Gene expression and anemia of chronic renal failure

描述(由申请人提供)： 这份修订后的申请书描述了一项严格的教育和调查培训计划，以促进申请者发展为慢性肾功能衰竭(CRF)贫血领域的独立内科科学家。申请者索尔·努尔科博士是克利夫兰诊所的一名肾科医生，对CRF及其后果有主要的临床兴趣。培训将由赞助商保罗·福克斯博士监督。他在系里的实验室。细胞生物学研究调节铁稳态的细胞和分子机制，以及导致贫血的缺陷。项目联合发起人琳达·格雷厄姆博士是一名血管外科医生，她将主要负责使用小鼠CRF外科模型进行研究的培训和表现。 贫血是晚期肾脏疾病的一种常见且令人衰弱的后果。重组人促红细胞生成素(EPO)疗法的问世极大地改变了肾病学家在这一人群中治疗贫血的方式，并为大多数患者带来了更好的结果和更高的生活质量。然而，对促红细胞生成素的反应是不同的，多达10%的透析人群对促红细胞生成素具有抗药性。EPO反应性的变化可能是由于生理因素，包括尿毒症、炎症、铁状态以及环境和遗传因素。我们建议研究CRF期间影响EPO反应的基因的表达。我们已经开始了一种基于微阵列的方法来识别在CRF诱导过程中或在EPO治疗后表达发生变化的候选基因。选择了手术诱发慢性肾功能衰竭的小鼠模型，其尿毒症和贫血的状态与人类疾病相似。在初步研究中，小鼠接受CRF 3wk，然后一半接受EPO治疗3wk。全身反应如前所述，即诱发严重尿毒症和贫血。EPO治疗部分恢复了大多数小鼠的血液学参数。处死后，从肝、脾和骨髓中分离出RNA。用Affymetrix基因芯片系统分析每组2只小鼠的肝脏RNA。CRF可诱导多种PPARα诱导基因的表达，包括细胞色素P450 A10和胰岛素样生长因子结合蛋白(IGFBP)-1。据我们所知，这是CRF激活肝脏PPAR-α的第一个证据。我们的初步研究提出了以下假设：CRF期间肝脏PPAR-α的激活，可能是通过血液中的尿毒症毒素，改变了特定肝脏基因的表达。这些基因的失调抑制了红细胞的生成，特别是对EPO的反应。我们将通过追求以下特定目标来验证这一假说：(1)确定CRF对PPAR-α诱导和红细胞生成相关基因在肝脏中表达的影响(2)确定CRF的程度和PPAR-α活性与CRF小鼠红细胞生成之间的关系，以及(3)利用PPAR-α阴性和氯贝特处理的小鼠，确定PPAR-α在CRF介导的基因表达变化中的体内作用。我们期望这些研究将有助于我们了解CRF期间EPO抵抗的分子机制，并可能导致克服这种抵抗并增强EPO活性的治疗药物的开发。

DESCRIPTION (provided by applicant): This revised application describes a rigorous training program of education and investigation to facilitate the applicant's development into an independent physician-scientist in the field of anemia of chronic renal failure (CRF). The applicant, Dr. Saul Nurko, is a nephrologist at the Cleveland Clinic, with a principal clinical interest in CRF and its consequences. Training will be supervised by the Sponsor, Dr. Paul Fox. His laboratory in the Dept. of Cell Biology investigates cellular and molecular mechanisms regulating iron homeostasis, and defects that lead to anemia. Dr. Linda Graham, project Co-Sponsor, is a vascular surgeon who will have primary responsibility for training and performance in studies using the surgical model of CRF in the mouse. Anemia is a common and debilitating consequence of advanced kidney disease. The advent of recombinant human erythropoietin (Epo) treatment significantly changed how nephrologists manage anemia in this population, and has led to better outcomes and a higher quality of life for most patients. However, the response to Epo is variable and up to 10 percent of the dialysis population are "Epo-resistant". Variations in Epo-responsiveness may be due to physiological factors including uremia, inflammation, iron status, and environmental and genetic factors. We propose to investigate the expression of genes influencing the response to Epo during CRF. We have begun a microarray-based approach to identify candidate genes with altered expression during CRF induction or after treatment with Epo. A mouse model of surgery-induced CRF was selected which gives a state of uremia and anemia comparable to human disease. In Preliminary Studies, mice were subjected to CRF for 3 wk, and then half were treated with Epo for an additional 3 wk. Systemic responses were as described previously, namely, induction of severe uremia and anemia. Treatment with Epo partially restored hematological parameters in most mice. At sacrifice, RNA was isolated from liver, spleen, and bone marrow. Liver RNA from 2 mice in each group was analyzed using the Affymetrix GeneChip system. CRF induced the expression of several peroxisome proliferator-activated receptor (PPAR)-alpha-inducible genes including cytochrome p450 type A10 and insulin-like growth factor binding protein (IGFBP)-1. To our knowledge this is the first evidence for activation of hepatic PPAR-alpha by CRF. Our preliminary studies have led us to propose the following hypothesis: Activation of liver PPAR-alpha during CRF, possibly via uremic toxins in blood, alters the expression of specific hepatic genes. Dysregulation of these genes inhibits erythropoiesis, particularly, in response to Epo. We will test this hypothesis by pursuing the following Specific Aims: (1) Determine the effects of CRF on the expression of PPAR-(-induced and erythropoiesis-related genes in liver (2) determine the relationship between the extent of CRF and PPAR-alpha activity and erythropoiesis in mice with CRF, and (3) determine the in vivo role of PPAR-alpha in CRF-mediated alteration of gene expression using PPAR-alpha-null and clofibrate-treated mice. We expect that these studies will help us to understand the molecular mechanisms involved in Epo-resistance during CRF, and possibly will lead to the development of therapeutic agents that overcome this resistance and potentiate the activity of Epo.