The Exocyst in Ciliogenesis and Acute Kidney Injury

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 支持性护理，对最严重的病例实施透析；然而，发病率和死亡率仍然存在 非常高。AKI缺乏可用的治疗方法的一个主要原因是对肾脏如何 肾小管细胞可从急性肾损伤中恢复，因此治疗方法有限。识别，一个 治疗目标和途径将通过允许合理的药物设计来满足主要的未得到满足的需求。目标是 这里是为了确定高度保守的八蛋白外囊运输复合体，特别是 Central Sec10(又名Exoc5)组件，可用于促进AKI后的恢复和/或防止受伤。 肾小管细胞损伤后，细胞开始失去极性，随后细胞死亡，细胞脱落。 管腔，然后活细胞的扩散和去分化，以覆盖裸露的区域，伴随着增殖， 分化和细胞极性的重建。极性或分泌途径对AKI的恢复至关重要， 和细胞功能，而外囊是已知的中介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) 途径调控肾小管的发生。我们还表明，外囊，特别是Sec10成分，位于中央 参与肾纤毛和肾小管的形成。具体地说，Sec10击倒被抑制，而Sec10 过度表达增加，纤毛生成和小管生成。这些截然不同的研究领域最近汇聚在一起， 我们发现，Sec10通过激活MAPK加速了氧化损伤的恢复，氧化损伤是一种缺血样损伤。 我们现在已经产生了Sec10fl/fl小鼠，并有初步数据显示在小鼠近端有Sec10缺失 肾小管可加重缺血再灌注损伤，抑制修复。此外，该基因的定点突变 人SEC10中高度保守的VxPx纤毛靶向序列抑制体外培养的人肾小管上皮细胞的微管形成 3D胶原蛋白凝胶，并阻止拯救sec10突变斑马鱼。拟议中的实验将测试 总体而言，假设Sec10通过EGF受体激活MAPK途径来防止损伤 和/或促进AKI后的肾脏恢复，这种作用是通过初级纤毛介导的，而Sec10 是一个治疗靶点。因此，我们将研究Sec10如何增加EGF受体的敏感性，即 激活MAPK以促进受伤后的恢复(目标1.1)。然后我们将调查Sec10和排泄物是如何 都与线粒体功能有关。在AKI中已发现的一个关键途径是原发性改变 肾小管代谢，通过降低三磷酸腺苷和三磷酸腺苷水平进而影响区域循环。 线粒体功能障碍。线粒体也与ADPKD有关，ADPKD是最常见的纤毛疾病，提示 纤毛和线粒体之间可能存在联系。在这里，我们将研究这一新的途径，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。 由不同的小GTP酶管理的贩运(目标1.2)。我们将测试AKI之后的Sec10保护是否 在小鼠中通过初级纤毛确定是否需要一种蛋白质Ift88的近端小管特异性敲除 对于纤毛形成，会加重损伤并阻止I/R后的恢复(目标2.1)。如果纤毛看起来居中 参与，我们将使用我们新产生的Sec10纤毛靶向序列突变小鼠和I/R来证实这一点 伤害(目标2.2)。无论纤毛受累如何，我们都将确认MAPK通路参与了Sec10- 使用小分子抑制剂对小鼠I/R损伤的中介保护作用。然后我们将获得原则上的证据 Sec10可以通过使用我们新产生的诱导性Sec10来促进康复和/或防止受伤- 过度表达小鼠并进行I/R损伤(目标3.1)。最后，我们将确定Sec10基因的传递是否可以 在I/R前后使用体内病毒和非病毒递送的Sec10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提供新的机械洞察力 并对开发治疗AKI的新方法产生重大影响。

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.