Novel mechanisms and Drosophila model of APOL1-HIV-1 nephropathies in children

儿童 APOL1-HIV-1 肾病的新机制和果蝇模型

• 批准号: 10202573
• 负责人: ZHE HAN
• 金额: $ 42.39万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202573
• 关键词: AIDS-Associated Nephropathy; APOL1 gene; Adult; Affect; Autophagocytosis; Cell Size; Cells; Cessation of life; Child; Childhood; Chronic Kidney Failure; Development; Drosophila genus; Dynamin; Ectopic Expression; Endocytosis; Experimental Models; Future; Genes; Genetic; Genetic Transcription; Genetic study; Guanosine Triphosphate Phosphohydrolases; HIV; HIV Envelope Protein gp120; HIV-1; Homeostasis; Impairment; Individual; Infection; Kidney; Kidney Diseases; Knowledge; Mediating; Medical Genetics; Modeling; Mus; Names; Nephrology; Organelles; Pathogenesis; Pathologic; Pathway interactions; Phenotype; Plasma; Positioning Attribute; Process; Proteins; Proteinuria; RNA Interference; Regulation; Reporter; Role; Sampling; Structure; System; TNF gene; Testing; Tissues; Transgenic Mice; Transgenic Organisms; Urine; Vacuole; Workload; base; clinically relevant; cytotoxic; fly; high risk; high throughput screening; in vivo; kidney cell; new therapeutic target; novel; novel therapeutics; overexpression; podocyte; programs; risk variant; screening; tat Genes; trafficking; transcriptome sequencing; young adult

PROJECT SUMMARY Individuals carrying two APOL1 risk alleles (RA) named G1 or G2 have ~ 30-fold higher odds of developing HIV-nephropathy (HIVAN) compared to HIV+ controls. Our current knowledge regarding the role of APOL1 in HIVAN is mostly derived from studies done in cultured renal cells or clinical genetic studies, and new experimental models are needed to gain a more in depth understanding of the interactions between HIV-1 and the APOL1-RA in vivo. Our pediatric nephrology program has been studying HIV-renal diseases (HIV- RD) for 25 years and is in a unique position to fill this gap. Recently, we developed Drosophila APOL1-G0 and G1 transgenic (Tg) lines, and found that ectopic expression of APOL1-G1 in nephrocytes (the equivalent of mammalian podocytes) enhanced the endocytic activity and size of these cells, impaired the acidification of intracellular vacuoles, and accelerated their death. In addition, we found that HIV-1 induces a low level productive infection of podocytes cultured from HIV+ children through a dynamin-dependent endocytosis mechanism that is independent of CD4, and increases the expression of APOL1-G1 in these cells. Furthermore, we found that overexpression of APOL1-G1 in Tg-flies and cultured podocytes increases the activity of GTPases involved in endocytosis, regulation of cytoskeletal networks, and cell trafficking. Based on these findings, we hypothesize that HIV-1 acts as a “second hit” to precipitate HIV-RD in children by infecting podocytes through an endocytosis mediated mechanism that increases the expression / activity of the APOL1-RA beyond their toxic threshold levels. In turn, these changes impair key endocytic, cell trafficking, acidification, and autophagy pathways that disrupt the homeostasis of podocytes and accelerate their dead, causing proteinuria and HIV-RD. To test this hypothesis, in aim 1 we will determine the phenotype of Drosophila Tg lines expressing APOL1-G0/G1/G2, and the HIV- genes Tat, Nef, and Vpr, specifically in nephrocytes, and assess how they affect their structure and function. In aim 2, we will define how APOL1-RA and HIV-genes interact in vivo to modulate the function of nephrocytes in dual Tg-fly lines, define whether APOL1-RA precipitate the death of nephrocytes expressing HIV-genes by affecting their autophagic flux, and perform RNA-Seq analysis to identify new transcriptional pathways that regulate these interactions. These findings will be validated in HIV-Tg26 mice carrying the autophagy reporter construct RFP-EGFP-LC3. In aim 3, we will select the best Drosophila model and perform an RNAi-based APOL1-HIV genetic interaction screening to identify new pathways that affect the function and survival of nephrocytes, determine how the APOL1-RA affect the infection of podocytes, and validate all clinical relevant findings in samples or tissues derived from children with HIV-RD. These studies will identify new mechanisms through which APOL1-RA and HIV-1 interact to infect podocytes and precipitate HIV-RD, and develop new clinically relevant Tg-fly models to a) study the pathogenesis of HIV-RD in children and young adults; b) develop a high throughput screening system to identify new genetic modulators of the APOL1-HIV interactions in vivo; and c) serve as future platforms to identify and screen new drug targets against HIV-RD.

项目摘要 携带两个名为G1或G2的APOL 1风险等位基因（RA）的个体发生RA的几率高约30倍。 与HIV+对照相比，HIV肾病（HIVAN）。我们目前关于APOL 1在以下方面作用的知识： HIVAN主要来自于在培养的肾细胞中进行的研究或临床遗传学研究， 需要实验模型来更深入地了解HIV-1之间的相互作用 和体内APOL 1-RA。我们的儿科肾脏病项目一直在研究艾滋病毒-肾脏疾病（艾滋病毒- 研发）25年，并处于填补这一空白的独特地位。最近，我们开发了果蝇APOL 1-G 0 和G1转基因（Tg）系，并发现APOL 1-G1在肾细胞中的异位表达（相当于 的哺乳动物足细胞）增强这些细胞的内吞活性和大小，削弱酸化 细胞内的空泡，并加速其死亡。此外，我们发现，HIV-1诱导低水平的 通过动力蛋白依赖性内吞作用从HIV+儿童培养的足细胞的生产性感染 这种机制不依赖于CD 4，并增加这些细胞中APOL 1-G1的表达。 此外，我们发现在TG果蝇和培养的足细胞中APOL 1-G1的过表达增加了细胞凋亡。 参与胞吞作用、细胞骨架网络调节和细胞运输的GTP酶活性。基于 根据这些发现，我们假设HIV-1是促使儿童发生HIV-RD的“第二次打击” 通过内吞作用介导的机制感染足细胞， APOL 1-RA的活性超过其毒性阈值水平。反过来，这些变化削弱了关键 内吞，细胞运输，酸化和自噬途径，破坏了体内平衡， 足细胞并加速其死亡，引起蛋白尿和HIV-RD。为了验证这一假设，在aim 1中， 我们将确定表达APOL 1-G 0/G1/G2和HIV-1基因的果蝇Tg系的表型。 达特，Nef和Vpr，特别是在肾细胞中，并评估它们如何影响其结构和功能。在aim中 2，我们将定义APOL 1-RA和HIV-基因如何在体内相互作用，以调节肾细胞的功能， 双TG-飞线，确定是否APOL 1-RA沉淀表达HIV基因的肾细胞的死亡， 影响其自噬通量，并进行RNA-Seq分析，以确定新的转录途径， 调节这些相互作用。这些发现将在携带自噬报告基因的HIV-Tg 26小鼠中得到验证。 构建RFP-EGFP-LC 3。在目标3中，我们将选择最好的果蝇模型，并进行基于RNAi的研究。 APOL 1-HIV遗传相互作用筛选，以确定影响HIV功能和存活的新途径 肾细胞，确定APOL 1-RA如何影响足细胞的感染，并验证所有临床相关的 来自HIV-RD儿童的样本或组织中的发现。这些研究将发现新的 APOL 1-RA和HIV-1相互作用感染足细胞并沉淀HIV-RD的机制，以及 开发新的临床相关Tg-fly模型，以a）研究儿童和青少年HIV-RD的发病机制 B）开发高通量筛选系统以鉴定APOL 1-HIV的新的遗传调节剂 体内相互作用;和c）作为未来的平台，以确定和筛选针对HIV-RD的新药物靶点。