An In Vitro Model for Studying the Role of Acidic Proteins in Randalls Plaque and

描述(申请人提供)：在过去的30年里，美国肾结石的发病率上升了60%以上，而且与肾结石相关的发病率也很高；因此，迫切需要确定结石形成的潜在机制(S)。这项应用的总体目标是建立一个体外模型系统来研究特发性肾结石的两个阶段：1)以兰德尔斑块(RP)形式沉积的磷酸钙(CAP)，以及随后的RP与草酸钙(CaOx)的过度生长，形成复合结石。这一提议的中心假设是，尿液和肾组织中存在的酸性蛋白质可能会诱导一种非经典的结晶过程，称为聚合物诱导液体前体(PILP)过程，该过程可能是结石中发现的许多结构特征的原因。开发体外模型系统的基本原理是，它可以用来筛选和测试从尿液和肾组织中分离出来的物种，看看它们单独或结合在一起如何影响“仿生结石”的形成。这样的模型系统然后可以用来帮助识别用于治疗或预防疾病的方案和/或治疗剂。具体目标1将决定能否开发出第一阶段的仿生快速原型系统。人工基底膜和间质组织将通过PILP过程进行矿化，以确定与RP相关的纳米结构特征，如多层球体和胶原矿化，是否可以在体外复制。特定的目标2将决定是否可以开发第二阶段的仿生结石模型系统，在该系统中，目标1中形成的斑块将作为CaOx过度生长的病灶。具体目标3将确定尿液条件对体内CaOx过度生长的影响，以及对放置在大鼠膀胱中的仿生斑块的影响，并将分析得到的结石的结构特征，以与I目标2中的仿生‘结石’和天然结石进行比较。在AIMS 2和AIMS 3中，将测试来自人类肾组织的天然斑块以进行比较。所有这三个目标的结合将使矿物学‘签名’的发展成为可能，然后这些签名可以被破译，以清楚地指出各种物种对石头形成机制的影响。这项拟议的研究具有创新性，因为它为大分子在结石形成中的作用提供了一个完全不同的视角，其中假设骨桥蛋白是PILP类型过程中的关键角色。此外，首次建立了一套包括特发性肾结石的I期(斑块形成)和II期(结石形成)的体外模型系统。这反过来又提供了测试各种物种对结石形成的影响所需的工具，并在该过程的多个阶段提供了信息。这项工作的意义在于，它将有助于开发用于评估结石形成机制的生物标记物(S)；开发治疗方案和/或治疗剂；以及能够导致预防结石发生和复发的机制理解。 公共卫生相关性：拟议中的研究与公共健康相关，因为它将提供与肾结石形成机制(S)相关的基本知识，据估计，10-15%的美国人口在其有生之年至少会患上一种肾结石，其发病率在过去30年中上升了60%以上。该项目与NIDDK的使命相关，因为它将提供一个体外模型系统，用于研究特发性结石形成的机制(S)，这将导致新的预防和治疗方法，降低医疗保健成本，并提高患有这种疾病的人的生活质量。

DESCRIPTION (provided by applicant): The incidence of nephrolithiasis in the USA rose by more than 60% over the last 3 decades, and there is considerable morbidity associated with kidney stones; therefore, there is a critical need to determine the underlying mechanism(s) involved in stone formation. The overall objective of this application is to develop an in vitro model system to study the two stages of idiopathic nephrolithiasis: 1) the deposition of calcium phosphate (CaP) in the form of Randall's plaque (RP), and 2) the subsequent overgrowth of RP with calcium oxalate (CaOx), forming a composite stone. The central hypothesis of this proposal is that the acidic proteins present in urine and renal tissues may induce a non-classical crystallization process, called the polymer- induced liquid-precursor (PILP) process, and that this process may be responsible for many of the structural features found in stones. The rationale for developing an in vitro model system is that it can be used to sort out and test species isolated from urine and renal tissue to see how they separately, or in combination, influence the 'biomimetic stone' formation. Such a model system can then be used to help identify protocols and/or therapeutic agents for treatment or prevention of the disease. Specific Aim 1 will determine if a biomimetic RP model system of stage I can be developed. Artificial basement membrane and interstitial tissue will be mineralized by the PILP process to determine if nanostructural features relevant to RP, such as multi- laminated spherules and collagen mineralization, can be duplicated in vitro. Specific Aim 2 will determine if a biomimetic stone model system of stage II can be developed, where the plaque developed in Aim 1 will serve as a nidus for overgrowth of CaOx. Specific Aim 3 will determine the influence of urinary conditions on the in vivo overgrowth of CaOx on the biomimetic plaque placed in a rat urinary bladder, and will analyze structural features of the resulting stone for comparison to the biomimetic 'stones' i Aim 2, and to native stones. In Aims 2 and 3, native plaque from human renal tissues will be tested for comparison. The combination of all three aims will enable the development of mineralogical 'signatures', which can then be deciphered to clearly point to the influence of various species on the formation mechanism of a stone. The proposed research is innovative because it provides an entirely different perspective on the role of macromolecules in stone formation, where it is hypothesized that osteopontin is a key player in a PILP type process. In addition, for the first time, an in vitro model system can be developed that encompasses both Stage I (plaque formation) and Stage II (stone formation) of idiopathic nephrolithiasis. This in turn provides the tools needed to test the influence of various species on the formation of stones, with information provided at multiple stages of the process. The significance of this work is that it will enable the development of biomarkers for evaluation of stone formation mechanism(s); development of treatment protocols and/or therapeutic agents; and a mechanistic understanding which can lead to preventive measures against the occurrence and recurrence of stones. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it will provide fundamental knowledge related to the formation mechanism(s) of kidney stones, for which it is estimated that 10-15% of the American population will have developed at least one kidney stone during their lifetime, and the incidence has risen by more than 60% over the past 30 years. The project is relevant to NIDDK's mission because it will provide an in vitro model system for investigating the mechanism(s) involved in idiopathic stone formation, which will lead to new methods of prevention and treatment, lowering the cost of health care and improving quality of life for those afflicted with this pathology.