CaMKⅡ/YAP在肾小管上皮细胞凋亡中的作用及其机制研究

Renal tubular epithelial cell apoptosis is one of the most important events in acute kidney injury (AKI). Thus, it is of great significance to further explore the underlying mechanism of renal tubular epithelial cell apoptosis to find the target for treating AKI. Previous study demonstrated that CaMKII played an important role in oxidative stress injury and cell apoptosis. Both pharmacological inhibition and genetic deletion of CaMKII have been shown to be protective against endoplasmic reticulum-stress induced cardiomyocytes apoptosis. Our previous studies showed that nuclear YAP was remarkably reduced in high glucose, lipopolysaccharide or adriamycin injured podocytes, thereby failed to inhibit NFAT mediated cell apoptosis. What is more, CaMK can promote the function of NFAT. Thus, in this study, co-immunoprecipitation, immunofluorescence, western blotting and flow cytometry will be applied to explore whether CaMKII is involved in renal tubular epithelial cell apoptosis, whether CaMKII binds to YAP in cytoplasm and restrains YAP into nuclear thereby promotes NFAT mediated cell apoptosis. Ischemia reperfusion injury or lipopolysaccharide induced AKI in CaMKII knock out and normal mice will be used to explore the role of CaMKII/YAP in renal function, pathology and renal tubular epithelial cell apoptosis. It is the first time that the role of CaMKII/YAP was demonstrated in renal tubular epithelial cell apoptosis. This study would provide new insight and potent target for treating AKI.

肾小管上皮细胞（RTEC）凋亡是急性肾损伤（AKI）的重要表现之一，对AKI中RTEC凋亡机制的深入研究以寻找治疗靶点对AKI的防治有着重大的意义。既往研究表明CaMKⅡ在细胞凋亡中发挥着重要作用。我们以往研究表明在脂多糖、阿霉素等有害刺激损伤时，胞核中YAP蛋白明显减少以致于对NFAT蛋白的抑制作用减弱从而使足细胞发生凋亡。CaMK可促进NFAT功能的发挥。因此，本研究拟运用免疫共沉淀、免疫荧光、免疫印迹、流式细胞术等技术，探讨CaMKⅡ是否参与RTEC凋亡，是否通过在胞浆中结合并阻止YAP入核而放任NFAT在细胞核中发挥促进细胞凋亡的作用；在CaMKⅡ基因敲除鼠和正常鼠中构建缺血再灌注损伤及脂多糖诱导AKI小鼠模型，整体探索CaMKⅡ/YAP对肾功能、肾脏病理的影响及在RTEC凋亡中的作用。本研究首次阐述CaMKⅡ/YAP在RTEC凋亡中的作用，为AKI的防治提供新的理论和治疗靶点。

肾小管上皮细胞（RTEC）凋亡是急性肾损伤（AKI）的重要表现之一。 在AKI中，对RTEC凋亡机制的深入研究以寻找治疗靶点对AKI的防治有着重大的意义。既往研究表明CaMKⅡ在细胞凋亡中发挥着重要作用。我们以往研究表明在脂多糖、阿霉素等有害刺激损伤时，胞核中YAP蛋白明显减少以致于对NFAT蛋白的抑制作用减弱从而使足细胞发生凋亡。CaMK可促进NFAT功能的发挥。因此，本研究运用免疫共沉淀、免疫荧光、免疫印迹、流式细胞术、细胞转染等技术，在体外培养RTEC实验中验证了CaMKⅡ参与细胞凋亡，并且是通过在胞浆中结合并阻止YAP入核而放任NFAT在细胞核中发挥促进细胞凋亡的作用；在正常鼠中构建阿霉素诱导AKI小鼠模型，发现其血肌酐升高、细胞凋亡增加、CaMKⅡ活性增加、胞核中抗凋亡蛋白YAP减少而促凋亡蛋白NFAT增加，抑制CaMKⅡ磷酸化可减轻以上这些作用。本研究首次阐述CaMKⅡ/YAP/NFAT在RTEC凋亡中的作用，为AKI的防治提供新的理论和治疗靶点。