Intracellular Ca ²⁺ Levels and Mitochondrial Bioenergetics in Proximal Tubule Cells Are Affected by Polyamines

Background: Polyamines are molecules with multiple amino groups; primary polyamines found in mammals are putrescine, spermidine, and spermine. Polyamines are involved in cell proliferation, redox balance, apoptosis, and regulatory interactions with nucleic acids. Dysregulation of polyamine level and handling has been observed in multiple cardiovascular and renal diseases. In the kidney, the proximal tubules (PTs) are key to the regulation of blood pressure and rely on their mitochondrial bioenergetics to support electrolyte reabsorption. We hypothesized that in the PT, polyamines regulate calcium influx and mitochondrial bioenergetics. Methods: Kidneys were collected from 3-month-old male and female Sprague Dawley (SD) rats for metabolomic analysis, mitochondrial calcium dynamics assay, and PT isolation. Mitochondria were isolated by differential centrifugation and challenged with a spectrofluorimetric calcium uptake protocol using CaGreen. Metabolic profiles of renal cortices were generated using UHPLC-HRMS; metabolites were identified in El-MAVEN using exact mass and retention time with an in-house library. MetaboAnalyst 6.0 was used for statistical analysis (cutoff fold change 1.1, p-<0.05). PTs were isolated by vibrodissociation, and NO production (DAF-FM) and intracellular Ca 2+ homeostasis (Calbright630) were tested. Cultured female OK PT cells were used to establish the effect the polyamines or their metabolism byproducts on calcium dynamics (Fluo8), peroxynitrite production (HPF), and mitochondrial ΔΨ (TMRM). OriginPro was used to statistical comparisons. Results: Metabolomic analysis identified 174 mitochondria-focused metabolites in the renal cortex, and 55 were differentially abundant between the sexes. Notably, there was a dramatic increase in a byproduct of polyamine biosynthesis, 5’methylthioadenosine (MTA), in male cortex compared to females (FC=41.9, p<0.05). Western blotting revealed abundant expression of the rate limiting enzyme in polyamine catabolism pathway (SSAT, p<0.01) in the female renal cortex, pointing to higher abundance polyamines in males. Of note, renal cortical mitochondria from female SD rats exhibited an earlier mPTP opening compared to males (p<0.01). Live Ca 2+ imaging experiments in OK cells demonstrated that acute application of 100 uM MTA decreased mitochondrial ΔΨ. Applications of up to 100 uM putrescine and spermine evoked a dose-dependent increase in intracellular Ca 2+ , elevated ΔΨ, increased peroxynitrite formation, and caused mitochondrial calcium uptake. MTT assay revealed reduced cell viability following 1 h incubation with > 10 uM putrescine, but not spermine. In PTs isolated from male SD kidneys, acute application of Ang II (20 µM) promoted a rapid depletion of cytosolic Ca 2+ level, which was restored by the application of spermine (100 µM), accompanied with an increase in NO production. Conclusions: We report sexual dimorphisms in renal polyamine handling. Furthermore, polyamines play a role in PT calcium handling and mitochondrial function. Further studies will be devoted to establishing the intracellular cascades downstream of the polyamines in the PT. Funding: 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.