Urethral stricture disease (USD) in males can result from trauma such as blast or straddle injuries, infection, inflammation, or iatrogenic/idiopathic etiologies. USD produces voiding and storage-related urinary complications, which can damage the bladder and then ultimately impair renal function. In Veterans, a total of 92,448 procedures were performed for USD in 5 years. Of the transurethral and open surgical interventions, a majority of Veterans (>95%) undergo non-surgical transurethral interventions for USD. These non-surgical interventions are not often permanently successful. Thus, the currently used non-surgical interventions are not only ineffective but also known to complicate surgical interventional outcomes. The analysis of urethral scar tissue has shown increased fibrosis of the urethral epithelium and surrounding corpus spongiosum. Our preliminary studies confirm these findings and further demonstrate that repeated transurethral interventions such as transurethral dilation (TUD) increased scarring. As seen in urethral scar tissues from stricture patients, scarring is mediated by upregulation of the fibrogenic network. We hypothesize (i) urinary microbiomes may play a role by altering mucosal permeability and (ii) that increased fibrosis after injury. A clear understanding of these molecular mechanisms involved in urethral fibrosis would enable identification of novel targets for development of innovative strategies in order to eventually prevent/treat this disorder. The specific aims of our studies are to determine: 1) mechanisms of increased tissue damage after repeated transurethral interventions; 2) the role of microbiomes and 3) causality using in vitro co-culture studies. We will use several novel approaches: 1) longitudinal measurement of stricture development in Veterans using non-invasive imaging; 2) detailed analytical studies in urine/scar tissues from stricture patients to identify cellular and molecular pathways. These evaluations will also include the role of microbiomes; 3) We will isolate bacteria from clean catch urine specimens and perform co-culture experiments using human urothelial cell culture (HUCC) model. The proposed studies will help to (i) identify the microbiomes that have the ability to adhere to and invade urethral mucosal cells (urothelium), induce fibrosis or cell (mucosal) damage and (ii) also to a test a mucosal barrier protection strategy. Mechanistic insights gained from these studies will not only examine the topic from the molecular level, but also unravel novel targets for further development of treatment strategies to prevent fibrosis. Thus, our proposal is both conceptually novel (role of microbiomes) and innovative (uses novel approaches, interventions, and tools such as MR-UTE to study fibrosis), paving the way for new therapies. This study has high potential for clinical translation (application of novel diagnostic tools and development of anti-fibrotic interventions) to maximize functional ...