{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T16:40:09Z","timestamp":1792428009147,"version":"4.5.0"},"reference-count":32,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T00:00:00Z","timestamp":1766361600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Science and Technology Council, Taiwan","award":["NSTC 113-2314-B-040-025"],"award-info":[{"award-number":["NSTC 113-2314-B-040-025"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 \u201cToxic Urine Hypothesis\u201d\u2014a 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-\u03baB 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-\u03baB activation than in the controls, with upregulated 14-3-3\u03b6 and phosphorylated NF-\u03baB p65 in bladder epithelial cells. These findings support the \u201cToxic Urine Hypothesis\u201d, revealing persistent NF-\u03baB-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.<\/jats:p>","DOI":"10.3390\/ijms27010130","type":"journal-article","created":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T16:19:26Z","timestamp":1766420366000},"page":"130","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Pilot Proteomic Analysis of Urinary Extracellular Vesicles Supports the \u201cToxic Urine Hypothesis\u201d as a Vicious Cycle in Refractory IC\/BPS Pathogenesis"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6122-6765","authenticated-orcid":false,"given":"Man-Jung","family":"Hung","sequence":"first","affiliation":[{"name":"Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung 402306, Taiwan"},{"name":"Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Chung Shan Medical University, Taichung 402306, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1543-906X","authenticated-orcid":false,"given":"Evelyn","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung 402306, Taiwan"},{"name":"Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Chung Shan Medical University, Taichung 402306, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tsung-Ho","family":"Ying","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung 402306, Taiwan"},{"name":"Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Chung Shan Medical University, Taichung 402306, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng-Ju","family":"Chien","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, Chung Shan Medical University, Taichung 402306, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying-Ting","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung 402306, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2283-1377","authenticated-orcid":false,"given":"Wen-Wei","family":"Chang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, Chung Shan Medical University, Taichung 402306, Taiwan"},{"name":"Department of Medical Research, Chung Shan Medical University Hospital, Taichung 402306, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.eururo.2007.09.019","article-title":"Diagnostic criteria, classification, and nomenclature for painful bladder syndrome\/interstitial cystitis: An ESSIC proposal","volume":"53","author":"Nordling","year":"2008","journal-title":"Eur. Urol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1055\/s-0038-1676089","article-title":"Interstitial Cystitis\/Bladder Pain Syndrome","volume":"36","author":"Marcu","year":"2018","journal-title":"Semin. Reprod. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/j.ogc.2014.05.004","article-title":"Interstitial cystitis: Epidemiology, pathophysiology, and clinical presentation","volume":"41","author":"McLennan","year":"2014","journal-title":"Obstet. Gynecol. Clin. N. Am."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Yu, W.R., Jhang, J.F., Jiang, Y.H., and Kuo, H.C. (2024). The Pathomechanism and Current Treatments for Chronic Interstitial Cystitis and Bladder Pain Syndrome. Biomedicines, 12.","DOI":"10.3390\/biomedicines12092051"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"866","DOI":"10.5534\/wjmh.240295","article-title":"Pentosan Polysulfate Sodium and Maculopathy in Patients with Interstitial Cystitis: A Systematic Review and Meta-Analysis","volume":"43","author":"Lee","year":"2025","journal-title":"World J. Men\u2019s Health"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1097\/JCMA.0000000000000489","article-title":"Application of hyaluronic acid in patients with interstitial cystitis","volume":"84","author":"Lee","year":"2021","journal-title":"J. Chin. Med. Assoc."},{"key":"ref_7","first-page":"161","article-title":"Intravesical chondroitin sulphate for interstitial cystitis\/painful bladder syndrome","volume":"84","author":"Downey","year":"2015","journal-title":"Ulst. Med. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1036","DOI":"10.1002\/nau.70036","article-title":"The Efficacy and Safety of Dimethyl Sulfoxide Into the Bladder for the Treatment of Interstitial Cystitis\/Bladder Pain Syndrome: A Systematic Review and Meta-Analysis","volume":"44","author":"Li","year":"2025","journal-title":"Neurourol. Urodyn."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Kuo, H.C. (2022). Clinical Application of Botulinum Neurotoxin in Lower-Urinary-Tract Diseases and Dysfunctions: Where Are We Now and What More Can We Do?. Toxins, 14.","DOI":"10.3390\/toxins14070498"},{"key":"ref_10","first-page":"1735","article-title":"How Intravesical Platelet-Rich Plasma Can Help Patients with Interstitial Cystitis\/Bladder Pain Syndrome: A Comprehensive Scoping Review","volume":"35","author":"Soliman","year":"2024","journal-title":"Int. Urogynecol. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"605","DOI":"10.23736\/S2724-6051.25.06232-9","article-title":"The efficacy and safety of intravesical platelet-rich plasma injections into the bladder for the treatment of interstitial cystitis\/bladder pain syndrome: A systematic review and meta-analysis","volume":"77","author":"Li","year":"2025","journal-title":"Minerva Urol. Nephrol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1372","DOI":"10.1016\/j.jand.2015.03.021","article-title":"Nutritional considerations for patients with interstitial cystitis\/bladder pain syndrome","volume":"115","author":"Gordon","year":"2015","journal-title":"J. Acad. Nutr. Diet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3055","DOI":"10.1038\/s41598-021-82676-5","article-title":"Lifestyle and behavioral modifications made by patients with interstitial cystitis","volume":"11","author":"Lin","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.urology.2017.02.053","article-title":"Clinical Efficacy of 1-Year Intensive Systematic Dietary Manipulation as Complementary and Alternative Medicine Therapies on Female Patients with Interstitial Cystitis\/Bladder Pain Syndrome","volume":"106","year":"2017","journal-title":"Urology"},{"key":"ref_15","first-page":"110","article-title":"Urine biomarker could be a useful tool for differential diagnosis of a lower urinary tract dysfunction","volume":"36","author":"Yu","year":"2024","journal-title":"Tzu Chi Med. J."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Lin, H.Y., Lu, J.H., Chuang, S.M., Chueh, K.S., Juan, T.J., Liu, Y.C., and Juan, Y.S. (2021). Urinary Biomarkers in Interstitial Cystitis\/Bladder Pain Syndrome and Its Impact on Therapeutic Outcome. Diagnostics, 12.","DOI":"10.3390\/diagnostics12010075"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.2174\/1570159X17666190617095844","article-title":"Partners in Crime: NGF and BDNF in Visceral Dysfunction","volume":"17","author":"Coelho","year":"2019","journal-title":"Curr. Neuropharmacol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Yu, W.R., Jiang, Y.H., Jhang, J.F., and Kuo, H.C. (2023). Use of Urinary Biomarkers in Discriminating Interstitial Cystitis\/Bladder Pain Syndrome from Male Lower Urinary Tract Dysfunctions. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241512055"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e12404","DOI":"10.1002\/jev2.12404","article-title":"Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches","volume":"13","author":"Welsh","year":"2024","journal-title":"J. Extracell. Vesicles"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1186\/s12964-024-01999-8","article-title":"On the road: Extracellular vesicles in intercellular communication","volume":"23","author":"Wessler","year":"2025","journal-title":"Cell Commun. Signal."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1111\/jcmm.17132","article-title":"Roles of mesenchymal stem cells and exosomes in interstitial cystitis\/bladder pain syndrome","volume":"26","author":"Wen","year":"2022","journal-title":"J. Cell. Mol. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1002\/jcb.29356","article-title":"Long noncoding RNA (MEG3) in urinal exosomes functions as a biomarker for the diagnosis of Hunner-type interstitial cystitis (HIC)","volume":"121","author":"Liu","year":"2020","journal-title":"J. Cell. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e1140","DOI":"10.1002\/iid3.1140","article-title":"MiR-9-enriched mesenchymal stem cells derived exosomes prevent cystitis-induced bladder pain via suppressing TLR4\/NLRP3 pathway in interstitial cystitis mice","volume":"12","author":"Cui","year":"2024","journal-title":"Immun. Inflamm. Dis."},{"key":"ref_24","first-page":"3974","article-title":"Dynamic transcriptome analysis unveils key proresolving factors of chronic inflammatory arthritis","volume":"130","author":"Kong","year":"2020","journal-title":"J. Clin. Investig."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"8836","DOI":"10.7150\/thno.63396","article-title":"Role of prostaglandin E2 in tissue repair and regeneration","volume":"11","author":"Cheng","year":"2021","journal-title":"Theranostics"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Marentette, J.O., Hurst, R.E., and McHowat, J. (2015). Impaired Expression of Prostaglandin E2 (PGE2) Synthesis and Degradation Enzymes during Differentiation of Immortalized Urothelial Cells from Patients with Interstitial Cystitis\/Painful Bladder Syndrome. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0129466"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1097\/MIB.0000000000000244","article-title":"Involvement of heat shock protein a4\/apg-2 in refractory inflammatory bowel disease","volume":"21","author":"Adachi","year":"2015","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Stoffers, P., Guckenbiehl, S., Welker, M.W., Zeuzem, S., Lange, C.M., Trebicka, J., Herrmann, E., and Welsch, C. (2022). Diagnostic and prognostic significance of cell death markers in patients with cirrhosis and acute decompensation. PLoS ONE, 17.","DOI":"10.1371\/journal.pone.0263989"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"854","DOI":"10.1369\/0022155415600498","article-title":"Human SERPINB12 Is an Abundant Intracellular Serpin Expressed in Most Surface and Glandular Epithelia","volume":"63","author":"Niehaus","year":"2015","journal-title":"J. Histochem. Cytochem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.dld.2019.08.018","article-title":"SERPINB12 as a possible marker of steroid dependency in children with eosinophilic esophagitis: A pilot study","volume":"52","author":"Oliva","year":"2020","journal-title":"Dig. Liver Dis."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e70022","DOI":"10.1002\/jex2.70022","article-title":"Uromodulin and the study of urinary extracellular vesicles","volume":"3","author":"Harding","year":"2024","journal-title":"J. Extracell. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e12184","DOI":"10.1002\/jev2.12184","article-title":"Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis","volume":"11","author":"Correll","year":"2022","journal-title":"J. Extracell. Vesicles"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/130\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T16:21:19Z","timestamp":1766420479000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/130"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,22]]},"references-count":32,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["ijms27010130"],"URL":"https:\/\/doi.org\/10.3390\/ijms27010130","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,22]]}}}