GLYCOSPHINGOLIPID STORAGE IN PROXIMAL TUBULAR CELLS

我们发现尿沉渣乳糖神经酰胺（LacCer）水平 在纯合子家族性高胆固醇血症（FH）患者中升高15倍 与正常人相比。 LacCer定位于细胞质中， FH尿近端小管细胞囊泡。 血浆置换治疗 门腔静脉分流术减少尿LacCer排泄 并清空近端小管（PT）细胞中的细胞质囊泡。 我们的总体目标是：1）表征近端肾小管细胞 维持在培养物中; 2）确定生物化学机制， FH纯合子中LacCer的尿水平升高; 3）分离和 表征PT细胞中的胞质囊泡。 14 C的吸收 葡萄糖神经酰胺对LDL和[3 H]半乳糖（半乳糖氧化酶[3 H] KBH 4 还原）标记的LacCer对LDL的作用， 评估LDL上的外源性LacCer是否有助于 直接与LacCer的细胞水平增加有关，或间接与 葡萄糖神经酰胺的摄取及其在尿中转化为LacCer 细胞 细胞掺入[14 C]标记半乳糖的能力， 将葡萄糖、丝氨酸和棕榈酸盐转化为LacCer和UDP-半乳糖：GlcCer， 将测量半乳糖基转移酶活性以评估半乳糖基转移酶活性。 这些细胞中LacCer的内源性合成。 近端肾小管细胞 通过匀浆，离心， 和蔗糖密度梯度离心。 细胞质囊泡将 采用标记酶测定、免疫化学技术 和电子显微镜。 无论细胞质小泡是否 能够合成LacCer或直接从LDL获得LacCer的人将 进行评估。 主要假设 纯合子FH中尿细胞LacCer水平升高 可能是由于LDL上外源性LacCer的非受体介导的摄取， 增加LacCer的内源性合成或两者。 这些研究将 提供了新的见解鞘糖脂和脂蛋白代谢， 正常和FH受试者的近端肾小管细胞及其病理生理学 在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.