RENAL CRYSTAL GROWTH INHIBITOR PROTEINS

肾晶体生长抑制蛋白

基本信息

批准号：
2444129
负责人：
JACK G KLEINMAN
金额：
$ 10.03万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 1998-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from the Applicant's Abstract): Urinary calculus formation is believed to be preceded by crystal nucleation in renal tubular fluid, growth and aggregation of these crystals, and subsequent attachment of the crystals of renal epithelium. Urinary proteins probably influence these processes, but the nature of the interaction between urinary proteins and calcium oxalate and phosphate crystals is only partly understood. Osteopontin (OPN) is a glycosylated phosphoprotein that is secreted by renal cortical cells in culture. It is a potent inhibitor of calcium oxalate crystal growth in vitro. OPN has been found in human urine, as well as in renal stone matrix; its role in renal stone formation is unclear. Regulation of OPN production has not been studied in the kidney; however, in other tissues production is regulated by a variety of hormones and growth factors, including PTH and 1,25(OH)2D3. The effect that these hormones have on the protein are complex, however, as they may change the types of post-translational modification that the protein undergoes, in a tissue-specific manner. For example, 1,25(OH)2D3 stimulates production of a non-phosphorylated form of OPN in one target tissue, and phosphorylation may change OPN's function. The investigator has isolated OPN from renal cortical cells in primary culture, and has used antibodies raised to the protein to localize the sites of OPN production in the kidney. This grant request seeks to address several questions related to the function of OPN in the kidney. Thus: 1) using the cell culture system, and assays for crystal growth, the nature of the post-translational modifications introduced into OPN by renal cells will be studied, as well as their effect on crystal growth. The effect of phosphorylation, glycosylation and sulfation will be investigated, as well as the post-translational processing of OPN by cleavage of a peptide from the C-terminal; 2) regulation of OPN production by hormonal stimuli will be studied in vitro and in vivo, using methods to detect hormonal effects on OPN message, as well as protein secretion. Effects on post-translational modifications will also be sought; and 3) a new assay will be characterized, that the applicant hopes will provide a sensitive way to detect effects of urinary macromolecules on crystal nucleation. It is likely that abnormalities of urinary crystal growth inhibitors contribute to the pathogenesis of renal stones in a subset of stone formers. A better understanding of the ways that proteins influence crystallization in fluids that are supersaturated with respect to calcium salts may lead to more effective therapy for these patients, and may also contribute to a better understanding of pathologic calcification occurring in other sites.

描述（改编自申请人的摘要）：尿路结石据认为，形成先于肾中的晶体成核。管状流体，这些晶体的生长和聚集，以及随后的肾上皮晶体的附着。尿蛋白可能会影响这些过程，但相互作用的本质尿蛋白与草酸钙和磷酸盐晶体之间是只理解了一部分。骨桥蛋白 (OPN) 是一种糖基化培养物中肾皮质细胞分泌的磷蛋白。它是体外草酸钙晶体生长的有效抑制剂。开放网络已在人类尿液以及肾结石基质中发现；它的作用肾结石的形成尚不清楚。 OPN 生产监管已尚未在肾脏中进行研究；然而，在其他组织中，生产受多种激素和生长因子的调节，包括 PTH 和 1,25(OH)2D3。这些激素对蛋白质的影响是然而，这很复杂，因为它们可能会改变翻译后的类型蛋白质以组织特异性方式进行的修饰。例如，1,25(OH)2D3 刺激非磷酸化的产生一种靶组织中 OPN 的形式，磷酸化可能会改变 OPN 功能。研究人员从肾皮质细胞中分离出 OPN 在原代培养物中，并使用针对该蛋白质产生的抗体来定位肾脏中 OPN 的产生部位。本次拨款申请旨在解决与 OPN 在网络中的功能相关的几个问题肾。因此：1) 使用细胞培养系统，并进行晶体分析生长，引入的翻译后修饰的性质将研究肾细胞对 OPN 的影响，以及它们对晶体生长。磷酸化、糖基化的影响将研究硫酸化以及翻译后通过从 C 末端切割肽来加工 OPN； 2）将在体外研究激素刺激对 OPN 产生的调节在体内，使用方法检测激素对 OPN 信息的影响，如以及蛋白质的分泌。对翻译后修饰的影响也会被寻求； 3) 一种新的测定方法将被表征，即申请人希望能够提供一种灵敏的方法来检测尿的影响大分子对晶体成核的影响。很可能出现异常尿晶体生长抑制剂有助于发病机制肾结石是结石形成者的一个子集。更好地理解蛋白质影响流体结晶的方式钙盐过饱和可能会导致更有效对这些患者进行治疗，也可能有助于更好地了解其他部位发生的病理性钙化。