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

描述(申请人提供)：血管内皮生长因子-A对血管生成是必不可少的，但其非血管作用尚不清楚。过量的血管内皮生长因子-A在糖尿病肾病和肾小球疾病中起致病作用，而糖尿病肾病和肾小球疾病是终末期肾病的主要原因。足细胞血管内皮生长因子-A是形成和维持肾小球滤过屏障所必需的。然而，足细胞血管内皮生长因子-A失调导致蛋白尿的分子机制尚不清楚。我们在体内研究了血管内皮生长因子-A对足细胞表型、细胞间相互作用和肾小球基底膜(GBM)动态平衡的调节作用。在成年小鼠中，足细胞VEGF164的适度过度表达模拟了糖尿病肾病，而在新生小鼠中，它导致了一种微小的变化性疾病。此外，成年小鼠足细胞血管内皮生长因子-A的沉默诱导了涉及肾小球滤过屏障所有成分的戏剧性表型。我们的数据也提出了血管内皮生长因子-A直接作用于足细胞的可能性；这些细胞在体内表达VEGFR2，而血管内皮生长因子-A在体外调节裂隙-横隔膜蛋白的表达。本研究的目的是阐明血管内皮生长因子-A调控足细胞表型和细胞-细胞相互作用的机制，并了解肾小球滤过屏障动态平衡的分子基础。我们的发现导致我们假设：1)足细胞VEGF164适度过表达导致足细胞减少；2)成年小鼠足细胞VEGF164过度表达足以诱导糖尿病肾病表型；3)血管内皮生长因子-A在裂隙-横隔膜信号复合体和基底膜成分中发挥调节作用；4)足细胞分泌血管内皮生长因子-A是一个受调控的过程。为了验证我们的假设，我们将检查转基因小鼠的表型，这些转基因小鼠以四环素调节的方式过度表达或沉默足细胞血管内皮生长因子-A，重点关注它们的可逆性和年龄相关特征。我们确定了足细胞中VEGFR2的相互作用伙伴，并将确定VEGF-A信号是否改变它们的表达、蛋白质-蛋白质相互作用和肌动蛋白结合特性。我们将通过脉冲追逐标记来确定血管内皮生长因子-A的分泌动力学，并检测钙离子和一氧化氮对血管内皮生长因子-A胞吐途径的调节。建议的实验将为糖尿病肾病的发病机制以及控制足细胞表型和血管内皮生长因子-A分泌的信号提供新的和重要的数据。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。公共卫生相关性糖尿病肾病中蛋白尿的分子机制尚不清楚，糖尿病肾病是导致肾功能衰竭的主要原因。这项建议侧重于一种重要的血管生成因子，即血管内皮生长因子(VEGF-A)，在控制肾滤器的结构和行为中的作用。拟议的转基因小鼠实验将提供新的信息，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。

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.