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.

项目总结