Female Sprague-Dawley rats were purchased from the animal facility of the Central Animal Breeding Service Center at Army Medical University (AMU, Third Military Medical University), Chongqing, China. All protocols and animal research procedures were approved by the Ethics Committee and conducted in accordance with the guidelines of the Animal Care and Use Committee of the Third Military Medical University. Rats were housed at room temperature under a 12-h light/dark cycle with free access to food and water.

The synthesized IR-780 powder was pre-dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mmol and stored at −20 °C for later use. Prior to each administration, the solution was diluted 50-fold with sterile PBS and thoroughly mixed. All operations described above were performed in the dark.

Anesthetize 8-week-old female Sprague-Dawley rats with isoflurane under surgical conditions and disinfect the suprapubic region. Lubricate and insert a sterile catheter with an outer diameter of approximately 1 mm into the urethra. Make a 1 cm incision in the midline of the lower abdomen with a surgical blade, bluntly dissect the muscle layer, expose the bladder neck and bladder, and ligate the bladder neck with 5–0 silk suture. After tying the knot, the catheter was removed. Rats in the sham group underwent the same surgical procedure without ligation and served as the control group for comparison.

The experiment comprised two phases: model validation and formal experimentation. During model validation, rats were divided into two groups: sham (control group, n = 6) and pBOO (surgical group, n = 6). One week post-surgery, bladder inflammation, structure, and molecular characteristics were assessed to validate modeling success and determine optimal treatment timing. The formal experimental phase randomly assigned rats to four groups: sham (control group, n = 8), sham + IR-780 (control group + IR-780, n = 8), pBOO (surgical group, n = 8), and pBOO + IR-780 (surgical group + IR-780, n = 8). One week post-surgery, rats received intraperitoneal injections of IR-780 (0.667 mg/kg) or an equivalent volume of PBS solution twice weekly for 3 weeks. This dosage was determined based on the results of our laboratory’s previously published research (Chen et al., 2024). Four weeks after pBOO surgery, each group underwent urodynamic testing. Blood samples were collected from the retroorbital sinus to analyze renal function changes. Rats were then humanely euthanized, and their major organs were harvested for ex vivo imaging. Finally, bladder inflammation, structure, molecular biology, and histological features were assessed, along with pBOO-induced reflux nephropathy. The experimental workflow is illustrated in Figure 1A. It is worth noting that the model validation phase and the formal experimental phase utilized two completely independent batches of animal samples. No animal was reused across experiments in different phases.

Rats were pretreated with IR-780 (0.667 mg/kg, i.p.) or PBS 24 h prior to near-infrared fluorescence imaging using the Kodak FX Professional Imaging System (New Haven, CT) to detect IR-780 distribution in organs.To examine the distribution of IR-780 in the bladder, bladder tissue was frozen and sectioned into 8 μm slices. IR-780 fluorescence signals were detected using Leica LAS AF Lite software (Leica, Wetzlar, Germany).To determine the subcellular localization of IR-780 in BSMCs, primary BSMCs were isolated from normal SD rats and cultured using the adherent culture method. Immunofluorescence staining with anti-α-SMA antibody (1:200, MB9212S, Abmart) was performed to identify the isolated cells as bladder smooth muscle cells. Cultured BSMCs were then seeded in 35 mm culture dishes and incubated with 20 μM IR-780 for 20 min, followed by incubation with 100 nmol/L MitoTracker Green (C1048, Beyotime, China) for 30 min. Cell nuclei were stained with Hoechst 33,342 (C1022, Beyotime, China) and imaged using a confocal microscope (Leica TCS SP5).

This study employed cystometry under anesthesia to evaluate urinary function in rats. Intraperitoneal injection of 1.2 g/kg urethane was used to induce anesthesia and immobilize rats 4 weeks post-surgery. Throughout the bladder pressure measurement process, anesthesia depth was assessed periodically via toe pinch reflex testing. Appropriate anesthesia was defined as complete absence of pain withdrawal reflexes while maintaining stable baseline vital signs (e.g., spontaneous respiration, heart rate). Based on reflex monitoring results, urethane was micro-titrated according to a pre-established dosing protocol to maintain stable light anesthesia. A constant-temperature heating pad was used throughout the experiment to maintain stable body temperature.A midline abdominal incision was made to expose the bladder, and a PE-50 catheter was inserted into the apex of the bladder dome. The other end of the catheter was connected via a three-way connector.One end of the three-way connector was connected to a pressure transducer (Laborie Medical Technologies Inc., Beijing, China) to record bladder pressure, while the other end was connected to an infusion pump. Sterile room-temperature saline was slowly infused at a constant rate of 18 mL/h. Pumping was immediately stopped upon observing urine in the external urethra and resumed once urination ceased. After the bladder pressure curve stabilized, it was automatically recorded by computer for 20 min.Immediately after urination, stop the infusion, withdraw the residual fluid from the bladder, and measure its volume to determine the residual urine volume (RV). In addition to residual urine volume, test parameters include urinary frequency within 20 min, maximum bladder capacity (MBC, volume of saline pumped prior to first voiding), maximum voiding pressure (MVP, peak pressure during the active voiding phase), voiding efficiency, and bladder compliance [BC, calculated as (MBC/TP - BP)]. Threshold pressure (TP) is defined as the intravesical pressure prior to voiding. Baseline pressure (BP) is defined as the minimum pressure between two voiding events.Measurements were taken on four rats per group. The average of three urination cycles per rat was used to eliminate any variation. This procedure was performed three times per rat.

Each group of rats was euthanized after measuring bladder wet weight. Bladder and kidney tissues were collected and rapidly frozen in liquid nitrogen or immersed in 4% paraformaldehyde. Following fixation and dehydration in 4% paraformaldehyde, tissues were paraffin-embedded, sectioned into 5 μm slices on slides, and subsequently subjected to H&E and Masson’s trichrome staining.

For immunofluorescence analysis of tissue sections, paraffin sections are deparaffinized and rehydrated, then treated with 3% hydrogen peroxide at room temperature for approximately 10 min to inactivate endogenous peroxidase activity.Then, place the sections in a container with antigen retrieval solution, heat in an autoclave until full pressure is reached for 3 min, followed by blocking with 1% goat serum albumin at room temperature for 30 min. Incubate overnight at 4 °C with anti-α-SMA (Abmart, 1:200), then incubate the sections with the corresponding secondary antibody at 37 °C for 1 h. After washing, stain cell nuclei with DAPI (C1006, Beyotime, China). Stained bladder sections were examined under an optical microscope (Olympus Corporation, Tokyo, Japan), and images were captured using a digital camera mounted on the microscope. All images were analyzed using ImageJ software.

A small portion of bladder tissue from each group was excised for total RNA extraction using Trizol reagent, followed by reverse transcription with the RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher). Real-time PCR (RT-PCR) was performed using SYBR Green qPCR Master Mix according to the manufacturer’s protocol. Primers are listed in Table 1 (Supplementary Material). Data were normalized to β-actin as an internal control using the ΔCT method.

In brief, extract total protein from each group of bladder tissue/kidney tissue on ice using RIPA buffer containing a mixture of protease and phosphatase inhibitors for 30 min. Depending on protein concentration, prepare samples using 5X loading buffer. Load an equal amount of protein from each sample onto a 4%–12% Tris-glycine SDS-PAGE gel, run electrophoresis, and transfer to a PVDF membrane. Blot the membrane, incubate overnight at 4 °C with the designated primary antibody (1:1000 dilution), and incubate for 1 h with HRP-conjugated secondary antibody (1:2000 dilution). Visualize and analyze band intensities using an enhanced chemiluminescence detection system (Bio-Rad Laboratories) and ImageJ software.

Following the instructions of the detection kit (C1090, Beyotime, China), after dewaxing the sections, add a drop of proteinase K working solution (20 μg/L) onto the tissue sections and incubate for 20 min. Then, add 50 μL of TUNEL mix solution as directed (the negative control group was treated only with 50 μL of fluorescein-labeled dUTP solution). Place the slides in a humid chamber and incubate at 37 °C in the dark for 1 h. Finally, add DAPI-containing anti-fluorescence quenching mounting medium and mount the slides. Imaging of stained sections was performed using a fluorescence microscope.

Fresh bladder tissue from each group was prepared into frozen sections and incubated briefly at 37 °C in the dark with Mito-Tracker Red, 2′,7′-dichlorodihydrofluorescein diacetate (DHE, 10 μM, Beyotime) and MitoSOX™ Red (10 μM, Invitrogen) for 30 min at 37 °C in the dark. This measured the relative number of active mitochondria in BSMCs, the in situ levels of intracellular ROS, and mitochondrial superoxide levels, respectively. Fluorescence intensity was quantified using ImageJ software.

Each group of fresh bladder smooth muscle tissue was trimmed into 1 × 1 × 3 mm tissue blocks, fixed overnight in 4% glutaraldehyde, fixed with 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in fresh 100% resin. Ultrathin sections cut from the blocks were stained with 3% uranium acetate in saturated alcohol and lead citrate for 8 min each, then examined by transmission electron microscopy at 100 kV. In all groups, bladder tissue samples for TEM analysis were uniformly obtained from the mid-layer smooth muscle region of the bladder body.

Each experiment was repeated at least three times. All statistical analyses were performed using SPSS software version 26.0 and GraphPad Prism software version 10.0. Pairwise comparisons were conducted using one-way or two-way analysis of variance (ANOVA) to determine statistical significance. P < 0.05 was considered statistically significant.

Seven days after surgery, rats in the pBOO group exhibited markedly distended and swollen bladders with slightly dilated ureters (Figure 1B). Upon removal, the bladders in the pBOO groups showed increased weight compared to those in the control groups and displayed macroscopically evident inflammatory changes (Figures 1B,C). Hematoxylin and eosin (HE) staining revealed thickened urothelium and increased inflammatory cell infiltration in the bladders of rats with pBOO. Masson’s trichrome staining demonstrated a thickened muscularis propria without significant collagen deposition. The primary histological features at this stage were inflammation and detrusor muscle hypertrophy (Figure 1D). We assessed the levels of proinflammatory cytokines in the bladder and found that pBOO group exhibited significantly elevated mRNA expression of IL-1β, IL-6, and TNF-α following obstruction (Figure 1E). These results confirm that the pBOO model was successfully established and is suitable for subsequent drug intervention studies.

We obtained key organs from the four rat groups for in vivo imaging. As expected, in the longitudinal comparison of organs within the same group, IR-780 demonstrated targeting properties in partially obstructed bladders. We observed significantly stronger NIR fluorescence in the bladder than in other organs such as the heart, liver, spleen, lungs, and intestines (Figure 2A). In a cross-group comparison, the pBOO + IR-780 group exhibited markedly higher NIR fluorescence in the bladder than the other three groups (Figure 2B).

To determine the distribution of IR-780 within the bladder wall, histological analysis was performed on fresh-frozen bladder sections obtained 1 day after the intraperitoneal injection of IR-780. Confocal microscopy revealed that IR-780 was primarily localized within the smooth muscle layer of the bladder (Figure 2C). Therefore, this study focused on exploring the role of IR-780 in BSMCs.

To determine the subcellular localization of IR-780, we isolated and cultured primary BSMCs from 8-week-old Sprague-Dawley rats. The cells were identified by immunofluorescence staining for α-smooth muscle actin (α-SMA), a specific smooth muscle cell marker (Supplementary Figure S1). As previously reported, co-localization of IR-780 with MitoTracker Green confirmed its targeted accumulation within BSMC mitochondria (Figure 2D). Collectively, these findings demonstrate that IR-780 can serve as a target dye for pBOO bladders.

Four weeks postoperatively, bladder pressure was measured in the rats, and the pressure curves are shown in Figure 3A. Analysis of the bladder pressure-time curves revealed that, compared with rats in the sham group, those in the pBOO group exhibited significantly increased MVP, MBC, RV, and voiding frequency (p < 0.0001, p < 0.001, p < 0.0001, p < 0.05), but decreased BC and VE (p < 0.001, p < 0.01), indicating typical pBOO decompensation. In contrast, the pBOO + IR-780 group showed decreased MVP, voiding frequency and increased compliance (p < 0.001, p < 0.01, p < 0.05 compared to pBOO group), and demonstrated certain improvements despite no significant differences in MBC, RV, or VE (Figures 3B–G). These data confirm that IR-780 administration exerts a protective effect by alleviating voiding-phase and storage-phase dysfunction in pBOO.

The mechanism of pBOO progression involves an ordered overlapping sequence from inflammation to fibrosis (Hughes et al., 2016). Multiple molecular mechanisms are involved. IL-6 and TNF-α, which are classic proinflammatory cytokines, mediate the activation and regulation of immune responses (He et al., 2021; Gheinani et al., 2017). The TGF-β1/Smad 3 pathway is closely associated with organ fibrosis (Meng et al., 2016), while the hypoxia-inducible factor-1α(HIF-1α) pathway represents an adaptive respond to hypoxic environments (Sun et al., 2023). Conversely, IL-10 suppresses proinflammatory responses and limits tissue damage caused by excessive inflammation (Ouyang et al., 2011).

We therefore examined the key molecules associated with pBOO to investigate the effects of IR-780 on several critical cytokines in the bladder tissues of rats with pBOO 4 weeks post-obstruction. In the pBOO group, both IL-1β and TNF-α expression in the bladder were significantly upregulated at the mRNA level (both p < 0.05 compared to the sham group). Following IR-780 treatment, IL-1β and TNF-α expression levels reduced (both p < 0.05 compared to the pBOO group) (Figures 4A,C). The bladder fibrosis factors TGF-β1, Collagen type I α1 chain (Col1a1), and Collagen type III (Col3) were significantly increased in the pBOO group (p < 0.05, p < 0.05, p < 0.001 compared to the sham group), and IR-780 reduced this expression (p < 0.05, p < 0.05, p < 0.01 compared to the pBOO group) (Figures 4F–H). These findings indicate that IR-780 exhibits anti-inflammatory effects and mitigates progressive fibrosis in pBOO-induced bladders.

In the early stages of BOO, mechanical stress induces the upregulation of α-SMA expression to enhance detrusor contractility, representing compensatory remodeling. However, when obstruction persists long-term, bladder wall fibrosis progressively worsens. Massive smooth muscle cell apoptosis occurs and is replaced by collagen fibers, whereas myofibroblast activation and proliferation enter an exhausted phase, leading to a significant decline in α-SMA synthesis capacity. At this point, the reduced α-SMA levels indicate that the bladder has transitioned from the compensatory phase to the decompensatory phase (Metcalfe et al., 2010; Salinas et al., 2004; Yoshida and Yamaguchi, 2014). After 4 weeks, the bladders in the pBOO group exhibited marked congestion and swelling, with increased weight compared to those in the sham group. However, the bladders in the pBOO + IR-780 group showed slightly reduced volume and weight relative to those in the pBOO group (Figures 5A,B). Compared with the control group, the surgical group exhibited thickened urothelium and inflammatory cell infiltration and progressive changes in the muscularis propria. Muscle bundles gradually thickened, then underwent disruption and dissolution, with widening of the interstitial spaces and subsequent structural disorganization. After 3 weeks of IR-780 intervention, the degree of damage to the bladder tissues showed significant improvement (HE staining) (Figure 5C). Masson’s trichrome staining revealed significantly increased collagen fiber deposition in the pBOO group compared to that in the sham group (P < 0.01). Although collagen deposition remained higher in the pBOO + IR-780 group than that in the control group, it significantly reduced in the pBOO + IR-780 group compared to the pBOO group. (P < 0.01) (Figures 5C,D). Additionally, we detected α-SMA expression in bladder tissues via Western blotting and immunofluorescence staining. Decreased α-SMA expression was observed in the pBOO group (vs.control group, p < 0.05), whereas the pBOO + IR-780 group showed increased α-SMA expression (vs.pBOO group, p < 0.05) (Figures 5E–H). This confirms the earlier emphasis on BSMCs as the focus of this study and indicates that IR-780 can restore the reduced smooth muscle content induced by pBOO.

BOO ultimately leads to severe complications such as vesicoureteral reflux, bladder dysfunction, bilateral renal damage, renal failure, bladder cancer, and bladder neck tumors (Cisek, 2017). Owing to the varying duration and progression of BOO lesions, many patients still develop progressive bladder dysfunction and renal impairment after surgery, which are difficult to treat (Wang et al., 2023). Therefore, we investigated the effects of IR-780 on pBOO-induced renal injury. Surprisingly, compared to sham rats without ureteral reflux, rats in the pBOO group exhibited markedly dilated ureters and renal pelvises, enlarged kidneys, and increased capsular tension, indicating the presence of vesicoureteral reflux. In contrast, the pBOO + IR-780 group showed a milder presentation: although the ureters were dilated, both kidneys exhibited relatively normal coloration with no significant hydronephrosis or perinephric fluid accumulation (Figure 6A). Serum was extracted from rats to measure serum creatinine (CREA), uric acid (UA), and urea levels. Compared to the sham group, the pBOO group exhibited significantly elevated CREA, UA, and UREA levels. However, IR-780-treated rats with pBOO exhibited reduced CREA, UA, and urea levels (p < 0.0001, p < 0.05, p < 0.01, compared to the pBOO group) (Figure 6B). HE and Masson staining revealed normal glomerular morphology without tubular dilatation in the sham group. The pBOO group exhibited glomerular deformation and atrophy, accompanied by tubular injury and marked glomerulosclerosis (Figure 6C). The pBOO group demonstrated the highest expression of α-SMA and vimentin protein using Western blotting (p < 0.01 and p < 0.001 compared to the sham group). The pBOO + IR-780 group α-SMA showed lower expression than the pBOO group but higher expression than the sham and sham + IR-780 groups; the pBOO + IR-780 group vimentin protein showed lower expression than the pBOO group (p < 0.001, p < 0.001 compared to the pBOO group), but higher expression than the sham and sham + IR-780 groups (p < 0.05 compared to the pBOO group) (Figures 6D,E). Immunofluorescence staining validated these findings (Figures 6F,G), suggesting that IR-780 may suppress pBOO-induced renal fibrosis. In summary, IR-780 treatment ameliorated pBOO-induced renal injury, potentially by reducing bladder injury or through direct protective effects on renal tissue.

TUNEL staining revealed a significant increase in apoptotic cells within the bladder smooth muscle of the pBOO group compared with the sham and pBOO + IR-780 groups (all P < 0.05). However, apoptosis was markedly reduced in the pBOO + IR-780 group (P < 0.05 compared to the pBOO group) (Figures 7A,B). Western blotting revealed that the protein levels of cleaved Caspase-3 were significantly elevated in the pBOO group compared to the sham group (P < 0.01), whereas they were markedly reduced in the pBOO + IR-780 group compared to the pBOO group (P < 0.01) (Figures 7C,D). These data indicate that IR-780 effectively ameliorates apoptosis in BSMCs from rats with pBOO. Based on the mitochondrial localization of IR-780 in BSMCs, we further examined proteins associated with the mitochondrial apoptotic pathway to investigate the relationship between IR-780 and apoptosis. Western blot analysis revealed that Bcl-2-associated X, Cytochrome C, and cleaved Caspase-9 significantly increased (p < 0.05, p < 0.01, p < 0.01 compared to the sham group), whereas Bcl-2 protein expression decreased (p < 0.01 compared to the sham group). However, IR-780 treatment blocked these changes (Figures 7E–I). Collectively, these data indicate that IR-780 inhibits mitochondrial-related apoptosis in the bladder tissues of rats with pBOO.

Frozen sections of bladder tissues from rats in each group were stained with DHE and MitoSOX to measure the cellular and mitochondrial superoxide levels. Quantitative analysis of DHE and MitoSOX staining revealed significantly elevated levels of cellular and mitochondrial superoxides in the pBOO group (P < 0.05 and P < 0.001 compared to the sham group), whereas IR-780 treatment suppressed the elevation in cellular and mitochondrial superoxide levels (P < 0.05 and P < 0.01 compared to the pBOO group) (Figures 8A,B,D,F). This demonstrates that IR-780 possesses certain antioxidant effects. Fluorescent staining with MitoTracker Red was used to assess mitochondrial damage in bladder tissues of each rat group. The results showed that the number of viable mitochondria in the bladder tissues of the pBOO group significantly reduced (P < 0.001 compared to the sham group), whereas the number of mitochondria increased after IR-780 treatment (P < 0.05 relative to the pBOO group) (Figures 8C,F). Transmission electron microscopy images revealed changes in mitochondrial structure and morphology. Cells in the sham group exhibited a higher number of mitochondria with normal morphology, showing a well-organized arrangement and a clear structure. In contrast, cells in the pBOO group demonstrated a reduced mitochondrial quantity with significantly diminished, fractured, and even dissolved cristae. Numerous mitochondria exhibited calcification and swelling, accompanied by decreased electron density in the mitochondrial matrix and vacuolar degeneration. Compared to the surgery group, the pBOO + IR-780 group exhibited a significant increase in mitochondrial quantity and cristae number, with the morphology approaching normal (Figure 8G). These findings indicate that IR-780 exerts a protective effect on mitochondria within BSMCs.

Based on the above results, we further investigated whether the antioxidant effects of IR-780 were mediated by Nrf2 activation. Western blotting analysis of representative proteins in four bladder groups revealed that compared to the sham group, the pBOO group exhibited decreased levels of HO-1, GPX1, and SOD1 (p < 0.05, p < 0.01 and p < 0.05) (Figures 9A,B,D–F), and increased Keap1 levels (p < 0.05) (Figures 9A,C). However, IR-780 treatment upregulated pBOO-induced Nrf2 protein expression (vs. pBOO group p < 0.05) while downregulating Keap1 (p < 0.01), reversing the decrease in the antioxidant proteins HO-1, GPX1, and SOD1 (p < 0.01, p < 0.05, and p < 0.05). These results suggest that the protective effect of IR-780 may be related to Nrf2 activation (Figure 10).