Nox4介导的H2O2产生在常染色体显性遗传多囊肾病发展中的作用及机制研究

ADPKD, one of the most common human monogenic diseases, is characterized by massive enlargement of fluid-filled cysts of renal tubular origin that compromise normal renal parenchyma and leads to renal failure. There is no approved drug therapy for ADPKD. Recent clinical studies demonstrate oxidative stress as present early in ADPKD. NADPH oxidases, a family of enzymes known as Nox, are also a main source of ROS production in kidney diseases. Nox4 has been shown to be the major source of ROS in the renal cells. However, the pathophysiological role of Nox4 in ADPKD remains uncharacterized. Our work had revealed that the expression of Nox4 and p22phox was increased in ADPKD kidney and H2O2 could promote MDCK cyst enlargement. Therefore, we predict that Nox4 mediated H2O2 production is very important to ADPKD development. To clarify this hypothesis, we use MDCK cyst model, embryonic kidney model and ADPKD mouse model combined with genetic technique and chemical intervention. We will further explore the role of Nox4 in ADPKD development and discover the impact of Nox4 and H2O2 on ADPKD renal cyst epithelia proliferation and fluid secretion. This research will shed a light on new drug target development for ADPKD treatment.

常染色体显性遗传多囊肾病患者的肾脏在发病早期即处于氧化应激状态，烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（Nox4）作为肾脏中产生氧自由基的主要代谢酶，在多种肾脏疾病中高表达并参与调解肾脏氧化应激，但Nox4与多囊肾囊泡发展的关系尚不清楚。最新研究发现活性氧能够促进囊液分泌；同时我们的前期工作表明，多囊肾囊泡内Nox4和p22phox高表达，且H2O2能够激活AMPK/mTOR信号通路促进囊泡增大。因此，我们提出假说：Nox4介导产生的H2O2通过抑制HIF1α的泛素化降解，上调CFTR和GLUT1转录，激活AMPK/mTOR信号通路，从而促进囊泡发展。本项目拟利用MDCK囊泡模型、胚胎肾囊泡模型和多囊肾小鼠模型，运用基因操纵和化学干预等方法，阐明Nox4对囊泡发展的影响，揭示Nox4介导的H2O2产生影响囊泡上皮细胞增殖和囊液分泌的具体机制，为多囊肾的治疗提供新的靶点和实验依据。

常染色体显性遗传多囊肾病以双侧肾脏多发性囊泡为主要特征，不断增大的囊泡压迫肾实质，导致终末期肾衰，目前尚无有效治疗药物。烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NOX4）作为肾脏中产生氧自由基的主要代谢酶，在多种肾脏疾病中高表达并参与调解肾脏氧化应激状态，但Nox4与多囊肾囊泡发展的关系目前还没有研究报道。为了揭示NOX4对多囊肾的影响及作用机制，我们建立3种多囊肾模型，即pkd1敲除小鼠模型，MDCK囊泡模型和胚胎肾囊泡模型，通过给予NOX4的抑制剂（GKT137831）或NOX4敲除方法评价抑制NOX4对囊泡发展的影响，并采用Western blot，免疫荧光，化学发光法等技术检测囊泡上皮细胞内相关蛋白的表达，以揭示NOX4促进囊泡发展的作用机制。研究结果发现，与同窝对照小鼠相比，多囊肾小鼠肾脏中NOX4和p22phox表达量明显增高，NRF2表达量明显降低，且囊泡上皮细胞中H2O2和8-OhdG含量明显增高；采用MDCK囊泡模型，胚胎肾囊泡模型和多囊肾小鼠3种囊泡模型，给予GKT137831能够有效延缓囊泡发展。体内和体外实验结果显示，GKT137831和NOX4敲除能够下调p22phox表达，上调NRF2的表达，并且抑制H2O2产生；进一步的研究发现，GKT137831和NOX4敲除能够抑制HIF1α和GLUT1表达，抑制AMPK/mTOR/ERK信号通路的激活，下调CFTR、PCNA和Ki67的表达，从而抑制囊液分泌和细胞增殖，发挥治疗多囊肾病的作用。研究成果揭示Nox4介导的H2O2产生影响囊泡上皮细胞增殖和囊液分泌的具体机制，为多囊肾的治疗提供新的靶点和实验依据。

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