Molecular Mechanism of APOL1 Associated Kidney Disease

描述（由申请人提供）：超过50万美国人患有终末期肾病（ESRD）。非裔美国人患ESRD的比率比欧洲人高4-5倍。最近，我们发现载脂蛋白L1（ApoL 1）基因的编码序列变异，仅存在于最近的非洲血统的个体中，解释了这种健康差异的很大一部分。这一点已被多项研究证实。ApoL 1变异体对不同类型的肾脏疾病有重要影响，包括高血压相关的ESRD、局灶节段性肾小球硬化和HIV相关肾病。具有两个变异ApoL 1等位基因的个体患肾脏疾病的风险增加7-30倍。3.5 100万非裔美国人可能有高风险的ApoL 1基因型。没有两个变异等位基因的非洲裔美国人有最小的额外风险。目前，对ApoL 1的生物学或其在肾脏中的作用知之甚少。ApoL 1在抵抗锥虫方面具有明确的作用，G1和G2变体似乎在非洲变得很常见，因为它们提供了对引起非洲睡眠病的锥虫形式的保护。在这里，我们的目标是阐明ApoL 1的功能及其在引起人类肾脏疾病中的作用，以了解肾脏疾病风险差异的生物学机制。ApoL 1是Bcl 2家族成员，具有BH 3-only结构域，这表明ApoL 1在细胞凋亡中可能起作用。我们在卵母细胞中的初步实验使我们相信，该途径可能是ApoL 1相关疾病中肾小球损伤的重要机制。我们建议：1.定义ApoL 1的细胞生物学和细胞生理学：我们将定义ApoL 1的基本细胞生物学，包括生物合成，运输，离子通道特性，内吞作用和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体ApoL 1生物学，以了解导致肾脏疾病的机制。2.检查ApoL 1对足细胞凋亡的特异性作用：我们假设ApoL 1风险变体通过N端的BH 3结构域促进足细胞凋亡，并且肾脏风险变体增强ApoL 1的促凋亡作用。我们将在不同ApoL 1基因型的足细胞培养和ApoL 1结构设计用于探测潜在的细胞凋亡机制的足细胞培养中检验这一假设。3.使用斑马鱼模型研究体内ApoL 1介导的肾损伤：我们将使用斑马鱼来了解ApoL 1变体如何促进肾小球损伤。我们已经证明，G1风险变异增加胚胎致死率，并导致肾小球病理，野生型ApoL 1可以逆转G1引起的病理表型。我们将研究异源ApoL 1表达在整个生物体中使用普遍存在的和组织特异性启动子的影响。我们将定义导致疾病的风险变异域和防止G1介导的损伤的野生型域。我们还将定义野生型，G1和G2形式的ApoL 1在体内之间的特定相互作用。最后，我们将研究ApoL 1变体在促进或预防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.