Mechanisms involved in uremic toxin mediated down regulation of ApoA-I expression

描述(由申请人提供)：在过去的几十年里，全球慢性肾脏疾病(CKD)的患病率出现了惊人的上升。动脉粥样硬化性心血管疾病是CKD患者死亡的主要来源。高密度脂蛋白水平降低和高密度脂蛋白介导的反向脂质转运缺陷是CKD人群动脉粥样硬化素质的主要因素。慢性肾脏病患者血清高密度脂蛋白水平降低的一个主要原因是高密度脂蛋白的主要结构和功能成分载脂蛋白A-L的减少。本实验室动物实验表明，尿毒症大鼠血清载脂蛋白A-L降低，肝组织中ApoA-ApoA的表达降低，肝组织ApoA-ApoA-此外，在尿毒症条件下，人肝细胞载脂蛋白A-L的表达、合成和分泌减少已被证实。鉴于大量证据表明载脂蛋白A-L缺乏与心血管事件和死亡风险增加有关，旨在破译慢性肾脏病载脂蛋白A-L缺乏的机制的研究具有至关重要的意义。我们推测慢性肾脏病患者血清中存在的尿毒症毒素是导致载脂蛋白A-L在转录水平下调的原因。利用载脂蛋白A-L启动子-荧光素酶的构建，我们发现暴露于尿毒症的肝细胞中载脂蛋白A-L启动子的活性降低。此外，我们认为这种抑制作用是通过载脂蛋白A-L启动子内的尿毒症反应元件(URE)介导的。因此，在载脂蛋白A-L基因启动子中，一个或多个转录因子与URE结合会导致转录活性降低。此外，我们假设尿毒症对URE的影响发生在肝脏和肠道细胞(这两种细胞负责产生血清载脂蛋白A-L)。利用缺失定位和启动子荧光素酶报告基因的构建，我们希望在载脂蛋白A-L启动子中找到一个Ure。随后，将利用定点突变来确定URE内的顺式调控位点。此外，可能参与载脂蛋白A-L调控的转录因子将通过计算分析来确定。最后，使用凝胶迁移率漂移和超漂移分析，鉴定的调控蛋白与载脂蛋白A-L启动子中的顺式调控元件的结合将得到证实。这些研究将在人类肝脏(HepG2)和肠道(Caco 2)细胞中进行。流行病学观察发现，载脂蛋白A-L降低与心血管死亡率增加有关，而慢性肾脏病是载脂蛋白A-L缺乏的一种状态，这使得该分子成为有吸引力的治疗靶点。一旦确定了导致慢性肾脏病载脂蛋白A-L缺乏的机制，就可以设计出更有效的治疗方法，专门针对纠正这些有害的机制。

DESCRIPTION (provided by applicant): During the past few decades there has been an alarming rise in the prevalence of chronic kidney disease (CKD) worldwide. Atherosclerotic cardiovascular disease is the major source of mortality in CKD patients. Decreased HDL levels and defective HDL-mediated reverse lipid transport are major contributors to the atherogenic diathesis in the CKD population. A major cause of decreased serum HDL level in CKD is the reduction in apoliprotein A-l, the major structural and functional component of HDL. Animal studies in our lab have shown decreased ApoA-l in serum and decreased mRNA expression in liver of uremic rats. Furthermore, decreased ApoA-l expression, synthesis and secretion by human liver cells under uremic conditions has been documented. Given the preponderance of evidence linking ApoA-l deficiency to increased risks of cardiovascular events and mortality, studies aimed at deciphering the mechanisms responsible for the ApoA-l deficiency of CKD are of vital importance. We hypothesize that uremic toxins present in the serum of CKD patients are responsible for down regulation of ApoA-l at the transcriptional level. Using an ApoA-l promoter-luciferase construct, we have shown decreased ApoA-l promoter activity in liver cells exposed to uremia. Furthermore, we believe this inhibitory effect is mediated through a uremia responsive element (URE) within the ApoA-l promoter. Therefore, binding of one or more transcription factors to URE in the promoter of the ApoA-l gene results in decreased transcriptional activity. In addition, we hypothesize that the effect of uremia on the URE occurs in both the liver and intestinal cells (the two cell types responsible for producing serum ApoA-l). Using deletion mapping and a promoter luciferase reporter construct, we hope to identify a URE in the ApoA-l promoter. Subsequently, site-directed mutagenesis will be utilized to identify cis-regulatory sites within the URE. Furthermore, transcription factors potentially involved in ApoA-l regulation will be identified using computational analysis. Finally, using electrophoretic mobility shift and supershift assays, binding of the identified regulatory proteins to the cis-regulatory elements in the ApoA-l promoter will be confirmed. These studies will be carried out in human liver (HepG2) and intestinal (Caco2) cells. The epidemiological observation that decreased ApoA-l is associated with increased cardiovascular mortality along with the fact that CKD is a state of ApoA-l deficiency makes this molecule an attractive target for therapy. Once the mechanisms responsible for ApoA-l deficiency in CKD have been identified, then more effective therapies aimed specifically at correcting those deleterious mechanisms can be devised.