MECHANISMS OF RENAL URATE TRANSPORT

肾脏尿酸盐转运机制

• 批准号: 3236166
• 负责人: RUTH G ABRAMSON
• 金额: $ 20.3万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3236166
• 关键词: affinity chromatography; basolateral membrane; brush border membrane; cell membrane; dogs; electrophoresis; histochemistry /cytochemistry; ion transport; laboratory rabbit; laboratory rat; magnesium; renal cortex; renal tubular transport; transport proteins; urate

The proposed studies are designed to test the hypotheses that uricase is a membrane-associated protein that transports urate across renal plasma membranes, and that at least two transporters, an anion exchanger and the putative transporter, uricase, are present in brush border and/or basolateral membranes of the renal proximal tubule cells of several mammalian species. The mechanism(s) of urate transport will be assessed in unstimulated and copper stimulated brush border and basolateral membrane vesicles of rat, dog, and rabbit renal cortex prepared with several techniques: free-flow electrophoresis, magnesium aggregation, calcium precipitation, and percoll gradients. Transport will be examined in the presence and absence of inwardly directed electrolyte gradients, and in the presence and absence of pH gradients. Kinetic studies will assess saturation, inhibition and temperature dependence of transport. The kinetic constants of transport in vesicles will be compared to those obtained for urate oxidation by membranes. To further characterize the mechanism(s) of urate transport, the phenomena of homeo and heteroexchange diffusion will be evaluated. Cytochemical studies will be performed on renal and hepatic tissue slices and renal membrane vesicles to localize intramembranous and intracellular sites of uricase. This technique is based on a coupled oxidation-peroxidation reaction between hydrogen peroxide, produced by enzymatic (uricase) oxidation of urate, and cerium chloride. The product of this reaction, cerium perhydroxide, forms fine electron dense deposits at the site of the reaction, thereby permitting electron microscopic mapping of the distribution of uricase. In species and renal membranes in which uricase is detected in transport and cytochemical studies, uricase will be isolated and purified. Solubilized membranes will be subjected to affinity chromatography. Purification will be assessed with gel filtration, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), gradient PAGE, and isoelectric focusing. When purified, polyclonal antibodies will be raised and the effect of the antibodies on urate transport in membrane vesicles will be examined. Finally, purified uricase will be reconstituted into artificial lipid membranes or biological membranes devoid of uricase (red blood cell ghosts). Urate transport and kinetic constants of transport and oxidation will then be examined in the reconstituted system. This collective data should provide a solid base for understanding the mechanism(s) of urate transport, and perhaps the transport of other endogenous and exogenous organic anions within the renal proximal tubule.

拟议的研究旨在检验尿酸酶是一种 跨肾血浆转运尿酸的膜相关蛋白 膜，以及至少两个转运体，一个阴离子交换器和一个 可能的转运体尿酸酶存在于刷子边缘和/或 肾近端小管上皮细胞的基侧膜 哺乳动物物种。尿酸盐转运机制(S)将在#年进行评估。 非刺激性和铜刺激性刷缘和基底外侧膜 大鼠、狗和兔肾皮质囊泡的制备 技术：自由流动电泳法、镁凝聚法、钙法 降水和Percoll梯度。交通运输将在 内向电解质梯度的存在和不存在，以及 是否存在pH梯度。动力学研究将评估 运输的饱和、抑制和温度依赖性。这个 囊泡中运输的动力学常数将与 通过膜氧化尿酸盐制得。为了进一步描述 尿酸盐转运机制、同位交换和异位交换现象(S) 将对扩散进行评估。细胞化学研究将在 肾、肝组织切片和肾膜囊泡定位 尿酸酶的膜内和细胞内部位。这项技术是 基于氢气之间的氧化-过氧化耦合反应 过氧化氢，通过酶(尿酸酶)氧化尿酸和铈而产生 氯化物。这个反应的产物，过氢氧化铈，形成细小的 电子致密沉积在反应部位，从而允许 尿酸酶分布的电子显微镜图谱。在物种上 和肾膜，其中尿酸酶被检测到运输和 细胞化学研究，尿酸酶将被分离和提纯。已溶解 膜将经过亲和层析。净化将会 用凝胶过滤、十二烷基硫酸钠(SDS)进行评估 聚丙烯酰胺凝胶电泳(PAGE)、梯度PAGE和等电点 集中注意力。纯化后，将会产生多克隆抗体，并且 抗体对膜泡中尿酸盐转运的影响将是 检查过了。最后将纯化的尿酸酶重组为人工尿酸酶。 不含尿酸酶(红细胞)的脂膜或生物膜 鬼魂)。尿酸盐转运与转运和氧化的动力学常数 然后将在重组的系统中进行审查。这些收集的数据 为理解尿酸盐的作用机理(S)提供了坚实的基础 运输，也许还有其他内源性和外源性的运输 肾近端小管内的有机阴离子。