Podocyte Dysfunction in Diabetic Glomerulopathy

糖尿病肾小球病中的足细胞功能障碍

• 批准号: 6623379
• 负责人: SHELDON CHEN
• 金额: $ 13.02万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623379
• 关键词: collagen; diabetes mellitus; glucose; growth factor receptors; kidney cell; kidney disorder; pathologic process; protein biosynthesis; receptor expression; tissue /cell culture; transforming growth factors; vascular endothelial growth factors; vascular endothelium

Dr. Sheldon Chen is currently conducting his research in the laboratory of Dr. Faud N. Ziyadeh at the University of Pennsylvania, which boasts a world-renowned biomedical community and provides state- of- the-art facilities, superb human resources, and outstanding technical support. By completing the proposed project, Dr. Chen hopes to acquire new technical expertise, improve his ability to synthesize data into a credible paradigm, hone his critical thinking skills, and afterwards pursue novel or related research that expands upon his findings. After an additional year of mentored fellowship research training, he will join the faculty of the Renal Division at the University of Pennsylvania, where he hopes to mature into a fully-independent academic physician-scientist. Diabetic kidney disease remains the number one cause of renal failure in the United States. As the defining metabolic abnormality in diabetes mellitus, high blood glucose undoubtedly plays an important role in the pathogenesis of diabetic nephropathy. Our laboratory has discovered that Transforming Growth Factor-beta (TGF-beta) mediates much of the injuries effect of hyperglycemia, and the two metabolic factors may conspire to cause even greater renal damage. The podocyte is a highly specialized epithelial cell in the glomerulus that synthesizes part of the glomerular basement membrane (GBM) and regulates the macromolecular permeability across the glomerular filtration barrier. Podocyte dysfunction secondary to the deleterious environment of the diabetic milieu may contribute to the GBM thickening and increased macromolecular permeability leading to proteinuria in diabetic glomerulopathy. Recent cell culture advances have allowed us to study the in vitro podocyte in its fully differentiated state. We propose to examine the effects of high glucose and exogenous TGF-beta on the cultured, differentiated mouse podocyte with respect to the altered expression of type IV collagen and vascular endothelial growth factor (VEGF), two proteins that are likely involved in the pathophysiology of GBM thickening and diabetic proteinuria. Northern analysis will be used to quantitate the gene expression, and immunoblotting and ELISA will be used to measure the protein production of the component alpha chains of collagen IV and the isoforms of VEGF in response to high glucose and TGF-beta treatment. Whether high glucose and TGF-beta have synergistic effects will also be investigated. The cellular mechanisms underlying the high glucose-induced expression of the TGF-beta signaling receptor (preliminary data), which may explain the hypothesized high glucose/ TGF- beta synergy, will be elucidated by the nuclear runoff technique, analysis of promoter activity by luciferase reporter assay, and electrophoretic mobility shift assay. Lastly, specific inhibitors of TGF-beta will be used to determine whether some of the high glucose effects may be mediated by the TGF-beta system in the podocyte.

Sheldon Chen博士目前正在宾夕法尼亚大学的Faud N. Ziyadeh博士的实验室进行研究，该大学拥有世界著名的生物医学界，并提供国有的设施，出色的人力资源和出色的技术支持。 通过完成拟议的项目，Chen博士希望获得新的技术专长，提高他将数据合成为可信范式的能力，磨练他的批判性思维能力，然后从事扩展其发现的小说或相关研究。 经过一年的指导研究培训，他将加入宾夕法尼亚大学肾部门的学院，在那里他希望成熟成为一个完全独立的学术医师科学家。糖尿病肾脏疾病仍然是美国肾衰竭的第一名。 作为糖尿病中定义的代谢异常，高血糖无疑在糖尿病肾病的发病机理中起着重要作用。 我们的实验室发现，转化生长因子-Beta（TGF-BETA）介导高血糖的许多损伤作用，而两个代谢因子可能会促进更大的肾脏损害。 足细胞是肾小球中高度专业的上皮细胞，它合成了肾小球基底膜（GBM）的一部分，并调节跨肾小球滤过屏障的大分子渗透率。 糖尿病环境有害环境继发的足细胞功能障碍可能有助于GBM增厚，并增加大分子渗透性，从而导致糖尿病性肾小球病的蛋白尿。 最近的细胞培养进展使我们能够研究其完全分化状态的体外足细胞。 我们建议检查高葡萄糖和外源性TGF-β对IV型胶原型和血管内皮生长因子（VEGF）的表达的改变，对培养的，分化的小鼠足细胞的影响，两种可能参与GBM增厚和糖尿病性蛋白质素的病理生理学的蛋白质。 北方分析将用于定量基因表达，免疫印迹和ELISA将用于测量胶原蛋白IV的成分α链的蛋白质产生以及VEGF的同工型，以响应高葡萄糖和TGF-β处理。 高葡萄糖和TGF-β是否具有协同作用。 TGF-BETA信号受体（初步数据）的高葡萄糖诱导表达的基础的细胞机制，这可以解释假设的高葡萄糖/ TGF-β协同作用，将通过核径流技术，Luciferase Reporter shiptersssay和电力迁移分析和电力迁移分析和电力迁移分析，并通过核径流技术来阐明。 最后，将使用特异性TGF-β的抑制剂来确定某些高葡萄糖作用是否可以由Podocyte中的TGF-β系统介导。