Effects of endoplasmic reticulum stress on lipid levels, inflammatory and apoptotic pathways in renal epitel cells

Objective: This study aimed to determine tunicamycin (TM)-related sphingolipid and polyunsaturated fatty acid (PUFA) changes in human kidney cells (HEK-293) and their effects on inflammatory and apoptotic pathways. Tunicamycin was employed to induce endoplasmic reticulum (ER) stress in HEK-293 cells and an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), was given to minimize cytotoxicity. Method: Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Sphingomyelin (SM), ceramide (CER) and PUFA levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Glucose-regulated protein 78-kd (GRP78), cleaved caspase-3 and cyclooxygenase-1 (COX-1) protein levels were assessed by immunofluorescence. Cytosolic phospholipase A2 (cPLA2), total COX and prostaglandin E2 (PGE2) were measured to evaluate changes in enzyme activity. Results: Decreased cell viability was observed in TM treated cells. Administration of TUDCA following TM treatment significantly increased cell viability compared to TM treatment alone. Tunicamycin-induced ER stress was confirmed by significantly increased protein levels of GRP78. A significant increase was observed in C18-C24 CERs and caspase-3 activity, while a significant decrease occurred in sphingosine-1-phosphate (S1P) and cPLA2 activity in cells treated with μg/ml versus controls. The decrease in cPLA2 activity was accompanied by significantly increased PUFA levels in TM-treated cells. Tauroursodeoxycholic acid treatment in conjunction with TM significantly decreased ER stress, C18-C24 CERs, caspase 3 activity, and increased S1P levels. Conclusion: Results show the build-up of long-chain CERs and PUFAs in HEK-293 cells undergoing ER stress alongside increased apoptotic activity. Tauroursodeoxycholic acid administration, along with TM treatment alleviated the build-up of CERs and TM-induced apoptotic activity in kidney epithelial cells.