Molecular Mechanisms of Albumin Trafficking in Podocytes

足细胞白蛋白运输的分子机制

• 批准号: 8566186
• 负责人: JUDITH T., BLAINE
• 金额: $ 7.73万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8566186
• 关键词: Affect; Albumins; Albuminuria; Alleles; American; Animal Model; Animals; Apoptosis; Apoptotic; Basal Cell; Biological Process; Cardiovascular system; Cell Death; Cell membrane; Cells; Cessation of life; Chronic Kidney Failure; Data; Degradation Pathway; Development; Disease; Disease Progression; End stage renal failure; Endocytosis; Endothelial Cells; Enzymes; Excretory function; Exposure to; Extravasation; Figs - dietary; Filtration; Foot Process; Foundations; Functional disorder; Future; Genetic Predisposition to Disease; Goals; Grant; Health; Human; Imaging Techniques; Inflammatory; Inflammatory Response; Intervention Studies; K-Series Research Career Programs; Kidney; Kidney Diseases; Kidney Failure; LAMP-1; LDL-Receptor Related Protein 2; Ligands; Link; Lysosomes; Mediating; Membrane; Microscopy; Molecular; Molecular Biology; Morbidity - disease rate; Nephrotic Syndrome; Oxidative Stress; Pathway interactions; Positioning Attribute; Production; Proteins; Renal function; Resistance; Risk; Side; Structure of thyroid parafollicular cell; Techniques; Testing; Toxic effect; Toxin; Tubular formation; Urine; Vacuole; Work; biological adaptation to stress; cytokine; enzyme activity; foot; glomerular basement membrane; glomerular filtration; glomerulosclerosis; in vivo; innovation; mortality; mouse model; mutant; novel; podocyte; public health relevance; receptor; regenerative; slit diaphragm; trafficking; uptake; urinary

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) is a national health problem that affects ~ 39 million Americans. Progression of chronic kidney disease to end stage kidney disease is associated with considerable cardiovascular morbidity and mortality. Albuminuria is one of the key features of chronic kidney disease and a marker of CKD progression. The mechanisms of albumin excretion in the urine are poorly understood. In normal subjects, resistance to albumin excretion in the urine is mediated by an intact glomerular filtration barrier which is comprised of glomerular endothelial cells, the glomerular basement membrane, podocyte foot processes and the sub-podocyte space. In chronic kidney disease and other proteinuric states these barriers are disturbed allowing significant quantities of albumin to escape into the urine. Podocytes can take up albumin but the mechanisms of endocytosis in podocytes are not known. Our overall hypothesis is that healthy podocytes endocytose albumin in a polarized and receptor-mediated manner and that in albuminuric states the uptake and disposal of albumin is overwhelmed, leading to toxicity to the podocyte and increased podocyte death. In Specific Aim 1 we will examine albumin endocytosis and degradation in healthy podocytes. Specifically, we will test the hypothesis that albumin endocytosis occurs at the basal cell membrane and is mediated by megalin and that albumin degradation occurs in the lysosome. In Specific Aim 2 we will examine albumin handling in podocytes in the nephrotic state. We hypothesize that in heavy albuminuria albumin uptake is disordered and albumin degradation is impaired. Specifically, we will examine whether in nephrotic states albumin uptake is no longer confined to the basal membrane and whether albumin overload leads to activation of lysosomal enzymes and disruption of lysosomal integrity. To test our hypotheses we will use a combination of molecular biology and advanced imaging techniques in cultured human podocytes and will extend our findings to a mouse model of nephrotic syndrome. Our proposal is novel in that it proposes a mechanistic link between impaired albumin handling and podocyte death. Increased podocyte loss due to albumin toxicity may be an important factor in kidney disease progression since podocytes are terminally differentiated cells with low regenerative capacity and podocyte loss is strongly correlated with progressive kidney failure. In addition, this proposal will lay the foundation for an R01 submission. Determination of the mechanisms of albumin uptake and disposal in normal podocytes will allow comparison of these mechanisms in APOL1 risk allele podocytes. We hypothesize that albumin acts as a "second hit" in APOL1 mutant podocytes that have a genetic predisposition to accelerated loss. In addition, identification of the pathways necessary for albumin uptake may allow for future intervention studies that limit albumin uptake by podocytes in heavy albuminuria, thereby decreasing podocyte death and slowing kidney disease progression.

描述（由申请人提供）：慢性肾脏疾病（CKD）是影响约3900万美国人的国家健康问题。慢性肾脏疾病到末期肾脏疾病的进展与相当大的心血管发病率和死亡率有关。蛋白尿是慢性肾脏疾病的关键特征之一，也是CKD进展的标志。尿液中白蛋白排泄的机制知之甚少。在正常受试者中，尿液中对白蛋白排泄的抗性是由完整的肾小球滤过屏障介导的，该肾小球滤波屏障由肾小球内皮细胞，肾小球基底膜，足细胞脚部过程和亚细胞增生空间组成。在慢性肾脏疾病和其他蛋白尿状态下，这些障碍受到干扰，使大量白蛋白逃入尿液中。足细胞可以吸收白蛋白，但尚不清楚足细胞中内吞作用的机理。我们的总体假设是，健康的足细胞内吞白蛋白以两极分化和受体介导的方式，在白蛋白状态下，白蛋白的摄取和处置不堪重负，导致对足细胞的毒性和足细胞的毒性增加。在特定目标1中，我们将检查健康足细胞中的白蛋白内吞和降解。具体而言，我们将检验以下假设：白蛋白内吞作用发生在基底细胞膜上，并由梅加林介导，而白蛋白降解发生在溶酶体中。在特定目标2中，我们将检查肾病状态下足细胞中的白蛋白处理。我们假设在沉重的蛋白尿中，白蛋白的摄取是无序的，白蛋白降解受损。具体而言，我们将检查在肾病状态下，白蛋白摄取是否不再局限于基底膜，以及白蛋白的过载是否导致溶酶体酶的激活和溶酶体完整性的破坏。为了检验我们的假设，我们将在培养的人足细胞中使用分子生物学和高级成像技术的组合，并将我们的发现扩展到肾病综合征的小鼠模型。我们的建议是新颖的，因为它提出了白蛋白处理和足细胞死亡受损之间的机械联系。由于小白蛋白毒性导致的足细胞损失增加可能是肾脏疾病进展的重要因素，因为足细胞是末端分化的细胞，其再生能力较低，并且足细胞损失与渐进性肾脏衰竭密切相关。此外，该提案将为R01提交奠定基础。确定正常足细胞中白蛋白摄取和处置机制的确定将允许比较APOL1风险等位基因足细胞中的这些机制。我们假设白蛋白在APOL1突变体足细胞中充当“第二击”，具有加速损失的遗传倾向。此外，鉴定白蛋白摄取所需的途径可能允许将来的干预研究限制在沉重的白蛋白尿尿中的podocytes摄取白蛋白的摄取，从而减少了足细胞死亡和肾脏疾病进展减缓。