BINDING OF CALCIUM CRYSTALS WITH RENAL CELLS

虽然肾结石是一种常见的疾病，但人们对晶体滞留在肾脏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，以防止引起疼痛、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。我们以前的研究表明，一水草酸钙(COM)晶体在几秒钟内就能与培养的肾上皮细胞顶端表面的阴离子、含唾液酸的糖蛋白结合(用来模拟肾小管)，这表明晶体可以保留在肾脏中的一种机制。鉴定肾细胞表面那些介导晶体结合的分子，以及它们的表达是如何被调控的，将极大地增加对肾结石发病机制的了解。我们的初步研究表明，肾细胞结合COM晶体的能力不是静态的，而是受外源性前列腺素E(PGE)和细胞内环磷酸腺苷(CAMP)的调节。此外，我们最近利用一种新的实验方案-晶体亲和层析的工作，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，包括一个明显新的细胞表面蛋白(CSP)，它已经被分离并部分测序。我们的具体目标是：1)通过研究PGE对羟基磷灰石(HA)晶体与细胞的黏附的影响及其在晶体与损伤单层黏附过程中的作用，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-晶体相互作用的响应；研究PGE对细胞内cAMP的调节；确定PGE对肾细胞蛋白质和糖蛋白合成的影响；以及明确PGE暴露后特定晶体受体蛋白的表达。2)鉴定和鉴定肾上皮细胞表面COM和HA晶体糖蛋白受体(S)；获得编码一种新的CSP COM晶体受体的全长c DNA克隆；研究CSP在肾脏细胞中的表达调控；确定CSP基因和蛋白在肾组织和非肾组织中的分布；以及鉴定其他尿晶体和不同类型细胞的细胞表面受体。这一结果将为控制尿晶黏附于肾上皮细胞表面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.