神经酰胺从头合成途径异常在糖尿病肾病足细胞损伤中的作用及其机理研究

Diabetic nephropathy is one of the leading causes of death in chronic renal failure and diabetes. Diagnosis and treatment in early stage of diabetic nephropathy is very important. More and more studies have shown podocyte injury is the key event at early stage, which causes many pathophysiological processes including mesangial matrix expansion, glomerular basement membrane thickening and glomerulosclerosis. Podocytes injury exhibits as cytoskeleton rearrangement, insulin resistance, and apoptosis, etc. However, the mechanism underlying podocyte injury is not clear. Sphingolipids are important cellular structure components and signaling molecules. In previous studies, we compared the profile of sphingolipids in renal cortex of diabetic rats and control rats using lipidomic strategy, and found ceramides, one group of sphingolipids, dramatically increased in diabetic rats. Furthermore, the expression of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramide, was elevated in diabetic rats, suggesting dysregulation of de novo synthesis of ceramide plays an important role in development of diabetic nephropathy. In many cell types, ceramide has been reported to regulate apoptosis, cytoskeleton rearrangement, and insulin resistance. Can ceramides lead to podocyte injury in diabetic nephropathy? What is the mechanism? Using type 2 diabetic rats and podocytes culture, we analyse the role of dysregulation of de novo synthesis of ceramide in podocyte apoptosis, cytoskeleton rearrangement, and insulin resistance in diabetic nephropathy. And we also use clinical kidney biopsy to study if SPT can be a marker for early diagnosis of diabetic nephropathy. The results of our study may provide new insight for understanding mechanism of development of diabetic nephropathy, and new targets for clinical treatment.

糖尿病肾病（DN）是导致慢性肾功能衰竭的主要病因之一，早期诊治意义重大。研究表明，足细胞损伤是其早期关键事件，可诱导基底膜增厚、系膜增生、肾小球硬化等过程。其损伤表现涉及细胞骨架重构、胰岛素抵抗、细胞凋亡等。鞘脂是重要的细胞结构成分和信号分子。我们利用脂类组学分析发现糖尿病大鼠肾皮质中鞘脂主要成分神经酰胺（Cer）表达增加，且调控Cer从头合成的关键酶SPT表达也增高，提示Cer从头合成异常在DN发生发展中可能起重要作用。研究表明Cer可调控细胞骨架重构、胰岛素抵抗、凋亡等，那么Cer是否可介导足细胞损伤？其相关机制如何？本课题拟利用大鼠模型和体外培养足细胞，从足细胞骨架重构、胰岛素抵抗及细胞凋亡等角度研究Cer从头合成途径异常在糖尿病肾病足细胞损伤中的作用及其机制，并利用临床肾活检标本研究SPT可否作为DN的早期诊断标志物，为DN的发生发展提供新的理论基础及治疗靶点。

糖尿病肾病（DN）是导致慢性肾功能衰竭的主要病因之一，早期诊治意义重大。我们利用脂类组学分析发现糖尿病大鼠肾皮质中鞘脂主要成分神经酰胺（Ceramide，Cer）表达增加，且调控Cer从头合成的关键酶丝氨酸棕榈转移酶（SPT）表达也增高，提示Cer从头合成异常在DN发生发展中可能起重要作用。研究表明Cer可调控细胞骨架重构、胰岛素抵抗、凋亡等。E3泛素化连接酶复合物Cul4B参与转录、细胞存活与增殖等多种细胞过程。本课题利用大鼠模型，Cul4b基因敲除小鼠和体外培足细胞和肾小管上皮细胞研究发现：糖尿病肾病大鼠肾组织切片中肾小管上皮细胞及足细胞Cul4B表达明显降低。体外Cer干扰肾小管上皮细胞导致Cul4B的改变。肾脏纤维化在糖尿病肾病的慢性化过程中起决定作用。本研究利用肾脏纤维化模型－小鼠单侧输尿管结扎（UUO）模型,研究显示结扎侧肾脏的Cul4B表达较对照侧肾脏显著降低；同时，8周龄的cul4b敲除小鼠肾脏电镜结果显示已经有明显的胶原纤维丝的积累，而同龄的野生型小鼠肾脏中则未发现，提示Cul4B极有可能在糖尿病肾病发生过程中抑制肾脏纤维化的发生。本研究发现了糖尿病肾病肾脏慢性纤维化的新的分子生物学机制。.顺铂肾损害是临床中常见并发症，极大的限制了顺铂的临床应用。其机制目前尚未完全清楚。近年研究显示：凋亡在其过程中发挥着重要作用。表没食子儿茶素没食子酸酯（EGCG）是茶叶中提取的生物活性物质，具有抗氧化，抗炎和抗肿瘤的作用。本研究利用顺铂肾损害小鼠模型研究EGCG对于顺铂肾损害的保护作用及其机制。结果显示：EGCG可以明显抑制CP导致的肾小管上皮细胞凋亡，从而发挥肾脏保护作用。