Role of APOL1 in HIVAN pathogenesis

描述（由申请人提供）：HIV-1相关肾病（HIVAN）是一种几乎完全局限于非洲裔个体的疾病。HIVAN的发病机制尚未完全清楚，然而，形成肾小球血液滤过屏障的足细胞的功能障碍和损伤对这种疾病的发展至关重要。HIV-1介导的足细胞损伤的机制尚不清楚，主要是由于这些细胞上缺乏经典HIV-1进入受体的表达。然而，HIV-1 Nef在实验模型中重现HIVAN表型的发现表明，足细胞可以被其他生产性感染细胞释放的Nef损伤。此外，局灶性节段性肾小球硬化症（FSGS）和HIVAN的频率的显著种族差异已经导致发现APOL 1基因的遗传变体，其使这些风险等位基因纯合的非裔美国人易患这些肾病。然而，野生型（WT）APOL 1蛋白的细胞功能和APOL 1风险突变体在HIV-1感染中对足细胞损伤的贡献仍然未知。本申请着眼于APOL 1途径，该途径可用于治疗HIVAN中Nef介导的足细胞损伤。我们发现，APOL 1在足细胞强烈共定位与溶酶体和自噬体，这表明APOL 1可能在自噬中发挥作用。我们还证明了APOL 1 WT刺激分化的足细胞中的自噬。由于APOL 1基因风险变体的遗传是完全隐性的，因此分析APOL 1对自噬的影响将需要APOL 1风险等位基因纯合的足细胞模型。我们已经建立了一种从尿液中分离和培养人足细胞的方法，该方法将用于探索APOL 1在足细胞中的作用。在本申请中，我们提出野生型APOL 1通过激活自噬通量和抵消Nef对自噬的抑制作用来保护HIVAN的发展。相反，缺乏这些功能的APOL 1风险变体允许Nef的有害作用。我们建议：1）通过免疫沉淀（IP）、蛋白质印迹和共聚焦显微镜比较APOL 1和高危双突变体（APOL 1 DM）之间的相互作用和自噬机制，剖析APOL 1和高危双突变体（APOL 1 DM）促进自噬起始的机制。2)为了研究APOL 1抵消HIV-1 Nef对自噬体成熟的抑制的机制，我们将测试APOL 1 WT是否与Nef竞争，从而刺激自噬体成熟。将鉴定Beclin 1/UVRAG/C-Vps复合物中Nef和APOL 1的结合伴侣。将使用新型串联荧光mRFP-EGFP-LC 3构建体分析自噬体与溶酶体的融合。肾致病性SF 2 Nef和突变体将用于辨别SF 2 Nef抑制自噬的机制以及APOL 1 WT如何克服Nef的抑制。我们相信，我们的建议的结果将导致一种新的治疗策略，旨在防止足细胞损伤HIVAN刺激自噬通量和靶向Nef在APOL 1风险等位基因的存在。 公共卫生相关性：艾滋病毒相关性肾病（HIVAN）几乎完全（>90%）限于非洲裔人。最近，研究人员发现了一种名为APOL 1的基因，该基因可能是导致黑人中HIVAN发病率异常高的原因。我们希望发现由该基因编码的蛋白质的正常功能，并发现这种蛋白质的变化（几乎只在黑人中发现）是如何引起HIV的。我们的研究可能有助于评估发展HIVAN的风险和开发针对这种疾病的新治疗方法。

DESCRIPTION (provided by applicant): HIV-1 associated nephropathy (HIVAN) is a disease almost entirely limited to individuals of African descent. The pathogenesis of HIVAN is not fully understood, however dysfunction and injury to podocytes that form the glomerular blood filtration barrier are essential to the development of this disease. The mechanism of HIV-1- mediated podocyte injury is unclear mostly due to the lack of expression of classical HIV-1 entry receptors on these cells. However, findings that HIV-1 Nef recapitulates a HIVAN phenotype in experimental models, suggest that podocytes can be injured by Nef that is released by other productively infected cells. Further, striking racial disparities in the frequency of focal segmentl glomerulosclerosis (FSGS) and HIVAN have led to the discovery of genetic variants of the APOL1 gene that predispose African Americans homozygous for these risk alleles to the development of these nephropathies. However, the cellular function of the wild type (WT) APOL1 protein and the contribution of APOL1 risk mutants to podocyte damage in the face of HIV-1 infection remain unknown. This application looks at the APOL1 path that could be therapeutically exploited to protect against Nef-mediated podocyte injury in HIVAN. We show that APOL1 in podocytes strongly colocalizes with lysosomes and autophagosomes, suggesting that APOL1 may play a role in autophagy. We also demonstrate that APOL1 WT stimulates autophagy in differentiated podocytes. Since inheritance of the APOL1 gene risk variants is fully recessive, analysis of the effect of the APOL1 on autophagy will require a podocyte model homozygous for the APOL1 risk alleles. We have developed a method for isolation and culture of human podocytes from urine that will be used to explore the role of APOL1 in podocytes. In this application, we propose that wild type APOL1 is protective against HIVAN development by activating autophagic flux and counteracting the inhibitory effect of Nef on autophagy. In contrast, APOL1 risk variants lacking these functions permit the deleterious effects of Nef. We propose: 1) To dissect the mechanism(s) by which APOL1 and a high risk double mutant (APOL1 DM) contribute to autophagy initiation by comparing the interactions between these two molecules and the autophagy machinery, using immunoprecipitation (IP), western blotting and confocal microscopy. 2) To investigate the mechanism(s) by which APOL1 counteracts the suppression of autophagosome maturation by HIV-1 Nef, we will test whether APOL1 WT competes with Nef and thus stimulates autophagosome maturation. Binding partners of Nef and APOL1 in Beclin1/UVRAG/C-Vps complexes will be identified. Autophagosome fusion with lysosomes will be analyzed using a novel tandem fluorescent mRFP-EGFP-LC3 construct. Renal pathogenic SF2 Nef and mutants will be used to discern mechanism(s) by which SF2 Nef inhibits autophagy and how APOL1 WT overcomes Nef's inhibition. We believe that results of our proposal will lead to a novel therapeutic strategy aimed at preventing podocyte injury in HIVAN by stimulation of autophagic flux and by targeting Nef in the presence of APOL1 risk alleles. PUBLIC HEALTH RELEVANCE: HIV-associated kidney disease (HIVAN) is almost entirely (>90%) limited to people of African descent. Recently researchers found a gene called APOL1 that may be responsible for the unusually high incidence of HIVAN in blacks. We wish to discover the normal function of the protein coded by this gene and to find how changes in this protein, seen almost exclusively in blacks, cause HIVAN. Our studies may help both assess the risk for developing HIVAN and for developing a novel treatment against this disease.