AB279. SPR-06 Fibrosis in the bladder in response to outlet obstruction is triggered through the NLRP3 inflammasome and the production of IL-1β

Objective: Bladder outlet obstruction (BOO), most commonly created by benign prostatic hyperplasia, promotes an inflammatory state that produces voiding dysfunction. Recently, we have shown that the NLRP3 inflammasome triggers this inflammation. Over time, inflammation promotes fibrosis. In this study we explore the role of NLRP3 (and IL-1β produced by activated NLRP3) in BOO-induced fibrosis.

Methods: Rats were divided into 5 groups: (I) control, (II) sham, (III) BOO + Vehicle (Veh; 1 mL, 40% EtOH, p.o.), (IV) BOO + glyburide (Gly; NLRP3 inhibitor; 10 mg/kg, p.o.) or (V) BOO + Anakinra (Ana; IL-1 receptor antagonist; 25 mg/kg, i.p.). BOO is constructed by inserting a 1-mm transurethral catheter, tying a suture around the urethra and removing the catheter. Medications were given prior to surgery and once daily for 12 days. Hypertrophy was assessed by bladder weight, fibrosis by collagen staining (Masson’s Trichrome Stain), IL-1 receptor 1 (IL-1R1), prolyl 4-hydroylase (P4H) and lysyl oxidase (LOX) localization by immunofluorescence and collagen secretion by Sirius Red.

Results: BOO increased production of collagen in the bladder which was blocked by either preventing NLRP3 activation with glyburide or blocking IL-1β’s action at its receptor, clearly implicating the NLRP3/IL-1β pathway in fibrosis during BOO. IL-1β directly triggers collagen synthesis in other tissues and we found that recombinant IL-1β stimulated pro-collagen production in control urothelial cells placed in culture. Consistent with urothelia as a source of pro-collagen production, isolated urothelial cells from BOO rats secreted significantly more IL-1β than control cells. In control rats, the IL-1β receptor, IL-1R1, was highly expressed in the basal layer of the urothelia with less staining in the umbrella cells and detrusor and no staining in the interstitium. P4H (a marker of pro-collagen synthesis) exhibited similar staining, although enhanced expression in the basal urothelia was not as prominent. LOX (marker of mature collagen fibril production) was also found in urothelia and detrusor but additionally in the interstitium (the site of mature collagen fibril production). In BOO rats, the hyperplastic urothelia contained multiple layers of cells that all expressed IL-1R1 with P4H and LOX. Gly and Ana prevented this change in IL-1R1 distribution.

Conclusions: NLRP3-derived IL-1β triggers fibrosis during BOO, most likely through an autocrine loop in which IL-1β acts back on urothelia to drive collagen production.

Funding Source(s): NIDDK: R01DK103534 (PI-Purves)

Keywords: Fibrosis; bladder; outlet obstruction; inflammasome; urothelium