Pathogenesis and therapeutic options of APOL1-associated renal diseasese

APOL1相关肾脏疾病的发病机制和治疗选择

• 批准号: 440399433
• 负责人: Privatdozent Dr. Tobias Hermle
• 金额: --
• 依托单位: Klinik für Innere Medizin IV - Nephrologie und Allgemeinmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/440399433?language=en
• 关键词: Pathogenesis; therapeutic; options; APOL1; associated

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.

终末期肾病在非裔美国人中的发病率大约是欧洲血统美国人的三倍。这种差异不能用社会经济因素来解释，但大多数过度风险可归因于APOL 1基因的两种风险变体。纯合子携带者有超过15%的终生风险发展为衰弱性肾脏疾病，如局灶节段性肾小球硬化症（FSGS）和高血压相关性肾脏疾病。没有具体的治疗方法。杂合携带者可以免受昏睡病的影响，这使得风险变异在非洲血统人群中极其常见，全球有数百万纯合携带者面临风险。由于在主要模式生物中缺乏直系同源物，APOL 1相关肾病的发病机制甚至APOL 1的细胞内功能仍不清楚。已经提出了几种可能的发病机制的基础上过表达模型，但报道的结果往往是相互矛盾的。研究内源性APOL 1在培养足细胞和转基因果蝇模型中的亚细胞定位，我们在内质网（ER）中检测到APOL 1。APOL 1在果蝇中的表达诱导IRE 1依赖的ER应激，这意味着可能的致病作用。APOL 1风险变体表达增加果蝇足细胞样肾细胞的内吞功能表达APOL 1的细胞从翅膀前体组织的上皮层顶部挤出，这种效应对于风险等位基因更为明显。为了研究APOL 1风险变体在不过度表达的情况下对足细胞生物学的作用，我们建议通过基因组编辑将风险变体G2同源地引入到培养的足细胞系（aim 1）中。为了获得体内条件的近似值，将足细胞分化并在暴露于干扰素-β的情况下保持在延长的培养物中，已知干扰素-β增强APOL 1的内源性表达。与不携带风险等位基因的对照足细胞系相比，该细胞系的功能分析将有助于区分过表达伪像和相关机制。为了在快速体内模型中进行补充研究，我们将在果蝇中采用人APOL 1变体的转基因表达（目的2）。 我们将研究几种APOL 1转基因在足细胞样肾细胞和翼盘上皮模型中的作用。特别是，我们将研究ER应激信号传导，并探索肾细胞功能增加和顶端细胞挤出的表型的机制，这些表型是在风险变体表达中特别观察到的。最后，使用G2-APOL 1诱导的肾细胞功能增加作为读数，我们将在整个动物药物筛选中筛选FDA批准的药物库（目的3）以鉴定保护性化合物。这项工作计划可能会阐明APOL 1的功能，并发现潜在的治疗选择。