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

描述（由申请人提供）：VEGF-A是血管生成所必需的，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起致病作用，这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失调诱导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-细胞相互作用和肾小球基底膜（GBM）稳态。足细胞VEGF 164在成年小鼠中的中度过表达模拟糖尿病肾病，而在新生小鼠中，它导致最小的变化样疾病。此外，成年小鼠足细胞VEGF-A沉默诱导了一个戏剧性的表型，涉及肾小球滤过屏障的所有组件。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VEGFR 2，而VEGF-A在体外调节裂膜蛋白的表达。本研究的目的是阐明VEGF-A调控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假设：1）足细胞VEGF 164的中度过表达诱导足细胞消失; 2）成年小鼠足细胞VEGF 164的过表达足以诱导糖尿病肾病表型; 3）VEGF-A在裂隙-隔膜信号复合物和GBM组成的调节中起作用; 4）足细胞中VEGF-A的胞吐是一个受调节的过程。为了验证我们的假设，我们将研究转基因小鼠的表型，过表达或沉默足细胞VEGF-A在四环素调节的方式，专注于其可逆性，和年龄依赖性的功能。我们确定了VEGFR 2在足细胞中的相互作用伙伴，并将确定VEGF-A信号是否改变其表达，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来确定VEGF-A分泌的动力学，并检查Ca+2和一氧化氮对VEGF-A胞外途径的调节。这些实验将为糖尿病肾病的发病机制以及控制足细胞表型和VEGF-A胞吐的信号提供新的重要数据。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。糖尿病肾病是肾功能衰竭的主要原因，其蛋白尿的分子机制尚不清楚。该建议的重点是一种重要的血管生成因子，称为血管内皮生长因子（VEGF-A），在控制肾脏过滤器的结构和行为中的作用。在转基因小鼠中的拟议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理解，以及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.