RENAL CRYSTAL GROWTH INHIBITOR PROTEINS

描述（改编自申请人摘要）：尿路结石 据信，在肾中晶体成核之前， 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，但相互作用的性质 草酸钙和磷酸盐晶体之间的联系 只是部分理解。骨桥蛋白（OPN）是一种糖基化的 在培养中由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。OPN 在人类尿液以及肾结石基质中发现;其作用 在肾结石形成中的作用尚不清楚。对OPN生产的监管 尚未在肾脏中进行研究;然而，在其他组织中， 由多种激素和生长因子调节，包括PTH和 1，25（OH）2D3。这些激素对蛋白质的影响是 复杂，然而，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经历的修饰。 例如，1，25（OH）2D 3刺激非磷酸化的 一种靶组织中的OPN形式，磷酸化可以改变OPN的 功能研究者从肾皮质细胞中分离出OPN 在原代培养中，并使用针对蛋白质的抗体， 定位肾脏中OPN产生的位点。这份补助金申请 寻求解决与OPN在联合国中的职能有关的几个问题， 肾因此：1）使用细胞培养系统，并测定晶体 生长，所引入的翻译后修饰的性质 将研究肾细胞对OPN的作用，以及它们对 晶体生长磷酸化、糖基化和 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骨桥蛋白产生的调节 以及在体内，使用检测激素对OPN信息的影响的方法， 以及蛋白质分泌。对翻译后修饰的影响 也将寻求;和3）一种新的测定将被表征， 申请人希望能提供一种灵敏的方法来检测尿 大分子对晶体成核的影响。很有可能异常 尿晶体生长抑制剂的作用 结石形成者中的肾结石。更好地理解 蛋白质影响液体结晶的方式 相对于钙盐过饱和可导致更有效的 治疗这些患者，也可能有助于更好地 了解其他部位发生的病理性钙化。

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.