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

尿毒症毒素介导 ApoA-I 表达下调的机制

基本信息

批准号：
8122373
负责人：
Hamid Moradi
金额：
$ 6.3万
依托单位：
SOUTHERN CALIFORNIA INST FOR RES/EDUC
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8122373
关键词：
Animals Antibodies Apolipoprotein A-I Apolipoproteins A Area Atherosclerosis Binding Biological Assay Cardiovascular system Cell Culture Techniques Cell Line Cells Characteristics Chronic Kidney Failure Complement Computer Analysis Consensus DNA DNA Sequence Dialysis patients Dialysis procedure Disease susceptibility Down-Regulation Electrophoretic Mobility Shift Assay Elements Enhancers Epidemiology Epithelial Cells Event Exhibits Fractionation Gene Expression Genes Genetic Transcription Hepatic Hepatocyte High Density Lipoproteins Human In Vitro Indium Inflammation Inflammatory Intestines Ion Exchange Link Liver Luciferases Maps Measurement Measures Mediating Methods Modeling Molecular Nuclear Nuclear Protein Nucleic Acid Regulatory Sequences Nucleotides Oligonucleotides Oxidative Stress Pathway interactions Patients Phosphotransferases Play Population Prevalence Prevention Process Production Promoter Regions Protein Binding Proteins Rattus Regulation Regulatory Element Reporter Reporting Response Elements Role SP600125 Serum Site Site-Directed Mutagenesis Small Interfering RNA Source System TNF gene Testing Tissues Toxin Transcriptional Regulation Ultrafiltration Uremia base cardiovascular risk factor cell type cytokine effective therapy genetic regulatory protein hepatoma cell interest kinase inhibitor knock-down lipid transport mRNA Expression mortality promoter pyrrolidine dithiocarbamate research study response reverse cholesterol transport transcription factor

项目摘要

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.

描述（由申请人提供）：在过去的几十年中，全球慢性肾脏疾病（CKD）的患病率呈惊人的上升趋势。动脉粥样硬化性心血管疾病是CKD患者死亡的主要原因。HDL水平降低和HDL介导的脂质反向转运缺陷是CKD人群致动脉粥样硬化素质的主要因素。CKD中血清HDL水平降低的主要原因是载脂蛋白A-I（HDL的主要结构和功能组分）的减少。本实验室的动物实验表明，尿毒症大鼠血清中ApoA-1水平降低，肝脏中ApoA-1 mRNA表达降低。此外，已经记录了在尿毒症条件下人肝细胞的ApoA-I表达、合成和分泌减少。鉴于将ApoA-I缺乏与心血管事件和死亡率的风险增加联系起来的证据的优势，旨在破译导致CKD的ApoA-I缺乏的机制的研究是至关重要的。我们假设存在于CKD患者血清中的尿毒症毒素负责在转录水平下调ApoA-I。使用ApoA-I启动子-荧光素酶构建体，我们已经显示暴露于尿毒症的肝细胞中ApoA-I启动子活性降低。此外，我们认为这种抑制作用是通过ApoA-I启动子内的尿毒症反应元件（URE）介导的。因此，一种或多种转录因子与ApoA-I基因启动子中的URE的结合导致转录活性降低。此外，我们假设尿毒症对URE的影响发生在肝细胞和肠细胞（负责产生血清ApoA-I的两种细胞类型）两者中。使用缺失作图和启动子荧光素酶报告构建体，我们希望鉴定ApoA-I启动子中的URE。随后，将利用定点诱变来鉴定URE内的顺式调节位点。此外，将使用计算分析来鉴定可能涉及ApoA-I调节的转录因子。最后，使用电泳迁移率变动和超变动测定，将确认所鉴定的调节蛋白与ApoA-l启动子中顺式调节元件的结合。这些研究将在人肝（HepG 2）和肠（Caco 2）细胞中进行。ApoA-I降低与心血管死亡率增加相关的流行病学观察结果沿着CKD是ApoA-I缺乏的状态这一事实使得该分子成为有吸引力的治疗靶标。一旦确定了导致CKD中ApoA-I缺乏的机制，则可以设计出更有效的治疗方法，特别是针对纠正这些有害机制。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Amelioration of nephropathy with apoA-1 mimetic peptide in apoE-deficient mice.

DOI：
10.1093/ndt/gfq274
发表时间：
2010-11
期刊：
Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association
影响因子：
0
作者：
N. Vaziri;H. J. Kim;H. Moradi;Farbod Farmand;K. Navab;M. Navab;S. Hama;A. Fogelman;Y. Quiroz;B. Rodriguez-Iturbe
通讯作者：
N. Vaziri;H. J. Kim;H. Moradi;Farbod Farmand;K. Navab;M. Navab;S. Hama;A. Fogelman;Y. Quiroz;B. Rodriguez-Iturbe

Skeletal muscle mitochondrial depletion and dysfunction in chronic kidney disease.