GLYCOSPHINGOLIPID STORAGE IN PROXIMAL TUBULAR CELLS

我们发现尿沉渣中乳酸神经酰胺(LacCer)水平 在纯合子家族性高胆固醇血症(FH)中升高15倍 人类受试者与正常人相比。LacCer定位于胞浆中。 FH近端肾小管上皮细胞的小泡。血浆置换治疗 门腔分流术减少尿LacCer排泄量 排空近端肾小管(PT)细胞内的胞质小泡。 我们的总体目标是：1)近端肾小管细胞的特征 在培养中保持；2)确定细菌生长的生化机制 FH纯合子尿LacCer水平升高；3)分离和 描述PT细胞的细胞质小泡的特征。~(14)C的摄取 葡萄糖神经酰胺对低密度脂蛋白和[~3H]半乳糖(半乳糖氧化酶)的影响 细胞对低密度脂蛋白的标记LacCer的研究 评估外源性LacCer对低密度脂蛋白是否有贡献 直接导致LacCer细胞水平的增加或间接地 尿液中葡萄糖神经酰胺的摄取及其向LacCer的转化 细胞。细胞结合[14C]标记半乳糖的能力， 葡萄糖、丝氨酸和棕榈酸转化为LacCer和UDP-半乳糖：GlcCer， 半乳糖基转移酶活性将被测量，以评估 在这些细胞中内源性合成LacCer。近端肾小管细胞 胞质囊泡将通过匀浆、离心法、 蔗糖密度梯度离心法。细胞质小泡将 利用标志物酶分析、免疫化学技术进行表征 和电子显微镜。无论细胞质小泡是不是 能够合成LacCer或直接从低密度脂蛋白中获取LacCer将 被评估。 重大假设。纯合子FH患者尿液细胞LacCer水平升高 可能是由于非受体介导的外源LacCer对低密度脂蛋白和 增加内源LacCer的合成或两者兼而有之。这些研究将 提供了对神经节苷脂和脂蛋白代谢的新见解 正常人和FH患者近端肾小管上皮细胞及其病理生理 FH中的LacCer存储。

We have found that the urinary sediment lactosylceramide (LacCer) levels are elevated 15 fold in homozygous familial hpypercholesterolemic (FH) human subjects compared to normal. LacCer was localized in the cytoplasmic vesicles of FH urinary proximal tubular cells. Plasma exchange treatment and portacaval shunt operation decreased the urinary excretion of LacCer and emptied the cytoplasmic vesicles in the proximal tubular (PT) cells. Our overall objectives are: 1) to characterize proximal tubular cells maintained in culture; 2) to determine the biochemical mechanism for the elevated urinary levels of LacCer in FH homozygotes; 3) to isolate and characterize the cytoplasmic vesicles in PT cells. The uptake of 14C glucosylceramide on LDL and [3H] galactose (galactose oxidase [3H] KBH4 reduction) labeled LacCer on LDL by cells will be studied in order to assess whether or not the exogenously derived LacCer on LDL contributes directly to the increased cellular levels of LacCer or indirectly following the uptake of glucosylceramide and its conversion to LacCer in the urinary cells. The capacity of cells to incorporate [14C] labeled galactose, glucose, serine and palmitate into LacCer and the UDP-galactose: GlcCer, galactosyltransferase activity will be measured in order to assess the endogenous synthesis of LacCer in these cells. The proximal tubular cell cytoplasmic vesicles will be isolated by homogenization, centrifugation, and sucrose density gradient centrifugation. The cytoplasmic vesicles will be characterized employing marker enzyme assays, immunochemical techniques and electron microscopy. Whether or not the cytoplasmic vesicles are capable of synthesizing LacCer or acquiring LacCer directly from LDL will be assessed. Major hypothesis. Increased urinary cell LacCer levels in homozygous FH may be due to a non-receptor mediated uptake of exogenous LacCer on LDL and increased endogenous synthesis of LacCer or both. These studies will provide new insights into glycosphingolipid and lipoprotein metabolism in proximal tubular cells from normal and FH subjects and the pathophysiology of LacCer storage in FH.