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

肾结石是一种常见和反复发作的疾病，超过10%的美国人患有肾结石。 但其潜在的病理生理学机制仍不完全清楚。因为肾结石发生了 美国退伍军人主要是在中年，受影响的男性多于女性，是这方面的高危群体 疾病。目前的治疗方法效果有限，结石复发率仅略有下降， 多年来，预防性治疗一直没有取得真正的进展。在以下方面取得突破 显然，理解是必要的。石头是草酸钙(CaOx)晶体的集合体，具有几层 有机物质，主要是尿蛋白，在单个晶体之间，这些蛋白质可能扮演着 在结石形成过程中起着关键作用。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。结石形成与正常尿液蛋白质组的比较 到目前为止，个体未能识别出关键的蛋白质差异，但我们最近的初步数据比较 CaOx结石基质中蛋白质的相对丰度与它们的尿液丰度相比，突出了2个小的 可能是关键蛋白质的亚群，特别是包括高度阴离子和高度 阳离子蛋白质。这样的蛋白质组合表明聚阴离子-聚阳离子聚集体在 触发结石形成，因为我们之前已经证明，这种聚集体刺激CaOx晶体 成核和聚集。因此，我们假设，蛋白质的相对丰度对 在CaOx石材基质中的成石过程将比在新鲜情况下看到的更快 同一患者排出的尿液，特别是包括高度阴离子和阳离子蛋白。 虽然拟议的实验不会阐明这些特定蛋白质成为 富含CaOx结石基质，证实这种特定的蛋白质混合物存在于大量 在以机械学为基础的研究探索蛋白质之间的联系之前，结石是必要的 凝聚和石头的形成是相关的。这项拟议的工作将识别和表征关键的蛋白质 石块的形成有两个特定的目的。在特定的目标1中，我们将确认在石头中富含关键蛋白质 用定量质谱法测定石材基质中蛋白质的相对丰度 40例CaOx结石患者的尿液标本。在具体目标2中，相对丰度 在稳定的健康条件下尿液中关键蛋白质的含量将在我们的结石前小组和 匹配正常人群以测试结石患者尿液中关键蛋白丰度的预期增加。 免疫印迹技术将被用来避开与定量质谱学相关的问题 低丰度蛋白质的特性。CaOx结石相关关键蛋白的鉴定 形成代表了石头研究中的一种新范式，可以指导未来对蛋白质的机制研究 结石形成的诱因，并有望启发新的治疗策略。

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.