Nicotine-mediated signaling in glomerular podocytes

Introduction: Although nicotine’s harmful effects on renal function are established, the precise cellular mechanisms of smoking-related damage are understudied. Smoking-Related Glomerulopathy (SRG) is a renal disease phenotype associated with smoking. This condition histologically mimics diabetic nephropathy, however, SRG patients present with proteinuria and renal insufficiency without diabetes. Here we investigated the acute and chronic nicotine-related oxidative and nitrosative stress in glomerular podocytes. We hypothesized that nicotine may promote nitrosative stress in the kidney cells through the rapid production of peroxynitrite (ONOO-) and a decline in nitric oxide (NO) bioavailability. Methods: To test the hypothesis, we used a conditionally immortalized human podocyte line and confocal imaging to detect the production of ONOO- (Hydroxyl Radical and Peroxynitrite Sensor; HPF), intracellular Ca2+ (Fluo-8), and NO (DAF-FM). The presence of nicotinic acetylcholine receptors (nAChR) receptor subunits in podocytes was confirmed with immunocytochemistry and live imaging using commercially available pharmacology. NOS activity was analyzed in response to Ang II with the specific blockers for NOS1 (NΩ-Propyl-L-arginine hydrochloride) and NOS2 (L-NIL) subunits. One-way ANOVA (OriginPro) was used for statistical analysis. Results: Immunostaining indicated that human and rat kidneys express nicotinic acetylcholine receptors (nAChR). Notably, we detected specific expression of α7 nAChR in glomerular podocytes. In podocytes, acute nAChR activation promoted the mobilization of intracellular Ca2+ controlled by intracellular store activation, and fast ONOO- transients. Multiple consecutive applications of nicotine resulted in repeated intracellular Ca2+ and ONOO- transients. Nicotine-mediated ONOO- response was efficiently blocked in the presence of superoxide dismutase (SOD). The application of specific α7 or α4β2, α2β4, α4β4 and α3β4 nAChR agonists elicited Ca2+ transients but did not reproduce the ONOO- response to nicotine, suggesting that nitrosative processes may occur independently from Ca2+ influx or nAChR function. Chronic exposure to nicotine (12-hrs) resulted in a significant decline in podocytes’ NO bioavailability (p<0.05). While under normal conditions, NO is produced primarily by NOS1 (70%), and the rest is attributed to NOS2, chronic nicotine exposure led to an elevation of NOS2 (75%) and a decline in NOS1 activity (p<0.001). We observed similar NOS remodeling under hyperglycemic conditions, suggesting similar nitrosative processes in response to nicotine and high glucose. Conclusions: Nicotine promotes superoxide-stimulated nitrosative stress and peroxynitrite formation in podocytes leading to decline in NO bioavailability and pathological activation of NOS2. R01 NIDDK DK126720 (OP), DK129227 (OP), HL148114 (DVI), NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (OP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.