Early cell‐specific changes in nitric oxide synthases, reactive nitrogen species formation, and ubiquitinylation during diabetes‐related bladder remodeling

BACKGROUND: Urinary bladder dysfunction in diabetes is a well-recognized phenomenon but the mechanisms involved and initiating events are not clear. The physiological production of nitric oxide (NO) plays an important role in bladder tone and local immune defense, and recent studies have shown that NO-derived reactive nitrogen species occur in many settings of chronic disease, including other diabetic complications. Here, we investigated the early time-dependent and cell-specific changes in the nitric oxide synthase isoforms (NOS1, 2, and 3), peroxynitrite, and ubiquitinylation in the well-documented streptozotocin-induced rat model of diabetes. METHODS: Immunohistochemical methods and automated digital imaging were used for the measurement of morphometric and histochemical analysis of the bladder tissue regions. Region-specific 3-nitrotyrosine (a biomarker of NO dysregulation and reactive nitrogen species formation) and ubiquitinylated protein prevalence (marker of proteasomal activity) were also investigated. RESULTS: Immunohistochemistry revealed early, time-dependent, and cell-specific alterations in the three isoforms of NOS. We also observed region-specific increases in protein nitration, demonstrating first-time evidence of reactive nitrogen species formation in this setting. The changes in nitration did not pattern changes in NOS2 induction or tissue ubiquitinylation, and these alterations preceded any detectable changes in bladder structure (3 days vs 2 weeks) in this same animal preparation. CONCLUSIONS: These data demonstrate that selective and regionally distinct changes in nitric oxide production and impaired nitric oxide control are early events during diabetic cystopathy and that mechanisms leading to increased oxidative stress and proteasomal activation may be key participants leading to organ dysfunction in this setting.