Podocyte Secreted Proteins

描述(申请人提供)：PI实验室最近发表的研究表明，在人类和实验性糖皮质激素敏感型肾病综合征的足细胞中，血管生成素样蛋白4(ANGPTL4)的表达增加。这种蛋白分泌到肾小球毛细血管环导致与肾小球基底膜(GBM)结合，导致肾病范围选择性蛋白尿、足突弥漫性消失和GBM电荷损失。在实验性微小病变病(MCD)中，足细胞分泌的ANGPTL4缺乏唾液酸残基，而唾液酸残基通常在糖基化位点结合。由于底物的相对缺乏或唾液酸生物合成途径的改变，这种低唾液状态影响了ANGPTL4与GBM蛋白的相互作用。给足细胞ANGPTL4转基因表达的大鼠喂饲唾液酸前体ManNAc 12天后，蛋白尿减少了40%以上，并显著增加了ANGPTL4的唾液酸化。我们假设足细胞分泌的ANGPTL4与GBM蛋白相互作用诱导蛋白尿，这一过程受ANGPTL4唾液酸化状态的强烈影响。在具体目标1中，我们通过研究足细胞分泌的ANGPTL4与N-糖链和/或O-链的掺入，以及在实验性MCD中唾液酸合成和掺入途径的变化来表征ANGPTL4的唾液酸化。在特定的目标2中，我们用体外平板法研究唾液酸化和低唾液酸化的ANGPTL4与硫酸肝素蛋白多糖的相互作用，并用邻近连接试验研究它们与组织切片中所有GBM蛋白的相互作用。在具体目标3中，我们研究了转录因子ZHX1上调实验性MCD中足细胞ANGPTL4的表达，并提出了研究显性负ZHX1结构是否可以在疾病状态下降低ANGPTL4表达的实验。本申请中提出的实验将有助于设计减少蛋白尿的治疗策略。 公共卫生相关性：该项目将研究血管生成素样蛋白4在蛋白尿和肾小球疾病发病机制中的作用。

DESCRIPTION (provided by applicant): Recent studies published from the PI's laboratory show increased expression of Angiopoietin-like 4 (Angptl4) in podocytes in human and experimental glucocorticoid sensitive nephrotic syndrome. Secretion of this protein into the glomerular capillary loop leads to binding to the glomerular basement membrane (GBM), and results in the development of nephrotic range selective proteinuria, diffuse effacement of foot processes, and loss of GBM charge. Angptl4 secreted from podocytes in experimental minimal change disease (MCD) is deficient in sialic acid residues that would normally be incorporated at glycosylation sites. This hypo-salivated state, caused either by a relative deficiency of substrate or concomitant changes in the sialic acid biosynthetic pathway, influences the interaction of Angptl4 with GBM proteins. Feeding sialic acid precursor ManNAc to rats with transgenic expression of Angptl4 from podocytes over a period of 12 day results in over 40% reduction in albuminuria, and significantly increases sialylation of Angptl4. We hypothesize that podocyte secreted Angptl4 interacts with GBM proteins to induce proteinuria, and this process is strongly influenced by the state of Angptl4 sialylation. In Specific Aim 1, we characterize the sialylation of podocyte secreted Angptl4 by studying its incorporation into N-glycans and / or O-glycans, and looking for changes in the sialic acid synthesis and incorporation pathway in experimental MCD. In Specific Aim 2, we study the interaction of sialylated and hypo-sialylated Angptl4 with heparin sulfate proteoglycans using in vitro plate assays, and with all GBM proteins in tissue sections using the Proximity Ligation Assay. In Specific Aim 3, we investigate the upregulation of podocyte Angptl4 expression in experimental MCD by transcriptional factor ZHX1, and propose experiments to study whether dominant negative ZHX1 constructs can be used to reduce Angptl4 expression in disease states. The experiments proposed in this application will help in the design of therapeutic strategies for the reduction of proteinuria. PUBLIC HEALTH RELEVANCE: This project will investigate the role of Angiopoietin-like 4 in the pathogenesis of proteinuria and glomerular disease.