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Accumulation of lipids and their intermediary metabolites under endoplasmic reticulum (ER) stress instigates metabolic failure, described as lipotoxicity, in the kidney. This study aimed to determine ER-stress-related sphingolipid and polyunsaturated fatty acid (PUFA) changes in human kidney cells. Tunicamycin (TM) was employed to induce ER stress and an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), was given to minimize cytotoxicity. Cell viability was determined by MTT assay. Sphingomyelin (SM), ceramide (CER), and PUFA levels were measured by LC–MS/MS. Glucose-regulated protein 78-kd (GRP78), cleaved caspase-3 and cyclooxygenase-1 (COX-1) levels were assessed by immunofluorescence. Cytosolic phospholipase A2 (cPLA2), total COX, and prostaglandin E2 (PGE2) were measured to evaluate changes in enzyme activity. 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 TM versus controls. The decrease in cPLA2 activity was accompanied by significantly increased PUFA levels in TM treated cells. TUDCA treatment in conjunction with TM significantly decreased ER stress, C18-C24 CERs, caspase 3 activity, and increased S1P levels. Results show the buildup of long chain CERs and PUFAs in kidney cells undergoing ER stress alongside increased apoptotic activity. TUDCA administration, along with TM treatment alleviated the buildup of CERs and TM-induced apoptotic activity in kidney epithelial cells.

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Prilocaine (PRL) is a common local anesthetic. Despite the successful use of regional anesthesia for intraocular surgery, there are associated side effects that may affect the retina in case of accidental intravitreal injection. This study examined the signal transduction pathways activated by PRL toxicity and determined the protective role of nitric oxide synthase‐2 (NOS2) inhibition in cultured human‐derived retinal pigment epithelial cells (ARPE‐19). Toxicity analysis was performed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay to detect the toxic dose of PRL and protective effectiveness of asperglaucide (ASP), an NOS2 inhibitor. Nuclear factor kappa B p65 (NF‐κB p65), phosphorylated NF‐κB p65, phospho‐protein kinase B (AKT), NOS2, nitrotyrosine, and cleaved caspase‐3 protein levels were evaluated by immunofluorescence staining and/or western blot analysis. Interleukin‐6 (IL‐6) and nitrated protein levels were quantified using an immunoassay, whereas caspase‐3 activity and nitrite/nitrate levels were measured using a fluorometric method. A significant increase in NF‐κB p65, and phosphorylated NF‐κB p65 and AKT levels due to PRL toxicity was observed. Similarly, IL‐6, NOS2, nitrite/nitrate, and nitrotyrosine levels were significantly higher in PRL‐treated cells than in control cells. Application of ASP to PRL‐treated cells reduced NF‐κB p65, and phosphorylated NF‐κB p65 and AKT to basal levels. IL‐6, NOS2, nitrite/nitrate, and nitrotyrosine levels also considerably decreased following ASP treatment in cells experiencing PRL‐induced toxicity. Moreover, the caspase‐3‐dependent apoptotic pathway was not activated. Our results indicate that ASP could ameliorate PRL‐induced activation of NF‐κB p65 that led to inflammation in cultured ARPE‐19 cells.