Protective Effect of Purinergic P2X7 Receptor Inhibition on Acrolein-Induced Urothelial Cell Damage

Abstract

Patients undergoing chemotherapy with cyclophosphamide experience cystitis due to excretion of a toxic metabolite, acrolein. Cystitis, an inflammation of the bladder, is associated with damage to the integrity of the urothelial barrier. The purinergic P2X7 receptor (P2X7R) is increasingly recognized for its role in inflammation and cell death. P2X7R is expressed abundantly on the bladder urothelium. The aim of this study was to investigate the role of P2X7R in acrolein-induced inflammatory damage in primary cultured porcine bladder urothelial cells. Confluent urothelial cells in culture were treated with acrolein to induce damage; also, with the P2X7R selective antagonist, A804598. Cell viability assay, immunocytochemistry, and trans-epithelial electrical resistance (TEER) studies were carried out to investigate the effect of treatments on urothelial cell function. Acrolein induced a significant reduction in urothelial cell viability, which was protected by the presence of A804598 (10 µM). The urothelial barrier function, indicated by TEER values, was also significantly reduced by acrolein, whereas pre-incubation with P2X7R antagonist significantly protected the urothelial cell barrier from acrolein-induced TEER reduction. The structure of urothelial cell tight junctions was similarly impacted by acrolein treatment, showing the fragmentation of zona occludens-1 (ZO-1) immunoreactivity. Pre-treatment of cells with A804598 countered against the actions of acrolein and maintained ZO-1 expression level and cell structure. The damaging effect of acrolein on urothelial cells integrity could be impaired by inhibition of P2X7R, therefore P2X7R blockade may be a possible therapy in patients with bladder cystitis caused by cyclophosphamide treatment.

Blocking of P2X7r Reduces Mitochondrial Stress Induced by Alcohol and Electronic Cigarette Exposure in Brain Microvascular Endothelial Cells

“…Further, functional barrier data support the idea that mitochondrial dysfunction, Ca 2+ intracellular and ATP extracellular release underlie BBB injury by ETH or 1.8% e-Cig, and prevention of these negative effects by P2X7r inhibition preserves integrity. A recent study demonstrated that urothelial barrier function was normalized by P2X7r selective antagonist, A804598, in acrolein-induced bladder urothelial cell damage in a porcine model [ 30 ]. The multifaced role of P2X7r in BBB regulation during neuroinflammatory diseases has been recognized [ 73 ].…”

“…In this report we treated hBMVECs with A804598 (10 μM), a novel and competitive antagonist of P2X7r that is known to have high affinity for blocking P2X7 receptors [ 29 , 30 ]. Our aim was to identify unifying mechanisms of brain endothelial injury caused by ETH, ALD, and e-Cig vape.…”

Cranberry, but not D-mannose and ibuprofen, prevents against uropathogenic Escherichia coli-induced cell damage and cell death in MDCK cells

“…The protocol for measuring trans-epithelial electrical resistance (TEER) was adapted from the one used for TEER measurement in porcine urothelial cells ( Taidi et al, 2022 ). In brief, once MDCK cells in T75 flasks had reached 80% confluence, they were passaged and plated (1 × 10 5 cells per well) onto Corning Transwell inserts and cultured in MEM supplemented with 10% FBS and 1% penicillin streptomycin.…”

Sustained drug delivery to reduce the extent of burn progression

“…In a study of primary cultures of porcine bladder urothelial cells, acrolein-induced inflammatory damage had increased cell toxicity, increased P2X7 expression, and disrupted the tight junction protein ZO-1. However, this effect was abolished with the treatment of the selective P2X7R antagonist A804598, thus retained cell integrity and prevented degradation of tight junctions (Taidi et al, 2022). Similarly, another study reported on porcine theca cells from antral follicles, extracellular ATP induced cell death and DNA fragmentation was mediated by P2X7.…”