APOL1 Nephropathy: Linking Genetics and Mechanisms

非裔美国人患肾衰竭的比率比欧洲人高4-5倍 美国人APOL 1基因的编码变异，只在最近的非洲血统的人中发现，驱动一个新的基因。 很大一部分的风险差异。我们将这些APOL 1变体命名为G1（S342 G和I384 M）， G2（del388N389Y）。风险遗传是隐性的：只有携带两个变异APOL 1等位基因的个体 患肾脏疾病的风险显著增加。在美国，大约13%的非洲裔美国人， 大约有400万人有高风险基因型。大多数隐性疾病都是由... 功能突变令人惊讶的是，有来自肾细胞、小鼠、果蝇和 支持APOL 1风险变体（RV）实际上是功能获得性突变的想法的酵母 当高水平表达时会导致毒性。本提案的总体目标是 了解APOL 1肾病的机制，因此需要两个RV， 这个隐性的、功能获得性毒性的基本问题。我们建议G 0，或野生- 型，APOL 1等位基因可以改变风险变体的行为，从而防止其毒性。在AIM 1中， 我们将使用同基因的Crispr工程APOL 1 BAC转基因小鼠来确定不同的 G 0和风险变异型APOL 1在肾小球完整复杂性中的特性。我们将测试 不同的APOL 1变异体的单独和组合，以回答有关基因剂量的问题， WT挽救风险变异毒性，以及两者作用机制的可能差异 APOL 1主要风险等位基因（G1和G2）。在AIM 2中，我们的目标是了解APOL 1靶向脂质， 水滴。我们观察到脂滴（LDs）显示显著的G 0，但很少或没有G1或G2 在细胞系统中的定位和G 0的共表达促进G1或G2移动到脂质上 水滴。我们将使用成像和生化分析来检查APOL 1定位的决定因素 确定APOL 1与其他LD相关蛋白的结合是否存在差异，以及 定义靶向LD的改变的APOL 1风险变体的细胞后果。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 APOL 1介导的细胞毒性。我们已经观察到，APOL 1 RV具有更强的 比G 0更容易聚集。我们的数据表明，RV聚集发生在线粒体。我们 假设聚集是RV毒性关键因素，G 0可干扰RV 聚合来在基于细胞的系统中，我们将表征APOL 1聚集体，确定是否 聚集对于APOL 1介导的细胞毒性是必不可少的，了解聚集体对细胞毒性的影响， 线粒体功能，并测试G 0是否可以逆转RV聚集和细胞毒性。体外 将在肾类器官、小鼠模型和人体组织中验证结果。

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.