NLRP3/Il-1β Mediates Changes in Urothelial Muscarinic Receptors M2 and M3 during Bladder Outlet Obstruction in Rats

Hughes F1, Jett A1, Pines S1, Tolbert S1, Jin H1, Purves T1

Research Type

Pure and Applied Science / Translational

Abstract Category

Overactive Bladder

Abstract 128
Open Discussion ePosters
Scientific Open Discussion Session 7
Wednesday 29th August 2018
12:15 - 12:20 (ePoster Station 7)
Exhibition Hall
Bladder Outlet Obstruction Overactive Bladder Physiology
1. Duke University Medical Center
Presenter
F

Francis Hughes

Links

Poster

Abstract

Hypothesis / aims of study
Bladder outlet obstruction (BOO) is a prevalent disorder mostly found in older men in response to benign prostatic hyperplasia (BPH), although it can occur in women with organ prolapse, children with posterior urethral valves or in any population due to stone formation.  Obstruction leads to repetitive bouts of high pressure, excessive stretch and transient hypoxia in the bladder. These insults initiate an inflammatory response that leads to overactive bladder (OAB) symptoms, fibrosis and denervation. Eventually these changes can cause bladder decompensation and organ failure.  Recently a critical role has been demonstrated for the NLRP3 inflammasome, found in the urothelia, in triggering the inflammation associated with BOO and controlling the downstream events of OAB, fibrosis and denervation [1]. The multimeric NLRP3 inflammasome forms in response to damage associated molecular patterns (DAMPs) released by cells in response to the repetitive pressure, stretch and hypoxia, and activates caspase-1. This protease then processes pro-IL-1b and pro-IL-18 to their mature forms which are released and function as pro-inflammatory cytokines to initiate the inflammatory response.
In addition to initiating the inflammatory reaction, the urothelium is now understood to influence many aspects of bladder function by altering afferent signaling from the bladder, muscle contractility and growth/differentiation of the entire organ [2].  It does this through expression of several receptors and soluble mediators, many of which demonstrate changes in expression during BOO. In this study we have chosen to examine expression of the muscarinic receptors, M2 and M3, for they have an increased expression during BOO that is thought to contribute to altered sensation and the pathological conditions of overactivity [3]. To gain insight into whether the NLRP3 inflammasome and its product IL-1b may play a role in triggering these changes, the expression level of M2 and M3 were measured in the presence and absence of a NLRP3 inflammasome inhibitor, glyburide, or an IL-1 receptor antagonist, anakinra.
Study design, materials and methods
Partial obstruction of the bladder outlet was performed in Sprague Dawley rats (≈200g, ≈45 day old) by inserting a catheter (P-50 tubing), tying a nondissolvable suture around the urethra, and removing the catheter. Treated rats were administered glyburide (10 mg/kg in 1 ml 40% ethanol; p.o.) or anakinra (25 mg/kg in 1.6 mM citric acid (pH 6.5), 1 mM EDTA, 133 µM polysorbate 80, 140 mM NaCl; i.p.) daily, starting one day prior to surgery with the second dose given 1-2 h before surgery. Animals were then treated for 12 days before sacrifice. For histological analysis bladders were weighed and then fixed in neutral buffered formalin overnight before being embedded in paraffin, sectioned (5 µm) and stained using standard techniques, including citrate antigen retrieval (pH 6.0), with a rabbit anti-M2 (Santa Cruz, cat# sc-9107; 1:50) or rabbit anti-M3 (Santa Cruz, cat# sc-9108, 1:50) primary antibody along with a goat-anti-rabbit-AF488 secondary antibody (Southern Biotech, 1:500).	
For flow cytometry, bladders were inverted, inflated with PBS and tied off with a purse string suture before submersion for 1 hr at 37 °C in 5 ml of a 1mg/ml collagenase P solution. Isolated cells (≈106) were then pelleted and fixed in 250 µL of Cytofix (BD Biosciences) (30 minutes, 4°C).  Pellets were washed (Cytoperm – wash buffer provided by manufacturer), blocked (1% BSA, 0.5 hr on ice) and incubated in the same primary (1:50 dilution in wash buffer) and secondary antibodies as above. Cells were then analyzed by flow cytometry.
Significance was assessed by a one-way analysis of variance (ANOVA) followed by a Tukey's post-hoc analysis using GraphPad InStat software (La Jolla, CA). Statistical significance was defined as p < 0.05.
Results
Bladder weight was increased in the BOO bladders relative to control. This increase was blocked to levels not significantly different from controls by treatment with glyburide or anakinra. Immunocytochemistry of control tissue (Figure 1) demonstrated that both M2 and M3 expression in the bladder was primarily localized to the urothelia.  BOO did not change this distribution pattern, although there was hyperplasia of the M2 and M3-expressing urothelial layer (arrow).  Treatment with glyburide or anakinra also did not change the expression pattern, although it did suppress the hyperplasia. Flow cystometric analysis (Figure 2) demonstrated a significant increase in the expression level per cell of M2 and M3 in the BOO urothelia over control. This increase was completely prevented by treatment with glyburide or anakinra.
Interpretation of results
Blocking the NLRP3/Il-1b pathway with either glyburide or anakinra has previously been shown to prevent inflammation-induced increases in bladder weight and this holds true with this particular cohort of rats.  M2 and M3 expression was found primarily localized to the urothelia and this distribution did not change with any manipulation, suggesting the urothelia may be the most relevant bladder cell type to study this receptor in.  The number of urothelia did increase with BOO and this was blocked with the inhibitors, as described before, while all urothelia retained expression.  Thus, one possible mechanism of altered sensation/overactivity in BOO (58) may be the NLRP3-dependent increase in the number of M2 and M3 expressing cells.  In addition, flow cystometric analysis also demonstrated that urothelia from BOO rats contained more M2 and M3 per cell, offering a potential second mechanism for altered sensation/overactivity. This change in expression per cell was also blocked by inhibitors of the NLRP3/Il-1b pathway, clearly further establishing the central importance of this pathway in the pathological alterations to the bladder in response to BOO
Concluding message
The novel results in the study provide greater insight into the central role the NLRP3/IL-1b pathway plays in the development of BOO pathologies and clearly indicate it as a potential target for therapeutic intervention.
Figure 1
Figure 2
References
  1. Hughes FM, Jr., Hill HM, Wood CM, Edmondson AT, Dumas A, Foo WC, Oelsen JM, Rac G, Purves JT. The NLRP3 Inflammasome Mediates Inflammation Produced by Bladder Outlet Obstruction. J Urol (2016) 195:1598.
  2. Birder L, Andersson KE. Urothelial signaling. Physiol Rev (2013) 93:653.
  3. Kim JC, Yoo JS, Park EY, Hong SH, Seo SI, Hwang TK. Muscarinic and purinergic receptor expression in the urothelium of rats with detrusor overactivity induced by bladder outlet obstruction. BJU Int. (2008) 101(3):371.
Disclosures
Funding National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, USA (R01DK103534 to JTP) and intramural funds from Duke University Medical Center, Department of Surgery, Division of Urology Clinical Trial No Subjects Animal Species Rat Ethics Committee Duke University Institutional Animal Care and use Committee
28/03/2024 07:38:52