BINDING OF CALCIUM CRYSTALS WITH RENAL CELLS

钙晶体与肾细胞的结合

• 批准号: 6600908
• 负责人: FREDERICK Gary TOBACK
• 金额: $ 10.78万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6600908
• 关键词: calcium; clinical research; cyclic AMP; human subject; hydroxyapatites; laboratory rat; membrane proteins; nephrolithiasis; oxalates; pathologic process; prostaglandin E; receptor

Although nephrolithiasis is a common condition, the sequence of events by which crystals are retained in the kidney and form stones is poorly understood. Therefore, additional knowledge is required to identify susceptible patients and formulate new therapeutic strategies to prevent kidney stones which cause pain, hematuria, urinary tract obstruction and infection, and to eliminate the need for expensive procedures such as extracorporeal shock wave lithotripsy or surgery. Our previous studies demonstrate that calcium oxalate monohydrate (COM) crystals bind within seconds to anionic, sialic acid-containing glycoproteins on the apical surface of kidney epithelial cells in culture (employed to model the tubule), suggesting one mechanism whereby crystals could be retained in the kidney. Identification of those molecules on the renal cell surface that mediate crystal binding, and how their expression is modulated, will greatly increase insight into the pathogenesis of nephrolithiasis. Our Preliminary Studies provide evidence that the capacity of renal cells to bind COM crystals is not static, but is regulated by exogenous prostaglandin E (PGE) and intracellular cyclic AMP. Additionally, our recent work utilizing a novel experimental protocol, crystal-affinity chromatography, provides new information about the nature of receptors on the surface of cultured renal cells that bind calcium oxalate crystals, including one apparently novel Cell Surface Protein (CSP) that has been isolated and partially sequenced. Our Specific Aims are to: 1) Define mechanisms by which PGE modulates renal cell crystal affinity by investigating its effect on adhesion of hydroxyapatite (HA) crystals to cells, and its role during crystal adhesion to wounded monolayers; study PGE release in response to renal cell-crystal interactions; study regulation of intracellular cAMP in response to PGE; define the effect of PGE on renal cell protein and glycoprotein synthesis; and define the expression of specific crystal receptor proteins after exposure to PGE. 2) Identify and characterize renal epithelial cell surface glycoprotein receptor(s) for COM and HA crystals; obtain a full-length cDNA clone encoding an apparently novel CSP COM crystal receptor; study regulation of CSP expression in renal cells; define CSP gene and protein distribution in renal and non-renal tissues; and characterize cell surface receptors for other urinary crystals, and in different types of cells. The results will provide new understanding of the mechanisms that control adhesion of urinary crystals to the surface of kidney epithelial cells.

尽管肾结石是一种常见疾病，但人们对晶体滞留在肾脏并形成结石的事件顺序知之甚少。因此，需要更多的知识来识别易感患者并制定新的治疗策略，以预防引起疼痛、血尿、尿路梗阻和感染的肾结石，并消除昂贵的手术（如体外冲击波碎石术或手术）的需要。我们之前的研究表明，一水草酸钙（COM）晶体在几秒钟内与培养物（用于模拟肾小管）肾上皮细胞顶端表面的阴离子、含唾液酸糖蛋白结合，这表明晶体可以保留在肾脏中的一种机制。鉴定肾细胞表面介导晶体结合的分子以及它们的表达如何调节，将大大增加对肾结石发病机制的了解。我们的初步研究提供的证据表明，肾细胞结合 COM 晶体的能力不是静态的，而是受到外源性前列腺素 E (PGE) 和细胞内环 AMP 的调节。此外，我们最近的工作利用一种新颖的实验方案——晶体亲和色谱法，提供了有关培养肾细胞表面结合草酸钙晶体的受体性质的新信息，其中包括一种已分离并部分测序的明显新颖的细胞表面蛋白（CSP）。我们的具体目标是： 1) 通过研究 PGE 对羟基磷灰石 (HA) 晶体与细胞粘附的影响，及其在晶体粘附到受伤单层过程中的作用，确定 PGE 调节肾细胞晶体亲和力的机制；研究 PGE 释放对肾细胞-晶体相互作用的反应；研究细胞内 cAMP 响应 PGE 的调节；定义 PGE 对肾细胞蛋白质和糖蛋白合成的影响；并定义暴露于 PGE 后特定晶体受体蛋白的表达。 2) 鉴定和表征肾上皮细胞表面糖蛋白受体的 COM 和 HA 晶体；获得编码明显新颖的CSP COM晶体受体的全长cDNA克隆；研究肾细胞中CSP表达的调节；定义肾组织和非肾组织中 CSP 基因和蛋白质的分布；并表征其他尿晶体和不同类型细胞的细胞表面受体。这些结果将为控制尿晶体粘附到肾上皮细胞表面的机制提供新的认识。