Adult female randomly cycling Sprague Dawley rats (175 - 199 g; Harlan, Prattville AL, USA) were used in all studies. All experimental protocols were approved by the Institutional Animal Care and Use Committee at our institution.

Animals were injected (i.p.) with 80 mg/kg CP or saline. We have previously shown that this dose induces changes in physiological bladder function indicative of HC within 24 h [10]. Following established protocols [11,12] the Mesna animals received Mesna at −0.5, 4 and 8 h relative to CP injection (Figure 1(a)). For caspase-1 inhibitor experiments Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethyl ketone (YVAD-AOM; EMD Chemicals, Rockland, MA, USA) was dissolved in DMSO (5 mg/ml), diluted in saline (to 200 µM), and 200 µl (or 200 µl of 2.5% DMSO) was instilled transurethrally in anesthetized rats (ketamine/xylazine; 90 mg/kg and 10 mg/kg i.p., respectively). Catheters were capped and left indwelling. 1 h later rats were injected i.p. with 80 mg/kg CP. One half hour later additional ketamine/xylazine (45 mg/kg, 5 mg/kg, respectively) was administered (i.p.). Two hours after CP injection rats were sacrificed. For anakinra experiments, rats were injected i.p. with 100 mg/kg anakinra (Amgen, Thousand Oaks, CA, USA) in saline (or saline alone) 1 h prior to injection of CP.

Urothelium were obtained by scraping, similar to what has been reported elsewhere [13,14]. Briefly, the isolated bladder was cut open from sphincter to dome. The dome ends were pinned and the sphincter held by forceps with a bent tip. The urothelium was gently scraped from the detrusor using a size 11 scalpel blade, twisting the blade to lift the cells. The cells were placed in ice-cold PBS, pelleted (800 × g, 10 min), resuspended in 25 µl of 10 mM MgCl₂, 0.25% Igepal CA-630, mixed with an equal volume of 40 mM Hepes (pH 7.4), 20 mM NaCl, 2 mM EDTA, 20% Glycerol and stored at −20˚C until analyzed (<1 week).

Caspase-3/7 activity was measured using the Caspase Glo system from Promega Inc. (Madison, WI, USA) and the manufacturer’s recommendations. Caspase-1 was analyzed similar to our previous studies [15]. Extracts (20 μl) were combined with 50 μl assay buffer (25 mM HEPES, 5% sucrose, 0.05% CHAPS (pH 7.5)), 10 μl 100 mM dithiothreitol and 20 μl 1 mM substrate (N-AcetylTyr-Val-Ala-Asp-7-amino-4-trifluoromethylcoumarin

(Ac-YVAD-AFC); Enzo Life Sciences, Inc., Farmingdale, NY, USA) in blacked-walled 96 well plates. Fluorescence (Excitation 400 nm, Emission 505 nm) was measured every 30 sec for 15 min and the slope determined. A standard curve of AFC was used to calculate specific activity.

Cells were resuspended in 1% paraformaldehyde and incubated in turn with 200 µl of BD Bioscience’s (San Jose, CA) FACS Permeabilization Solution-2 (10 min), 1 ml of 0.5% BSA in PBS (30 min), 100 µl of anti-PARP (cleaved p85) rabbit monoclonal antibody (Epitomics, Burlingame, CA, USA; 1:50 dilution; 30 min) and 200 µl Alexa Fluor 488 conjugated goat anti-rabbit IgG (H + L); Invitrogen (Life Technologies, Carlsbad, CA, USA; 1:500 dilution, 30 min), with washing in between before being analyzed by flow cytometry.

To test for differences over time (Figure 2), we modeled the log of percent difference from baseline (time = 0) using linear regression with fixed effects for hours from baseline, treated as categorical variables. Regression model assumptions were evaluated using residual plots and QQ-plots. p-values represent tests that percent changes from baseline were different from 0, and p values < 0.05 were considered significant. Other comparisons were conducted using non-parametric Wilcoxon rank sum tests or Student’s t-test, as appropriate, and were also considered significant if p < 0.05.

Caspase-3/7 activity in urothelium was followed for 72 h after the injection of 80 mg/kg CP. As shown in Figure 2(a), we detected a biphasic induction of apoptosis with an initial peak at 2 h, followed by a return to baseline at 8 h. At 16 h, levels had increased again and continued to go up until at least the 48 h time point. While we have previously shown that this dosing paradigm induces changes in physiological bladder function indicative of HC at 24 h [10], the peak at 2 h is novel and is not associated with gross morphological change (data not shown). To confirm the 2 h result, we have examined expression of an independent apoptotic marker, cleaved PARP [16]. As shown in Figures 2(b) and (c), there was a 21.2% increase in the percentage of the urothelium cells with cleaved PARP at 2 h (% of cells under the M1 gate).

To access the contribution of acrolein to the two waves of apoptosis, rats were pretreated with Mesna 0.5 h prior to CP administration. Due to the short half-life of Mesna [17], rats assessed at 24 h also received additional doses of Mesna at 4 and 8 h [11,12] (Figure 1(a)). As shown in Figure 1(b), Mesna significantly (p < 0.05) reduced the caspase-3/7 activity in urothelium at 24 h but only slightly and non-significantly reduced the activity at 2 h, indicating that the early peak of apoptosis is mostly acrolein-independent while the latter wave is largely dependent on this metabolite.

Damage to a cell or tissue often results in the activation of caspase-1. As shown in Figure 3(a), we detect significant activation of caspase-1 two hours after CP administration, further indicating urothelium damage at this time point. Working upstream, caspase-1 can cleave caspase-3/7 directly [18,19] or stimulate apoptosis through production of IL-1β [20,21]. Direct luminal instillation of the caspase-1 inhibitor YVAD-AOM blocked the increase in caspase-3/7 activity stimulated by CP after 2 h (Figure 3(b)). Caspase-3/7 activity was significantly (p < 0.05) stimulated by CP in rats that were not pretreated with anakinra (an IL-1 receptor antagonist) and in ones that were pretreated (Figure 3(c)). In addition, there was no significant (p = 0.28) difference noted between the anakinra pretreated and saline rats which did not receive CP, and there was no significant (p = 0.10) difference noted between the anakinra pretreated and saline rats which did receive CP. In other words, pretreatment of rats with the IL-1β receptor antagonist anakinra had no effect (Figure 3(c)).