Influence of Urinary Macromolecules on Crystal Aggregation

描述（由申请人提供）：大多数肾结石在尿液大分子的影响下形成草酸钙一水合物（COM）晶体的聚集体，但目前，没有尿液检查可以前瞻性地识别结石形成者。我们实验室先前的工作表明，与正常健康成人相比，来自结石患者的尿液大分子混合物具有较低的净负电荷。此外，研究表明，当净电荷接近于零时，大分子聚集发生；通过混合相同比例的聚阳离子和聚阴离子，或通过减少聚阴离子上每条链的负电荷数量，如tam - horsfall蛋白，大分子聚集形成导致COM晶体聚集。这一建议验证了一个假设，即结石患者可以通过尿液大分子中低净负电荷的存在来识别，这种情况与大分子聚集、晶体聚集和结石形成有关。本提案将通过以下两个特定目标的研究方案来验证这一假设：(1)验证尿大分子中低净负电荷作为结石患者的疾病标志物，并与表型相关。我们将对复发性COM型结石患者和正常对照者的尿液大分子进行分离，并通过胶体滴定法对其净负电荷进行表征，以验证低净负电荷是结石疾病的标志，并确认我们实验室最近开发的简化样品处理程序。这些相同的样品也将充分表征其对COM晶体成核，聚集和生长的影响，以定义表型队列；可能在尿大分子中有共同缺陷的患者亚群，以便在Specific Aim 2下进行识别。家族队列也将为此目的进行研究，因为它们可能具有单一缺陷的特征。(2)鉴定与低净负电荷和结石形成相关的大分子结构缺陷。使用各种蛋白质组学分析技术，特别是二维凝胶电泳方法和MALDI-TOF质谱法，将在结石患者中确定导致特定功能缺陷的特定大分子成分（或其组合）。这些方法将确定在结石患者的尿液大分子混合物中观察到的低净负电荷是否来自1)存在额外的聚阳离子大分子，2)缺乏临界聚阴离子大分子，或3)与正常健康成人尿液中相比，临界聚阴离子大分子的净电荷减少，从而确定疾病的原因。我们预计将发现不止一种类型的缺陷，因为许多不同的大分子与肾结石有关。公共卫生相关性：该提案的成功完成将产生一种诊断方法，特别是胶体滴定法，用于前瞻性地识别结石形成者，并允许饮食和生活方式咨询，以预防或延迟疾病的发作。鉴定导致胶体滴定试验中观察到的变化的蛋白质结构改变将有助于确认我们的结石形成过程模型，为智能设计新的治疗药物提供基础，真正预防疾病复发，而不是像目前的治疗方法那样减少疾病复发。

DESCRIPTION (provided by applicant): Most kidney stones form as aggregates of calcium oxalate monohydrate (COM) crystals under the influence of urinary macromolecules, but currently, no urine test can prospectively identify stone formers. Prior work in our laboratory has shown that the urinary macromolecular mixtures from stone formers have low net negative charge, compared to normal healthy adults. Also, it has been shown that macromolecular aggregation occurs when the net charge is near zero; either by mixing equal protions of polycations and polyanions or by reducing the number of negative charges per chain on polyanions, such as Tamm-Horsfall Protein, and that macromolecular aggregate formation causes COM crystal aggregation. This proposal tests the hypothesis that stone formers can be identified by the presence of low net negative charge in their urinary macromolecules, and that this condition is linked to macromolecular aggregation, crystal aggregation, and stone formation. This proposal will test this hypothesis by following a research protocol with 2 Specific Aims: (1) Verification of low net negative charge in urinary macromolecules as the disease marker for stone formers, and correlation with phenotype. Urinary macromolecules from recurrent COM stone formers and normal controls will be isolated and characterized for their net negative charge by Colloidal Titration to verify that low net negative charge is a marker for stone disease, and confirm a simplified sample handling procedure recently developed in our laboratory. These same samples will also be fully characterized with respect to their effect on COM crystal nucleation, aggregation, and growth to define phenotypic cohorts; subsets of patients that are likely to share a common defect in their urinary macromolecules to facilitate identification under Specific Aim 2. Familial cohorts will also be studied for this purpose, since they are likely characterized by a single defect. (2) Identification of the macromolecular structural defect(s) associated with low net negative charge and stone formation. Specific macromolecular components (or combinations thereof) responsible for specific functional defects will be identified in stone formers using various techniques of proteomic analyses, specifically 2 dimensional gel electrophoresis methods and MALDI-TOF mass spectrometry. These methods will identify whether the low net negative charge observed in the urinary macromolecular mixtures of stone formers derives from 1) the presence of additonal polycationic macromolecules, 2) the absence of critical polyanionic macromolecules, or 3) reduction of net charge on critical polyanion macromolecules compared to those in the urine from normal healthy adults, and therefore identify the cause(s) of disease. We anticipate that more than one type of defect will be found, since many different macromolecules are associated with kidney stones. PUBLIC HEALTH RELEVANCE: The successful completion of this proposal will yield a diagnostic method, specifically Colloidal Titration, for prospectively identifying stone formers, and allowing for dietary and lifestyle counselling to prevent or delay onset of disease. Identification of altered protein structures leading to the changes observed in the Colloidal Titration assay will help to confirm our model of the stone forming processes, providing the basis for the intelligent design of new therapeutic agents that truly prevent disease recurrence, rather than reduce it, as in current treatments.