Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis.
Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis.
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Cdk5 或 p35（Cdk5 激活剂）表达的敲低导致足细胞凋亡
DOI：
10.1371/journal.pone.0160252
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Gao YC
中科院分区：
文献类型：
--
作者：
Zheng YL;Zhang X;Fu HX;Guo M;Shukla V;Amin ND;E J;Bao L;Luo HY;Li B;Lu XH;Gao YC
足细胞是终末分化的肾小球上皮细胞。足细胞丢失在许多肾脏疾病中都有发现。Cdk5是一种主要受p35调控的周期蛋白依赖性蛋白激酶。为了研究Cdk5/p35在足细胞存活中的作用，我们首先应用western blotting （WB）分析确认了Cdk5和p35在肾脏发育和培养的永生化小鼠足细胞中的时间过程表达。我们还通过足细胞中p35的过表达证明p35在促进足细胞分化中起重要作用。为了解除小鼠足细胞中Cdk5或p35的表达调控，我们使用RNAi技术分别分析了足细胞特异性标志物Wilms Tumor 1 （WT1）和cleaved caspase 3的细胞功能和凋亡测定。使用细胞计数试剂盒-8对活细胞进行计数。我们发现Cdk5缺失导致WT1表达降低和细胞凋亡。然而，值得注意的是，p35的下调降低了Cdk5的活性，但对cleaved caspase 3的表达没有影响。然而，它确实降低了转录因子WT1的表达，并产生足细胞畸形。另一方面，通过TUNEL分析和cleaved caspase3抗体的免疫荧光染色，可以检测到p35失调足细胞中凋亡的增加。Cdk5和p35敲低细胞的足细胞活力均下降。通过RNAi敲除Cdk5或p35基因不会影响细胞周期素I的表达，细胞周期素是足细胞中另一种Cdk5激活因子。我们得出结论，Cdk5和p35在维持足细胞分化和存活中起着至关重要的作用，并建议这些蛋白作为足细胞损伤肾病治疗干预的靶点。
Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases.