The following experiments were performed in accordance with institutional and national guidelines and regulations. The University of Vermont Institutional Animal Care and Use Committee approved all experimental protocols (no. X9-020) involving animal use. Animal care was under the supervision of the University of Vermont's Office of Animal Care and Management in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress, or distress.

Female Wistar rats (n = 5-9 per treatment group, unless otherwise noted) and female C57/BL6 mice (n = 5-6 per treatment group) were bred locally at the Larner College of Medicine at the University of Vermont or purchased from Charles River Laboratories. Females were used as subjects due to experiments involving drug administration via external urethral catheterization. Rats and mice were maintained under standard laboratory conditions: they were housed two per cage and maintained on a 12:12-h light-dark cycle, with free access to food and water. Estrous cycle status was not determined in these studies.

Rats were anesthetized with isoflurane (2-3%) and externally catheterized using a transurethral catheter. This was performed using a lubricated 22-gauge gavage needle and gently inserting it through the urethra and into the bladder, such that it did not have contact with the bladder wall. Yoda1 (0.4 mg/kg) was infused into the bladder and remained there for 30 min (10, 12, 17, 23). A vehicle solution of 5% DMSO in 95% saline was also infused in a separate cohort of rats. After the 30-min infusion, the bladder was manually voided. The rats recovered from anesthesia for 30 min before being put into the Urovoid system to study functional bladder outcomes.

Metabolic cages and the Urovoid system (Med Associates, St. Albans Vermont) were used to study functional voiding parameters in rats. On the day of functional testing, rats (n = 6-7 per group) were placed into individual metabolic cages, situated above a flask on a balance, connected to a computer with Urovoid software. Rats remained in these cages with ad libitum access to food and water for 24 h (h). Over that time, their urine was caught in a flask on a balance, and the computer recorded the time and weight of each void to obtain functional voiding parameters such as number of voids, intermicturition interval (the time between each void to determine voiding frequency) and the mass of each void. Volume of water consumed by each rat in 24 h was also recorded and analyzed.

Rats received two voiding assessments using the Urovoid system. First, a baseline assessment was done 7-10 days before Yoda1/vehicle infusion to assess each rat's normal voiding function. Before the start of this baseline Urovoid assessment, the rats were anesthetized with isoflurane (2-3%), externally catheterized and infused with saline for 30 min. For the second assessment, 7-10 days later at the same time of day, the same rats were intravesically infused for 30 min with Yoda1 (0.4 mg/kg) or vehicle while the rats were anesthetized (2-3% isoflurane). The voiding assessment began 30 min after the infusion. By using this method of repeated assessments, individual differences and variability were minimized since rats are compared to their own baseline voiding function. Furthermore, since baseline assessments were done after administration of anesthesia and a transurethral catheter, the effects seen in the experimental groups are not due to these variables. Rats were excluded if their volume of water intake in the second (i.e., experimental) assessment was increased by over 20% compared to baseline consumption to ensure that changes in voiding frequency from baseline were not due to changes in water consumption.

Rats/mice were lightly anesthetized with isoflurane (2–3%) and given a single injection of CYP (150 mg/kg, i.p. for rats; 200 mg/kg, i.p. for mice) to induce acute and intermediate cystitis, or three injections (75 mg/kg, i.p. for rats and mice) every 72 h to induce chronic cystitis. For acute experiments, a cohort of rats/mice were euthanized (5% isoflurane and thoracotomy) 4 h after the CYP injection. For the intermediate experiments, rats/mice were euthanized 24 h after the CYP injection. Lastly, for the chronic CYP-treated cohort, rats/mice were euthanized 24 h after the last CYP injection on day (d) 8. The urinary bladder was immediately dissected after euthanasia.

Following the tissue harvest, the urinary bladder was quickly dissected under RNase-free conditions. The bladder was cut open along the midline and pinned to a Sylgard-coated dish, and the urothelium, including the suburothelium, was removed from the detrusor muscle with fine forceps and iris scissors. The urothelium and detrusor were evaluated separately.

We first extracted total RNA from samples (n = 5-9) using Stat-60 total RNA/mRNA isolation reagent (Tel-Test B Labs, Friendswood, TX). RNA samples (2 μg) were then used to synthesize cDNA using a mixture of random hexamers and oligo(dT) primers with M-MLV reverse transcriptase (Promega). Primers (Table 1) were designed using Oligo Primer Analysis Software 7.0. We prepared the quantitative PCR standard for each transcript using the amplified cDNA products ligated directly into the pCR2.1 TOPO vector using the TOPO TA cloning kit (Invitrogen). To verify the nucleotide sequences of the inserts, we used automated fluorescent dideoxy dye terminator sequencing (Vermont Integrative Genomics Resource). All cDNA templates were diluted 10-fold (to limit the inhibitory effects of the reaction components) and assayed using Luna universal quantitative PCR master mix (New England Biolabs). We performed qPCR (7500 Fast real-time PCR system, Applied Biosystems, Foster City, CA) under standard conditions as outlined: (24) serial heating at 94°C for 2 min and (19) amplification over 45 cycles at 94°C for 15 s and 60°C for 30 s. The amplified product was exposed to SYBR green I melting analysis by ramping the temperature of the reaction samples from 60° to 95°C. We ensured the creation of a single DNA melting profile under the outlined dissociation assay conditions to confirm the amplification of a unique, single product that was free of primer dimers and other anomalous products.

For data analysis, we constructed a standard curve by amplification of serially diluted plasmids containing the target sequence. The data were analyzed at the termination of each assay utilizing sequence detection software (version 1.3.1, Applied Biosystems, Norwalk, CT). In standard assays, default baseline settings were selected and the increase in SYBR green I fluorescence intensity (ΔRn) was plotted as a function of cycle number. The threshold cycle was determined by the software as the amplification cycle at which ΔRn first intersects the established baseline. Normalized data are expressed as the relative quantity of the gene of interest normalized to the relative quantity of the housekeeping gene L32 or an average of 18S and L32 housekeeping genes.

Mice were used for Ca²⁺ imaging experiments due to difficulties loading Ca²⁺ fluorescent dye into urothelial sheets procured from rats.

Harvested bladders from naïve (no CYP)/4 h/24 h/chronic (8 d) CYP-treated mice were placed in cold HEPES solution composed of (mM): 134 NaCl, 6 KCI, 10 glucose, 10 HEPES, 1 MgCl₂, 2 CaCl₂, 10 glucose and adjusted to pH 7.4 with NaOH. The bladder was cut open along the midline, and pinned to a Sylgard coated dish where the urothelium was separated from the suburothelium (i.e., lamina propria) and the detrusor using fine forceps and iris scissors (25). To detect Ca²⁺ events, the urothelial sheets were loaded with a Ca²⁺ sensitive fluorescent dye (10 μM Cal 520; AAT Bioquest, Inc.) for 90 min at 37°C before being placed in a physiological saline solution (PSS) composed of (mM): 119 NaCI, 4.7 KCI, 24 NaHCO₃, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 MgSO₄, 7 glucose and constantly bubbled with Biological Gas (95% O₂, 5% CO₂) to maintain pH at 7.4.

To measure Ca²⁺ activity following PIEZO1 activation, the urothelial sheets were first imaged for 90 s to establish baseline Ca²⁺ activity. The urothelium was then superfused for 10 min with Yoda1 (20 μM) followed by a 90 s recording of the same region. In cell-based assays, Yoda1 has an EC₅₀ of 10-50 μM (12) and we found that a concentration of 20 μM optimized solubility of the compound and response.

Changes in Ca²⁺ activity after PIEZO1 activation were then compared to baseline Ca²⁺ activity. Vehicle controls (DMSO 0.1%) were also conducted in a separate cohort of mice (no CYP) to control for laser damage and any cellular effects of DMSO. Images were collected at a rate of 16 images/second using a Noran Oz laser scanning confocal microscope (26) and a Nikon 60x water immersion fluorescent objective (NA 1.2). Cal 520 was excited at 488 nm and the emitted fluorescence was collected at >500 nm (26). Analysis of Ca²⁺ events was performed with custom-written software (Volumetry G9: Grant Hennig).

Image sequences of the urothelium were captured (512 × 512 at a spatial resolution 0.689 μm per pixel and temporal resolution of 0.625 s). The files were saved as multislice TIFF files and imported into custom-written software (Volumetry G9d, GWH). Motion stabilization and background intensity normalization (debleaching) was applied as necessary followed by temporal averaging (±2 frames: total time 3.13 s) and spatial smoothing (Gauss 5 × 5 pixel, sd = 1.0) to reduce noise.

Loading of Cal520 into the urothelium is often uneven with cells displaying bright and dim baseline fluorescence intensities. This creates issues when measuring Ca²⁺ event amplitudes using the traditional F/F0 ratio, as cells with high background intensities will have smaller calculated amplitudes compared to cells with low background intensities, even though the absolute change in intensity during Ca²⁺ events may be similar.

To circumvent this limitation, we calculated Ca²⁺ event amplitude using a signal-to-noise approach where changes in intensity during Ca²⁺ events are put in relation to the variation of intensity during quiescent periods (27). The use of spatio-temporal (ST) maps calculated from active sites is used descriptively in this study to demonstrate patterns of Ca²⁺ events but was not used quantitatively due to the ovoid nature of urothelial cells that was not suited to dimensional averaging. To better measure the extent and intensity of Ca²⁺ events in ovoid cells, pixel binning (10 × 10 pixel average) was used to spatially reduce noise, then a conservative quiescence estimator (20% of the 15 m:40 s recording time = ~3 min) was used to demarcate time periods in which a cell was not active. A histogram of the intensities throughout the recording at each pixel was calculated, and then the standard deviation (SD) of the dimmest 20% of pixels was calculated (SDqmin). Quiescent periods were defined statistically as those portions of the recording that had intensities <11.0 SDqmin. Then, within the defined quiescent periods, standard deviation and average intensity (AVGq) was recalculated (SDq) enabling the movie intensities to be converted to Z-scores (Zscr: (intensity-AVGq)/SDq). A threshold was used to define active Ca²⁺ events (≥2.7 Zscrs) and remaining noise was filtered using a particle filter (≤ 3 pixels).

Contiguous pixels in Ca²⁺ events were coalesced into coordinate-based particles in each frame, then an overlap function was used between successive frames to determine the time course of Ca²⁺ events and create ST objects. All spatial, temporal, and intensity information was extracted from Ca²⁺ event ST objects including duration (s), area (μm²), maximum intensity (Zscr) and an integrated measure of elevated overall active Ca²⁺ presence or “output” in the cytoplasm of all cells in the field of view (FOV) we abbreviated to Zum²s (Zscr ∙ μm² ∙ s.min⁻¹). This measure multiplied number of active pixels by their size (μm²), their intensity (Zscr) and their duration (s). This value was normalized to per minute and is a useful way to summarize changes in Ca²⁺ activity in the entire FOV.

Values are presented as means ± SEM. Power and sample size were determined using G*Power statistical software. Data were compared using One-Way ANOVA, mixed model repeated measures ANOVA, and Student's paired t-test, as appropriate, with SAS and GraphPad Prism statistical software. For the mixed model repeated measures ANOVA, statistical test assumptions were not met, therefore, a square root transformation was used to compress variance in the analysis of the number voiding events, and a natural log transformation was used on the raw data in the analysis of intermicturition interval and void mass. Differences with P ≤ 0.05 are considered statistically significant.

Previous studies in unconscious rats have shown that blockade of PIEZO1 using the non-selective MSC antagonist, GsMTx4 (2.5 μM), increases the intermicturition interval (i.e., the time between voiding events) when infused directly into the bladders of naïve rats, and rats chronically treated with CYP (17). Thus, we hypothesized that activating PIEZO1 via intravesical infusion of Yoda1 directly into the bladder would recapitulate the hallmark symptom of increased voiding frequency commonly seen in patients with IC/BPS and animal models using CYP-treatment.

Changes in voiding parameters after Yoda1 (n = 6) were compared to changes after vehicle (n = 7) by testing the group^*condition interaction at each timepoint to evaluate the difference between the effects at each timepoint. Significant differences that are noted indicate a significant difference between the effects (baseline vs. vehicle compared to baseline vs. Yoda1 at each timepoint). 0-4 h after baseline infusion, rats voided an average of 3 times, which significantly increased to an average of 5 times after Yoda1 infusion (P ≤ 0.05; Figure 1A). From 4-8 h after Yoda1 infusion, voiding events increased to an average of 9 compared to 5 voids at baseline (P ≤ 0.05). From 8 to 12 and 12 to 16 h after Yoda1 infusion, rats voided an average of 9 and 8 times which were significantly more than their baseline voiding levels during each respective time period (P ≤ 0.05).

These changes in voiding frequency after infusion of Yoda1 corresponded with decreases in the IMI compared to vehicle such that 0-4 h after Yoda1 infusion, the IMI decreased from baseline by 50.6% (P ≤ 0.01). From 4 to 8 h the IMI decreased 45.7% (P ≤ 0.001), from 8 to 12 h the IMI decreased 34.5% (P ≤ 0.01), and from 12 to 16 h, the IMI decreased 44.2% compared to baseline (P ≤ 0.01; Figure 1B). There were no significant changes in the number of voiding events or IMI compared to baseline 16-20 or 20-24 h after Yoda1 infusion. Interestingly, even with the increase in voiding frequency, there was no statistical difference in void mass from baseline after Yoda1 administration compared to vehicle at any timepoint tested (Figure 1C). However, we did find a statistical difference in the total void mass after 24 h in rats treated with Yoda1 (28.9 g ± 11.4) compared to baseline void mass (19.1 g ± 11.4; P ≤ 0.05).

Due to the established importance of urothelial signaling in mechanotransduction and subsequent afferent nerve terminal activation, we hypothesized that there would be bladder tissue-specific regulation of MSC mRNA during varying durations of CYP-induced cystitis in the urothelium vs. the detrusor. Contrary to this, we observed similar regulation of all MSCs tested (Trpv1, Trpv4, Piezo1, and Piezo2) in both the urothelium (Figures 2A–D) and the detrusor (Figures 2E–H). Specifically, we found that compared to naïve (no CYP-treatment), chronic CYP-treatment led to increases in the mRNA expression of all MSCs examined: Trpv1 (P ≤ 0.0001), Trpv4 (P ≤ 0.01), Piezo1 (P ≤ 0.05), and Piezo2 (P ≤ 0.01) in the urothelium. Similarly, in the detrusor after chronic CYP-induced cystitis, mRNA expression of Trpv1 (P ≤ 0.01), Trpv4 (P ≤ 0.01), Piezo1 (P ≤ 0.05), and Piezo2 (P ≤ 0.001), were all upregulated. Additionally, after 24 h of CYP treatment, Trpv1 and Trpv4 gene expression was increased in the urothelium (P ≤ 0.01 and P ≤ 0.05, respectively) and Trpv4 mRNA was upregulated in the detrusor (P ≤ 0.05). No changes in MSC expression were observed in acute (4 h) CYP treatment in the urothelium or the detrusor.

Given the changes in MSC gene expression, we utilized Ca²⁺ imaging to determine CYP-induced functional changes in PIEZO1 activity. To ensure any observed effect of Yoda1 was due to the drug itself, we first tested whether the drug vehicle (0.1% DMSO) or time affected Ca²⁺ activity. There was no change in overall Ca²⁺ activity compared to baseline after a 10-min incubation period in the vehicle solution (Figure 3A). In our experimental groups, we found that Yoda1 elicited differences in urothelial cell Ca²⁺ activity dependent on duration of CYP-treatment. In urothelial sheets from mice not treated with CYP, we found no significant difference (P = 0.06) in Ca²⁺ activity after Yoda1 administration (Figure 3B).

In urothelial sheets from mice that were treated with acute (4 h; Figure 3C) and intermediate (24 h; Figure 3D) durations of CYP, we found significant increases in Ca²⁺ activity compared to baseline after Yoda1 treatment. Specifically, in the 4 h CYP treatment group, the Ca²⁺ activity increased by 1.54 × 10⁶ Zum²s, which represents a 2.67-fold increase compared to baseline (P ≤ 0.05). Similarly, in the 24 h CYP treatment group, the Ca²⁺ activity increased by 7.02 × 10⁵ Zum²s, a 1.5-fold increase from baseline (P ≤ 0.05). We observed noticeable within-group variability in the Ca²⁺ activity of the chronic CYP-treated group and ultimately, there was no difference between Ca²⁺ activity at baseline and after Yoda1 in that group (Figure 3E).

Though we only demonstrated statistical significance with Yoda1 administration in two of the four experimental groups (4 and 24 h CYP treatment), we did see medium to large effect sizes, as measured by Cohen's d values (28), for three of the four groups compared to baseline: no CYP (d = 0.88), 4 h CYP (d = 1.86), and 24 h CYP (d = 0.71). Notably, these effect sizes were larger than both the vehicle control (d = 0.26) and the chronic CYP (d = 0.20) treatment groups which suggests physiological relevance of PIEZO1 activation and calcium activity in three of the four experimental groups.

Since there were no statistically significant changes in Ca²⁺ activity after Yoda1 in the naïve (no CYP) group, and yet we saw functional voiding changes after intravesical infusion of Yoda1 in vivo of naïve animals, we wanted to investigate other possible molecular changes following PIEZO1 activation with Yoda1. Previous research suggests a relationship between urothelial stretch, like with distention, and tight junction dynamics (29, 30). Therefore, we hypothesized that activation of PIEZO1 may be disrupting tight junction organization or expression in the urothelium. In this experiment we investigated gene expression changes in tight junctions that have previously been shown to be expressed in the urothelium (zona occludens-1 and claudin-2,−4, and−8) (24), as well as claudin-1 which has recently been shown to be regulated by PIEZO1 in the intestinal epithelium (30).

Compared to rats treated with vehicle, rats treated with Yoda1 showed significant decreases in the expression of claudin-1 (Cldn1), Cldn8, and zona occludens-1 (ZO-1) mRNA in the urothelium (Figure 4). Specifically, Cldn1 decreased by 43.6% (P ≤ 0.05), Cldn8 decreased by 48.2% (P ≤ 0.01), and ZO-1 decreased by 45.0% (P ≤ 0.0001). Gene expression of Cldn2 and Cldn4 were statistically unregulated by PIEZO1 activation when compared to vehicle.