MicroRNA-195通过靶向抑制SLC2A3干扰足细胞葡萄糖摄取并导致足细胞损伤的机制研究

MicroRNAs play an important role in the pathophysiologic processes of podocyte injury and cellular repair in diabetic nephropathy. Our earlier work demonstrated that microRNA-195(mir-195) induced apoptosis and F-actin rearrangement in podocyte, and it was partially responsible for albuminuria in diabetic nephropathy. We also found that SLC2A3 was abundant in podocytes and it contributed greatly to cellular glucose transportation. Moreover, SLC2A3 is one of target genes of mir-195. Nevertheless, we do not know whether mir-195 inhibit cellular glucose uptake of podocyte in high glucose surroundings or not. There are no answers available from the domestic and international literatures, neither. Herein we put forward the following hypothesis: over-expression of mir-195 reduces podocyte glucose uptake through the inhibition of SLC2A3, which makes the podocyte in great energy shortage though it located in high glucose environment. Thus those podocytes may face greater survival pressure and function impairment that may result in albuminuria or diabetic nephropathy. In contrast, mir-195 of appropriate amount inhibitors may play a protective role in podocyte. To prove this hypothesis, we designed the present project. Firstly, the immortalized mouse podocyte was cultured in high glucose medium. Mir-195 transfection, siRNA of SLC2A3 gene, inhibitors of SLC2A3 and BCL2 protein were used to intervene mir-195 expression and glucose metabolism of podocyte at different levels. Then the activity of hexokinase, the synthesis of ATP, ADP and AMP, the cytoskeleton, the autophagy in different groups of podocytes were evaluated, respectively. The expression of podocyte specific functional proteins were also detected. Further, model of diabetic nephropathy was induced by intraperitoneal injection of streptozocin in DBA/2 mouse. In the model, mir-195 and SLC2A3 in situ expression of podocyte in paraffin section of renal cortex will be quantitated after the intervention of mir-195 via vena caudalis injection. Correlation analysis between plasma glucose level, mir-195 or SLC2A3 expression and albuminuria quantitation were conducted, too. Thus we test our hypothesis in vivo and in vitro to elucidate the mir-195 mediated regulation mechanism of cellular glycometabolism in podocyte and it's effect on diabetic nephropathy, which may shed light on new strategies for the prevention and treatment of diabetic nephropathy.

葡萄糖转运是影响细胞糖摄取和糖代谢的关键限速步骤。我们前期发现足细胞膜上高表达葡萄糖转运体SLC2A3且该转运体受microRNA-195（简称mir-195）靶向抑制后足细胞出现结构和功能异常。在此基础上提出假设：mir-195 过量表达会导致足细胞葡萄糖摄取不足和能量缺乏，继而出现细胞损伤。本课题拟高糖培养足细胞并建立小鼠糖尿病肾病（DN）模型，用mir-195转染，siRNA干扰，SLC2A3抑制剂刺激细胞，尾静脉注射mir-195等方法从多环节干预足细胞，通过比较糖代谢酶活性、中间产物、ATP合成及自噬、细胞超微结构、细胞损伤指标，检测足细胞功能蛋白，分析肾脏病理、蛋白尿与肾小球足细胞mir-195和SLC2A3表达相关性等手段从分子、细胞和整体水平探讨mir-195对糖转运的调节机制并验证该假设。找到该通路上的关键分子靶点并进行干预有望减轻足细胞损伤，从而延缓DN 的发生和进展。

DN是糖尿病常见的慢性并发症，由此带来的医疗费用始终居高不下。DN是多因素综合作用引起的疾病。然而多年的临床实践证明，尽管降血糖、控制血压、调脂以及应用ACEI和ARB降低肾小球内灌注压等综合治疗措施不断完善，当前的治疗仍不能获得满意效果，仍有约20%-30%的患者最终发展到ESRD并且不得不依赖透析。由此可见，DN的发病机制仍有很多不明之处，仍是我国亟待解决的关键科学问题之一。.肾脏中足细胞，肾小球系膜细胞，肾小球毛细血管内皮细胞和肾小管上皮细胞等固有细胞的功能调节和结构改变等均有miRNA参与。这提示miRNAs在调控基因表达和多种肾脏疾病方面可能发挥着非常重要的作用。前期我们证实microRNA-195可以调控肾脏固有细胞凋亡。我们通过信息检索发现microRNA-195靶向抑制20余种细胞膜转运体基因。由于葡萄糖为水溶性，必须借助葡萄糖转运体才能通过脂溶性的细胞膜。故葡萄糖转运是细胞内糖代谢的关键限速步骤。葡萄糖转运体异常与糖尿病及其并发症的发生发展具有极为密切的关系。本课题通过高糖培养足细胞并建立小鼠DN模型，用mir-195转染，siRNA干扰，SLC2A3抑制剂刺激细胞，尾静脉注射mir-195等方法从多环节干预足细胞，通过比较糖代谢酶活性、中间产物、ATP合成及自噬、细胞超微结构、细胞损伤指标，检测足细胞功能蛋白，分析肾脏病理、蛋白尿与肾小球足细胞mir-195和SLC2A3表达相关性等手段从分子、细胞和整体水平探讨mir-195对糖转运的调节机制。证实该通路上的关键分子靶点SLC2A3并进行干预有望减轻足细胞损伤，从而延缓DN的发生和进展。我们发现阻断SLC2A3后仅D-葡萄糖消耗量明显减少。我们发现正常情况下高糖环境中的足细胞表达microRNA-195会代偿性增高以限制过多的葡萄糖进入细胞。但如果“矫枉过正”，microRNA-195表达过高，对SLC2A3的抑制作用过强，则导致细胞内葡萄糖缺乏和病理损伤。此时细胞处于应激状态，面临更大的生存压力，自噬增多，其正常功能受损。此时如果适当阻抑microRNA-195表达则使足细胞更能适应其所处的环境，从而对足细胞起到保护作用。.我们的研究在一定程度上丰富了糖尿病肾病的发病机制，进而为开发更有针对性的小分子化合物拓展糖尿病肾病的防治策略提供了新思路。