The Exocyst in Ciliogenesis and Acute Kidney Injury

纤毛发生和急性肾损伤中的胞外囊

• 批准号: 10164562
• 负责人: JOSHUA H LIPSCHUTZ
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10164562
• 关键词: 3-Dimensional; Acute; Acute Kidney Tubular Necrosis; Acute Renal Failure with Renal Papillary Necrosis; Admission activity; Affect; Alanine; Area; Autosomal Dominant Polycystic Kidney; Blood Circulation; Cell Culture Techniques; Cell Death; Cell Line; Cell Polarity; Cell Survival; Cell physiology; Cells; Chronic Kidney Failure; Cilia; Collagen; Complex; Critical Pathways; Data; Dependovirus; Development; Dialysis procedure; Docking; Drug Design; EGF gene; ERBB2 gene; Epidermal Growth Factor Receptor; Gel; Gene Delivery; Goals; Homeostasis; Hospitalization; Hospitals; Human; Human Cloning; Injury; Intensive Care Units; Ischemia; Kidney; Knock-out; Knowledge; Ligands; Link; MAPK1 gene; Mediating; Mediator of activation protein; Medical; Membrane Proteins; Messenger RNA; Metabolism; Mitochondria; Mitogen-Activated Protein Kinase Inhibitor; Mitogen-Activated Protein Kinases; Monomeric GTP-Binding Proteins; Morbidity - disease rate; Mus; Mutant Strains Mice; Mutate; Mutation; Organelles; Pathogenesis; Pathway interactions; Patients; Process; Proline; Proteins; Recovery; Recovery of Function; Renal tubule structure; Reperfusion Injury; Reperfusion Therapy; Research; Sagittaria; Secretory Vesicles; Site; South Carolina; Speed; Supportive care; Technology; Testing; Tubular formation; Veterans; Viral; Zebrafish; cell injury; ciliopathy; cilium biogenesis; erbB-2 Receptor; experimental study; in vivo; injury recovery; insight; kidney cell; knock-down; mitochondrial dysfunction; mortality; mutant; novel; novel strategies; overexpression; oxidative damage; prevent; protective effect; protein transport; receptor; receptor sensitivity; repaired; small molecule inhibitor; therapeutic target; trafficking; transduction efficiency; vector

Acute kidney injury (AKI) is a significant and increasing problem. Medical management currently consists of supportive care, with dialysis implemented for the most severe cases; however, morbidity and mortality remain very high. A major reason for the lack of available treatments for AKI is a gap in the knowledge of how kidney tubule cells recover from AKI, which has, therefore, limited possible approaches for treatment. Identifying, a therapeutic target and pathway would meet a major unmet need by allowing for rational drug design. The goal here is to determine whether the highly conserved eight-protein exocyst trafficking complex, and particularly the central Sec10 (aka Exoc5) component, can be used to enhance recovery, and/or prevent injury, following AKI. After renal tubule cell injury, there is initial loss of cell polarity, followed by cell death and sloughing of cells into the lumen, then spreading and dedifferentiation of viable cells to cover the denuded area, with proliferation, differentiation, and reestablishment of cell polarity. The polarity, or secretory, pathway is crucial for AKI recovery, and cell function, and the exocyst is known for mediating the targeting and docking of secretory vesicles carrying membrane proteins. Over the past twenty years, we showed that the mitogen-activated protein kinase (MAPK) pathway regulates tubulogenesis. We also showed that the exocyst, especially the Sec10 component, is centrally involved in renal ciliogenesis and tubulogenesis. Specifically, Sec10 knockdown inhibited, and Sec10 overexpression increased, ciliogenesis and tubulogenesis. These distinct research areas recently converged, as we showed that Sec10 speeded recovery from oxidative damage, an ischemia-like injury, by activating MAPK. We have now generated Sec10fl/fl mice, and have preliminary data showing Sec10 deletion in murine proximal tubules worsens ischemia and reperfusion (I/R) injury, and inhibits repair. Furthermore, site-specific mutation of the highly-conserved VxPx ciliary targeting sequence in human SEC10 inhibits tubulogenesis in cells grown in 3D collagen gels, and prevents the rescue of sec10 mutant zebrafish. The proposed experiments will test the overall hypothesis that Sec10 activates the MAPK pathway, through the EGF receptor, to prevent injury and/or enhance renal recovery following AKI, that this effect is mediated via primary cilia, and that Sec10 is a therapeutic target. Accordingly, we will investigate how Sec10 increases EGF receptor sensitivity, which activates MAPK to enhance recovery from injury (Aim 1.1). We will then investigate how Sec10 and the exocyst are involved in mitochondrial function. A critical pathway that has been identified in AKI is alterations in primary tubular metabolism, which secondarily affect the regional circulation through decreased levels of ATP and mitochondrial dysfunction. Mitochondria are also involved in ADPKD, the most common ciliopathy, suggesting a possible link between cilia and mitochondria. Here we will investigate this novel pathway, and the possibility that the exocyst could be the mediator between cilia and mitochondria function, possibly by differential protein trafficking regulated by different small GTPases (Aim 1.2). We will test if Sec10 protection following AKI is mediated via primary cilia by determining in mice if proximal tubule-specific knockout of Ift88, a protein necessary for ciliogenesis, worsens injury and prevents recovery following I/R (Aim 2.1). If cilia appear to be centrally involved, we will confirm this using our newly-generated Sec10 ciliary targeting sequence mutant mice and I/R injury (Aim 2.2). Regardless of ciliary involvement, we will confirm that the MAPK pathway is involved in Sec10- mediated protection from I/R injury in mice using small molecule inhibitors. We will then obtain proof of principle that Sec10 can enhance recovery, and/or prevent injury, by using our newly-generated inducible Sec10- overexpressing mice and performing I/R injury (Aim 3.1). Finally, we will determine if Sec10 gene delivery can prevent injury, and/or enhance recovery, using viral and non-viral delivery of Sec10 in vivo prior to, and after, I/R (Aim 3.2). Successful completion of these experiments will provide novel mechanistic insights into AKI pathogenesis and recovery, and have a major impact on the development of new approaches to treat AKI.

急性肾损伤（阿基）是一个重要且日益严重的问题。医疗管理目前包括 支持性治疗，对最严重的病例进行透析;然而， 非常高。缺乏阿基可用治疗方法的一个主要原因是对肾脏如何影响AKI的认识存在空白。 肾小管细胞从阿基中恢复，因此限制了可能的治疗方法。识别，a 治疗靶点和途径将通过允许合理的药物设计来满足主要的未满足的需求。目标 这是为了确定是否高度保守的八蛋白外囊运输复合物，特别是 中心Sec 10（aka Exoc 5）组分，可用于增强阿基后的恢复和/或预防损伤。 肾小管细胞损伤后，最初细胞极性丧失，随后细胞死亡和脱落， 管腔，然后活细胞扩散和去分化以覆盖裸露区域，伴随增殖， 分化和细胞极性的重建。极性或分泌途径对于阿基恢复至关重要， 和细胞功能，外囊是已知的介导的靶向和对接的分泌囊泡携带 膜蛋白在过去的二十年里，我们发现丝裂原活化蛋白激酶（MAPK） pathway调节tubulogenesis管发生。我们还表明，外囊，特别是Sec 10组分，是中央的， 参与肾纤毛发生和肾小管发生。具体而言，Sec 10敲低抑制，Sec 10敲低抑制， 过度表达增加，纤毛发生和小管发生。这些不同的研究领域最近融合， 我们发现Sec 10通过激活MAPK加速了氧化损伤（一种缺血样损伤）的恢复。 我们现在已经产生了Sec 10 fl/fl小鼠，并且初步数据显示在小鼠近端中Sec 10缺失， 肾小管抑制缺血再灌注（I/R）损伤，抑制修复。此外， 人SEC 10中高度保守的p53 Px纤毛靶向序列抑制生长在 3D胶原蛋白凝胶，并阻止救援sec 10突变斑马鱼。拟议的实验将测试 Sec 10通过EGF受体激活MAPK通路以防止损伤的总体假设 和/或增强阿基后的肾恢复，这种作用是通过初级纤毛介导的，并且Sec 10 是一个治疗目标。因此，我们将研究Sec 10如何增加EGF受体的敏感性， 激活MAPK以促进损伤的恢复（目的1.1）。然后我们将研究Sec 10和外囊 与线粒体功能有关。在阿基中已经确定的一个关键途径是原发性高血压的改变。 肾小管代谢，通过降低ATP水平， 线粒体功能障碍线粒体也参与了ADPKD，最常见的纤毛病变，这表明 纤毛和线粒体之间的联系在这里，我们将研究这一新的途径，以及这种可能性， 外囊可能是纤毛和线粒体功能之间的中介体，可能是通过差异蛋白实现的 受不同的小型全球贸易点管制的贩运活动（目标1.2）。我们将测试阿基后的Sec 10保护是否 通过在小鼠中确定是否近端小管特异性敲除Ift 88（一种必需的蛋白质）， 对于纤毛发生，抑制损伤并阻止I/R后的恢复（目的2.1）。如果纤毛出现在 我们将使用我们新产生的Sec 10纤毛靶向序列突变小鼠和I/R 伤害（目标2.2）。无论睫状体参与，我们将证实MAPK途径参与Sec 10- 使用小分子抑制剂在小鼠中介导的I/R损伤保护。然后我们将获得原理证明 Sec 10可以通过使用我们新产生的诱导型Sec 10来增强恢复和/或预防损伤， 过表达小鼠和进行I/R损伤（目的3.1）。最后，我们将确定Sec 10基因递送是否可以 在I/R之前和之后使用Sec 10的病毒和非病毒体内递送预防损伤和/或促进恢复 (Aim 3.2）。这些实验的成功完成将为阿基提供新的机制见解 发病机制和恢复，并对治疗阿基的新方法的开发产生重大影响。