Podocyte Secreted Proteins

描述（由申请人提供）：PI实验室最近发表的研究显示，在人类和实验性糖皮质激素敏感性肾病综合征中，足细胞中血管生成素样4（Angptl 4）的表达增加。这种蛋白质分泌到肾小球毛细血管袢中导致与肾小球基底膜（GBM）结合，并导致肾病范围选择性蛋白尿、足突弥漫性消失和GBM电荷损失的发展。在实验性微小病变疾病（MCD）中，从足细胞分泌的Angptl 4缺乏通常会在糖基化位点掺入的唾液酸残基。由底物的相对缺乏或唾液酸生物合成途径中的伴随变化引起的这种低唾液分泌状态影响Angptl 4与GBM蛋白的相互作用。在12天的时间段内向具有来自足细胞的Angptl 4的转基因表达的大鼠喂食唾液酸前体ManNAc导致超过40%的白蛋白尿减少，并且显著增加Angptl 4的唾液酸化。我们假设足细胞分泌的Angptl 4与GBM蛋白相互作用以诱导蛋白尿，并且该过程受到Angptl 4唾液酸化状态的强烈影响。在具体目标1中，我们通过研究其掺入N-聚糖和/或O-聚糖中来表征足细胞分泌的Angptl 4的唾液酸化，并在实验MCD中寻找唾液酸合成和掺入途径的变化。在特定目标2中，我们使用体外平板测定法研究了唾液酸化和低唾液酸化Angptl 4与硫酸肝素蛋白聚糖的相互作用，并使用邻近连接测定法研究了其与组织切片中所有GBM蛋白的相互作用。在具体目标3中，我们研究了转录因子ZHX 1对实验MCD中足细胞Angptl 4表达的上调，并提出了研究显性阴性ZHX 1构建体是否可用于降低疾病状态中Angptl 4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。

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.