二甲双胍与2-脱氧葡萄糖抑制小型猪常染色体显性多囊肾病囊肿形成的机制研究

Autosomal dominant polycystic kidney disease（ADPKD）is a genetic disorder. The pathogenesis of ADPKD is not clear, and there are no effective therapies. Cyst epithelial cells in ADPKD are similar to tumor cells and mainly depend on aerobic glycolysis to generate ATP. The activity of AMP-activated protein kinase (AMPK), a key energy metabolism enzyme, is inhibited in cyst epithelial cells of ADPKD. Metformin can inhibit proliferation of tumor cells by activating AMPK and 2-deoxyglucose can suppress proliferation of tumor cells by blocking glycolysis. Thus, we speculate that combined use of both drugs may effectively inhibit over-proliferation of cyst epithelial cells and attenuate ADPKD progression. However, it is not clear so far that the molecular mechanisms by which both drugs inhibit proliferation of cyst epithelial cells in ADPKD. Compared with small animals (such as rodents), large animals (such as mini-pigs) are more similar to humans with respect to genetics，metabolism, renal structure and function，and are ideal animal models for research of human kidney diseases. Therefore, in this study, for the first time, we investigate the inhibiting action of both drugs on the progress of cyst formation in ADPKD，observe the effects of both drugs on glucose metabolism, cell proliferation, cell apoptosis, cell polarity in cyst epithelial cells and explore the effects of both drugs on the activities of proliferation-associated signal pathways AMPK-mTOR and PKA-Raf-MEK-ERK, so that to illuminate the cellular and molecular mechanisms of treatment action of both drugs for ADPKD and to lay a theoretical foundation for future clinical use of these two drugs for treatment of human ADPKD.

常染色体显性多囊肾病是遗传性肾病，发病机制不清楚，无有效治疗药物。多囊肾囊肿上皮细胞与肿瘤细胞类似，主要依赖糖酵解产生ATP，能量代谢关键酶AMPK活性被抑制。二甲双胍和2-脱氧葡萄糖可分别通过激活AMPK及阻断糖酵解抑制肿瘤细胞增殖。因此，我们推测两者能有效抑制多囊肾上皮细胞增殖，阻止多囊肾进展。目前对两者抑制多囊肾上皮细胞增殖的分子机制并不清楚。与鼠类相比，小型猪在遗传、代谢、肾脏结构功能等方面与人更加类似，是研究人类肾脏疾病的理想动物模型。因此，本项目首次使用小型猪多囊肾模型观察了联合应用二甲双胍和2-脱氧葡萄糖对多囊肾囊肿形成的抑制作用；分析了两者对多囊肾囊肿上皮细胞糖代谢、细胞增殖、细胞凋亡、细胞极性的影响；观察了两者对增殖相关信号通路AMPK-mTOR与PKA-Raf-MEK-ERK活性的影响，以阐明两者治疗作用的细胞分子机制，为今后临床应用这两种药物治疗人多囊肾奠定理论基础。

常染色体显性多囊肾病 (ADPKD)是一种常见的由肾小管上皮细胞异常增殖导致的遗传性肾病，可缓慢进展至尿毒症，尚无有效的治疗方法。近年发现多囊肾病囊肿上皮细胞与肿瘤细胞类似，其糖代谢模式发生了明显的改变。二甲双胍是治疗2型糖尿病的一线药物，2-脱氧葡萄糖（2-Deoxyglucose，2DG) 是糖酵解的阻断剂。研究发现二甲双胍和2-脱氧葡萄糖可有效地抑制肿瘤细胞的增殖。因此，我们推测两者能有效抑制多囊肾上皮细胞异常增殖，阻止多囊肾病囊肿的形成。但是目前对两种药物抑制多囊肾囊肿上皮细胞增殖的机制并不清楚。因此，本项目首次从能量代谢的角度入手，观察联合或单独使用代谢药物二甲双胍和2-脱氧葡萄糖对多囊肾病小型猪模型的治疗效果，揭示两种药物治疗多囊肾病的细胞分子作用机制。 . 本研究中，我们发现在小型猪多囊肾病肾组织糖代谢表型发生了明显的改变，糖酵解关键酶HK1、PFK1和PKM2表达水平、乳酸脱氢酶活性及乳酸含量明显升高；线粒体呼吸链酶复合物I和IV活性明显降低，呼吸链酶NADH1和COX IV表达显著降低。发现联合使用二甲双胍和2-脱氧葡萄糖治疗可显著抑制肾囊肿形成，明显改善多囊肾小型猪肾功能，显著抑制肾囊泡上皮细胞的异常增殖，降低细胞周期蛋白Cyclin D1、CDK4、CDK6表达水平，抑制增值相关mTOR信号通路分子mTOR、p70S6K、4EBP1及ERK通路分子MEK和ERK1/2活性，抑制糖酵解关键酶HK1、HK2、PFK1和PKM2表达，降低ATP和乳酸产生水平，激活代谢关键酶AMPK活性。二甲双胍治疗组和联合用药组，线粒体氧化磷酸化酶NADH1表达水平显著降低，SDHA表达水平显著升高。在二甲双胍和联合干预组Complex I酶活性显著降低，2DG治疗组Complex I酶明显升高。ComplexⅡ酶活性在二甲双胍和联合干预组中明显升高。发现二甲双胍和2-脱氧葡萄糖治疗没有明显的肝、心、脑毒性。 .通过本项目实施，我们在国际上首次阐明了二甲双胍和2-脱氧葡萄糖通过调控糖代谢异常而抑制小型猪多囊肾病囊肿形成的分子细胞机制，研究成果为今后临床应用这些代谢药物治疗人类多囊肾病奠定了理论基础，为多囊肾病的治疗开辟了新的途径。