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

描述（由申请人提供）：在过去30年中，美国肾结石的发病率上升了60%以上，并且存在与肾结石相关的相当多的发病率;因此，迫切需要确定结石形成的潜在机制。本申请的总体目标是开发一种体外模型系统，以研究特发性肾结石的两个阶段：1）磷酸钙（CaP）以Randall斑块（RP）的形式沉积，以及2）随后RP与草酸钙（CaOx）过度生长，形成复合结石。该提议的中心假设是，存在于尿液和肾组织中的酸性蛋白质可能诱导非经典结晶过程，称为聚合物诱导的液体前体（PILP）过程，并且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，它可用于筛选和测试从尿液和肾组织中分离的物种，以了解它们如何单独或组合影响“仿生结石”的形成。这样的模型系统然后可以用于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将确定是否可以开发阶段I的仿生RP模型系统。人工基底膜和间质组织将通过PILP过程矿化，以确定与RP相关的纳米结构特征，例如多层小球和胶原矿化，是否可以在体外复制。具体目标2将确定是否可以开发阶段II的仿生结石模型系统，其中目标1中开发的斑块将用作CaOx过度生长的病灶。具体目标3将确定泌尿条件对放置在大鼠膀胱中的仿生斑块上的CaOx的体内过度生长的影响，并将分析所得结石的结构特征以与目标2中的仿生“结石”和天然结石进行比较。在目标2和3中，将对来自人肾组织的天然斑块进行检测以进行比较。所有这三个目标的结合将使矿物学“签名”的发展，然后可以解释清楚地指出各种物种对石头形成机制的影响。拟议的研究是创新的，因为它提供了一个完全不同的视角，大分子在结石形成中的作用，假设骨桥蛋白是PILP型过程中的关键角色。此外，第一次，在体外模型系统可以开发，包括第一阶段（斑块形成）和第二阶段（结石形成）的特发性肾结石。这反过来又提供了测试各种物种对石头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，它将能够开发用于评价结石形成机制的生物标志物;开发治疗方案和/或治疗剂;以及可以导致针对结石发生和复发的预防措施的机制理解。

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.