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，不太可能由单一因素导致整个疾病谱系。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重要的，但对这种疾病的研究知之甚少。介导草酸盐和com晶体在肾上皮细胞中的作用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。在五年的资助期内，我们证明草酸盐和com晶体激活p38 MAP激酶途径和NF-kB转录因子。我们还观察到，暴露于草酸盐和com晶体的人肾上皮细胞会导致IL-6（一种促炎细胞因子）的产生增加，这表明炎症信号的产生。然而，这些作用的机制尚不清楚。这些研究将评估p38 MAP激酶介导的NF-kB信号在草酸盐和com晶体的介导作用以及调节细胞反应中发挥核心作用的假设。我们观察到草酸盐和com晶体暴露导致肾上皮细胞中p38 MAP激酶和NF-kB的快速和强烈激活，这一假设得到了证实。然而，草酸盐和com晶体引起NF-kB活化的确切顺序尚不清楚。目前的研究有四个目标：(1)阐明草酸盐/ com晶体诱导NF-kB通路激活的分子机制；(2)研究NF-kB通路在草酸盐/ COM-晶体作用中的介导作用；(3)评估草酸/ com晶体调控肾上皮细胞IL-6水平的分子机制，重点研究p38 MAP激酶途径和NF-kB转录因子激活的作用；(4)评估p38 MAP激酶信号通路和NF-kB激活在高血氧性肾结石动物体内模型中的作用。了解介导草酸盐和COM晶体在肾上皮细胞中的作用的特定信号传导过程将有助于我们制定抑制这些药物病理作用的策略，防止晶体保留和肾结石形成。我们预计，拟议的研究以及我们的初步数据将确定p38 MAP激酶途径和NF-kB作为预防晶体保留的基于机制的靶标。作为一种转化方法，这些研究的长期目标是确定和建立p38 MAP激酶和NF-kB抑制剂在预防和治疗人类肾结石疾病中的有效性。公共卫生相关性：肾结石疾病是一种多因素疾病，单一因素不太可能导致所有结石患者的亚群。尽管如此，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，了解草酸盐和com晶体与肾小管细胞相互作用后介导其作用的信号机制，对于我们理解与这种疾病相关的分子事件至关重要。我们已经获得证据表明，在体外细胞培养系统中，草酸盐和com晶体相互作用可激活肾细胞中的p38 MAP激酶和NF-kB转录因子。拟建研究的目标是利用人肾细胞原代培养进一步确定这一途径，并验证“p38 MAP激酶途径介导的NF-kB转录因子通过调节基因表达在介导草酸盐和COM-晶体的作用中起核心作用”的假设。我们还寻求在目前的石病实验动物模型中扩展这些观察结果。此外，我们将在实验动物中测试p38 MAP激酶和NF-kB的药理学抑制剂在预防草酸盐和com晶体诱导的病理生理事件中的可行性。综上所述，我们的研究重点是表征p38 MAP激酶途径和NF-kB作为干预尿石症的分子靶点。因此，本研究方案完全满足项目公告中提出的要求。

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.