., Seattle, WA), anti-PGP9.5 (1:200) (catalog num- independently approved protocol, and only female mice ber 381000; Thermo Fisher Scientific, Waltham, MA), 432 NLRP3 Promotes Diabetic Bladder Dysfunction Diabetes Volume 68, February 2019 anti-neurofilament 200 (1:200) (NF-200; Ad-fibers; cat- to measure voided volume. The catheter was connected alog number N4142; Sigma-Aldrich), or anti–calcitonin to a syringe pump via an in-line pressure transducer gene-related peptide (1:80) (CGRP; C-fibers; catalog num- and sterile saline infused at 15 mL/min for 60–120 min.