Urinary tract infection (UTI) is the most common type of bacterial infection, with up to 60% of women experiencing UTI during their lifetime and the most common organism causing UTI is uropathogenic E. coli (UPEC) [1]. UPEC cause damage to the typical urothelial layer structure and the failure of the urothelial barrier in protecting the underlying layers of the bladder can largely contribute to the symptoms associated with UTI [2]. A better understanding of how bacteria modify urothelial permeability may lead to more efficient therapeutic strategies. The most benefit goes to the treatment of antibiotic-resistant UTI. UTI has been shown to induce the release of ATP from urothelial cells [3]. All P2X purinergic receptors can be activated by increased extracellular ATP. Therefore, P2X purinergic receptors can be an exciting target to investigate in patients suffering from UTI.
In this study, we aimed firstly to determine the effect of acute UPEC infection on urothelial barrier integrity in porcine primary cultured urothelial cells. Secondly, to determine if purinergic receptor antagonist could inhibit the effect of UPEC on the urothelium.

In this study, the effect of UPEC strain, UTI89 on the integrity of urothelial cell monolayer was investigated using porcine primary urothelial cells. UTI89 (OD600: 0.4) was incubated with the urothelial cells, and UPEC-induced urothelial damage was determined by measuring the cell viability, trans-epithelial electrical resistance (TEER) and immunocytochemistry of ZO-1 tight junction protein. We have also further investigated the mechanism of UPEC-induced damage to urothelial cells by measuring the level of intracellular reactive oxygen species (ROS) using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. To investigate the role of P2X purinergic receptors, urothelial cells were incubated with a non-selective P2X antagonist, PPADS (100 µM). To further narrow down which P2X receptor is primarily responsible for this effect, cells were also incubated with a selective P2X7 receptor antagonist, A804598 (10 µM).

UTI89 caused a significant disruption to urothelial cell barrier integrity as indicated by a decrease in TEER after only 2h incubation (P < 0.01, two-way ANOVA followed by Bonferroni’s multiple comparisons test, compared to control, n = 6) up to 24h treatment period (P < 0.0001) (Figure 1A). Pre-incubation of cells with PPADS (100 μM) reduced the effect of UTI89 on TEER at 2h and 4h but not at 24h (P < 0.0001, two-way ANOVA, compared to UTI89-treated) (Figure 1A). ZO-1 immunoreactivity (IR) was observed on cell membranes tightly connected to neighbor cells in control urothelial cells (Figure 1B).  Following 4h treatment with UTI89, there was a noticeable reduction in ZO-1IR. A partial protective action was demonstrated on UTI89-induced damage to ZO-1IR when cells were treated with PPADS (100 M). However, the P2X7 receptor antagonist, A804598 (10 µM), showed no effect on UTI89-induced changes in TEER and ZO-1IR (Figure 1A and 1B).   

UTI89 significantly reduced the viability of urothelial cells at 4h up to 12h incubations (P < 0.05, P < 0.0001, t-test, n=5) (Figure 2A). The pre-treatment of urothelial cells significantly inhibited the cytotoxic effect of UTI89 with PPADS (100 µM, P < 0.05) at 4h but there was no effect at 12h incubation (Figure 2B). Furthermore, the level of ROS was significantly increased following treatment with UTI89 (P < 0.001, two-way ANOVA) (Figure 2 C&D). This effect was inhibited by the treatment of cells with PPADS (100 µM) at 2h (P < 0.001), 4 h (P < 0.01) and 6h (P < 0.05) (Figure 2D). Again, A804598 (10 µM), did not affect UTI89-induced cytotoxicity, TEER, ZO-1 and ROS at any time point (Figure 2C).

The malicious actions of UTI89 on cultured urothelial cells include disrupted ZO-1 tight junctions, decreased TEER, reduced cell viability and increased ROS production. Although we have previously shown that the selective P2X7 receptor antagonist, A804598, can abolish acrolein-induced urothelial damage, A804598 was not effective to overcome bacterial-induced urothelial damage. Nevertheless, the non-selective purinergic antagonist, PPADS, exhibited partial protection against UTI89-induced damage. Therefore, purinergic receptors other than P2X7 may play a role in UPEC-induced bacterial cystitis.

This study demonstrated that UTI89 caused serious damage to urothelial barrier integrity and tight junctions in cultured porcine urothelial cells. The broad spectrum P2X receptor antagonist PPADS showed partial protection against these effects, but it was independent of P2X7R. Other purinergic P2X receptors may be involved in this protective action.

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