Pilot Proteomic Analysis of Urinary Extracellular Vesicles Supports the “Toxic Urine Hypothesis” as a Vicious Cycle in Refractory IC/BPS Pathogenesis

Despite treatments such as pentosan polysulfate, hyaluronic acid, botulinum toxin A, and platelet-rich plasma, many interstitial cystitis/bladder pain syndrome (IC/BPS) patients experience persistent symptoms. Urinary extracellular vesicles (uEVs) carry molecular cargo reflecting disease pathophysiology, yet their proteomic profiles in treated IC/BPS remain unexplored. This pilot study examined uEV proteomics in refractory IC/BPS cases to test the "Toxic Urine Hypothesis"-a vicious cycle, whereby urothelial dysfunction enables EV-mediated toxin penetration, triggering inflammation that further impairs the bladder barrier. Urinary EVs were isolated from six female IC/BPS patients on active treatments and four healthy female controls. Mass spectrometry-based proteomics identified differential protein expressions, followed by pathway enrichment analysis and functional validation using NF-κB reporter assays in HEK293T cells and Western blot in primary human bladder epithelial cells. IC/BPS EVs exhibited 31 upregulated proteins (including HPGD, KRT8, HSPA4, 14-3-3 family members) and 19 downregulated proteins (including neutrophil granule proteins MPO and ELANE), indicating suppressed acute neutrophil inflammation but enriched homeostatic, metabolic, and regenerative pathways. Patient EVs induced significantly higher NF-κB activation than in the controls, with upregulated 14-3-3ζ and phosphorylated NF-κB p65 in bladder epithelial cells. These findings support the "Toxic Urine Hypothesis", revealing persistent NF-κB-mediated chronic epithelial stress despite suppressed acute inflammation in treated IC/BPS patients, suggesting that therapies targeting inflammation and regeneration may help break this vicious cycle.