Molecular Mechanisms of Albumin Trafficking in Podocytes

描述（由申请人提供）：慢性肾脏疾病（CKD）是影响约3900万美国人的全国性健康问题。慢性肾脏疾病进展到终末期肾脏疾病与相当大的心血管发病率和死亡率相关。蛋白尿是慢性肾脏疾病的主要特征之一，也是CKD进展的标志。尿液中白蛋白排泄的机制尚不清楚。在正常受试者中，对尿液中白蛋白排泄的抵抗是由完整的肾小球滤过屏障介导的，该屏障由肾小球内皮细胞、肾小球基底膜、足细胞足突和亚足细胞间隙组成。在慢性肾病和其他蛋白尿状态下，这些屏障被破坏，使大量白蛋白逃逸到尿液中。足细胞可摄取白蛋白，但其内吞作用机制尚不清楚。我们的总体假设是，健康的足细胞以极化和受体介导的方式内吞白蛋白，而在蛋白尿状态下，白蛋白的摄取和处理被淹没，导致足细胞毒性和足细胞死亡增加。在特异性目标1中，我们将检查白蛋白在健康足细胞中的内吞作用和降解。具体来说，我们将验证白蛋白内吞作用发生在基底细胞膜上并由巨噬蛋白介导的假设，以及白蛋白降解发生在溶酶体中。在特异性目标2中，我们将检查肾病状态下足细胞中的白蛋白处理。我们假设在重白蛋白尿中，白蛋白摄取紊乱，白蛋白降解受损。具体来说，我们将研究在肾病状态下白蛋白摄取是否不再局限于基膜，白蛋白超载是否导致溶酶体酶的激活和溶酶体完整性的破坏。为了验证我们的假设，我们将在培养的人类足细胞中结合分子生物学和先进的成像技术，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受损和足细胞死亡之间的机制联系。白蛋白毒性引起的足细胞损失增加可能是肾脏疾病进展的一个重要因素，因为足细胞是终末分化的细胞，再生能力低，足细胞损失与进行性肾衰竭密切相关。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理的机制将允许在APOL1风险等位基因足细胞中比较这些机制。我们假设白蛋白在APOL1突变足细胞中起着“第二次打击”的作用，这些突变足细胞具有加速丢失的遗传易感性。此外，确定白蛋白摄取的必要途径可能有助于未来的干预研究，以限制重度白蛋白尿中足细胞对白蛋白的摄取，从而减少足细胞死亡并减缓肾脏疾病的进展。

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.