MECHANISMS OF RENAL URATE TRANSPORT

肾脏尿酸盐转运机制

• 批准号: 3236164
• 负责人: RUTH G ABRAMSON
• 金额: $ 19.84万
• 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/3236164
• 关键词: affinity chromatography; basolateral membrane; brush border membrane; cell membrane; electrophoresis; histochemistry /cytochemistry; ion transport; magnesium; renal cortex; renal tubular transport; 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假设 跨肾血浆转运尿酸盐的膜相关蛋白 膜，并且至少有两个转运蛋白、一个阴离子交换蛋白和 假定的转运蛋白尿酸酶存在于刷状缘和/或 几种肾近曲小管细胞的基底外侧膜 哺乳动物物种。 尿酸盐转运机制将在以下方面进行评估： 未刺激和铜刺激的刷状缘和基底外侧膜 大鼠、狗和兔肾皮质的囊泡，用几种方法制备 技术：自由流动电泳、镁聚集、钙 降水量和 Percoll 梯度。 运输将在 存在和不存在向内定向的电解质梯度，以及 pH 梯度的存在和不存在。 动力学研究将评估 运输的饱和度、抑制和温度依赖性。 这 囊泡中运输的动力学常数将与那些进行比较 通过膜氧化尿酸盐获得。 为了进一步表征 尿酸盐转运机制、同源交换和异质交换现象 将评估扩散。 细胞化学研究将进行 肾和肝组织切片和肾膜囊泡定位 尿酸酶的膜内和细胞内位点。 这种技术是 基于氢之间的耦合氧化-过氧化反应 过氧化物，由尿酸盐和铈的酶促（尿酸酶）氧化产生 氯化物。 该反应的产物过氢氧化铈形成精细的 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。 就物种而言 和肾膜，其中尿酸酶在运输和运输过程中被检测到 细胞化学研究中，尿酸酶将被分离和纯化。 溶解的 膜将进行亲和层析。 净化会 通过凝胶过滤、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 聚焦。 纯化后，将产生多克隆抗体， 抗体对膜囊泡中尿酸盐转运的影响 检查了。 最后，纯化的尿酸酶将被重构为人工尿酸酶 不含尿酸酶（红细胞）的脂质膜或生物膜 鬼）。 尿酸盐转运以及转运和氧化的动力学常数 然后将在重构的系统中进行检查。 本次集体数据 应该为理解尿酸盐的机制提供坚实的基础 运输，也许还有其他内源性和外源性的运输 肾近曲小管内的有机阴离子。