肾小管上皮细胞"增殖-死亡"恶性循环推动肾间质纤维化进展的分子机制阐释

Tubulointerstitial fibrosis (TIF) is one of the final common outcomes of chronic allograft nephropathy. Several hypotheses were developed to elucidate the progression of TIF, including inflammation, epithelial-mesenchymal transition (EMT), senescence, chronic hypoxia and reactive oxygen species (ROS). Indeed, these mechanisms were involved in the progression of TIF, we have recently found that TIF is promoted by the "damage - proliferation - death" pattern, therefore a new hypothesis is proposed: the common mechanism of TIF is the establishment of "proliferation - death" vicious circle of renal tubular epithelial cells (RTEC) under the chronic stresses conditions. Based on the hypothesis, under the pressure of chronic injury we plan to verify the "proliferation - death" vicious cycle is common process in the pathophysiology of TIF by mutiple cellular and animal model. Through inhibiting or enhancing the proliferation, we want to evidence the cell death is associated with the enterance of cell cycle. The conflicting molecular mechanisms between the activation of proliferation signal and death pathway in "proliferation - death" pattern is try to interpreted by DNA damage respone (DDR). Using ATM gene modified the cell line and animal or p21 knockout cell line and animal, we will study the DDR and it primered apoptosis pathway in those TIF model, to reveal the DDR is the true reason of cell death happened during the cell cycle. Additionally, specimens of clinical renal biopsy will be studied to further emphasis the validity of the hypothesis in TIF.This investigation proposes a novel hypothesis, emphasizing that the mechanisms of cell proliferation, under the persistent stresses, may have a negative impact on the pathphysiological processes of TIF, and attempts to reveal the molecular mechanisms of “proliferation – death” vicious cycle. Future success of the study will bring significant impact on prognosis of laboratory, delaying the progression of TIF, and protecting residual renal function.

肾间质纤维化（TIF）是肾脏慢性病变的最终结局。针对TIF有炎症、转分化、缺氧、衰老和氧自由基等假说。我们研究发现：损伤导致的代偿性增殖，使肾小管上皮细胞对损伤因素敏感，更易发生细胞死亡。就此提出新的假说：慢性损伤条件下，“增殖-死亡”恶性循环推动TIF进展；并试图阐明其机制。拟通过多个细胞和动物模型证明此恶性循环普遍存在于TIF的病理生理过程；采用促进和抑制增殖的手段，揭示 “增殖-死亡”恶性循环的细胞死亡发生在细胞周期进程中；通过ATM基因修饰和p21基因敲除的细胞和动物模型证实DNA损伤反应（DDR）激活的凋亡信号途径启动是细胞死亡的真正原因。本研究提出全新假说，强调了持续损伤条件下过度的增殖对TIF进程的负面影响，揭示了“增殖-死亡”恶性循环的分子机制为DDR启动了凋亡。经器官、细胞和分子水平的研究，将在TIF的实验室诊断、延缓TIF进展和保护残余肾功能等转化医学领域产生积极影响。

背景：纤维化是各种器官在持续损伤以后的最终结局。我们的前期研究发现：损伤会导致代偿性增殖，细胞进入分裂周期对外界的压力敏感，更易发生细胞损伤和死亡。因此提出假说：持续压力条件下，激活“增殖-死亡”恶性循环，推动器官损伤、炎症、纤维化发展，但对机制研究还处于空白。该恶性循环启动细胞周期和损伤，可能涉及增殖及DNA损伤反应（DDR）的信号通路的激活；抑制该此两通路的关键分子ATM与P21或将有助于改善细胞损伤，减少细胞死亡。.主要研究内容：体外试验中，抑制DDR通路或加快细胞周期，检测细胞凋亡和增殖的变化。体内试验中，建立重急性胰腺炎肾损伤模型，分别通过雷帕霉素及ATM抑制剂（KU 55933）作用，抑制增殖和DDR，观察细胞凋亡、增殖，分析DDR信号通路与肾损伤的联系；建立UUO、缺血再灌注肾损伤模型，使用抗炎物质垂盆草提取物（SSBE）、Notch 1抑制剂DAPT进行干预，行WB、免疫荧光、RT-PCR、基因分析等检测细胞凋亡、增殖、上皮-间质转化的标志分子及差异基因的表达。.重要结果和关键数据：HK 2损伤后，细胞增殖、凋亡上调（Bax、caspase3），细胞增殖信号PCNA表达增加，证明了在慢性损伤条件下细胞“凋亡-增殖”恶性循环的发生；干预ATM或加快细胞周期，损伤后增殖、凋亡增多，证明增殖导致的凋亡与DNA损伤有关。胰腺炎肾损伤模型中，炎症水平、凋亡、增殖均上调，雷帕霉素干预抑制细胞周期后，指标明显好转，凋亡明显下降，证明凋亡发生与细胞周期有关；其分子机制研究中，以KU 55933抑制DDR通路，结果发现凋亡、增殖更明显，说明DNA损伤参与了“凋亡-增殖”的恶性循环。Gene Ontology结果表明损伤导致Ki 67、Tgfb1、Tgfb2、Tgfb3 以及Gli1相关基因均上调，SSBE及DAPT能有效下调细胞增殖，和EMT、Col3α1和α-SMA的下调，上皮-间质转化被抑制，表明抑制增殖可阻断“增殖-凋亡”恶性循环推动的纤维化的发生。.科学意义：该研究证明了“增殖-凋亡”恶性循环的存在，而通过下调损伤因素（炎症）、抑制增殖能改善该恶性循环；通过ATM抑制剂发现，DDR参与到此恶性循环中，本研究为损伤的治疗提供了新的治疗靶点有望应用于临床。