Department of Nephrology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Gene. 2011 Sep 15;484(1-2):18-25. doi: 10.1016/j.gene.2011.05.025. Epub 2011 Jun 12.
Proteinuria is an exacerbating factor of chronic kidney diseases, leading to glomerulosclerosis. However, the molecular mechanisms mediating protein overload-induced podocyte injury are poorly understood. Recent studies have shown that apoptosis mediated by endoplasmic reticulum (ER) stress participated in the progression of a variety of kidney diseases. In the present study, we investigated the role of CD2-associated protein (CD2AP) in protein overload-induced ER stress and subsequent podocyte apoptosis. Conditionally immortalized mouse podocytes were cultured in vitro and treated with different concentrations of bovine serum albumin (BSA). In addition, CD2AP eukaryotic expression vector or siRNA was transfected into podocytes before exposed to BSA. Albumin endocytosis and podocyte apoptosis were visualized by confocal microscopy. The subcellular organelles were observed by transmission electron microscopy. The expressions of GRP78, caspase-12 and CD2AP were detected by RT-PCR or Western blot analysis. It was found that albumin was endocytosed by podocytes in a time-dependent manner. Accumulation of albumin in podocytes induced ER stress and apoptosis in a concentration-dependent manner as indicated by upregulation of GRP78 and caspase-12. Meanwhile, the subcellular organelles were disrupted and the expression of CD2AP was downregulated by high concentration of albumin. Transfection of CD2AP eukaryotic expression vector into podocytes increased CD2AP expression, depressed GRP78 and caspase-12 expressions, and inhibited podocyte apoptosis. In contrast, transfection of CD2AP siRNA deteriorated the above changes induced by BSA. It is concluded protein overload induces podocyte apoptosis via ER stress and CD2AP may play a crucial role in albumin overload-induced ER stress and apoptosis in podocytes.
蛋白尿是慢性肾脏病的加重因素，可导致肾小球硬化。然而，介导蛋白过载诱导的足细胞损伤的分子机制尚不清楚。最近的研究表明，内质网（ER）应激介导的细胞凋亡参与了多种肾脏疾病的进展。在本研究中，我们研究了 CD2 相关蛋白（CD2AP）在蛋白过载诱导的 ER 应激和随后的足细胞凋亡中的作用。体外培养条件永生化的小鼠足细胞，并以不同浓度的牛血清白蛋白（BSA）处理。此外，在 BSA 处理前，将 CD2AP 真核表达载体或 siRNA 转染到足细胞中。通过共聚焦显微镜观察白蛋白内吞和足细胞凋亡。用透射电子显微镜观察亚细胞器官。通过 RT-PCR 或 Western blot 分析检测 GRP78、caspase-12 和 CD2AP 的表达。结果发现白蛋白以时间依赖性方式被足细胞内吞。白蛋白在足细胞中的积累诱导 ER 应激和细胞凋亡，这表现为 GRP78 和 caspase-12 的上调。同时，高浓度白蛋白导致亚细胞器官破坏，CD2AP 表达下调。将 CD2AP 真核表达载体转染到足细胞中，增加了 CD2AP 的表达，下调了 GRP78 和 caspase-12 的表达，并抑制了足细胞凋亡。相反，CD2AP siRNA 的转染恶化了 BSA 诱导的上述变化。结论是蛋白过载通过 ER 应激诱导足细胞凋亡，而 CD2AP 可能在蛋白过载诱导的 ER 应激和足细胞凋亡中发挥关键作用。
