Podocyte-specific Rap1 agonism for treatment of glomerular disease

项目摘要/摘要： 蛋白尿肾病的发病率正在增加，全世界的病例远远超过5亿例。 在过去的二十年里，已经变得明显的是，被称为足细胞的特殊肾脏细胞调节 肾脏滤过，在所有形式的蛋白尿疾病中都会受到损害，无论其病因如何。尽管如此， 保护足细胞和减缓慢性肾脏疾病进展的有针对性的有效疗法是 完全缺乏。在此之前，我们曾报道过小GTP酶Rap1调节基础 足细胞中的生物过程通过在不活跃的GDP结合形式和活跃的GTP结合形式之间循环。 我们证明足细胞Rap1的激活状态受许多上游因素控制，两者都 积极和消极，这两个因素汇聚在一起，控制与GTP和GDP绑定的形式的比例。最重要的 这些上游调节因子的作用被人类家族性肾病综合征的存在所强调 由其中几个基因的突变引起，包括新发现的功能突变的获得 负性Rap1调控因子Rap1GAP。这些突变中的每一种都有共同之处，都会导致亲属 足细胞RAP1-GTP耗尽。在当前的提案中，我们证明了足细胞Rap1-GTP的水平 在包括糖尿病肾病(DKD)在内的人类肾小球疾病中也会减少。 足细胞Rap1激活水平的提高在基因或药物上保护足细胞 短期损伤模型。通过针对足细胞特定的上游RAP1调节通路，我们 开发新的RAP1激动剂化合物，可以激活足细胞中的RAP1，但不能激活其他类型的细胞。 这种方法允许足细胞特异性的药理学Rap1激活，并避免了潜在的 全身毒性。目前拨款的目标是探索RAP1的治疗潜力。 足细胞疾病中的激动剂，包括DKD。我们通过三个具体目标来实现这一点：i)测试 利用新技术研究增强足细胞Rap1活性是否会减轻慢性肾小球损伤 可诱导的足细胞特异性成分活性Rap1转基因小鼠。二)描述肾小球的特征 表达双错义Rap1GAP的新型Rap1GAP敲入小鼠模型的表型 然后利用两种新的小鼠模型来阐明特定的RAP1 建立足细胞损伤易感性与保护性的下游效应器。三)合成 足细胞靶向RAP1激动剂化合物及其修复足细胞损伤能力的筛选 转基因RAP1-GTP转基因斑马鱼。先导化合物的药效将在小鼠身上进行测试 包括DKD在内的肾脏疾病模型。我们目前的先导化合物BT-529是一种Rap1-Rap1GAP 相互作用拮抗剂，诱导足细胞激活RAP1，但不诱导其他肾脏细胞激活 极大地保护培养的足细胞免受伤害。总体而言，这项工作为急需的 针对肾脏疾病的足细胞靶向新疗法。

Project Summary/Abstract: The incidence of proteinuric kidney diseases are increasing with well over 500 million cases worldwide. Over the last two decades, it has become clear that specialized kidney cells, called podocytes, regulate kidney filtration and are injured in all forms of proteinuric diseases regardless of etiology. Despite this, targeted effective therapies that protect podocytes and slow chronic kidney disease progression are completely lacking. Previously, we reported that the small GTPase Rap1 regulates fundamental biological processes in podocytes by cycling between inactive GDP-bound and active GTP-bound forms. We demonstrate that the podocyte Rap1 activation state is controlled by many upstream factors, both positive and negative, that converge to control the ratio of GTP- and GDP-bound forms. The essential role of these upstream regulators is emphasized by the presence of human familial nephrotic syndrome caused by mutations to several of these genes, including newly identified gain of function mutations in the negative Rap1 regulator, Rap1GAP. Each of these mutations has in common that they cause relative depletion of podocyte Rap1-GTP. In the current proposal, we show that levels of podocyte Rap1-GTP are also diminished in human glomerular diseases including in diabetic kidney disease (DKD). Augmenting levels of podocyte Rap1 activation genetically or pharmacologically protects podocytes in short term injury models. By targeting podocyte-specific upstream Rap1 regulatory pathways, we are developing novel Rap1 agonist compounds that activate Rap1 in podocytes, but not in other cell types. Such an approach allows for podocyte-specific pharmacological Rap1 activation and avoids potential systemic toxicities. The goals of the current grant are to explore the therapeutic potential of Rap1 agonists in podocyte diseases including in DKD. We accomplish this via three specific aims: i) Test whether enhanced podocyte Rap1 activation will mitigate chronic glomerular injury utilizing novel inducible podocyte-specific constitutively active Rap1 transgenic mice. ii) Characterize the glomerular phenotype of a novel Rap1GAP knock-in mouse model that expresses a double missense Rap1GAP human disease-associated variant and then utilize both novel mouse models to elucidate specific Rap1 downstream effectors that establish podocyte injury susceptibility versus protection. iii) Synthesize podocyte-targeted Rap1 agonist compounds and screen their ability to rescue podocyte injury in transgenic Rap1-GTP deficient zebrafish. The efficacy of lead compounds will be tested in mouse models of kidney disease including in DKD. Our current lead compound, BT-529, a Rap1-Rap1GAP interaction antagonist, induces Rap1 activation in podocytes but not in other kidney cells and dramatically protects cultured podocytes from injury. Overall, this work sets the stage for urgently needed new podocyte-targeted therapies for kidney diseases.