Understanding the contribution of inflammatory exosomes towards mediating Nicotine-Induced Podocyte Injury and Glomerular Sclerosis

Background: Recent studies demonstrate the role of NLRP3 inflammasome activation in nicotine-induced podocyte injury, making it possible to target the intracellular machinery of inflammation for treatment of end stage renal disease. However, since Nlrp3 inflammasomes are activated in the cytosol and their products may not be secreted out of podocytes via a classical and Golgi apparatus-mediated delivery pathway, it remains unclear how NLRP3 inflammasome activation-derived products such as HMGB1, IL-1 β or IL-18 are released out to trigger the inflammatory response and ultimately cause podocyte injury or glomerular injury. Hence, we tested the hypothesis whether acid sphingomyelinase (Asm) contributes to robust release of inflammatory exosomes in podocytes and contributes to the glomerular injury or sclerosis. Methods: Exosome morphology and structural characterization was analyzed using transmission electron microscopy (cryo-TEM). Cytek Aurora flow cytometry was utilized to analyze and quantify the exosome release. Nanoparticle Tracking Analysis (NTA) was done using NanoSight NTA3.2 to determine exosome particle size distribution. Uninephrectomized Asm knockout (Asm -/- ) and wild type (Asm +/+ ) mice were fed a normal chow for 5 weeks with or without nicotine treatment to produce glomerular injury. Results: By confocal microscopy, exosome release in glomeruli was confirmed by colocalization of exosome markers (annexin A2 vs. alkaline phosphate, ALP) in glomeruli of nicotine treated Asm+/+ mice, but not in Asm-/- mice. Nanoparticle Tracking Analysis (NTA) showed a largely increased urinary exosomes or microvesicles (90-120 nm) in Asm+/+ mice upon nicotine treatment, but not in Asm-/- mice. Biochemical analyses showed that Nlrp3 inflammasome product, HMGB1 or IL-1β was significantly increased in glomeruli of nicotine treated Asm+/+ mice, but not in Asm-/- mice. In functional studies, we found that urinary protein excretion was significantly higher in nicotine treated Asm+/+ mice compared to vehicle treated mice. The Asm -/- mice had significantly decreased nicotine-induced increase in urinary protein excretion. In addition, nicotine treated Asm+/+ mice produced significant morphological changes in the glomeruli, such as mesangial expansion, capillary collapse and increased cellularity, which was significantly attenuated in the Asm-/- mice. Furthermore, the nicotine-induced glomerular damage index (GDI) was significantly attenuated in Asm- /- mice. In invitro studies, using nanoparticle tracking analysis (NTA) we found that nicotine stimulation increased exosomes secretion (60-120 nm) from podocytes in dose dependent manner. Cytek Aurora flow cytometry analysis showed that nicotine stimulation increased the CD9/CD63 expression (exosome markers) in podocytes compared to control cells. However, prior treatment Asm inhibitor, amitriptyline blocked the nicotine-induced exosome secretion (CD9/CD63 expression) in podocytes. In conclusion, our observations demonstrate that nicotine enhanced Nlrp3 inflammasomes activation and at the same time increased exosome release from podocytes, which work together to trigger podocyte glomerular inflammation and injury. This project was supported by NIH grant, R01HL148711. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.