APOL1 Nephropathy: Linking Genetics and Mechanisms

APOL1 肾病：遗传学和机制的联系

基本信息

批准号：
10540233
负责人：
David J Friedman
金额：
$ 47.82万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-14 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10540233
关键词：
Affinity Africa African American population African Trypanosomiasis African ancestry Alleles American Bacterial Artificial Chromosomes Behavior Binding Biochemical Biological Biological Models Biology Caring Cell Death Cell physiology Cells Code Data Disease Disparity Dose Drosophila genus Elements Endoplasmic Reticulum Engineering European Foot Process Gene Dosage Genes Genetic Genome Genotype Goals Heterozygote Human Image In Vitro Individual Inflammatory Inherited Interferons Kidney Kidney Diseases Kidney Failure Label Lead Link Lipids Literature Mediating Mitochondria Mitochondrial Matrix Molecular Movement Mus Names Organoids Pathway interactions Persons Phenotype Primates Property Proteins Proteinuria Public Health Publishing Reproducibility Resistance Risk Risk Behaviors Role Seminal Stains Stimulus Support System System Testing Toxic effect Transcript Transgenic Mice Variant Work Yeasts cytotoxic cytotoxicity disorder risk endophenotype experimental study gain of function gain of function mutation genetic variant high risk human tissue improved in vivo insight kidney cell loss of function loss of function mutation mitochondrial dysfunction mouse model overexpression prevent racial health disparity recessive genetic trait recruit risk variant trafficking

项目摘要

SUMMARY African Americans develop kidney failure at rates 4-5 fold higher than European Americans. Coding variants in the APOL1 gene, found only in people of recent African ancestry, drive a large fraction of this risk disparity. We have named these APOL1 variants G1 (S342G and I384M) and G2 (del388N389Y). Risk inheritance is recessive: only individuals carrying two variant APOL1 alleles have a markedly increased risk of kidney disease. In the U.S. approximately 13% of African Americans, or about 4,000,000 people, have a high-risk genotype. Most recessive diseases are caused by loss-of- function mutation. Surprisingly, there is data from model systems in kidney cells, mice, drosophila, and yeast that support the idea that the APOL1 risk variants (RV) are actually gain-of-function mutations that cause toxicity when expressed at high levels. The overarching goal of this proposal is to understand the mechanism of APOL1 kidney disease such that two RVs are required, providing insight into this fundamental question of recessive, gain-of-function toxicity. We propose that the G0, or wild- type, APOL1 allele can alter the behavior of the risk variants and thus prevent their toxicity. In AIM 1, we will use isogenic, Crispr-engineered APOL1 BAC transgenic mice to determine the differing properties of G0 and risk-variant APOL1 in the full complexity of the glomerulus. We will test the effects of different APOL1 variants singly and in combination in order to answer questions about gene dosage, WT rescue of risk-variant toxicity, and possible differences in mechanism of action between the two major APOL1 risk alleles (G1 and G2). In AIM 2, our goal is to understand APOL1 targeting to lipid droplets. We have observed that lipid droplets (LDs) show prominent G0 but little or no G1 or G2 localization in cell systems and that co-expression of G0 facilitates the movement of G1 or G2 onto lipid droplets. We will use imaging and biochemical analyses to examine determinates of APOL1 localization to LDs, determine if there are differences in the binding of APOL1 to other LD-associated proteins, and define the cellular consequence(s) of altered APOL1 risk variant targeting to the LD. We will test the relevance of these experiments with in vivo studies. In AIM 3, we will determine the role of aggregation in APOL1-mediated cytotoxicity. We have observed that the APOL1 RVs have much stronger propensity than G0 to aggregate. Our data indicate that RV aggregation occurs in mitochondria. We hypothesize that aggregation is a key element in RV toxicity, and that G0 can interfere with RV aggregation. In cell-based systems we will characterize the APOL1 aggregates, determine whether aggregation is essential for APOL1-mediated cytotoxicity, understand the effect of aggregates on mitochondrial function, and test whether G0 can reverse RV aggregation and cytotoxicity. In vitro results will be validated in kidney organoids, a mouse model, and in human tissues.

摘要非洲裔美国人以比欧洲高4-5倍的肾衰竭发展美国人。 APOL1基因中的编码变体仅在最近的非洲血统的人们中发现这种风险差异的很大一部分。我们命名了这些apol1变体G1（s342g和i384m）和 G2（DEL388N389Y）。风险继承是隐性的：只有携带两个变体Apol1等位基因的个体肾脏疾病的风险显着增加。在美国，大约13％的非裔美国人，或大约4,000,000人，具有高风险基因型。大多数隐性疾病是由 - 功能突变。令人惊讶的是，肾细胞，小鼠，果蝇和支持APOL1风险变体（RV）实际上是功能收益突变的酵母高水平表达时会导致毒性。该提议的总体目标是了解APOL1肾脏疾病的机制，因此需要两种RV，提供洞察力进入隐性，功能获得毒性的基本问题。我们建议G0或野生类型，Apol1等位基因可以改变风险变异的行为，从而防止其毒性。在AIM 1中，我们将使用ISEOGENIC，CRISPR工程的Apol1 BAC转基因小鼠来确定不同的 G0和风险变化apol1的性质在肾小球的全部复杂性中。我们将测试效果为了回答有关基因剂量的问题，不同的apol1变体的不同 WT营救风险变化的毒性，以及两者之间的作用机理差异主要的Apol1风险等位基因（G1和G2）。在AIM 2中，我们的目标是了解apol1靶向脂质飞沫。我们观察到脂质液滴（LDS）显示出明显的G0，但G1或G2几乎没有在细胞系统中的定位以及G0的共表达促进了G1或G2的运动飞沫。我们将使用成像和生化分析来检查APOL1定位的确定性到LDS，确定APOL1与其他LD相关蛋白的结合是否存在差异，并且定义靶向LD的APOL1风险变体变化的细胞后果。我们将测试这些实验与体内研究的相关性。在AIM 3中，我们将确定聚合的作用在Apol1介导的细胞毒性中。我们已经观察到Apol1 RV的强大倾向比G0汇总。我们的数据表明RV聚集发生在线粒体中。我们假设聚集是RV毒性中的关键要素，并且G0可以干扰RV 聚合。在基于单元的系统中，我们将表征APOL1聚集体，确定是否是否聚集对于Apol1介导的细胞毒性至关重要，了解聚集体对线粒体功能，并测试G0是否可以逆转RV聚集和细胞毒性。体外结果将在肾脏器官，小鼠模型和人体组织中进行验证。