内质网钙稳态在镉钼联合致鸭肾组织自噬中的调控作用

镉、钼是江西省境内重要的工业和环境重金属污染物，肾脏是其毒性作用的主要靶器官。内质网作为胞内钙库，其钙稳态在调控细胞自噬中发挥关键作用。课题组前期研究发现镉钼联合可致鸭肾组织损伤和自噬并伴随胞浆钙超载，但内质网钙稳态在此过程中的调控机制并不清楚。本课题在建立鸭镉钼联合亚慢性中毒病例和鸭肾小管上皮细胞培养模型的基础上，对原代细胞进行特定基因沉默与过表达，或经特异性抑制剂处理，再对模型细胞进行镉钼染毒，应用免疫印迹、荧光定量PCR和流式细胞等技术，检测CaMKKβ/AMPK/mTOR、Calpain/Atg5 活化通路、PLC-IP3-IP3R信号通路、钙缓冲蛋白及Beclin-1蛋白等参与调控内质网钙稳态过程中关键信号分子的表达、细胞自噬及胞内不同区域钙分布等，阐明内质网钙稳态在镉钼联合致鸭肾组织自噬中的调控作用，为从内质网钙稳态保护的角度防治镉钼的肾毒性提供理论依据。

Cadmium (Cd) and molybdenum (Mo) have been recognized to be two of most important environmental and industrial heavy metal pollutions in Jiangxi Province. The kidney is the main target organ of Cd and Mo when exposed. Endoplasmic reticulum (ER) is an important intracellular calcium store and ER calcium homeostasis plays a key part in the regulation of autophagy. Our research group have found that co-exposure to Cd and Mo could cause damage and autophagy in duck kidney tissue accompanied by cytosolic calcium overload. However, the role of ER calcium homeostasis in this process has not been clarified yet. In this study, the models of Mo, Cd alone and their combination of subchronic toxicity of ducks in vivo and as well the primary cultures of duck proximal tubular cells in vitro will be established. The RNA inference method and gene overexpression technology are used to make some genes silenced and overexpressed in the primary cultures of duck proximal tubular cells. In addition, some duck proximal tubular cells will be incubated with specific inhibitors. Then all of the model cells will be exposed to different doses of Cd or/and Mo, respectively. The study uses immunoblot assays and real-time quantitative PCR analyses to detect key signaling molecules expression levels in regulation of ER calcium homeostasis mediated by CaMKKβ/AMPK/mTOR, Calpain/Atg5 activated pathways, PLC-IP3-IP3R signaling pathway, calcium buffer proteins and Beclin-1 protein and autophagy-related genes expression levels in duck kidney tissue and duck proximal tubular cells, respectively. Likewise, calcium distribution in different domains in duck proximal tubular cells will be identified by flow cytometry. This study will elucidate the regulatory role of ER calcium homeostasis in Cd or/and Mo induced autophagy of duck kidney tissue, which can provide the theoretical evidence for prevention of nephrotoxicity induced by Cd or/and Mo from the perspective of protection of ER calcium homeostasis.