Kidney disease mechanisms associated with human genetic variation

描述(由申请人提供)：慢性肾脏疾病(CKD)，像许多复杂的疾病表型一样，反映了遗传易感性和环境因素之间的相互作用。在非洲血统的患者中，APOL1变异解释了晚期非糖尿病CKD的大部分额外风险。然而，只有一些具有风险基因的患者会患上肾脏疾病，这表明遗传上位性或环境应激是触发APOL1依赖的变异肾损伤的必要条件。为了研究APOL1变异如何导致CKD，我们将重点放在HIV相关肾病(HIVAN)上，这种疾病与APOL1风险单倍型最相关，并明显依赖于环境因素，HIV-1感染。我们的初步数据表明，APOL1在足细胞中表达，APOL1的过度表达激活了自噬。尽管依赖mTOR抑制核心自噬可能会导致糖尿病肾小球病变，这是一种与APOL1变异无关的肾脏病理，但我们提供的证据表明，APOL1与CKD的遗传关联是由于APOL1依赖于对不同的、选择性的自噬途径的调节，从而导致病原体的破坏。我们假设APOL1是一个自噬接头，它将显示在溶酶体上的对接陷阱(VAMP8)拴在自噬蛋白LC3-II上。APOL1的拴系功能是通过结合一个触发分子，HIV蛋白Nef来激活的，并导致携带HIV病毒颗粒和蛋白质的自噬小体的选择性降解。变异的APOL1蛋白是有功能缺陷的，允许艾滋病毒在感染细胞中持续存在，并继续合成包括Nef在内的病毒蛋白。NEF阻止病毒的自噬消除，对HIVAN的发展至关重要。我们提出的实验解决了关于APOL1在CKD中的功能的三个悬而未决的新问题。APOL1是在肾脏中原位合成的吗？它的亚细胞家园是什么？循环或肾脏表达的APOL1变异体是否参与了肾脏疾病？对特定环境应激反应激活的自噬通路的失调是否会导致APOL1风险基因型患者的肾脏疾病？这些问题的解决有以下特定的目的：1.确定APOL1在正常肾脏中的表达和细胞内定位，并确定APOL1的定位是否因危险基因和/或疾病诊断而异；2.为研究APOL1的功能建立体内模型；3.表征APOL1与VAMP8和HIV蛋白Nef之间的蛋白质相互作用，并检测细胞系和培养的足细胞中APOL1对自噬的正常和变异调节。这些研究可以产生新的、基于机制的治疗方法，改善非裔美国人患者的健康差异，并确定人类慢性肾脏病的新机制。

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD), like many complex disease phenotypes, reflects dependent interactions between genetic susceptibilities and environmental factors. APOL1 variants explain much of the excess risk of advanced non-diabetic CKD in patients of African ancestry. However, only some patients with risk genotypes develop kidney disease, suggesting that genetic epistasis or environmental stress is required to trigger variant APOL1-dependent kidney injury. To investigate how APOL1 variants result in CKD, we have focused on HIV- associated nephropathy (HIVAN), the disease most robustly associated with APOL1 risk haplotypes and clearly dependent on an environmental factor, HIV-1 infection. Our preliminary data demonstrate APOL1 is expressed in the podocyte, and APOL1 overexpression activates autophagy. Although mTor-dependent suppression of core autophagy may cause diabetic glomerulopathy, a renal pathology not associated with APOL1 variants, we provide evidence that the genetic association of APOL1 with CKD results from APOL1- dependent regulation of distinct, selective autophagy pathways, which result in the destruction of pathogens. We hypothesize that APOL1 is an autophagic adaptor that tethers a docking SNARE (VAMP8) displayed on lysosomes to the autophagosomal protein LC3-II. The tethering function of APOL1 is activated by binding a triggering molecule, the HIV protein Nef, and results in the selective degradation of autophagosomes carrying HIV viral particles and proteins. Variant APOL1 proteins are functionally defective, permitting HIV to persist in the infected cell and continue synthesis of viral proteins including Nef. Nef blocks autophagic elimination of the virus and is critical for development of HIVAN. Our proposed experiments address three unanswered and novel questions regarding APOL1 function in CKD. Is APOL1 synthesized in situ in kidney and what is its subcellular home? Does circulating or renal-expressed variant APOL1 mediate kidney disease? Does dysregulation of autophagy pathways activated in response to specific environmental stresses result in kidney disease in patients with APOL1 risk genotypes? These questions are addressed with the following Specific Aims: 1. Determine APOL1 expression and intracellular location in normal kidney, and determine if APOL1 localization varies with risk genotype and/or disease diagnosis; 2. Generate in vivo models for the study of APOL1 function; 3. Characterize protein interactions between APOL1 with VAMP8 and the HIV protein Nef and examine normal and variant APOL1 regulation of autophagy in cell lines and cultured podocytes. These studies can result in novel, mechanism-based therapies, improve health disparities in African American patients and identify novel mechanisms of human CKD.