Pathogenesis and therapeutic options of APOL1-associated renal diseasese

与欧洲血统的美国人相比，非洲裔美国人中终末期肾病的发病率大约高出三倍。这种差异不是由社会经济因素来解释的，但大多数额外的风险可归因于APOL1基因的两个风险变种。纯合子携带者一生有超过15%的风险患上衰弱的肾脏疾病，如局灶性节段性肾小球硬化(FSGS)和高血压相关的肾脏疾病。目前还没有具体的治疗方法。杂合子携带者不会患昏睡病，这使得风险变异在非洲血统的人群中非常常见，全世界有数百万纯合子携带者面临风险。在主要的模式生物中缺乏同源基因，APOL1相关肾脏疾病的发病机制甚至APOL1的细胞内功能仍不清楚。基于过度表达模型，已经提出了几种可能的致病机制，但报道的结果往往相互矛盾。在研究内源性APOL1在培养的足细胞和转基因果蝇模型中的亚细胞定位时，我们在内质网(ER)中检测到APOL1。APOL1在果蝇中的表达诱导了IRE1依赖的内质网应激，暗示了可能的致病作用。APOL1风险变异体表达增强了果蝇足细胞样肾细胞的内吞功能。表达APOL1的细胞从翅膀前体组织的上皮层顶部挤出，这一影响对于风险等位基因更为明显。为了研究APOL1风险变异体在足细胞生物学中的作用而不过度表达，我们建议通过纯合子基因组编辑将风险变异体G2引入培养的足细胞系(AIM1)。为了达到体内条件的近似性，足细胞将被分化，并在干扰素-的作用下长期培养，众所周知，干扰素-DNA可以增强载脂蛋白1的内源性表达。对该细胞系与未携带风险等位基因的对照足细胞系进行功能分析，将有助于区分过度表达的伪影和相关机制。为了在快速活体模型中进行补充研究，我们将在果蝇中使用人APOL1变体的转基因表达(目标2)。我们将研究几种APOL1转基因在足细胞样肾细胞和翼盘作为上皮模型中的作用。特别是，我们将研究内质网应激信号，并探索与Risk Variant表达特别观察到的肾细胞功能增强和心尖细胞挤出的表型相关的机制。最后，使用G2-APOL1诱导的肾细胞功能增加作为读出，我们将在整个动物药物筛选(AIM 3)中筛选FDA批准的药物文库以识别保护性化合物。这项工作计划可能阐明APOL1的功能并发现潜在的治疗方案。

End-stage renal disease is approximately three times more common in African Americans compared to Americans of European ancestry. This disparity is not explained by socio-economic factors but most of the excess risk is attributable to two risk variants of the APOL1 gene. Homozygous carriers have a life-time risk exceeding 15% to develop debilitating renal disease such as focal-segmental glomerulosclerosis (FSGS) and hypertension-associated renal disease. Specific therapies are unavailable. Heterozygous carriers are protected from sleeping sickness making the risk variants exceedingly common in populations of African ancestry with millions of homozygous carriers at risk worldwide. Lacking orthologs in the major model organisms, the pathogenesis of APOL1-associated renal disease and even the intracellular function of APOL1 remain unclear. Several possible pathogenetic mechanisms have been proposed based on overexpression models but the reported findings are often in conflict. Studying subcellular localization of endogenous APOL1 in cultured podocytes and in a transgenic Drosophila model, we detected APOL1 within the endoplasmic reticulum (ER). APOL1-expression in Drosophila induced IRE1-dependent ER stress implying a possible pathogenetic role. APOL1 risk variant expression increased endocytic function of the podocyte-like nephrocytes in fly. APOL1-expressing cells are extruded apically from the epithelial layer of a wing precursor tissue, an effect that was more pronounced for the risk allele. To study the role of APOL1 risk variants for podocyte biology without overexpression, we propose to introduce the risk variant G2 via genome editing homozygously into a line of cultured podocytes (aim1). To attain an approximation of conditions in vivo, podocytes will be differentiated and kept in prolonged culture under exposure to interferon- which is known to enhance endogenous expression of APOL1. A functional analysis of this cell line compared to a control podocyte line without carrying the risk allele will help to distinguish between overexpression artefacts and the relevant mechanisms. To perform a complementary investigation in a rapid in vivo model, we will employ transgenic expression of human APOL1 variants in Drosophila (aim 2). We will study the effects of several APOL1 transgenes in the podocyte-like nephrocytes and the wing disc as an epithelial model. In particular, we will study ER stress signaling and explore the mechanisms underlying the phenotypes of increased nephrocyte function and apical cell extrusion that were specifically observed with the risk variant expression. Finally, using G2-APOL1-induced increase of nephrocyte function as a read-out, we will screen a library of FDA-approved drugs in a whole animal drug screen (aim 3) to identify protective compounds. This work program is likely to elucidate APOL1 function and discover potential therapeutic options.