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（AKI）是一个严重且日益严重的问题。医疗管理目前包括 支持治疗，对最严重的病例进行透析；然而，发病率和死亡率仍然 非常高。缺乏 AKI 可用治疗方法的一个主要原因是对肾脏如何发挥作用的认识存在差距。 肾小管细胞从 AKI 中恢复，因此限制了可能的治疗方法。识别，一个 治疗靶点和途径将通过合理的药物设计来满足未满足的主要需求。目标 这里的目的是确定高度保守的八蛋白外囊运输复合体，特别是 中央 Sec10（又名 Exoc5）成分，可用于在 AKI 后促进恢复和/或预防损伤。 肾小管细胞损伤后，细胞极性最初丧失，随后细胞死亡并脱落 管腔，然后活细胞扩散和去分化以覆盖裸露区域，并进行增殖， 分化和细胞极性的重建。极性或分泌途径对于 AKI 恢复至关重要， 和细胞功能，并且外囊因介导携带的分泌囊泡的靶向和对接而闻名 膜蛋白。在过去的二十年里，我们发现丝裂原激活蛋白激酶（MAPK） 途径调节肾小管发生。我们还表明，外囊，特别是 Sec10 成分，集中在 参与肾纤毛发生和肾小管发生。具体来说，Sec10 敲低受到抑制，并且 Sec10 过度表达增加、纤毛发生和管状发生。这些不同的研究领域最近融合在一起，如 我们发现 Sec10 通过激活 MAPK 来加速氧化损伤（一种缺血样损伤）的恢复。 我们现在已经生成了 Sec10fl/fl 小鼠，并有初步数据显示小鼠近端 Sec10 缺失 肾小管会加重缺血和再灌注 (I/R) 损伤，并抑制修复。此外，位点特异性突变 人 SEC10 中高度保守的 VxPx 纤毛靶向序列抑制生长在 3D 胶原蛋白凝胶，并阻止 sec10 突变斑马鱼的救援。拟议的实验将测试 总体假设 Sec10 通过 EGF 受体激活 MAPK 通路，以防止损伤 和/或增强 AKI 后的肾脏恢复，这种作用是通过初级纤毛介导的，并且 Sec10 是一个治疗靶点。因此，我们将研究 Sec10 如何增加 EGF 受体敏感性，这 激活 MAPK 以促进损伤恢复（目标 1.1）。然后我们将研究 Sec10 和 exocyst 参与线粒体功能。 AKI 中已确定的一个关键途径是原发性基因的改变 肾小管代谢，通过 ATP 水平降低和 线粒体功能障碍。线粒体也与 ADPKD（最常见的纤毛病）有关，这表明 纤毛和线粒体之间可能存在联系。在这里，我们将研究这条新途径，以及 外囊可能是纤毛和线粒体功能之间的中介，可能是通过差异蛋白实现的 不同小 GTP 酶调控的贩运（目标 1.2）。我们将测试 AKI 后的 Sec10 保护是否有效 通过初级纤毛介导，通过确定小鼠中是否近端小管特异性敲除 Ift88（一种必需的蛋白质） 纤毛发生，加重损伤并阻碍 I/R 后的恢复（目标 2.1）。如果纤毛看起来位于中央 涉及，我们将使用我们新生成的 Sec10 纤毛靶向序列突变小鼠和 I/R 来确认这一点 伤害（目标 2.2）。无论纤毛是否参与，我们都会确认 Sec10- 中涉及 MAPK 通路 使用小分子抑制剂介导小鼠免受 I/R 损伤的保护。然后我们将获得原理证明 通过使用我们新生成的诱导型 Sec10，Sec10 可以增强恢复和/或预防损伤 过度表达小鼠并造成 I​​/R 损伤（目标 3.1）。最后，我们将确定 Sec10 基因传递是否可以 在 I/R 之前和之后在体内使用 Sec10 的病毒和非病毒递送来预防损伤和/或促进恢复 （目标 3.2）。这些实验的成功完成将为 AKI 提供新的机制见解 发病机制和恢复，并对 AKI 治疗新方法的开发产生重大影响。