Oleate Co-Treatment Prevents Palmitate-Induced Lipotoxicity and Enhances Lipid Droplet Formation in Human Renal Tubular Epithelial Cells

Saturated fatty acid-induced lipotoxicity, particularly from palmitate, contributes to the development of kidney injury, especially in individuals with obesity and metabolic disorders. In contrast, unsaturated fatty acids, such as oleate, promote the formation of lipid droplets (LDs), which sequester excess fatty acids and reduce cellular stress. LDs are key indicators of a cell’s capacity to buffer lipotoxicity, and the lipid droplet–associated protein Perilipin 2 (PLIN2) plays a central role in stabilizing and regulating these structures. This study investigated whether oleate co-treatment reduces palmitate-induced lipotoxicity in renal tubular epithelial cells (HK-2) and whether this effect is associated with increased PLIN2 expression and lipid droplet accumulation. HK-2 cells were treated with bovine serum albumin (BSA)-conjugated palmitate (0, 150 µM, 300 µM, or 450 µM) with or without oleate (150 µM) for 24 h. Palmitate-induced lipotoxicity was first evaluated by measuring the levels of extracellular and total ATP, using the RealTime-GloTM Extracellular ATP and CellTiter-GloTM Luminescent Cell Viability assay kits. Western blot analysis was performed to further evaluate the protein levels of mitochondrial markers of fusion (mitofusin 2, MFN2) and fission (dynamin-related protein 1, DRP1), cell apoptotic markers [cleaved poly (ADP-ribose) polymerase-1 (PARP1) and C/EBP homologous protein (CHOP)], and lipid accumulation marker PLIN2. Immunofluorescence staining was used to visualize lipid droplet accumulation. Exposure of HK-2 cells to palmitate at high concentrations (300–450 µM) triggered a robust release of extracellular ATP (eATP), a damage-associated molecular pattern signal, which was detectable as early as 8 hours and peaked at 16 hours, in a dose-dependent manner. Additionally, a dose-dependent decrease in ATP production was detected in palmitate-treated cells at 24 hours. This cytotoxic response was accompanied by disrupted mitochondrial dynamics, characterized by a marked reduction in MFN2 and the accumulation of small, fragmented mitochondria. Consistently, palmitate also elevated the expression of cytotoxicity markers, cleaved PARP-1, and CHOP. Notably, palmitate-induced eATP increase and total ATP reduction were abolished in the presence of oleate co-treatment. Additionally, co-treatment attenuated palmitate-induced mitochondrial fragmentation, PARP-1 cleavage, and CHOP upregulation, which was accompanied by a significant upregulation of PLIN2 protein and pronounced accumulation of larger lipid droplets. These findings suggest that oleate mitigates palmitate-induced mitochondrial dysfunction and lipotoxicity in human renal tubular epithelial cells, potentially through PLIN2-mediated lipid droplet formation. This abstract was presented at the American Physiology Summit 2026 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.