Pathogenesis of HIVAN: the role of Nef

HIVAN 的发病机制：Nef 的作用

• 批准号: 7071831
• 负责人: John Cijiang He
• 金额: $ 12.88万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071831
• 关键词: HIV infections; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell differentiation; cell proliferation; clinical research; contact inhibition; cyclins; genetically modified animals; human tissue; immunoprecipitation; integrins; kidney disorder; laboratory mouse; medical complication; microfilaments; microtubules; northern blottings; pathologic process; protein protein interaction; protein structure function; site directed mutagenesis; tissue /cell culture; virus protein; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): HIV-associated nephropathy (HIVAN) is the third leading cause of the end-stage renal disease in African Americans between ages 20-64. One of the pathologic features of HIVAN is collapsing focal segmental glomerulosclerosis (FSGS). Glomerular podocytes, the key cells involved in FSGS, exhibit a disease phenotype characterized by proliferation and dedifferentiation in both human and mouse kidneys with HIVAN. We reported that HIV-1 mRNA were expressed in podocytes of HIVAN patients and of HIV-1 transgenic mice. Studies from transgenic mice suggest that Nef, one of the HIV accessory proteins, plays an important role in the pathogenesis of HIVAN. We found that Nef induces podocyte proliferation and loss of contact inhibition in vitro. In addition, we found that Nef induces podocyte dedifferentiation and the rearrangement of actin filaments. Our preliminary data also suggest that Nef activates Src-Stat3, Ras-MAPK, and Rac1/CDC42 pathways in kidney podocytes. In this study, we propose the following specific aims: 1. To determine in podocytes: a) how Nef induces cell proliferation and loss of contact inhibition by examining the effects of Nef on expressions of cyclins, cyclin inhibitors, integrins, and adhesion molecules; b) how Nef affects the expression of differentiation markers, the organization of actin filaments and microtubules, and the arrangement of actin-associated molecules such as synaptopodin, Ezrin, and CD2AP. 2. To determine in podocytes: a) what are the potential Nef-interactive proteins as well as their down-stream signaling pathways; b) to define the relationships between Nef-induced signaling pathways and Nef-induced phenotypic changes using dominant negative mutants for Src, Stat3, Ras, Rac1/CDC42 or mutants for different Nef binding motifs. 3. To verify that these in vitro findings have in vivo relevance by determining: a) whether Nef induces similar signaling pathways and phenotypic changes in primary cultures of murine podocytes; b) whether these phenotypic changes and signaling pathway activations are observed in kidney podocytes of HIV transgenic mice and HIV patients with HIVAN in vivo. These studies should elucidate essential mechanism responsible for the development of HIV-associated nephropathy and how Nef plays a role in this pathologic process.

描述（由申请人提供）： HIV 相关肾病 (HIVAN) 是 20-64 岁非裔美国人终末期肾病的第三大原因。 HIVAN 的病理特征之一是塌陷性局灶节段性肾小球硬化症 (FSGS)。肾小球足细胞是 FSGS 中涉及的关键细胞，在感染 HIVAN 的人类和小鼠肾脏中表现出以增殖和去分化为特征的疾病表型。我们报道了 HIV-1 mRNA 在 HIVAN 患者和 HIV-1 转基因小鼠的足细胞中表达。转基因小鼠的研究表明，Nef（HIV 辅助蛋白之一）在 HIVAN 的发病机制中发挥着重要作用。我们发现 Nef 在体外诱导足细胞增殖和接触抑制丧失。此外，我们发现 Nef 诱导足细胞去分化和肌动蛋白丝的重排。我们的初步数据还表明，Nef 激活肾足细胞中的 Src-Stat3、Ras-MAPK 和 Rac1/CDC42 通路。在本研究中，我们提出以下具体目标： 1. 确定足细胞中：a) 通过检查 Nef 对细胞周期蛋白、细胞周期蛋白抑制剂、整合素和粘附分子表达的影响，确定 Nef 如何诱导细胞增殖和失去接触抑制； b) Nef 如何影响分化标记物的表达、肌动蛋白丝和微管的组织以及肌动蛋白相关分子（如突触蛋白、Ezrin 和 CD2AP）的排列。 2. 确定足细胞中： a) 潜在的 Nef 相互作用蛋白及其下游信号通路是什么； b) 使用 Src、Stat3、Ras、Rac1/CDC42 的显性失活突变体或不同 Nef 结合基序的突变体来定义 Nef 诱导的信号通路和 Nef 诱导的表型变化之间的关系。 3. 通过确定以下各项来验证这些体外研究结果是否具有体内相关性： a) Nef 是否在小鼠足细胞原代培养物中诱导类似的信号传导途径和表型变化； b) 在HIV转基因小鼠和体内携带HIVAN的HIV患者的肾足细胞中是否观察到这些表型变化和信号通路激活。这些研究应阐明 HIV 相关肾病发生的基本机制以及 Nef 如何在这一病理过程中发挥作用。