A two-hit model for NLRP3-mediated pyroptosis in human bladder epithelial cells: Hyperglycemia primes, infection triggers

Diabetic bladder dysfunction (DBD) affects over half of all diabetics, although its pathogenesis remains unexplained. Animal studies have connected NLRP3 inflammasome-mediated pyroptosis to DBD. It is uncertain whether hyperglycemia directly activates this pathway in human bladder epithelial cells. Immunohistochemistry was used to identify NLRP3 and GSDMD expression in bladder cancer tissues and adjacent normal epithelial tissues from six non-diabetic patients with bladder cancer. Human immortalized bladder epithelial cells (SV-HUC-1) were treated with high glucose (30 mM) or LPS (1 µg/mL) for 24–48 hours. Cell survival, pyroptosis, motility, and expression of NLRP3/Caspase-1/IL-1β were evaluated. In non-diabetic bladder cancer patients, there was no significant difference in NLRP3 or GSDMD expression between the malignant and normal tissues (P > 0.05). Both high glucose and LPS significantly enhanced the expression of NLRP3, Caspase-1, and IL-1β proteins in SV-HUC-1 cells (approximately 1.7- to 2.1-fold increase, all P < 0.001). High glucose by itself showed no discernible effects on these functional characteristics, while LPS treatment alone significantly decreased cell viability (70.8% vs control, P < 0.05), increased pyroptosis rate (9.6% vs 2.7%, P < 0.05), and impeded migratory capability (73.3 vs 165.0, P < 0.05). This study offers the first proof that elevated glucose levels in human bladder epithelial cells are sufficient to trigger the NLRP3 inflammasome pathway but not enough to cause pyroptosis. A crucial "second hit" that converts pathway activation into functional cellular damage is LPS. These findings support a "two-hit" model for DBD etiology and provide fresh insights into early intervention options that target the priming phase of NLRP3 activation.