• HIGHLIGHTS
- This study used urothelial proteomics to investigate the molecular mechanisms of overactive bladder (OAB) associated with bladder outlet obstruction.
- Differentially expressed proteins showed dysregulated cytoskeletal organization, neurotransmitter signaling, and immune‑related pathways in patients with OAB.
- These molecular alterations suggest structural disorganization and aberrant urothelial signaling as key mechanisms in bladder outlet obstruction‑related OAB and highlight potential targets for biomarker development and therapy.
INTRODUCTION
Overactive bladder (OAB) is a storage symptom syndrome defined by the International Continence Society as urgency, with or without urgent urinary incontinence, usually accompanied by increased daytime frequency and nocturia [1]. OAB is highly prevalent, affecting more than 45% of women older than 65 years [2], and has a significant impact on quality of life by increasing the risk of falls, fractures, anxiety, depression, and social isolation, as well as imposing a considerable economic burden [3].
Although OAB has traditionally been considered more common in women, large epidemiological studies have shown a similar prevalence in men [4]. In older men, OAB is frequently associated with benign prostatic hyperplasia (BPH) and bladder outlet obstruction (BOO). Indeed, 50%–75% of patients with BOO present with OAB, and symptoms persist in up to 38% of patients after treatment [5]. BOO, most often caused by BPH, can induce detrusor overactivity through ischemic denervation, structural remodeling, and altered neuromodulation [6]. Given the high prevalence of BPH in aging men, BOO-related OAB is an increasingly important clinical problem.
The pathophysiology of OAB is multifactorial. Historically, increased detrusor smooth muscle contractility was emphasized [7], forming the rationale for antimuscarinic therapy. However, recent evidence highlights altered urothelial signaling and enhanced afferent nerve activity [8,9]. The urothelium is now recognized as an active sensory organ that communicates bidirectionally with adjacent nerves [10,11], and dysregulation of these pathways may represent a novel therapeutic target.
Proteomic approaches enable comprehensive protein profiling and may provide mechanistic insights into disease pathophysiology [12]. In this study, we applied quantitative proteomics using liquid chromatography–tandem mass spectrometry (LC–MS/MS) to examine urothelial protein expression in patients with BOO-related OAB, with the goal of identifying candidate proteins that could elucidate disease mechanisms and suggest novel diagnostic or therapeutic strategies.
MATERIALS AND METHODS
Patients and Procedures
All study procedures were conducted with approval from the Institutional Review Board of Chungnam National University Hospital (CNUH 2013-08-036-002), and written informed consent was obtained from all participants. A total of 22 men over 50 years of age with BPH scheduled to undergo transurethral resection of the prostate (TURP) were enrolled. Each patient underwent a comprehensive urological evaluation, including detailed history taking, physical examination, the International Prostate Symptom Score (IPSS), and the Overactive Bladder Symptom Score (OABSS). Exclusion criteria included any history of hematuria due to other genitourinary conditions, urinary incontinence unrelated to OAB, active urinary tract infection (UTI), neurogenic bladder, renal disease, urological malignancy, or previous pelvic radiation therapy. After screening, several patients were excluded because they withdrew consent, did not meet the diagnostic criteria, or met one of the predefined exclusion criteria. Consequently, bladder tissue specimens were collected from 11 patients and processed for further analysis.
OAB was defined according to established criteria as an urgency score ≥2 and a total OABSS ≥3 [13]. Based on the OABSS, patients were classified into 2 groups. The BPH/OAB group (n=6) consisted of patients who met the OAB diagnostic criteria and had no evidence of UTI. The BPH/non-OAB group (n=5) consisted of patients without urgency symptoms, with an OABSS <2.
Bladder tissue specimens were obtained during TURP under general or regional anesthesia using a cold cup biopsy technique, which was selected to minimize protein denaturation during tissue collection. Biopsies were taken from the posterior bladder wall, approximately 2 cm above the ureteral orifice. To avoid bladder perforation, only mucosa layers were harvested. Immediately after collection, samples were snap-frozen and stored at -70°C until further biochemical and proteomic analyses.
Protein Extraction and Preparation
For proteomics analysis, bladder urothelial tissue samples were processed by grinding them with micropestles in 700 μL of radio‑immunoprecipitation assay buffer (Sigma-Aldrich, USA). Tissue remnants were eliminated by centrifugation at 2,000×g for 10 minutes at low speed, and the resulting supernatants were extracted. Protein concentration was determined using the bicinchoninic acid assay (Pierce, Thermo Fischer Scientific, USA). The prepared samples were stored at -70°C for subsequent analysis.
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and In-Gel Digestion
Protein samples (15 μg each) were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad, USA). Coomassie Brilliant Blue R-250 (Bio-Rad) was applied to stain the gels. A previously described method was used for ingel digestion. The gels were sectioned into 8 parts based on molecular weight. After desalting the gel fragments, cysteine was reduced and alkylated, followed by trypsin digestion (Sigma-Aldrich) for 16 hours at 37°C. An extraction solution (composed of 50mM ammonium bicarbonate, 50% acetonitrile, and 5% trifluoroacetic acid) was used to extract digested peptides. These were then dissolved in 10 μL of a sample solution containing 0.02% formic acid and 0.5% acetic acid (Sigma-Aldrich).
Protein Identification by Mass Spectrometry
Tryptic peptide samples (5 μL) were separated and identified using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). Peptides were enriched, and contaminants were removed on a trapping column (75-μm inner diameter, packed with 5-μm C18 particles, Acclaim Pep-Map100, Thermo Fisher Scientific). Concentrated peptide solutions were then analyzed using a 15-cm analytical column packed with 2-μm C18 particles (Acclaim PepMap RSLC, Thermo Fisher Scientific). Reversed-phase chromatography was performed on an Ultimate 3000 RSLC nano system (Thermo Fisher Scientific) with a binary solvent system of 0.1% formic acid (solvent A) and 80% ACN in 0.1% formic acid (solvent B). Peptides were separated using a linear gradient of solvent B from 5% to 95% at a flow rate of 300 nL/min for 100 minutes. MS and MS/MS spectra were acquired in data-dependent mode using a LTQ-Velos ESI ion trap mass spectrometer (Thermo Fisher Scientific). Each full MS scan (m/z range 300-2,000) was followed by 3 MS/MS scans of the most abundant precursor ions with dynamic exclusion. MS/MS analysis was performed thrice for each sample. Database searches used the following parameters: enzyme specificity: trypsin/P; maximum missed cleavages: 1; carboxymethyl (C) as a static modification; oxidation (M) and N-terminal acetylation as dynamic modifications; precursor mass tolerance: 0.8 Da; MS/MS mass tolerance: 0.8 Da. The MS/MS data were filtered using an false discovery rate criterion of <1%. Protein identification was performed using the UniProt human proteome database (UP 000005640_9606). Relative quantitation and significance were provided for all identified proteins using MaxQuant (v2.6.4). Protein quantification was based on the label‑free quantification value [14]. Differential expression analysis was performed in Perseus (ver. 2.1.1) using a SAM (significance analysis of microarrays)-based t-test with an s₀ parameter set to 2.
Bioinformatics Analysis
Ingenuity Pathway Analysis (Ingenuity Systems, USA) was employed to conduct functional annotation analysis and identify the most significant biological functions and/or diseases related to the dataset. The analysis relied on the Ingenuity Knowledge Base. To determine the likelihood that each biological function and/or disease associated with the dataset occurred by chance, Fisher exact test was used to calculate a P-value. The most relevant pathways in the dataset were identified through canonical pathway analysis using the Ingenuity Pathway Knowledge Base. Significance was established by calculating the ratio of proteins from the dataset mapping to a specific canonical pathway divided by the total number of proteins mapped to that pathway using Fisher exact test. Focus genes were superimposed on a global molecular network derived from the Ingenuity Pathway Knowledge Base to create networks based on individual protein connectivity. Concurrently, protein clusters were formed, and differentially expressed genes were categorized. Gene Ontology Enrichment analysis was performed using EnrichR [15].
RESULTS
Patients’ Backgrounds and Symptom Scores
The baseline clinical characteristics of the study participants are summarized in Table 1. During cystoscopic collection, any specimens in which smooth muscle fibers were visually identified or suspected were excluded. A total of 11 bladder tissue specimens were obtained for analysis, but due to limited tissue size, reliable protein extraction was possible in only 6 samples, which were subsequently included in the final proteomic analysis.
No significant differences were observed in age, prostate volume (total volume on transrectal ultrasound), transition zone volume, or maximum flow rate between the OAB group (n=6) and the non-OAB group (n=5). In contrast, storage symptom scores differed significantly. The total OABSS was significantly higher in the OAB group compared with the non-OAB group (6.93±2.24 vs. 0.63±0.52, P<0.001). Among the OABSS subscores, frequency, nocturia, and urgency were significantly elevated in the OAB group. Similarly, the IPSS storage subscore was significantly worse in the OAB group (9.00±3.55 vs. 1.88± 1.89, P<0.001), whereas voiding subscores did not differ between groups. In the quality-of-life category, scores were significantly worse in the OAB group than in the non-OAB group (4.00±1.47 vs. 2.63±1.51, P=0.042), indicating that OAB had a more detrimental impact on patient well-being.
Proteomic Analysis of Human BOO-Related OAB
Proteomic analysis was conducted using liquid chromatography– tandem mass spectrometry (LC–MS/MS) on urothelial tissue specimens obtained from 3 patients with BOO‑related OAB and 3 non‑OAB control patients. A total of 1,510 proteins were identified across all samples. The sample correlation heatmap demonstrated distinct clustering of the OAB group apart from the non-OAB group, suggesting that the urothelial proteome of patients with OAB exhibited alterations compared to that of non-OAB individuals. Statistical analysis identified 133 differentially expressed proteins between patients with and without OAB (Fig. 1A). Of these, 64 proteins were upregulated in the OAB cohort, whereas 69 were downregulated compared with the non-OAB group (Fig. 1B).
Overall, 8.8% of the identified proteins were classified as differentially expressed proteins, despite the differential expression criteria of P-value<0.05 and |FC (fold change)| ≥1.5. Thus, only a limited subset of proteins exhibited alterations in the bladder epithelial cells of patients with the OAB group compared with the non-OAB group. Nonetheless, given that OAB significantly impairs the quality of life, the differentially expressed proteins identified in this study are likely to yield valuable insights into the pathophysiology of OAB.
Comparison of Canonical Pathways Between BOO-Related OAB and Non-OAB Controls
To elucidate the molecular characteristics of patients with OAB, pathway analysis of differentially expressed proteins was performed. The analysis revealed significant involvement of multiple cellular processes (Fig. 2A). These included pathways linked to cell structure maintenance, such as actin cytoskeleton signaling, smooth muscle contraction, and Rho family GTPase signaling. Additionally, pathways associated with serotonin receptor signaling and immune system responses were identified (Table 2, Fig. 2B). During normal micturition, the detrusor muscle contracts in response to parasympathetic nervous system stimulation, and actin facilitates adaptive responses associated with detrusor muscle contraction by interacting with the cytoskeleton [16]. Among neurotransmitter pathways, serotonin affects bladder function through its receptors expressed on the detrusor muscle and neural pathways, modulating both contraction and relaxation involved in micturition control [17]. However, GNAO1, which encodes the inhibitory G-protein α subunit Gαo and participates in G‑protein coupled receptor (GPCR) pathways activated by serotonin receptors, was downregulated in patients with OAB. This finding suggests aberrant neurotransmitter signaling in the bladder epithelial cell tissue of patients with OAB (Table 2, Fig. 2B). Conversely, GNA13 (Gα13), which links GPCR activation to Rho GTPase-dependent cytoskeletal remodeling, was upregulated in patients with OAB. Despite this increase, most proteins associated with actin cytoskeleton and Rho GTPase signaling—implicated in cytoskeletal reorganization in patients with OAB—demonstrated reduced expression (Fig. 2B). Specifically, ACTA2, CFL2, MYLK, and PPP1R12B, typically associated with both signal transduction pathways, were downregulated in the urothelial cells of patients with OAB, suggesting that alterations in cellular structure were not efficiently executed. Neutrophil degranulation, another significant pathway, was overexpressed in patients with OAB (Table 2, Fig. 2B). Neutrophil degranulation is a crucial component of the inflammatory response, and its excessive occurrence may contribute to tissue damage and chronic inflammation. Inflammatory response has been identified as a substantial factor in the pathophysiology of OAB.
Functional Analysis of Differentially Expressed Proteins
Gene Ontology (GO) enrichment analysis was conducted to identify significantly enriched biological processes among the differentially expressed proteins between the OAB and non-OAB groups. Consistent with the pathway analysis, the GO results confirmed significant changes in cell structure and neurotransmitter catabolic processes (Fig. 3). In the OAB group, proteins associated with catecholamine and dopamine catabolism were significantly enriched. MOXD1 and MAOA, 2 proteins involved in both processes, play critical roles in the biosynthesis and degradation of neurotransmitters. Consistent with the results of pathway analysis, neurotransmission processes were found to be activated in patients with OAB. In the immune response category, IGHG3, IGHG1, and IGHA1, which are components of the B-cell receptor signaling pathway, were overexpressed. This finding suggests persistence of an inflammatory condition, wherein both the initial immune response (e.g., neutrophil degranulation) and subsequent acquired immune responses are activated. Another significantly enriched functional category was the negative regulation of peptidase activity, which prevents tissue damage caused by inflammatory responses, and appeared to be activated as a compensatory mechanism to counterbalance the chronic inflammatory state in patients with OAB. Conversely, biological processes enriched in downregulated proteins were predominantly cell structure-related categories, indicating that the results of the functional analysis were consistent with those of the pathway analysis (Fig. 3).
Structural Disorganization of Proteins in BOO-Related OAB Urothelium
Proteomic analysis revealed changes in cell-cell interactions within urothelial tissue samples from patients with OAB. Specifically, the levels of proteins associated with cell-cell contact and focal adhesion formation were significantly altered in the OAB group compared with the non-OAB group (Table 2, Fig. 4), suggesting potential disruption of epithelial barrier integrity. Focal adhesions facilitate the transduction of extracellular signals to the intracellular environment and influence cell-cell contact processes through cytoskeletal regulators such as Rho GTPases [18]. During micturition, bladder function is modulated by mechanotransduction pathways that include integrin/focal adhesion signaling in both the urothelium and detrusor smooth muscle [19]. In the present study, this pathway appeared to be overactivated in bladder epithelial cells of patients with OAB. The activation of focal adhesion kinase has been shown to contribute to detrusor contraction, and altered GPCR-Rho signaling has been implicated in hypercontractility and impaired relaxation in bladder dysfunction [20,21]. However, the proteomic analyses suggested aberrations in the molecular processes underlying muscle relaxation. Conversely, the attenuation of cell-cell contacts may result in diminished intercellular junctions, causing cells to adhere more firmly to the substrate. This phenomenon can reduce intercellular signaling and impair the capacity to respond to external signals appropriately, ultimately leading to abnormal functional decline.
DISCUSSION
The prevalence of OAB is approximately 15% worldwide, and many patients are affected [22]. Although OAB does not directly threaten life, patients experience psychological, social, and economic challenges due to frequent urination and urgency [23]. Current therapeutic approaches are largely conservative and focus on symptomatic relief rather than addressing the underlying molecular abnormalities. To date, no approved treatments directly target the pathophysiologic mechanisms involved in OAB. Existing interventions primarily include behavioral modifications, antimuscarinic agents, β3-adrenergic agonists, and invasive procedures for patients with severe symptoms [24]. However, because of individual variability in therapeutic efficacy, adverse effects of pharmacological interventions, disease recurrence, and prolonged treatment duration, achieving a cure remains challenging. Most therapeutic approaches therefore focus on symptom management. To fundamentally address OAB, a more comprehensive understanding of its pathogenesis is essential. Previous studies have proposed several molecular mechanisms, including aberrant acetylcholine secretion, neurotransmitter imbalance, detrusor abnormalities, and elevated adenosine triphosphate release [25,26]. However, a significant proportion of these investigations have been conducted in model organisms, and studies using bladder cells or tissues from patients remain insufficient. In this study, we identified protein alterations in the bladder epithelial cells of patients with OAB to elucidate molecular mechanisms underlying the disease. Neurotransmitters implicated in OAB include acetylcholine, norepinephrine, and serotonin. The predominant hormonal factors associated with OAB are acetylcholine hyperactivity and muscarinic receptor overactivation; however, these factors were not identified in this study. In contrast, proteins associated with noradrenaline and serotonin were identified as top-ranking proteins in the canonical pathway analysis. Generally, neurotransmitters are present in very low concentrations and have short half-lives, necessitating targeted methods for their detection. In the present study, only bladder epithelial cells were isolated and subjected to global proteomic analysis; consequently, these neurotransmitters were not identified. Previous studies have also reported that reduced serotonin levels are associated with OAB [27]. Although the concentration of serotonin could not be determined in this study, we demonstrated that GNAO1, which encodes the inhibitory G-protein α subunit and participates in GPCR pathways including serotonin receptors, was downregulated. In contrast, GNA13, a G-protein α subunit that mediates GPCR-to-Rho GTPase signaling and contributes to cytoskeletal remodeling, was upregulated. While GNA13 is known to activate Rho-specific guanine nucleotide exchange factors, which in turn activate RHOC, our results showed that RHOC was downregulated in the bladder epithelial cells of patients with OAB. RHOC promotes the formation of actin stress fibers and influences cell shape and movement [28]. Proteomic analysis therefore confirmed the reduced expression of RHOC in the bladder epithelial cells of patients with OAB, along with decreased expression levels of other proteins associated with cellular structural changes. These findings suggest that, in addition to hormone concentrations, patients with OAB may exhibit functional abnormalities in nerve signaling pathways, warranting further investigation. Because bladder urothelial tissues were obtained by cold-cup cystoscopic resection, some specimens were extremely small and insufficient for reliable protein extraction, which is an inherent limitation of this approach. During cystoscopic biopsy, smooth muscle areas can generally be identified and avoided under direct magnified visualization. Nevertheless, additional histological confirmation such as hematoxylin and eosin staining for every sample was not feasible due to ethical and Institutional Review Board restrictions, as multiple biopsies would increase bleeding risk and require additional coagulation. These methodological and ethical constraints are inherent to human urothelial research. Although the small sample size inevitably restricts the generalizability of our findings, proteomic approaches can yield meaningful results even from limited material. Accordingly, our analysis provides valuable preliminary insights into the molecular alterations associated with BOO-related OAB. Consequently, the results of this study were limited in their ability to fully elucidate the mechanisms underlying OAB in all patients. This study presents a patient-derived bladder epithelial proteomic analysis that offers preliminary insights into the molecular mechanisms of OAB pathogenesis.
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