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.
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