Disulfide high mobility group box-1 causes bladder pain through bladder Toll-like receptor 4

Abstract

Background: Bladder pain is a prominent symptom in several urological conditions (e.g. infection, painful bladder syndrome/interstitial cystitis, cancer). Understanding the mechanism of bladder pain is important, particularly when the pain is not accompanied by bladder pathology. Stimulation of protease activated receptor 4 (PAR4) in the urothelium results in bladder pain through release of urothelial high mobility group box-1 (HMGB1). HGMB1 has two functionally active redox states (disulfide and all-thiol) and it is not known which form elicits bladder pain. Therefore, we investigated whether intravesical administration of specific HMGB1 redox forms caused abdominal mechanical hypersensitivity, micturition changes, and bladder inflammation in female C57BL/6 mice 24 hours post-administration. Moreover, we determined which of the specific HMGB1 receptors, Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), mediate HMGB1-induced changes.

Results: Disulfide HMGB1 elicited abdominal mechanical hypersensitivity 24 hours after intravesical (5, 10, 20 μg/150 μl) instillation. In contrast, all-thiol HMGB1 did not produce abdominal mechanical hypersensitivity in any of the doses tested (1, 2, 5, 10, 20 μg/150 μl). Both HMGB1 redox forms caused micturition changes only at the highest dose tested (20 μg/150 μl) while eliciting mild bladder edema and reactive changes at all doses. We subsequently tested whether the effects of intravesical disulfide HMGB1 (10 μg/150 μl; a dose that did not produce inflammation) were prevented by systemic (i.p.) or local (intravesical) administration of either a TLR4 antagonist (TAK-242) or a RAGE antagonist (FPS-ZM1). Systemic administration of either TAK-242 (3 mg/kg) or FPS-ZM1 (10 mg/kg) prevented HMGB1 induced abdominal mechanical hypersensitivity while only intravesical TLR4 antagonist pretreatment (1.5 mg/ml; not RAGE) had this effect.

Conclusions: The disulfide form of HMGB1 mediates bladder pain directly (not secondary to inflammation or injury) through activation of TLR4 receptors in the bladder. Thus, TLR4 receptors are a specific local target for bladder pain.

Keywords: HMGB1; RAGE; TLR4; abdominal mechanical hypersensitivity; bladder pain; urothelium.

Fig. 1

Disulfide and all-thiol HMGB1 dose response effects on abdominal mechanical thresholds. (a) vehicle control, (b) 1 μg (n = 4) and (c) 2 μg (n = 3) disulfide HMGB1 did not affect abdominal mechanical threshold. (d) 5 μg (n = 5) disulfide HMGB1 significantly increased abdominal sensitivity using a 0.07 g filament. (e) 10 μg (n = 6) and (f) 20 μg (n = 3) disulfide HMGB1 significantly induced abdominal hypersensitivity using all four von Frey filaments. None of the all-thiol HMGB1 doses (g) 1 μg (n = 3), (h) 2 μg (n = 3), (i) 5 μg (n = 4), (j) 10 μg (n = 5) and (k) 20 μg (n = 3) changed abdominal mechanical sensitivity. *p < 0.05, **p < 0.01 compared with pre-instillation

Fig. 2

Bladder histology after disulfide HMGB1 and pretreatment with intravesical or intraperitoneal HMGB1 receptor antagonists. (a) Intravesical vehicle control instillation (n = 5), (b) intravesical TAK242 (n = 4), (c) intraperitoneal TLR4 antagonist TAK242 (n = 5), (d) 10 μg disulfide HMGB1 induced submucosal fibrosis with lamina propria expansion (black arrows) (n = 6), (e) intravesical FPS-ZM1 pretreatment (n = 6) or (f) intraperitoneal RAGE antagonist FPS-ZM1 (n = 3)

Fig. 3

TLR4 or RAGE antagonist pretreatment prevented abdominal mechanical hypersensitivity induced disulfide HMGB1. (a) 10 μg disulfide HMGB1 significantly increased abdominal mechanical sensitivity (percent responses) using all four von Frey filaments (n = 6). (b) Intravesical TLR4 antagonist TAK242 blocked disulfide HMGB1 induced abdominal hypersensitivity (n = 4). (c) Intraperitoneal TAK242 reduced mechanical hypersensitivity induced by 10 μg disulfide HMGB1 (n = 5). (d) Intravesical infusion of RAGE antagonist PSF-ZM1, however, did not affect abdominal mechanical hypersensitivity induced by disulfide HMGB1 (n = 6). (e) Intraperitoneal injection of PSF-ZM1 prevented disulfide HMGB1 induced mechanical hypersensitivity (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 Twenty-four hours post-instillation compared with pre-instillation

Fig. 4

Role of HMGB1 in PAR4 induced bladder pain. Activation of PAR4 receptors on urothelial cells elicits release of urothelial macrophage migration inhibitory factor (MIF). MIF binds to urothelial MIF receptors (CD74/CXCR4) to mediate release of urothelial HMGB1. Disulfide HMGB1 (ds HMGB1) may bind to TLR4 receptors in urothelium and/or nerve terminal innervating the bladder to mediate bladder pain