Hippo通路在急性肾损伤发病中的作用及其机制

When normal kidney tubular epithelial cells encountered ischemia/reperfusion (I/R) injury, there was a marked increase in cell death by necrosis and apoptosis. Different from the heart or brain, the kidney could achieve completely recovery by the proliferation and differentiation of the surviving cells if the insult was mild or did not last very long; tipical cases could experience several stages after acute kidney injury (AKI), including injury (<48h), repair (48h-5d) and late repair (5d-7d) stage. however, the repair could be maladaptive if the injury was too severe. This could result in varying degrees of chornic kidney diseases; some might eventually progress to end stage renal disease and rely on dialysis or transplantation. Hippo pathway, with mammlian Ste-20 like kinase 1(Mst1)as one of its most critical core kinases, has recently been found to be an significant signaling pathway that balance the proliferation and apoptosis of cells in organism.Our prelimilary studies in rat kidney I/R model showed that renal Mst1 level increased significantly in the injury stage but desreased significantly in the repair stage, which was identical to the change of renal function and the extent of the damage of the renal tissue. Thus, we assumed that hippo pathway could play different role in the injury and repair stages of I/R AKI. In this study, we will first observe the expression of core kinase of Hippo pathway, Mst1, to identify its role in different stages of I/R AKI. At the same time, we will detect the expression of its critical down stream target YAP and genes related to proliferation and apoptosis. We will also use kidney specific conditional knockout mice Mst1 Cre/Loxp to further explore the role of Mst1 in I/R AKI. Finally, we will clarify the mechanism of Hippo pathway in regulating I/R AKI, which might provide new target and method in treating AKI. The expression of Mst1 in normal and I/R NRK-52E cells will be regulated, and its role in renal injury/repair and its effect on down stream signaling pathway will be systematically studied. Finally, adnovirus vector will be constructed and injected into I/R rats through renal vein to down- or up-regulate the expression of Mst1 in injury stage or repair stage, respectively, to evaluate the influence on renal injury and repair.

急性肾损伤（AKI）后减少小管细胞凋亡、促进其再生对AKI的治疗具有重要意义。近年研究发现发育相关的Hippo通路在器官组织损伤后修复中具重要作用。前期研究在I/R AKI大鼠发现损伤期肾组织Mst1表达显著升高，修复期却显著降低，与肾功能和组织损伤程度变化一致。因而我们推测Hippo通路在I/R AKI损伤期和修复期可能具有不同的调控作用。拟进一步通过大鼠AKI模型和肾脏特异性条件性基因敲除小鼠Mst1 Cre/Loxp观察AKI各期Mst1以及下游YAP和关键增殖凋亡相关基因、细胞周期蛋白差异表达；体外调控Mst1在正常和I/R大鼠NRK-52E细胞内表达，研究其在肾损伤修复中的作用，并对其下游信号通路进行系统研究。构建腺病毒载体，经肾静脉注射导入动物模型体内，观察损伤期下调和修复期上调Mst1对肾损伤和修复的影响，探讨Mst1调控AKI分子机制，为治疗提供新的靶点和手段。

迄今为止，有关Hippo 通路在AKI 病程及发病机制中的作用，尤其是肾组织慢性化进展中的作用及其调控研究仍鲜有报道。本研究通过夹闭实验大鼠双侧肾动脉30 分钟或45 分钟构建了轻度和重度两种损伤程度缺血再灌注（ischemia/reperfusion, I/R） AKI 模型。缺血30 分钟组大鼠肾组织形态在再灌注后4 w 可完全恢复，但缺血45 分钟组动物肾组织出现了慢性化改变。主要表现为小管间质组织纤维化和肾小管分化异常。. 肾组织蛋白印迹结果显示研究发现以pYAP/YAP 为核心的Hippo 信号通路各蛋白在缺血30 min 和45 min I/R AKI 模型大鼠肾组织均有显著升高，其中缺血30 min呈先增高再降低；而缺血45 min 呈持续升高。同时，与缺血30 min 组细胞活化和成熟细胞标志蛋白表达恢复正常水平有所不同，缺血45 min 组动物肾组织细胞活化标志蛋白表达升高，成熟肾小管细胞标志性蛋白低于正常。肾组织免疫组化见YAP蛋白阳性染色主要位于肾小管上皮细胞胞浆与胞核。. 进一步细胞学实验在HK-2 细胞中过表达YAP 蛋白可显著促进细胞增殖，反之则可起减慢增殖的作用；此外，YAP 过表达还可引起纤维化相关蛋白I、III、IV 型胶原mRNA 表达水平升高；其下游调控的直接靶点CTGF 表达水平也显著升高，同时CTGF 也是肾纤维化病理生理中重要的因子之一。此外，YAP 过表达还可显著增加HK-2 细胞G2/M 期细胞的比例。 . 最后，根据既往学者结构生物学筛选结果使用YAP激动剂洋地黄毒苷Digitoxin腹腔注射用于I/R AKI 大鼠，结果显示pYAP/YAP 比例降低、YAP 活性增强可减少AKI 修复期后活化细胞向静止状态分化，促进肾组织细胞增殖，加重纤维化。. 综上所述，Hippo 通路参与了I/R AKI 损伤后的再生修复过程，其中通路的核心蛋白YAP 具有促进肾小管细胞增殖的有益作用，但同时其表达水平的持续升高以及蛋白核内表达的增多也具有促纤维化、抑制活化细胞向正常转化的有害作用，有望成为治疗的靶点。