Characterization of urethral fibrosis in a rabbit model: Potential roles of Wnt-ß catenin pathway and epithelial to mesenchymal transition.

AIM: To elucidate the precise cellular and molecular mechanisms that underlie urethral fibrogenesis. METHODS: Endoluminal electrocautery injury (using Karl Storz 10 Fr. Pediatric urethroscope) was employed in male rabbits (n = 6) to create mucosal injury. Retrograde urethrogram (RUG) and endoluminal ultrasound techniques were used to assess severity and changes in luminal cross-sectional area. Six control rabbits were subjected to sham injury, in which the electrocautery was inserted but not powered. Urethral tissues were harvested 30 days postinjury and subjected to RNA sequencing and quantitative polymerase chain reaction (qPCR) to determine changes in gene expression. Histological, immunostaining, and Western blot studies were used to determine changes in protein expression of known markers of fibrosis (eg, collagen, Integrinαv, GIV/Girdin, transforming growth factor-ß (TGF-ß), and pSMAD1,2,3). RESULTS: Trichrome staining confirmed increased connective tissue in urethral scar tissues. Immunostaining revealed a potential role for epithelial to mesenchymal cell transition (EMT) and positive labeling for all fibrotic markers (eg, collagen-1, Integrin αv, GIV/Girdin, transforming growth factor-ß (TGF-ß), and SMAD1,2,3). Western blot analysis confirmed increased protein levels of these fibrotic markers. CONCLUSION: Our RNA sequencing and qPCR studies, in conjunction with our protein data, suggest that urethral mucosal fibrogenesis may be mediated by novel fibrogenic signaling pathways involving Wnt-ß catenin, TGF-ß, GIV/Girdin, and EMT which lead to increased collagen deposition. Therapeutic strategies targeting these pathways may be beneficial in attenuating fibrogenesis and stricture progression.

A Novel Endoluminal Ultrasound Imaging Technique to Determine Urethral Luminal Cross-Sectional Area.

INTRODUCTION AND OBJECTIVES: Retrograde urethrogram (RUG) and voiding cystourethrogram (VCUG) are currently the gold standard imaging technique for diagnosis of urethral stricture and determination of stricture location. However, RUG and VCUG have multiple limitations. These techniques require exposure to ionizing radiation, the quality is operator and patient dependent, there is a moderate degree of invasiveness with urethral catheterization, can have artifacts because of patient positioning that underestimates stricture length. The development of novel imaging modalities without ionizing radiation to accurately evaluate the presence, location, length, and lumen cross-sectional area (CSA) of the urethral stricture would be of great value. The objective of this study was to develop a novel endoluminal ultrasound (ELUS) imaging technique that permits the accurate quantitation of urethral stricture. METHODS: Urethral strictures were created in rabbits (n = 5) by electrocautery and an ELUS technique was developed for subsequent luminal imaging. A 3.2F 40 MHz ultrasound (US) probe was introduced transurethrally and infused with US contrast agent. Images were recorded as the catheter was pulled back at a constant speed to acquire tomographic images. Lumen CSA over the entire urethral length was calculated using a custom methodology and validated in our laboratory. RESULTS: Urethral luminal CSA over the entire length of urethra before and after experimental stricture development was quantified including the length of stenosis. Intra- and interobserver variability (r = 0.99 for both) was excellent. CONCLUSIONS: Feasibility of ELUS as a quantitative technique to determine healthy urethral lumen and stricture CSA was demonstrated. The translational potential for a nonionizing imaging modality to better describe CSA, length, location, and uninvolved urethral CSA of the stricture is a significant improvement over current methodology.