Mechanisms of Kidney Diseases Associated With APOL1 Variation

APOL1 变异相关肾脏疾病的机制

• 批准号: 10607630
• 负责人: Leslie A Bruggeman
• 金额: $ 70.13万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-02 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607630
• 关键词: APOL1 gene; Adhesions; African; African ancestry; Amino Acids; Apoptosis; Biochemical; Biological Assay; Biotin; Biotinylation; Black Populations; Black race; Cell Culture Techniques; Cell Death; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Cessation of life; Chronic Kidney Failure; Data; Defect; Disease; Donor person; Drosophila genus; Ectopic Expression; Environment; Esters; European; Family; Focal Adhesions; Focal and Segmental Glomerulosclerosis; Genetic; Genotype; Golgi Apparatus; Haplotypes; Hela Cells; Human; Inflammatory; Injury; Interferon Type II; Interferons; Kidney; Kidney Diseases; Label; Mass Spectrum Analysis; Mediating; Membrane; Methods; Microscopic; Microscopy; Modeling; Morphology; Mus; Neighborhoods; Organoids; Pathogenicity; Pathway interactions; Patients; Peroxidases; Phenotype; Population; Prevalence; Protein Isoforms; Proteins; Proteome; Proteomics; Publishing; Risk; Stable Isotope Labeling; Stress; Structural Racism; Surface; System; Techniques; Testing; Toxic effect; Translating; Variant; Vesicle; Weight; Work; Zebrafish; black patient; candidate identification; caucasian American; cost; cytotoxicity; disorder risk; experimental study; gain of function; genetic association; genetic variant; high risk; high risk population; human disease; induced pluripotent stem cell; loss of function; mutant; non-diabetic; novel; overexpression; podocyte; promoter; protein complex; risk variant; trafficking

ABSTRACT Severe, progressive kidney diseases (CKD) are more common among Black patients than in other populations. The excess risk for non-diabetic CKD in this population is explained, in part, by the presence of genetic variants in the APOL1 gene, which are unique to some African ancestral populations. The APOL1 high risk (HR) genotype, defined as the presence of two risk alleles (G1 or G2), is strongly associated with some idiopathic proteinuric podocytopathies, primarily FSGS. The pathogenic mechanisms responsible for the genetic association remain poorly understood, although over-expression systems suggest kidney diseases result from HR variant-dependent cytotoxicity. However published data suggest that both loss-of protection and gain of toxic function pathways may mediate APOL-associated kidney diseases. To develop hypotheses about mechanisms, we characterized the G0 and G2 spatial proteomes using peroxidase-catalyzed proximity labelling and mass spectrometry to identify candidate APOL1-regulated pathways in HeLa cells with regulated APOL1 expression. APOL1 induction did not cause cell death as assayed by a method agnostic to death mechanism or by an apoptosis-specific assay. The compositions of the G0 and G2 spatial proteomes are markedly enriched in secretory pathway membrane and luminal proteins but their protein neighborhoods diverge in the Golgi where G0 and HR-APOL1s are loaded into a subset of RAB6+ vesicles. The G0 and G2 spatial proteomes suggest that cargos of these vesicles are APOL1-isoform specific, with G0 but not G2 being in proximity of cell surface proteins. Using a N-hydroxysuccinimide biotin ester enrichment method, we demonstrated G2 expression altered cell surface proteins with functional consequences. Interference reflectance microscopy suggests G2 expressing but not G0 expressing cells fail to maintain adhesion. Importantly, these phenotypes are recapitulated in isogenic IPSC-derived podocytes with G0, G1 and G2 genotypes. Based on these data, we hypothesize that APOL1 regulates podocyte surfaceome to maintain podocyte adhesion and differentiated functions. G2 fails to do this resulting in podocyte depletion and progressive podocytopathy. We propose the following experiments to better understand APOL1 reference and kidney risk variant function: Aim 1: Use stable isotope labelling by amino acids in cell culture (SILAC) and mass spectrometry to characterize the surface proteome of podocytes expressing APOL1-G0, G1 and G2 variants in the presence and absence of Interferon-γ. Aim 2: Characterize the RAB6A+ post-Golgi vesicular compartment using biochemical and microscopic methods to define the differential trafficking of RAB6A+ vesicles and the associated cargo that is dependent upon APOL1 genotype. Aim 3: Prove that the APOL1 isoform-specific cargos in RAB6+ vesicles regulate the podocyte surfaceome and define the APOL1–dependent mechanisms for the podocyte adhesion defect.

抽象的 严重的进行性肾脏疾病（CKD）在黑人患者中比其他人群更常见。 该人群中非糖尿病 CKD 风险过高的部分原因是基因变异的存在 存在于 APOL1 基因中，这是某些非洲祖先人群所特有的 APOL1 高风险 (HR)。 基因型，定义为两个风险等位基因（G1 或 G2）的存在，与某些特发性密切相关 蛋白尿性足细胞病，主要是 FSGS 的致病机制与遗传有关。 尽管过度表达系统表明肾脏疾病是由 HR 变异依赖性细胞毒性然而，已发表的数据表明，保护的丧失和毒性的增加。 功能途径可能介导 APOL 相关的肾脏疾病。 我们使用过氧化物酶催化的邻近标记和质量来表征 G0 和 G2 空间蛋白质组 光谱法来鉴定 HeLa 细胞中 APOL1 表达受调节的候选 APOL1 调节途径。 通过与死亡机制无关的方法或通过 G0 和 G2 空间蛋白质组的组成显着富集。 分泌途径膜和腔蛋白，但它们的蛋白质邻域在高尔基体中存在分歧 G0 和 HR-APOL1 被加载到 RAB6+ 囊泡的子集中。G0 和 G2 空间蛋白质组表明： 这些囊泡的货物是 APOL1 同工型特异性的，G0 但不是 G2 靠近细胞表面 使用 N-羟基琥珀酰亚胺生物素酯富集方法，我们证明 G2 表达发生改变。 具有功能后果的细胞表面蛋白。干涉反射显微镜表明 G2 表达。 但表达 G0 的细胞则不能维持粘附。重要的是，这些表型在同基因中得到了概括。 IPSC 衍生的具有 G0、G1 和 G2 基因型的足细胞 根据这些数据，我们使用了 APOL1。 调节足细胞表面组以维持足细胞粘附，而 G2 无法做到这一点。 导致足细胞耗竭和进行性足细胞病，我们建议进行以下实验以更好地进行。 了解 APOL1 参考和肾脏风险变异功能：目标 1：使用氨基酸稳定同位素标记 在细胞培养 (SILAC) 和质谱法中表征足细胞表达的表面蛋白质组 存在和不存在干扰素-γ 时的 APOL1-G0、G1 和 G2 变体 目标 2：表征 RAB6A+。 使用生化和显微镜方法确定高尔基体后囊泡区室的差异 RAB6A+ 囊泡和依赖于 APOL1 基因型的相关货物的运输 目标 3：证明。 RAB6+ 囊泡中的 APOL1 亚型特异性货物调节足细胞表面组并定义 足细胞粘附缺陷的 APOL1 依赖性机制。