Influence of Urinary Macromolecules on Crystal Aggregation

尿液大分子对晶体聚集的影响

• 批准号: 7730851
• 负责人: Jeffrey A Wesson
• 金额: $ 23.27万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-17 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730851
• 关键词: Adult; Agreement; Biological Assay; Calcium Oxalate; Calcium Oxalate Monohydrate; Calculi; Characteristics; Charge; Chemicals; Counseling; Crystal Formation; Crystallization; Defect; Development; Diagnostic Procedure; Disease; Disease Marker; Event; Exhibits; Family member; Funding; General Population; Genes; Genetic; Growth; In Vitro; Inheritance Patterns; Kidney Calculi; Laboratories; Lead; Life Style; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular Structure; Nature; Onset of illness; Pathway interactions; Patients; Phase; Phenotype; Play; Population; Post-Translational Protein Processing; Procedures; Process; Proteins; Proteomics; Protocols documentation; Publications; Recurrence; Regulation; Research; Risk; Role; Sampling; Screening procedure; Site; Techniques; Testing; Therapeutic Agents; Titrations; Two-Dimensional Gel Electrophoresis; UMOD gene; Urine; Work; base; cohort; design; glycosylation; macromolecule; novel therapeutics; polyanion; polycation; prevent; promoter; protein structure; public health relevance; urinary

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.

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为一水草酸钙（COM）晶体的聚集体，但目前，没有尿液检测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，结石形成者的尿大分子混合物具有较低的净负电荷。此外，已经表明，当净电荷接近零时，通过混合等量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，如Tamm-Horsfall蛋白质，发生大分子聚集，并且大分子聚集体的形成导致COM晶体聚集。该提案检验了结石形成者可以通过其尿大分子中存在低净负电荷来识别的假设，并且这种情况与大分子聚集、晶体聚集和结石形成有关。本提案将通过遵循具有2个特定目的的研究方案来验证这一假设：（1）验证尿大分子中的低净负电荷作为结石形成者的疾病标志物，以及与表型的相关性。将分离复发性COM结石形成者和正常对照的尿大分子，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物，并确认我们实验室最近开发的简化样本处理程序。还将充分表征这些相同样本对COM晶体成核、聚集和生长的影响，以定义表型队列;可能在其尿液大分子中具有共同缺陷的患者子集，以便于根据特定目标2进行识别。也将为此目的研究家族性队列，因为它们可能以单一缺陷为特征。(2)鉴定与低净负电荷和结石形成相关的大分子结构缺陷。将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，在结石形成者中鉴定导致特定功能缺陷的特定大分子组分（或其组合）。这些方法将鉴定在结石形成者的尿大分子混合物中观察到的低净负电荷是否来源于1）额外聚阳离子大分子的存在，2）关键聚阴离子大分子的缺乏，或3）与正常健康成人的尿液中的那些相比，关键聚阴离子大分子上的净电荷减少，并因此鉴定疾病的原因。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：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，为真正预防疾病复发的新治疗药物的智能设计提供基础，而不是像目前的治疗方法那样减少疾病复发。