Urolithiasis: Oxalate Interactions with the Renal Cells

描述(由申请人提供)：尿石症是一种多因素疾病，不太可能是单一因素导致这种疾病的整个谱系。尽管如此，草酸盐和草酸钙晶体(COM晶体)与肾上皮细胞的相互作用以及随之而来的细胞反应是这种疾病的重要但知之甚少的主题。在肾上皮细胞中介导草酸和共晶体效应的信号转导机制是肾结石的关键决定因素，也是我们研究的主要焦点。在五年的资助期间，我们证明了草酸盐和复合晶体激活了p38 MAP激酶通路和核因子-kB转录因子。我们还观察到，人肾上皮细胞暴露于草酸盐和COM晶体会导致IL-6的产生增加，这是一种促炎细胞因子，表明炎症信号的产生。然而，这些行动的机制仍不清楚。建议的研究将评估p38 MAP激酶介导的NF-kB信号在草酸和钴晶体的影响以及调节细胞反应中发挥核心作用的假说。这一假说是由我们的观察推动的，即草酸和钴晶体暴露导致肾上皮细胞中p38蛋白激酶和核因子-kB的快速而强劲的激活。然而，草酸盐和COM晶体引起NF-kB激活的确切序列/S尚不清楚。本研究有四个目的：(1)阐明草酸/共晶体诱导的核因子-kB途径激活的分子机制；(2)探讨核因子-kB通路在草酸/共晶体调节肾上皮细胞IL-6水平中的作用；(3)评价草酸/共晶体调节肾上皮细胞IL-6水平的分子机制，特别是p38 MAP激酶通路和核因子-kB转录因子激活的作用；以及(4)在高草酸肾结石动物模型中评价p38MAP信号转导和核因子-kB激活的作用。了解介导草酸和COM晶体对肾上皮细胞影响的特定信号过程将有助于我们制定策略，抑制这些药物的病理效应，防止晶体滞留和肾结石形成。我们预计，结合我们的初步数据，拟议的研究将确定p38 MAP激酶通路和NF-kB作为预防晶体滞留的机制靶点/S。作为一种翻译方法，这些研究的长期目标是定义和确定p38蛋白激酶和核因子-kB抑制剂在预防和治疗人类肾结石疾病方面的有效性。PUBLIC健康相关性：肾结石疾病是一种多因素疾病，不太可能由单一因素导致结石患者的所有亚型。然而，结石疾病发生的一个重要因素是结晶物质在肾小管中的滞留。因此，了解草酸和COM晶体与肾小管细胞相互作用后的信号机制对于我们理解与这种疾病相关的分子事件是至关重要的。我们已经获得的证据表明，在体外细胞培养系统中，草酸和COM晶体相互作用激活了肾细胞中p38MAPK和NF-kB转录因子。这项研究的目的是利用原代培养的人肾细胞进一步确定这一途径，并检验这一假设，即p38 MAP激酶途径介导的NF-kB转录因子通过调节基因表达在草酸和COM晶体的作用中发挥核心作用。我们还试图将这些观察扩展到目前的结石病实验动物模型中。此外，我们将测试一种p38 MAP激酶和核因子-kB的药理抑制剂在预防草酸和共晶诱导的实验动物病理生理事件中的可行性。综上所述，我们的研究重点是确定p38 MAP激酶通路和核因子-kB作为干预尿石症的分子靶点。因此，这项拟议的研究完全满足计划公告中提出的要求。

DESCRIPTION (provided by applicant): Urolithiasis is a multi-factorial disease and it is unlikely that a single factor will be responsible for the entire spectrum of this disorder. Nonetheless, oxalate and calcium-oxalate crystal (COM-crystal) interactions with renal epithelial cells, and the cellular responses that follow are important, but poorly understood subjects in this disorder. Signal transduction mechanisms that mediate the effects of oxalate and COM-crystals in renal epithelial cells are critical determinants in nephrolithiasis and have been the primary focus of our investigation. During the five year funding period, we demonstrated that oxalate and COM-crystals activate p38 MAP kinase pathway and NF-kB transcription factor. We also observed that exposure of human renal epithelial cells to oxalate as well as COM-crystals results in increased production of IL-6, a pro-inflammatory cytokine, indicating generation of inflammatory signals. However, the mechanism of these actions remains unclear. The proposed studies will evaluate the hypothesis that p38 MAP kinase mediated NF-kB signaling plays central role in mediating the effects of oxalate and COM-crystals, and in modulating the cellular responses. This hypothesis is driven by our observations that oxalate and COM-crystal exposure caused a rapid and robust activation of p38 MAP kinase and NF-kB in renal epithelial cells. However, the precise sequence/s through which oxalate and COM-crystals cause NF-kB activation is not understood. The present studies have four objectives: (1) to elucidate the molecular mechanisms involved in oxalate/COM-crystal induced activation of NF-kB pathway; (2) to investigate the roles played by NF-kB pathway in mediating the effects of oxalate/ COM- crystals; (3) to evaluate the molecular mechanisms by which oxalate/ COM-crystals regulates IL-6 levels in renal epithelial cells with special emphasis on the role of p38 MAP kinase pathway and NF-kB transcription factor activation; and (4) to evaluate the role of p38 MAP kinase signaling and NF-kB activation in vivo animal models of hyperoxaluric nephrolithiasis. Understanding the specific signaling processes that mediate the effects of oxalate and COM crystals in renal epithelial cells will help us develop strategies to inhibit the pathologic effects of these agents and prevent crystal retention and renal stone formation. We anticipate that proposed studies together with our preliminary data will identify p38 MAP kinase pathway and NF-kB as a mechanism-based target/s for the prevention of crystal retention. As a translational approach, the long-range goal of these studies is to define and establish the usefulness of p38 MAP kinase and NF-kB inhibitors for the prevention and therapy of human kidney stone disease.PUBLIC HEALTH RELEVANCE: Kidney stone disease is a multifactorial disorder and it is unlikely that a single factor will be responsible for all the subsets of stone patients. Nonetheless, one important factor in genesis of stone disease is retention of crystalline materials in the renal tubules. Therefore, understanding the signaling mechanisms that mediate the effects of oxalate and COM-crystals following their interaction with renal tubular cells are essential in our understanding the molecular events associated with this disorder. We have obtained evidence indicating activation of p38 MAP kinase and NF-kB transcription factor in renal cells upon oxalate and COM-crystal interaction in vitro cell culture systems. The goal of the proposed studies is to further define this pathway using primary cultures of human kidney cells and to test the hypothesis that, "p38 MAP kinase pathway mediated NF-kB transcription factor plays a central role in mediating the effects of oxalate and COM- crystals by regulating gene expression". We also seek to extend these observations in current experimental animal models of the stone disease. Moreover, we will test feasibility of a pharmacological inhibitors of p38 MAP kinase and NF-kB in preventing oxalate and COM-crystal induced pathophysiological events in the experimental animals. Taken together our studies focus on characterizing p38 MAP kinase pathway and NF-kB as a molecular targets for intervention in urolithiasis. Thus this proposed study completely satisfies the requirements set forth in the Program Announcement.