SPHINGOMYELIN ACCUMULATION IN PROXIMAL TUBULAR CELLS

在与庆大霉素孵育的人肾近端小管(PT)细胞中，我们 已经发现细胞表面结合和内化增加 ~(125)I-低密度脂蛋白(LDL)，但仍有~(125)I-低密度脂蛋白的降解 保持不变。随之而来的是公司的4倍增长。 [~(14)C]醋酸盐转化为鞘磷脂及其螺纹状的出现 庆大霉素处理细胞中的“髓样小体”。我们的总体目标 1)评估外源性脂类对血浆的贡献 庆大霉素处理的PT细胞中神经鞘脂脂上的脂蛋白； 2)评估导致这些物质积累的生化机制 庆大霉素处理细胞中的鞘磷脂；3)分离“髓样小体” 来自庆大霉素处理的PT细胞。第一，~3H-标记的摄取 神经鞘磷脂对低密度脂蛋白(LDL)的影响 将对庆大霉素处理的细胞进行研究，以调查 外源性磷脂对脂蛋白的直接作用 庆大霉素处理的细胞中鞘磷脂水平增加。活动 神经酰胺胆碱磷酸转移酶与棕榈酰辅酶A：丝氨酸棕榈酰 接下来将测定转移酶，以评估庆大霉素 刺激这些酶，进而增加内源合成 RT细胞中神经鞘脂脂的含量。神经鞘磷脂酶的水平将是 在对照组和庆大霉素处理的细胞中测量NEXT，以便评估 抑制鞘磷脂溶酶体降解是否有助于 神经鞘磷脂的细胞水平升高。最后，髓系小体 将从庆大霉素处理的PT细胞中分离并鉴定 使用生化技术和电子显微镜。 重大假设。庆大霉素处理的RT细胞中鞘磷脂的增加 可能是由于鞘磷脂分解代谢减少，摄取增加 外源性鞘磷脂对脂蛋白或内源性合成的促进作用 庆大霉素处理的PT细胞中鞘磷脂的表达。这些研究将 为理解鞘磷脂代谢和 庆大霉素肾毒性中脂蛋白代谢的研究 上皮细胞。我们对这一文化系统的研究将提供一个 在从动物数据到人类的推断中有价值的工具，可以使用 作为一种体外模型，用于未来许多药物诱导的人类研究 肾毒性。

In human kidney proximal tubular (PT) cells incubated with gentamicin, we have found that there is increased cell surface binding and internalization of 125I-low density lipoproteins (LDL), but 125I-LDL degradation remains unchanged. This is accompanied by a 4 fold increase in the incorporation of [14C] acetate into sphingomyelin and the appearance of whorl-like "myeloid bodies" in the gentamicin-treated cells. Our overall objectives are: 1) to assess the contribution of exogenously derived lipids on plasma lipoproteins on the cellular sphingolipids in gentamicin-treated PT cells; 2) to assess the biochemical mechanisms responsible for the accumulation of sphingolipids in gentamicin-treated cells; 3) to isolate "myeloid bodies" from gentamicin-treated PT cells. First, the uptake of 3H-labeled sphingomyelin on low density lipoproteins (LDL) by control cells and gentamicintreated cells will be studied so as to investigate whether exogenously derived phospholipids on lipoproteins contribute directly to increased sphingomyelin levels in gentamicin-treated cells. The activities of ceramide choline phosphotransferases and palmitoyl-CoA: serine palmitoyl transferase will be measured next in order to assess whether gentamicin stimulated these enzymes, which in turn increased the endogenous synthesis of sphingolipids in RT cells. The levels of sphingomyelinase will be measured next in control and gentamicin-treated cells in order to assess whether inhibition of lysosomal degradation of sphingomyelin contributes to the increased cellular level of sphingomyelin. Finally, the myeloid bodies will be isolated from gentamicin-treated PT cells and characterized employing biochemical techniques and electron microscopy. Major hypothesis. Increased sphingomyelin in gentamicin-treated RT cells may be due to decreased sphingomyelin catabolism, increased uptake of exogenous sphingomyelin on lipoproteins or increased endogenous synthesis of sphingomyelin in gentamicin-treated PT cells. These studies will provide new insights in understanding sphingomyelin metabolism and lipoprotein metabolism in gentamycin-induced nephrotoxicity in human renal epithelial cells. Our studies on this culture system will provide a valuable tool in the extrapolation from animal data to man and can be used as an in vitro model for a host of future studies on drug-induced human nephrotoxicity.