BINDING OF CALCIUM CRYSTALS WITH RENAL CELLS

尽管肾结石是一种常见的疾病，但晶体在肾脏中保留并形成结石的事件顺序尚不清楚。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、尿路阻塞和感染的肾结石，并消除对昂贵的手术（如体外冲击波碎石或手术）的需要。我们之前的研究表明，在培养的肾上皮细胞（用于模拟小管）的顶端表面，草酸钙一水合物（COM）晶体可以在几秒钟内与阴离子、含唾液酸的糖蛋白结合，这表明晶体可以在肾脏中保留的一种机制。鉴别肾细胞表面那些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了证据，证明肾细胞结合COM晶体的能力不是静态的，而是由外源性前列腺素E （PGE）和细胞内环AMP调节的。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，提供了关于培养的肾细胞表面结合草酸钙晶体的受体性质的新信息。包括一种明显新颖的细胞表面蛋白（CSP），该蛋白已被分离并部分测序。我们的具体目标是：1)通过研究PGE对羟基磷灰石（HA）晶体粘附细胞的影响，以及PGE在晶体粘附损伤单层过程中的作用，确定PGE调节肾细胞晶体亲和性的机制；研究PGE释放对肾细胞-晶体相互作用的响应；研究PGE对细胞内cAMP的调节作用；确定PGE对肾细胞蛋白和糖蛋白合成的影响；并确定暴露于PGE后特定晶体受体蛋白的表达。2)鉴别和表征肾上皮细胞表面COM和HA晶体的糖蛋白受体；获得编码CSP - COM晶体受体的全长cDNA克隆；肾细胞CSP表达调控的研究；明确CSP基因和蛋白在肾和非肾组织中的分布；并描述其他尿晶体和不同类型细胞的细胞表面受体。该结果将为控制尿晶体与肾上皮细胞表面粘附的机制提供新的认识。

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.