以Cdk5为靶点探讨其在糖尿病肾病足细胞凋亡中的作用及机制

The podocyte injury is closely related to the protein urine and the development and progression of diabetic nephropathy. Cyclin-dependent kinase 5 expresses in podocytes of glomeruli, and plays an important role in maintaining the podocyte normal morphology and function. Our early studies have demonstrated that the expression and activity of Cdk5 was significantly increased under the high glucose environments, and it was also associated with podocyte apoptosis. However, the specific mechnism of podocyte apoptosis is still unclear. The p38MAPK and JNK signaling pathways can be activated by phosphorylation of extracellular stress signal through a cascade, and involved in cell apoptosis. Our research focus on under the high glucose condition, whether the activated Cdk5 can phosphorylate and activate the pathway upstream molecules MEKK1 and (or) ASK1, followed by the activation of p38MAPK and (or) JNK pathway and podocyte apoptosis. The project intends to explore the signalling mechanism of podocyte apoptosis induced by Cdk5 under high glucose environment in vivo and in vitro, using the gene transfection, shRNA interference and specific inhibitors. Cdk5 is an important medium between high glucose and podocyte apoptosis. Our study will further enrich the theoretical basis that the inhibition of Cdk5 activity can reduce podocyte apoptosis, and provide a new target for the prevention and treatment of diabetic nephropathy.

足细胞损伤与糖尿病肾病蛋白尿的发生和病情进展关系密切。细胞周期素依赖性蛋白激酶5（Cdk5）表达于肾小球的足细胞，对维持足细胞正常的形态结构和功能具有重要作用。我们前期研究发现，高糖状态下，足细胞内Cdk5表达与活性增高，并同细胞凋亡有关，但其诱导凋亡的具体机制尚不清楚。p38MAPK和JNK通路可被细胞外应激信号通过级联通路磷酸化激活，参与了凋亡的发生。高糖状态下，活化的Cdk5是否可以通过磷酸化激活此两条通路的上游分子MEKK1和（或）ASK1，进而活化p38MAPK和（或）JNK通路诱导足细胞凋亡是本课题研究的重点内容。本项目拟利用基因转染、shRNA干扰及特异性抑制剂，体外、体内深入探讨高糖状态下Cdk5诱导足细胞凋亡的细胞内信号机制。作为高糖和足细胞凋亡之间的重要中间介质Cdk5，我们的研究将进一步充实抑制其活性减少足细胞凋亡的理论依据，为糖尿病肾病的有效防治提供新的干预靶点。

足细胞损伤与糖尿病肾病蛋白尿的发生和病情进展关系密切。细胞周期素依赖性蛋白激酶5（Cdk5）表达于肾小球的足细胞，对维持足细胞正常的形态结构和功能具有重要作用。本项目旨在探讨Cdk5在糖尿病足细胞损伤中的作用及机制。研究结果显示，利用siRNA和Roscovitine抑制糖尿病小鼠体内Cdk5的表达/活性，可显著改善糖尿病小鼠肾功能，减轻肾小球损伤及肾间质纤维化程度，并降低肾组织中p-MEKK1、p-JNK等蛋白的表达水平。高糖可显著升高足细胞内Cdk5的表达和激酶活性，并增强p-JNK的表达水平，利用Cdk5 siRNA转染或Roscovitine处理，抑制Cdk5表达/活性可显著降低高糖刺激足细胞内p-JNK的表达，并降低足细胞的凋亡水平。高糖可诱导足细胞内质网应激的发生，增强GRP78、Caspase-12及CHOP的表达。抑制Cdk5的表达/活性，可显著降低内质网应激诱导剂Tunicamycin（TM）诱导的足细胞凋亡及GRP78、Caspase-12及CHOP的表达。高糖/TM可增高足细胞内MEKK1的磷酸化水平，免疫共沉淀及磷酸化作用检测显示MEKK1磷酸化同Cdk5密切相关。抑制Cdk5的表达/活性后，MEKK1的磷酸化水平显著降低；而抑制MEKK1的表达/磷酸化可显著降低高糖/TM诱导的p-JNK表达及足细胞凋亡。以上结果提示高糖、内质网应激等因素可通过激活Cdk5/MEKK1/JNK通路诱导足细胞损伤及凋亡，参与糖尿病肾病的发生。