Hypothesis / aims of study
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a urinary bladder disorder with an unknown etiology. It is known that cyclophosphamide can cause cystitis and bladder pain in patients who undergo chemotherapy, a condition induced by acrolein, the highly toxic metabolite of cyclophosphamide. The mechanism by which acrolein causes cystitis is likely due to its direct damage to the urothelium, leading to subsequent inflammatory responses (1). The role of purinergic P2X7 receptor (P2X7R) in mediating bladder inflammation has recently come into attention. Previous studies have shown that P2X7R antagonists inhibited acrolein-induced inflammatory changes in the bladder such as oedema, haemorrhage, loss of urothelium and apoptosis (2, 3) and restored bladder function (3).
This study aimed to determine whether high concentrations of extracellular ATP can induce P2X7R-dependent large pore formation on urothelial cell membranes of the porcine bladder. The study also sought to explore the effect of acrolein on the integrity of urothelial cells and whether inhibition of P2X7R activities can protect against acrolein-induced damage and maintain urothelial barrier function.
Study design, materials and methods
The property of P2X7R-dependent large pore formation was investigated by measuring high concentrations of ATP- and Bz-ATP-evoked YO-PRO-1 uptake into cultured porcine urothelial cells. YO-PRO-1 is a fluorescent dye with a molecular weight of 630 Daltons that can only enter cells via abnormal large pores on the membrane, followed by binding to the nucleic acid of the cells and generating a fluorescence signal. Urothelial cells (50000 cells/well) isolated from freshly dissected female porcine bladder were plated in 96-well plates and incubated at 37 ºC, 5% CO2 for 72 h. Prior to performing the assay, urothelial cells were pre-incubated with ATP (300 µM) or Bz-ATP (300 μM) for 30 min, and then the culture media was gently replaced with 180 μl of the HEPES buffer. ATP or Bz-ATP (both at 300 μM) was added into the wells and the plate was immediately read with the FLUOstar OPTIMA Plate Reader at 30 s intervals for 15 readings (485 nm excitation and 520 nm emission). 20 μl of YO-PRO-1 iodide (491/509) (2 μM) was added between the 3rd and 4th reading. P2X7R antagonists, AZ11645373 (100 μM) or A804598 (1 μM) were added 30 min before the addition of agonists.
The effect of acrolein on the integrity of urothelial cells was studied by measuring the trans-epithelial electrical resistance (TEER), a well-known indicator of cell monolayer integrity. Urothelial cells (100000 cells/well) were plated in permeable transwell inserts and cultured in DMEM media. TEER values were measured using an EVOM epithelial volt/ohm meter. When a stable TEER reading was achieved, the cells were treated with acrolein (50 µM) and TEER values were measured at the time of treatment (taken as the baseline) and then in 2, 4, 24 and 48 h after the treatment. To check the involvement of P2X7R, cells were incubated with A804598 (10 μM), 30 min before and together with acrolein. Furthermore, immunocytochemistry of ZO-1 tight junction protein was performed on urothelial cells treated similarly as the TEER experiment.
Interpretation of results
In this study, we have demonstrated that the activation of P2X7R with high concentrations of agonists can open large pore on urothelial cells, a property that can lead to the release of inflammatory cytokines and apoptotic cell death. Inhibition of P2X7R activity by its antagonists can significantly protect the cells from pore formation. The study has also shown the damaging effect of acrolein on the barrier function of porcine primary urothelial cells, and the protective effect of P2X7R blockade against the damage. This result points out the important role of P2X7R in urinary bladder cystitis which is a major side effect of the chemotherapy agent cyclophosphamide and indicates the possible therapeutic role for P2X7R antagonists in this type of bladder cystitis.