Kidney disease mechanisms associated with human genetic variation

描述（由申请人提供）：慢性肾脏疾病（CKD）与许多复杂的疾病特征一样，表现为遗传易感性和环境因素之间的依赖相互作用。最近描述的与CKD和非洲血统相关的APOL1风险等位基因就是一个例子，其中只有一部分遗传易感患者发展为肾脏疾病，这表明需要第二次打击或压力才能触发变异型APOL1依赖性肾损伤。为了研究APOL1变异在CKD中的机制，我们选择关注hiv相关肾病（HIVAN），这种疾病与APOL1风险单倍型最密切相关，并且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，并且APOL1过表达激活自噬。尽管mtor诱导的核心自噬途径与糖尿病肾病的肾小球损伤有关（一种与APOL1变异无关的肾脏病理），但我们有证据表明，涉及病原体破坏的选择性自噬途径可能由APOL1唯一调节。我们假设APOL1通过结合内体上显示的对接SNARE （VAMP8）和自噬体蛋白LC3-II来发挥自噬细胞接头的作用。APOL1的自噬接头功能通过在感染后结合触发分子（如HIV蛋白Nef）而激活，并导致vamp8标记的内体货物（包括病毒颗粒和蛋白质）的选择性降解。变异APOL1蛋白在功能上存在缺陷，允许HIV在被感染的细胞中持续存在，并支持Nef的新合成，Nef是HIV蛋白的关键。我们提出的实验将解决CKD中APOL1功能的三个未解之谜和新问题。APOL1是在肾脏原位合成的吗？它的亚细胞家园是什么？循环或肾脏表达的APOL1变体介导肾脏疾病吗？在APOL1风险基因型患者中，响应特定环境应激激活的自噬通路失调会导致肾脏疾病吗？这些问题将以下列具体目标加以解决：测定正常肾脏中APOL1的表达和细胞内定位，并确定APOL1定位是否随风险基因型和/或疾病诊断而变化；2. 建立体内模型，研究APOL1的功能；3. 表征APOL1与VAMP8和HIV蛋白Nef之间的蛋白相互作用。检测正常和变异APOL1表达对细胞系和培养足细胞自噬的影响。关注APOL1调节自噬的独特途径将最有可能产生新的、基于机制的治疗方法。确定APOL1变异如何与人类肾脏疾病相关，将为了解常见慢性肾脏疾病的致病途径提供一个独特的机会，利用这些知识可以开发新的治疗、预防和风险分层策略。

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD), like many complex disease traits, manifests from dependent interactions between genetic susceptibilities and environmental factors. The recently described APOL1 risk alleles associated with CKD and African Ancestry is one example, where only a portion of genetically-susceptible patients develop kidney disease, suggesting a second hit or stress is required to trigger variant APOL1- dependent kidney injury. To investigate the mechanism of APOL1 variants in CKD, we have chosen to focus 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-induced core autophagy pathways have been implicated in the glomerular injury of diabetic nephropathy -- a renal pathology not associated with APOL1 variants -- we have evidence that selective autophagy pathways involving destruction of pathogens may be uniquely regulated by APOL1. We hypothesize that APOL1 functions as an autophagocytic adaptor by binding to both a docking SNARE (VAMP8) displayed on endosomes and to the autophagosomal protein LC3-II. The autophagocytic adaptor function of APOL1 is activated by binding a triggering molecule, such as the HIV protein Nef after infection, and results in the selective degradation of VAMP8-labeled endosomal cargo including viral particles and proteins. Variant APOL1 proteins are functionally defective, permitting HIV to persist in the infected cell, and supporting the new synthesis of Nef, the HIV protein critical for development of HIVAN. Our proposed experiments will 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 will be 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. Examine the effect of normal and variant APOL1 expression on autophagy in cell lines and cultured podocytes. Focusing on the unique pathways though which APOL1 regulates autophagy will most likely result in novel, mechanism-based therapies. Determining how APOL1 variation is associated with human kidney diseases will provide a unique opportunity to understand pathways causing common chronic kidney disease and use of that knowledge can be exploited to develop new strategies for therapy, prevention and risk stratification.