Nicotine Disrupts Calcium Regulation and Induces Oxidative Stress in Proximal Tubular Cells

Introduction: Nicotine consumption through vaping, cigarettes, or nicotine replacement therapy poses major risks for renal health, especially in individuals already diagnosed with kidney disease. Nicotine amplifies cardiovascular damage in hypertensive individuals. A gap in knowledge remains regarding the mechanisms leading to the harmful effects of nicotine, especially in renal epithelial cells. Proximal tubule (PT) epithelial cells are prone to oxidative stress and are key to the development of renal disease due to their high energy demands. We hypothesize that in the renal PTs nicotine promotes dysregulated calcium handling and oxidative stress, thus leading to cell damage. Methods: Nicotine was infused chronically into Dahl SS rats (0.2 mg/kg/day s.c.) fed 0.4% NaCl diet for 21 days. Electron microscopy was employed to assess mitochondrial ultrastructure and damage. Live confocal fluorescence imaging was performed on cultured opossum kidney (OK) PT cells; HPF, Fluo8, and TMRM fluorescent dyes were used to detect ONOO - , Ca 2+ transients, and to evaluate mitochondrial membrane potential (ΔΨm) after acute applications of nicotine (100 nM to 5 mM). Rhodamine Phalloidin (Rh-Ph) staining was employed to label actin cytoskeleton rearrangements in response to varying nicotine concentrations. MTT assay was done to measure cellular viability in response to nicotine. OriginPro was used for statistical analysis. Results: Histological analysis of the Masson’s trichrome stained kidneys in the Dahl SS rats after nicotine infusion demonstrated increased PT damage characterized by brush border loss, tubular swelling, protein cast formation, and signs of necrosis in nicotine infused rats. Blind scoring of EM images of mitochondria in the PTs revealed mitochondrial swelling (0.46 ± 0.02 vs. 0.54 ± 0.02 μm 2 in vehicle and nicotine groups, respectively, p<0.05), cristae loss and other signs of damage (2.4 ± 0.1 vs. 2.6 ± 0.1 a.u., p<0.05). In cultured OK cells acute applications of nicotine promoted dose-dependent intracellular ONOO - transients (average 1.6 ± 0.1 a.u increase from baseline of 1.0 a.u, for 300 μM of nicotine, p<0.05, n>50 cells) and Ca 2+ responses (average 2.3 ± 0.7 a.u increase from baseline, for 100 μM of nicotine, p<0.05, n>50 cells). Furthermore, 300 μM nicotine applied acutely evoked a decrease in mitochondrial membrane potential (0.9 ± 0.1 a.u. from baseline, p<0.01). Rh-Ph staining showed stress fiber formation upon exposure to nicotine (300 μM of nicotine for 16 hours). MTT assay revealed a decrease in viability proportionate to nicotine concentration (cell death at ~20% within the range of 100 nM to 300 μM of nicotine, with a decline down to 45% cell death at 5 mM; 30 min exposure). Conclusion: In PT cells, nicotine reduces cell viability, promotes acute ONOO - formation, calcium dysregulation, redox stress, and mitochondrial ΔΨ decrease. Understanding the mechanistic impact of nicotine on PT is crucial for developing therapies against smoking and vaping-related renal pathologies. R01 DK126720, R01 DK129227, R01 HL148114, AHA 24TPA1283630, U54HL169191, T32 HL155011 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.