Interstitial cystitis (IC) is characterised by urinary urgency, frequency, and pain in the bladder and surrounding pelvic regions. A chronic inflammatory response underlies the pathology of IC resulting in the loss of urothelial barrier functions, activation of mast cells, increased ATP release, and release of pro-inflammatory cytokines. A key contributor to inflammation and cell apoptosis is pannexin-1, an ATP release channel [1]. The aim of this study was to develop an in vitro model of cytokine induced urothelial damage. The second aim is to study the protective effect of the pannexin-1 inhibitor, 10Panx1 on the cytokine induced urothelial cell damage

Cell culture model of inflammation: Freshly isolated porcine urothelial cells were cultured in RPMI culture media for 8-10 days before being transferred onto transwell inserts. Inflammation and subsequent urothelial cell damage were induced by culture of the porcine urothelial cells with the pro-inflammatory cytokines TNFα and IL-1β (both at 100 ng/mL in DMEM culture media).

The degree of epithelial damage was assessed by measuring the transepithelial electrical resistance (TEER), a measure of electrical resistance across an epithelial monolayer (Figure 1A). TEER is widely used for the study of the integrity and permeability of the epithelial monolayer in the intestine. A reduction in TEER is used to indicate inflammatory damage. TEER of the cultured porcine urothelial cells was measured 4, 24 and 48 hours after the addition of the pro-inflammatory cytokines. The pannexin-1 blocking peptide 10Panx1 (100 µM) was included to examine the protective effects of this agent on the inflammation induced reduction in TEER. 

Immunocytochemistry of urothelial cell tight junctions: To determine if the inflammatory cytokines disrupt the urothelial cell tight junctions, immunocytochemistry of zonula occludens-1 (ZO-1) expression was examined. ZO-1 has been previously shown to be decreased in patients with IC [2]. ZO-1immunoreactivity on urothelial cells was examined in the presence and absence of 10Panx1.

Primary porcine urothelial cells after 8-10 days of culture yielded a satisfactory TEER (925 - 2500 Ωcm2). Pre-incubation of urothelial cells with TNFα and IL-β induced a significant reduction of TEER values at both 24 and 48 hrs (P < 0.01, n = 8, compared to TEER in control cells, Figure 1B), indicating damage to the urothelial barrier. When urothelial cells were exposed to cytokines in the presence of the pannexin-1 channel blocker, 10Panx1, the decrease in TEER values was significantly less than the cytokines only group (Figure 1B). 

ZO-1 immunoreactivity (ZO-1-IR) was expressed on urothelial cell membranes and in the cytoplasm (Figure 2A). Treatment of urothelial cells with cytokines for 24 hrs reduced ZO-1-IR and disrupted the tight junction integrity at cell-cell borders (Figure 2B). The damage of urothelial integrity was prevented by the incubation of urothelial cells with 10Panx1 (Figure 2C), as the intensity and cellular distribution pattern of ZO-1-IR in the cells of this group appeared very similar to that seen in control cells.

The present study has established a novel in vitro model of inflammation using an isolated urothelial cell culture system to study the barrier functions of the urothelium. The results demonstrated a significant increase in the permeability of the urothelial cell monolayer in the presence of TNFα and IL-1β, as indicated by a decrease in TEER measurements. Urothelial cell permeability is increased in IC; therefore, these results suggest a crucial role of cytokines in the pathogenesis of this condition. 

TNFα and IL-1β also induced degradation of the tight junction associated protein ZO-1 around the periphery of urothelial cells, indicating that pro-inflammatory cytokines disturb the urothelial permeability barrier by damaging tight junctions. 

Perhaps the most important finding of this study was the attenuation of cytokines-induced tight junction disruption and TEER reduction in the presence of 10Panx1.This implies a regulatory role for pannexin-1 in urothelial permeability barrier functions.

In conclusion, TNFα and IL-1β can disrupt urothelial tight junctions and increase urothelial permeability. The destructive effect of TNFα and IL-1β was attenuated in the presence of the 10Panx1, which may indicate a role for pannexin-1 channel blockers in the treatment of bladder inflammatory conditions. Similar ATP release channel blockers such as connexin 43 blocking peptides are in clinical trials for reducing inflammation and accelerating wound healing in conditions such as chronic diabetic foot ulcers, and acute corneal wounds [3].

Adamson & Leitinger (2014). The role of pannexin1 in the induction and resolution of inflammation. FEBS Lett, 588(8), 1416-1422.
Lee & Lee (2014). Decreased expression of zonula occludens-1 and occludin in the bladder urothelium of patients with interstitial cystitis/painful bladder syndrome. J Formos Med Assoc, 113(1), 17-22.
Grek et al (2015). Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: A multicenter, randomized trial. Wound Repair Regen 23(2), 203-212.