Interstitial Cystitis/Bladder pain syndrome (IC/BPS) is a clinical complex condition that presents with persistent pain, pressure or discomfort arising from the bladder accompanied by other lower urinary tract symptoms such as frequency or urgency and affects several million women and men in the US (Berry et al., 2011; Suskind et al., 2013). Different classification schemes have been developed yet patients are commonly divided between those having inflammatory disease in the bladder (“classic” IC/BPS with Hunner lesions) and those without (about 90% of patients) (Hanno et al., 2015; Akiyama and Hanno, 2019; Whitmore et al., 2019). Bladder pain/discomfort remains the cardinal symptoms of this disease and currently there is no consistent and effective treatment. Greater understanding of the mechanisms of bladder pain may lead to the discovery of potential new therapeutic targets.

We have studied the mechanisms of bladder pain using a rodent model that results in minimal damage to the organ while still producing bladder pain (Kouzoukas et al., 2015, 2016; Ma et al., 2018, 2019b; Ye et al., 2021). In this model, activation of urothelial protease activated receptors leads to release of urothelial MIF. Released MIF is then responsible for triggering release of urothelial HMGB1 to cause pain (Ma et al., 2017b).

MIF is a pro-inflammatory cytokine that is stored pre-formed in immune and non-immune cells (such as urothelial cells) (David, 1966; Jankauskas et al., 2019). In addition, there is accumulating evidence that MIF is involved in mediating or modulating nociception (Wang et al., 2011; Alexander et al., 2012). There is also ample evidence that MIF is involved in nitro-oxidative stress (Jüttner et al., 1998; Alam et al., 2012; Chuang et al., 2012; Cutrullis et al., 2017). In fact, a recent study showed that MIF induced reactive-oxygen species (ROS) generation, ERK phosphorylation and HMGB1 release in vitro (Lv et al., 2016). HMGB1 is nuclear protein recently associated with inflammation and pain (Agalave and Svensson, 2014; Tsujita et al., 2020; Sato et al., 2022).

Interestingly, thrombin induces ROS in vitro through PAR4 (Carrim et al., 2015) and activation of PAR1 and PAR4 receptors induced ROS in hepatocellular cancer cells (Mußbach et al., 2015). Therefore based on the evidence just reviewed we decided to investigate whether bladder ROS and ERK phosphorylation also participated in our model of PAR4-induced bladder pain.

The primary objective of these studies was to determine whether intravesical antagonism of ROS, ERK activation, or HMGB1 release had an effect on PAR4-induced bladder pain in mice. Secondarily, we examined the effects of these antagonists on PAR4-induced HMGB1 release in the bladder and the relationship of released bladder HMGB1 and bladder pain.

All animal experiments were approved by the Lexington VA Health Care System Institutional Animal Care and Use Committee (VER-19-005-AF) and performed according to the guidelines of the National Institutes of Health. C57BL/6 female mice (Jackson Laboratory, Bar Harbor, ME) were 12–14 weeks of age at the time of the experiments and were housed in standard rodent cages in rooms with a 14 h on (7 a.m.); 10 h off (9 p.m.) light cycle with ad libitum access to food Envigo 2018 Teklad global 18% protein rodent diet and water.

PAR4 receptors are expressed in bladder urothelial cells and peripheral nerve endings (D'Andrea et al., 2003). In fact, PAR4 peptides activated bladder afferents in mice causing a significant and sustained increase in afferent firing (Daly, 2011). Thus, intravesical PAR4 peptides may stimulate bladder afferent nerves directly to result in hyperalgesia. However, since PAR4-AP did not cause BHA in MIF KO mice (Ma et al., 2017a) we consider it likely that there is an effect on urothelial cells directly.

Intravesical administration of NACA (an analog of NAC with greater membrane permeability and used as an antioxidant by reducing ROS levels) (Grinberg et al., 2005), partially reduced PAR4-induced BHA (Figure 2B) and supporting our hypothesis. The exact trigger of ROS accumulation remains to be determined since activation of PAR4, MIF and/or TLR4 receptors can induce ROS accumulation (Carrim et al., 2015; Lv et al., 2016; Yin et al., 2019). NAC was reported to inhibit inflammatory bladder pain (Hiramoto et al., 2020) and our current results lend further support for a role for ROS in mediating PAR4-induced bladder pain. Decreased production of ROS has been proposed as a possible novel treatment for pain from IC/BPS (Hagn et al., 2019).

MIF binds to its canonical receptor CD74 to activate signaling pathways through ERK1/2/MAPK phosphorylation (Mitchell et al., 1999) while MIF can also bind other receptors including CXCR4 (Jankauskas et al., 2019). Intravesical blockade of either CD74 or CXCR4 prevented PAR4-induced BHA (Ye et al., 2021). Our current result that pre-treatment with an ERK1/2 specific inhibitor (FR180204) was not effective in blocking PAR4-induced bladder pain (Figure 2B) was not expected. It is possible that solubility issues or current doses of FR180204 were not sufficient to block ERK1/2 phosphorylation. It is also possible that PAR4-induced bladder pain may not be ERK1/2 dependent and may be eliciting ROS production through other signaling pathways.

We previously showed that PAR4 elicits release of HMGB1 from the urothelium in vivo and from urothelial cells (primary or immortalized) in vitro (Kouzoukas et al., 2016; Ye et al., 2021). EP is well-documented to inhibit active HMGB1 release in different cell systems and injury models (Chung et al., 2008; Davé et al., 2009; Kim et al., 2016; Irie et al., 2017; Seo et al., 2019; Zhang et al., 2019). Intravesical pre-treatment with EP was highly effective in blocking PAR4-induced BHA (Figure 2B) and resulted in a modest reduction in intravesical HMGB1 levels (245 ± 31.4 pg/mL) compared to PBS-PAR4 (285 ± 40.1 pg/mL). This change was not statistically significant (using ANOVA) but may be sufficient to trigger the effect on PAR4-induced bladder pain. Since EP also has anti-oxidant effects (Karabeyoǧlu et al., 2008; Shin et al., 2012) we cannot conclude that effects seen here on PAR-induced BHA are solely due to its ability to block HMGB1 release from the urothelium. However, the correlation between intraluminal HMGB1 levels and BHA (Figure 3) strongly supports a pivotal role for HMGB1 levels in the intravesical fluid (presumably released from the urothelium) in our model of bladder pain (Ma et al., 2017a,b). Other investigators have also noted that HMGB1 plays a role in other (inflammatory) models of bladder pain (Tanaka et al., 2014; Hiramoto et al., 2020) and so it is likely that HMGB1 is involved in bladder nociception.

Patients with IC/BPS are broadly divided into two categories: those with Hunner lesions (detected by cystoscopy) and a robust bladder inflammatory state and those without Hunner lesions or any obvious bladder pathology (approximately 90% of IC/BPS patients) (Whitmore et al., 2019; Homma et al., 2020). In our experiments we rely on a rodent model of bladder pain that results in minimal or no injury to the bladder to isolate pain mechanisms and not in resolution of organ injury.

We noted minimal edema and inflammation during histological examination in the PBS pre-treated and scrambled peptide treated group. This is our “no pain” control group and histological findings likely represents catheter induced changes. None of the other treatments (including intravesical PAR4-AP to induce BHA) caused a significant increase in histological changes over the control group. Moreover, we recently reported that intravesical PAR4 activation was not associated with increases in protein levels or mRNA upregulation of inflammatory cytokines (IL-1β; TNF-α) (Ye et al., 2021), an effect often seen in other inflammatory models of bladder pain (Malley and Vizzard, 2002; Smaldone et al., 2008; Sakura et al., 2009). Thus, several avenues of investigation lead us to conclude that 24 h after PAR4 administration, while mice are still experiencing BHA, there are no demonstrable signs of bladder inflammation. In addition, we did not see any changes in micturition frequency or volume in any of the treatment groups (even those that had untreated BHA) in this study or in any previous study (Kouzoukas et al., 2015, 2016; Ma et al., 2018, 2019b; Ye et al., 2021).

There are other urinary symptoms in IC/BPS (e.g., frequency, reduced micturition volume, urgency) aside from pain at urination. In fact, both Hunner and non-Hunner patients show increased frequency when compared to controls (Peters et al., 2011). Our rodent model can be viewed as limited since we do not find accompanying changes in micturition frequency or volume in mice that are experiencing BHA. On the other hand, this can be viewed as a strength since it focuses on pain mechanisms primarily. Other rodent models commonly used to mimic IC/BPS often result in significant bladder injury with associated increase in edema and inflammation (Bon et al., 1998; Bjorling et al., 2007).

We have evidence that activation of urothelial PAR4 receptors elicits the release of urothelial MIF in vivo and in vitro (Kouzoukas et al., 2016; Ye et al., 2021). We also reported that activation of PAR4 receptors results in BHA that persisted 24 h post insult (Kouzoukas et al., 2015, 2016; Ma et al., 2018, 2019b; Ye et al., 2021). Released MIF then binds to MIF receptors CD74 and CXCR4 to elicit urothelial HMGB1 release (Ye et al., 2021). Released HMGB1 binds to intravesical TLR4 receptors to mediate bladder pain (Ma et al., 2017b). Our current findings that ethyl pyruvate (known to block active HMGB1 release and also an antioxidant) prevented PAR4-induced BHA confirms our previous results. In addition, we now provide evidence that oxidative stress is involved in PAR4-induced BHA. Oxidative stress has been implicated in the pathogenesis of IC/BPS (Logadottir et al., 2013; Ener et al., 2015). Therefore, further dissecting PAR4-induced signaling pathways may lead to discover new therapeutic targets for bladder pain conditions, including IC/BPS.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Lexington VA Health Care IACUC Committee.

PV, SY, FM, and KM-S designed the experiments. SY, FM, and DM performed experiments and collected experimental data. PV, SY, FM, and DM performed data analysis. All authors contributed to manuscript preparation and review.

This study was funded by NIH (DK121695, PV; AR049610, RB). The material was the result of work supported with the resources and facilities at the Lexington (Kentucky) VA Health Care System.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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