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.

摘要 严重的进行性肾病（CKD）在黑人患者中比其他人群更常见。 这一人群中非糖尿病性CKD的过度风险部分是由于遗传变异的存在 在APOL 1基因中，这是一些非洲祖先群体所特有的。APOL 1高风险（HR） 基因型，定义为存在两个危险等位基因（G1或G2），与一些特发性 蛋白尿性足细胞病，主要是FSGS。致病机制负责遗传 相关性仍然知之甚少，尽管过度表达系统表明肾脏疾病是由 HR变体依赖性细胞毒性。然而，已发表的数据表明，无论是失去保护和获得有毒 功能通路可能介导APOL相关的肾脏疾病。为了发展关于机制的假设， 我们使用过氧化物酶催化的邻近标记和质量分析来表征G 0和G2空间蛋白质组。 在HeLa细胞中，通过质谱法鉴定具有调节的APOL 1表达的候选APOL 1调节途径。 APOL 1诱导不引起细胞死亡，如通过对死亡机制不可知的方法或通过免疫组织化学方法所测定的。 抗肿瘤特异性测定。G 0和G2空间蛋白质组的组成明显富集于 分泌途径膜和腔蛋白，但它们的蛋白质邻域在高尔基体中分叉， 将G 0和HR-APOL 1加载到RAB 6+囊泡的子集中。G 0和G2空间蛋白质组表明， 这些囊泡的运载物是APOL 1亚型特异性的，G 0而不是G2靠近细胞表面 proteins.使用N-羟基琥珀酰亚胺生物素酯富集方法，我们证明G2表达改变， 具有功能性后果的细胞表面蛋白。干涉反射显微镜显示G2表达 而不是表达G 0的细胞不能维持粘附。重要的是，这些表型在同基因组中重现。 具有G 0、G1和G2基因型的IPSC衍生的足细胞。基于这些数据，我们假设APOL 1 调节足细胞表面基因组以维持足细胞粘附和分化功能。G2无法做到这一点 导致足细胞耗竭和进行性足细胞病。我们提出以下实验来更好地 了解APOL 1参考和肾脏风险变异功能：目的1：使用氨基酸稳定同位素标记 在细胞培养（SILAC）和质谱法表征足细胞表达的表面蛋白质组 在存在和不存在干扰素-γ的情况下，APOL 1-G 0、G1和G2变体。目标2：表征RAB 6A + 后高尔基囊泡区室使用生化和显微镜的方法来定义的差异 RAB 6A+囊泡和相关货物的运输依赖于APOL 1基因型。目标3：证明 RAB 6+囊泡中的APOL 1亚型特异性货物调节足细胞表面组，并定义了 足细胞粘附缺陷的APOL 1依赖性机制