IDENTIFYING KEY PROTEINS IN CALCIUM OXALATE KIDNEY STONE FORMATION USING STONE MATRIX PROTEOMICS

肾结石是一种常见的和复发性疾病，影响超过10%的美国人， 但其潜在的病理生理学仍不完全清楚。因为肾结石的发生 主要发生在中年，男性多于女性，美国退伍军人是这种情况的高危人群。 疾病目前的治疗方法有效性有限，结石复发率仅适度降低， 并且多年来在预防性治疗方面没有真实的进展。的突破口 理解显然是必要的。结石形成为草酸钙（CaOx）晶体的聚集体， 有机物质，主要是尿蛋白，在各个晶体之间，这些蛋白质可能起着重要的作用。 在结石形成中起重要作用。蛋白质在结石形成中的作用被大量的 结石基质中发现的尿蛋白。结石患者与正常人尿蛋白质组的比较 迄今为止，个体未能确定关键的蛋白质差异，但我们最近的初步数据比较 CaOx结石基质中蛋白质相对于其尿丰度的相对丰度突出了2个小的 可能是关键蛋白质的蛋白质子集，特别是包括高阴离子和高阴离子蛋白质 阳离子蛋白质蛋白质的这种组合表明聚阴离子-聚阳离子聚集体在 触发结石形成，因为我们以前已经表明，这种聚集体刺激CaOx晶体 成核和聚集。因此，我们假设，关键蛋白质的相对丰度， 在CaOx结石基质中的结石形成过程将比在新鲜的 来自同一患者的排泄尿，特别是包括高度阴离子和阳离子蛋白质。 虽然所提出的实验将不会阐明这些特定蛋白质成为 富含CaOx结石基质，证实了这种特异性蛋白质混合物在大量 在基于机制的研究探索我们所建议的蛋白质之间的联系之前， 聚集和结石形成是相关的。拟议的工作将确定和表征关键蛋白质， 石的形成有两个具体的目的。在具体目标1中，我们将确认结石中关键蛋白质的富集 基质使用定量质谱法测定结石基质中蛋白质的相对丰度 和40例CaOx结石形成患者的尿样。在具体目标2中， 在稳定的健康状况下，尿液中的关键蛋白质将在我们的结石形成小组和 匹配正常人群以测试结石形成者尿液中关键蛋白质丰度的预期增加。 免疫印迹技术将用于规避与定量质谱相关的问题 低丰度蛋白质的表征。CaOx结石相关关键蛋白的鉴定 蛋白质的形成代表了结石研究的一个新范式，可以指导未来蛋白质的机理研究 触发结石形成，并有望激发新的治疗策略。

Kidney stones are a common and recurrent disease affecting more than 10% of Americans in their lifetime, but the underlying pathophysiology remains incompletely understood. Since kidney stones occur predominantly during middle age and affect more men than women, US veterans are a high risk group for this disease. Current therapies have limited effectiveness, with only a modest reduction in stone recurrence rates, and there has been no real advancement in preventive therapy for many years. A breakthrough in understanding is clearly needed. Stones form as aggregates of calcium oxalate (CaOx) crystals with layers of organic material, principally urinary proteins, between the individual crystals, and these proteins likely play a critical role in stone formation. The role of proteins in stone formation is obscured by the large number of urinary proteins found within stone matrix. Comparison of urine proteomes from stone forming and normal individuals has failed to identify critical protein differences to date, but our recent Preliminary Data comparing the relative abundances in proteins in CaOx stone matrix to their urinary abundances has highlighted 2 small subsets of proteins that are likely to be key proteins, specifically including both highly anionic and highly cationic proteins. Such a combination of proteins suggests a role for polyanion-polycation aggregates in triggering stone formation, because we have previously shown that such aggregates stimulate CaOx crystal nucleation and aggregation. Therefore, we hypothesize that the relative abundance of proteins critical to the stone forming process will be increased in CaOx stone matrix compared to that seen in freshly voided urine from the same patient, specifically including both highly anionic and cationic proteins. While the proposed experiments will not elucidate the mechanism whereby these specific proteins become enriched in CaOx stone matrix, confirming the presence of this specific protein mixture in a large number of stones is necessary before mechanistic based studies exploring our suggested link between protein aggregation and stone formation are relevant. The proposed work will identify and characterize key proteins in stone formation in two Specific Aims. In Specific Aim 1, we will confirm enrichment of key proteins in stone matrix using quantitative mass spectrometry to determine the relative abundance of proteins in stone matrix and urine samples obtained from 40 CaOx stone forming patients. In Specific Aim 2, the relative abundances of key proteins in urine under stable health conditions will be compared between our stone former panel and a matching normal population to test for the expected increase in key protein abundance in stone former urine. Immunoblot techniques will be used to circumvent problems associated with quantitative mass spectrometry characterization of low abundance proteins. Identification of key proteins associated with CaOx stone formation represents a new paradigm in stone research that can direct future mechanistic studies on protein triggers of stone formation, and hopefully inspire new therapeutic strategies.