Molecular Mechanism of APOL1 Associated Kidney Disease

描述(申请人提供)：超过500,000名美国人患有终末期肾病。非洲裔美国人患ESRD的比率是欧洲裔美国人的4-5倍。最近，我们发现载脂蛋白L1(APOL1)基因的编码序列变异只存在于最近有非洲血统的个体中，解释了这种健康差异的很大比例。这一点现在已经被多项额外的研究所证实。APOL1变异对不同类型的肾脏疾病有重大影响，包括高血压相关性终末期肾病、局灶节段性肾小球硬化和艾滋病毒相关性肾病。携带两个APOL1变异等位基因的个体患肾脏疾病的风险增加7-30倍。350万非裔美国人可能有高危APOL1基因。没有两个变异等位基因的非裔美国人的超额风险最小。目前，对APOL1的生物学及其在肾脏中的作用知之甚少。APOL1在对锥虫的抗药性中具有明确的作用，G1和G2变种似乎在非洲变得常见，因为它们提供了对导致非洲睡眠病的锥体形式的保护。在这里，我们的目标是阐明APOL1的功能及其在导致人类肾脏疾病中的作用，以了解肾脏疾病风险存在巨大差异的生物学机制。APOL1是Bcl2家族成员，具有BH3结构域，提示其在细胞凋亡中可能起作用。我们在卵母细胞上的初步实验使我们相信，这一途径可能是APOL1相关疾病中肾小球损伤的重要机制。1.定义APOL1的细胞生物学和细胞生理学：我们将定义APOL1的基本细胞生物学，包括生物合成、运输、离子通道特性、内吞作用以及对细胞器结构的影响 和功能。我们将比较野生型和肾脏风险变异的APOL1生物学，以了解导致肾脏疾病的机制。2.检测载脂蛋白1对足细胞凋亡的特异性影响：我们假设载脂蛋白1风险变异体通过N端的BH3结构域促进足细胞凋亡，肾脏风险变异体增强了载脂蛋白1‘S促凋亡作用。我们将在不同APOL1基因类型的培养足细胞中验证这一假设，并通过将APOL1构建物导入足细胞来探索潜在的凋亡机制。3.用斑马鱼模型研究APOL1介导的活体肾损伤：我们将利用斑马鱼来了解APOL1变异体如何促进肾小球损伤。我们已经证明，G1风险变异体增加胚胎致死率并导致肾小球病变，野生型APOL1可以逆转G1引起的病理表型。我们将使用普遍存在的和组织特异性的启动子来研究异源APOL1在整个生物体中表达的影响。我们将定义导致疾病的风险变异结构域和防止G1介导的损伤的野生型结构域。我们还将定义野生型、G1和G2形式的APOL1在体内的具体相互作用。最后，我们将研究APOL1变异体在促进或阻止肾小球细胞凋亡中的作用。

DESCRIPTION (provided by applicant): More than 500,000 Americans suffer from end-stage renal disease (ESRD). African Americans develop ESRD at rates 4-5 fold higher than European Americans. Recently we showed that coding sequence variants in the ApolipoproteinL1 (ApoL1) gene, present only in individuals with recent African ancestry, explain a very large proportion of this health disparity. This has now been confirmed by multiple additional studies. ApoL1 variants have a major impact on different types of kidney disease including hypertension-associated ESRD, focal segmental glomerulosclerosis, and HIV-associated nephropathy. Individuals with two variant ApoL1 alleles have a 7-30 fold increased risk for kidney disease. 3.5 million African Americans likely have the high risk ApoL1 genotype. African Americans without two variant alleles have minimal excess risk. At present, little is known about the biology of ApoL1 or its rol in the kidney. ApoL1 has a defined role in resistance to trypanosomes, and the G1 and G2 variants appear to have become common in Africa because they confer protection against the forms of trypanosomes that cause African Sleeping Sickness. Here, our goal is to elucidate the function of ApoL1 and its role in causing human kidney disease in order to understand the biological mechanisms underlying this large disparity in kidney disease risk. ApoL1 is a Bcl2 family member with a BH3-only domain, suggesting a possible role in apoptosis. Our initial experiments in oocytes lead us to believe that this pathway may be an important mechanism of glomerular injury in ApoL1-associated disease. We propose to: 1. Define the cell biology and cell physiology of ApoL1: We will define the basic cellular biology of ApoL1 including biosynthesis, trafficking, ion channel properties, endocytosis, and effects on organellar structure and function. We will compare wild type and renal risk variant ApoL1 biology to understand the mechanisms that lead to kidney disease. 2. Examine specific effects of ApoL1 on apoptosis in podocytes: We hypothesize that ApoL1 risk variants promote podocyte apoptosis via a BH3 domain in the N- terminus and that the renal risk variants enhance ApoL1's pro-apoptotic effects. We will test this hypothesis both in cultured podocytes of different ApoL1 genotypes and by transfecting podocytes with ApoL1 constructs designed to probe the underlying apoptotic mechanisms. 3. Use a zebrafish model to study ApoL1- mediated renal injury in vivo: We will use zebrafish to understand how ApoL1 variants promote glomerular injury. We have demonstrated that the G1 risk variant increases embryonic lethality and causes glomerular pathology, and that wild type ApoL1 can reverse the pathologic phenotype caused by G1. We will examine the effects of heterologous ApoL1 expression in a whole organism using ubiquitous and tissue-specific promoters. We will define the risk variant domains that cause disease and the wild type domains that prevent G1-mediated injury. We will also define the specific interactions between the wild-type, G1, and G2 forms of ApoL1 in vivo. Lastly, we will examine the role of ApoL1 variants in promoting or preventing apoptosis of glomerular cells.