Functions of VEGF-A in podocytes:implications for renal disease

足细胞中 VEGF-A 的功能：对肾脏疾病的影响

• 批准号: 7822694
• 负责人: Alda Tufro
• 金额: $ 34.82万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7822694
• 关键词: Actins; Address; Adult; Angiogenic Factor; Behavior; Binding; Blood Vessels; Cell Communication; Cell Line; Cell-Cell Adhesion; Cells; Child; Complex; Data; Defect; Development; Diabetic Nephropathy; Disease; Down-Regulation; End stage renal failure; Endothelial Cells; Exocytosis; Foot Process; Genetic; Growth Factor Overexpression; Homeostasis; Human; In Vitro; Kidney; Kidney Diseases; Kidney Failure; Kinetics; Label; Maintenance; Mediating; Molecular; Mus; Newborn Infant; Nitric Oxide; Pathogenesis; Pathway interactions; Phenotype; Physiologic pulse; Physiological; Play; Process; Property; Proteinuria; Regulation; Renal glomerular disease; Role; Signal Transduction; Structure; Testing; Tetanus Helper Peptide; Tetracyclines; Time; Transgenes; Transgenic Mice; Tumor Angiogenesis; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; age related; angiogenesis; autocrine; base; cell type; design; glomerular basement membrane; glomerular filtration; improved; in vivo; innovation; nephrin; novel; overexpression; paracrine; podocyte; protein expression; protein protein interaction; public health relevance; research study; response; slit diaphragm; treatment strategy

DESCRIPTION (provided by applicant): VEGF-A is essential for angiogenesis but its non-vascular roles are not well understood. Excessive VEGF-A plays a pathogenic role in diabetic nephropathy and glomerular disorders, the leading causes of end-stage renal disease. Podocyte VEGF-A is required for the development and maintenance of the glomerular filtration barrier. However, the molecular mechanisms whereby the dysregulation of podocyte VEGF-A induces proteinuria are unknown. We determined that VEGF-A regulates podocyte phenotype, cell-cell interactions and glomerular basement membrane (GBM) homeostasis in vivo. Moderate podocyte VEGF164 overexpression in adult mice mimics diabetic nephropathy, whereas in newborn mice it causes a minimal change-like disease. Moreover, podocyte VEGF-A silencing in adult mice induces a dramatic phenotype involving all components of the glomerular filtration barrier. Our data also raise the possibility that VEGF-A acts directly in podocytes; these cells express VEGFR2 in vivo, and VEGF-A regulates slit-diaphragm protein expression in vitro. The objectives of this proposal are to elucidate the mechanisms of VEGF-A control of podocyte phenotype and cell-cell interactions, and to understand the molecular basis of glomerular filtration barrier homeostasis. Our findings led us to hypothesize that 1) moderate overexpression of podocyte VEGF164 induces podocyte effacement; 2) podocyte VEGF164 overexpression in adult mice is sufficient to induce a diabetic nephropathy phenotype; 3) VEGF-A plays a role in the regulation of the slit-diaphragm signaling complex and GBM composition; 4) VEGF-A exocytosis in podocytes is a regulated process. To test our hypotheses, we will examine phenotypes from transgenic mice that overexpress or silence podocyte VEGF-A in a tetracycline regulated manner, focusing on their reversibility, and age-dependent features. We identified VEGFR2 interacting partners in podocytes and will determine whether VEGF-A signaling alters their expression, protein-protein interactions and actin binding properties. We will define the kinetics of VEGF-A secretion by pulse-chase labeling, and examine the regulation of VEGF-A exocytic pathway by Ca+2 and nitric oxide. The proposed experiments should provide novel and important data on the pathogenesis of diabetic nephropathy, and on the signals that control podocyte phenotype and VEGF-A exocytosis. Understanding the molecular basis of renal diseases should enable us to design new strategies for treatment. PUBLIC HEALTH RELEVANCE The molecular mechanisms of proteinuria in diabetic kidney disease, the leading cause of renal failure, are poorly understood. This proposal focuses on the role of an important angiogenic factor called vascular endothelial growth factor (VEGF-A), in the control of the structure and behavior of the kidney filters. The proposed experiments in transgenic mice should provide new information to improve our understanding of diabetic nephropathy in adults and kidney diseases in children, as well as enable us to design new strategies for treatment of diabetic nephropathy and glomerular diseases.

描述（由申请人提供）：VEGF-A 对于血管生成至关重要，但其非血管作用尚不清楚。过量的 VEGF-A 在糖尿病肾病和肾小球疾病中发挥致病作用，而肾小球疾病是终末期肾病的主要原因。足细胞 VEGF-A 是肾小球滤过屏障发育和维持所必需的。然而，足细胞 VEGF-A 失调引起蛋白尿的分子机制尚不清楚。我们确定 VEGF-A 在体内调节足细胞表型、细胞间相互作用和肾小球基底膜 (GBM) 稳态。成年小鼠中足细胞 VEGF164 中度过度表达类似于糖尿病肾病，而在新生小鼠中它会引起类似微小病变的疾病。此外，成年小鼠足细胞 VEGF-A 沉默会诱导涉及肾小球滤过屏障所有组成部分的显着表型。我们的数据还提出了 VEGF-A 直接作用于足细胞的可能性；这些细胞在体内表达 VEGFR2，而 VEGF-A 在体外调节狭缝隔膜蛋白的表达。该提案的目的是阐明 VEGF-A 控制足细胞表型和细胞间相互作用的机制，并了解肾小球滤过屏障稳态的分子基础。我们的研究结果使我们做出假设：1）足细胞 VEGF164 的中度过度表达会诱导足细胞消失； 2）成年小鼠足细胞VEGF164过度表达足以诱发糖尿病肾病表型； 3）VEGF-A在调节裂隙隔膜信号复合物和GBM组成中发挥作用； 4)足细胞中VEGF-A的胞吐作用是一个受调控的过程。为了检验我们的假设，我们将检查以四环素调节方式过度表达或沉默足细胞 VEGF-A 的转基因小鼠的表型，重点关注其可逆性和年龄依赖性特征。我们鉴定了足细胞中的 VEGFR2 相互作用伙伴，并将确定 VEGF-A 信号传导是否改变其表达、蛋白质-蛋白质相互作用和肌动蛋白结合特性。我们将通过脉冲追踪标记定义 VEGF-A 分泌动力学，并检查 Ca+2 和一氧化氮对 VEGF-A 胞吐途径的调节。拟议的实验应该为糖尿病肾病的发病机制以及控制足细胞表型和 VEGF-A 胞吐作用的信号提供新颖且重要的数据。了解肾脏疾病的分子基础应该使我们能够设计新的治疗策略。公众健康相关性 糖尿病肾病（肾衰竭的主要原因）中蛋白尿的分子机制尚不清楚。该提案重点关注一种重要的血管生成因子，即血管内皮生长因子（VEGF-A）在控制肾过滤器的结构和行为中的作用。拟议的转基因小鼠实验应该提供新的信息，以提高我们对成人糖尿病肾病和儿童肾脏疾病的了解，并使我们能够设计治疗糖尿病肾病和肾小球疾病的新策略。