内质网钙稳态在铅镉联合诱导大鼠肾小管上皮细胞凋亡中的作用

Endoplasmic reticulum (ER) is an important intracellular calcium store and ER calcium homeostasis plays a key part in the regulation of apoptosis. Cytoplasm calcium overload occurred in the apoptosis of primary cultures of rat proximal tubular (rPT) cells induced by co-exposure to lead and cadmium in previous study. However, the role of ER calcium homeostasis in this process has not yet been defined explicitly. In this project, the primary cultures of rPT cells will be treated with RNA inference method and gene overexpression technology to make some genes silenced and overexpressed. In addition, some rPT cells will be incubated with specific inhibitors. Then all of the model cells will be exposed to different doses of lead and/or cadmium, respectively. After the exposure, immunoblot assays and real-time quantitative PCR analyses will be performed on rPT cells to assess key signaling molecules expression levels in regulation of ER calcium homeostasis mediated by Calpain/caspase-12 activated pathways, IP3R signaling pathways, calcium buffer proteins and Bcl-2 family proteins, respectively. Likewise, apoptosis and calcium distribution in different domains in rPT cells will be identified by flow cytometry and confocal fluorescent microscopy, respectively. Therefore, the role of ER calcium homeostasis in rPT cells exposed to lead and cadmium will be clarified. Furthermore, this project will contribute significantly to signal transduction mechanism implicated in combined toxic effects of lead and cadmium on rat kidney from the cellular and molecular level.

内质网是重要的胞内钙库，且内质网钙稳态在调控细胞凋亡过程中发挥重要作用。前期研究表明铅镉联合可诱导大鼠肾小管上皮细胞凋亡并伴随胞浆钙超载，但内质网钙稳态在此过程中的调控机制尚不明确。本项目在应用RNA干扰与基因过表达技术对原代大鼠肾小管上皮细胞进行特定基因沉默与过表达处理的基础上，结合特异性信号转导抑制剂处理，分别对模型细胞进行铅、镉单独与联合暴露。染毒后，采用免疫印迹技术、荧光定量PCR技术、流式细胞术及激光共聚焦显微技术检测Calpain/caspase-12活化通路、IP3R信号通路、钙缓冲蛋白及Bcl-2家族蛋白参与调控内质网钙稳态过程中关键信号分子的表达、细胞凋亡及胞内不同区域钙分布。本项目将阐明内质网钙稳态在铅镉联合诱导大鼠肾小管上皮细胞凋亡中的作用，进而从细胞和分子水平探讨铅镉联合所致肾毒性损伤的信号转导机制。

内质网是重要的胞内钙库，且内质网钙稳态在调控细胞凋亡过程中发挥重要作用。前期研究表明铅镉联合可诱导大鼠肾小管上皮细胞凋亡并伴随胞浆钙超载，但内质网钙稳态在此过程中的调控机制尚不明确。本项目在应用RNA干扰与基因过表达技术对原代大鼠肾小管上皮细胞进行特定基因沉默与过表达处理的基础上，结合特异性信号转导抑制剂处理，分别对模型细胞进行铅镉染毒。染毒后，检测Ca2+依赖性Calpain/caspase-12活化途径、IP3R信号通路、钙缓冲蛋白及Bcl-2家族蛋白参与调控内质网钙稳态过程中关键信号分子的表达、细胞凋亡及胞内不同区域钙分布。取得如下研究结果：①铅镉导致胞内不同区域钙重分布，即胞浆钙超载的同时伴随内质网钙外流和线粒体钙超载，胞浆钙超载激活Calpain/caspase-12而启动细胞凋亡；②铅镉所致胞浆钙超载是三种钙缓冲蛋白(GRP78、GRP94、CRT)表达上调的原因，钙缓冲蛋白上调是内质网应激凋亡途径活化的重要标志；③铅镉上调细胞内第二信使IP3含量，激活内质网钙释放受体IP3R1和IP3R2的表达，促使内质网钙过度释放至胞浆内，引起胞浆钙超载和线粒体钙超载；④铅染毒可磷酸化修饰CaMKII，磷酸化修饰的CaMKII是内质网钙外流和胞内钙超载的重要靶蛋白；⑤铅镉染毒下调Bcl-2和Bcl-xL表达水平而上调Bax表达水平，但Bcl-2家族蛋白对胞内钙分布无明显的调控作用。本项目明确了铅镉通过活化内质网钙释放受体而触发内质网钙稳态失衡的作用机制，阐明了CaMKII磷酸化修饰在内质网钙稳态失衡中的关键作用，证实了内质网钙过度释放所致胞浆钙超载是铅镉诱导内质网应激凋亡途径的重要原因。本项目从内质网钙稳态的角度详细阐明了铅镉致肾小管上皮细胞凋亡的分子机制，为探讨铅镉致肾毒性损伤提供了重要的理论依据。