Influence of Urinary Macromolecules on Crystal Aggregation

描述(申请人提供)：大多数肾结石在尿液大分子的影响下形成一水草酸钙(COM)晶体的聚集体，但目前还没有尿液测试可以前瞻性地识别结石形成。我们实验室先前的工作表明，与正常的健康成人相比，结石形成者的尿大分子混合物具有较低的净负电荷。此外，研究还表明，当净电荷接近零时，通过混合等比例的聚阳离子和聚阴离子，或者通过减少聚阴离子(如Tamm-Horsfall蛋白质)上每条链上的负电荷数量，大分子聚集发生，并且大分子聚集形成导致COM晶体聚集。这一建议验证了这样一种假设，即结石形成者可以通过其尿液大分子中存在的低净负电荷来识别，并且这种情况与大分子聚集、晶体聚集和结石形成有关。这项建议将通过以下两个具体目标的研究方案来验证这一假说：(1)验证尿大分子中低净负电荷作为结石形成的疾病标志及其与表型的相关性。对复发性结石患者和正常对照组的尿液大分子进行分离，并用胶体滴定法测定其净负电荷，以证实低净负电荷是结石疾病的标志，并确认我们实验室最近建立的简化的样品处理程序。这些相同的样本还将根据它们对COM晶体成核、聚集和生长的影响进行充分表征，以确定表型队列；可能具有共同尿大分子缺陷的患者亚群，以便于在特定目标2下进行识别。为此目的，也将研究家族性队列，因为他们可能具有单一缺陷的特征。(2)与低净负电荷和成石有关的大分子结构缺陷(S)的鉴定。将使用各种蛋白质组学分析技术，特别是二维凝胶电泳法和MALDI-TOF质谱仪，在石制品中识别导致特定功能缺陷的特定大分子成分(或其组合)。这些方法将确定在尿中观察到的结石形成物大分子混合物中的低净负电荷是否源于1)加成的聚阳离子大分子的存在，2)临界多阴离子大分子的缺乏，或3)临界多阴离子大分子上的净电荷与正常健康成年人尿液中的相比减少，从而确定疾病的原因(S)。我们预计会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：这项提议的成功完成将产生一种诊断方法，特别是胶体滴定，用于前瞻性地识别结石形成者，并允许进行饮食和生活方式咨询以预防或推迟疾病的发生。识别导致在胶体滴定分析中观察到的变化的改变的蛋白质结构将有助于确认我们的结石形成过程模型，为智能设计真正防止疾病复发而不是像目前的治疗方法那样减少疾病复发的新治疗剂提供基础。

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.