Çeker Tuğçe, Yılmaz Çağatay, Kırımlıoglu Esma, Aslan Mutay
Department of Medical Biochemistry, Akdeniz University, Faculty of Medicine, Antalya 07070, Turkey.
Department of Histology and Embryology, Akdeniz University, Faculty of Medicine, Antalya 07070, Turkey.
Toxicol Res (Camb). 2022 Jul 22;11(4):683-695. doi: 10.1093/toxres/tfac041. eCollection 2022 Aug.
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 A (cPLA), total COX, and prostaglandin E (PGE) 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 cPLA activity in cells treated with TM versus controls. The decrease in cPLA 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.
内质网（ER）应激下脂质及其中间代谢产物的积累会引发肾脏代谢功能衰竭，即脂毒性。本研究旨在确定人肾细胞中与内质网应激相关的鞘脂和多不饱和脂肪酸（PUFA）的变化。采用衣霉素（TM）诱导内质网应激，并给予内质网应激抑制剂牛磺熊去氧胆酸（TUDCA）以最小化细胞毒性。通过MTT法测定细胞活力。通过液相色谱-串联质谱法（LC-MS/MS）测量鞘磷脂（SM）、神经酰胺（CER）和PUFA水平。通过免疫荧光评估葡萄糖调节蛋白78-kd（GRP78）、裂解的半胱天冬酶-3和环氧化酶-1（COX-1）水平。测量胞质磷脂酶A（cPLA）、总COX和前列腺素E（PGE）以评估酶活性的变化。在TM处理的细胞中观察到细胞活力下降。与单独的TM处理相比，TM处理后给予TUDCA显著提高了细胞活力。GRP78蛋白水平显著升高证实了衣霉素诱导的内质网应激。与对照相比，TM处理细胞中的C18-C24神经酰胺和半胱天冬酶-3活性显著增加，而鞘氨醇-1-磷酸（S1P）和cPLA活性显著降低。cPLA活性的降低伴随着TM处理细胞中PUFA水平的显著增加。TUDCA与TM联合处理显著降低了内质网应激、C18-C24神经酰胺、半胱天冬酶3活性，并提高了S1P水平。结果表明，内质网应激的肾细胞中长链神经酰胺和PUFA积累，同时凋亡活性增加。给予TUDCA并结合TM处理可减轻肾上皮细胞中神经酰胺的积累和TM诱导的凋亡活性。
