BINDING OF CALCIUM CRYSTALS WITH RENAL CELLS

钙晶体与肾细胞的结合

• 批准号: 6349623
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349623
• 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.

虽然肾结石是一种常见的疾病，但对晶体保留在肾脏中并形成结石的顺序知之甚少。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，并消除对昂贵手术（如体外冲击波碎石术或手术）的需要。我们以前的研究表明，草酸钙一水合物（COM）晶体结合在几秒钟内的阴离子，唾液酸的糖蛋白在培养（用于模拟肾小管）的肾上皮细胞的顶面，这表明一种机制，晶体可以保留在肾脏。鉴定肾细胞表面介导晶体结合的分子，以及它们的表达是如何被调节的，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晶体的能力是不是静态的，而是由外源性前列腺素E（PGE）和细胞内的环AMP调节。此外，我们最近的工作利用一种新的实验方案，晶体亲和层析，提供了新的信息的性质上的培养的肾细胞，结合草酸钙晶体，包括一个显然是新的细胞表面蛋白（CSP），已被分离和部分测序的表面受体。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以及其在晶体粘附至损伤单层中的作用来确定PGE调节肾细胞晶体亲和力的机制;研究响应于肾细胞-晶体相互作用的PGE释放;研究响应于PGE的细胞内cAMP的调节;确定PGE对肾细胞蛋白质和糖蛋白合成的影响;并确定暴露于PGE后特异性晶体受体蛋白的表达。2)鉴定和表征COM和HA晶体的肾上皮细胞表面糖蛋白受体;获得编码明显新型CSP COM晶体受体的全长cDNA克隆;研究肾细胞中CSP表达的调节;确定CSP基因和蛋白质在肾和非肾组织中的分布;表征其他尿晶体和不同类型细胞中的细胞表面受体。这一结果将为了解尿结晶在肾上皮细胞表面的粘附机制提供新的认识。