MDM2介导的成纤维细胞活化在肾小管间质纤维化中的作用及其机制

The accumulation of extracellular matrix (ECM) with the expanded interstitial region is the key pathological feature of tubulointerstitial fibrosis (TIF). The pathologic ECM mainly comes from the proliferative and transdifferentiated fibroblast. However the mechanisms of the activation of fibroblast (proliferation, transdifferentiation and ECM overproduction) are not completely understood. . Murine Double Minute 2(MDM2) is an E3 ubiquitin ligase which regulates cell cycle, differentiation, inflammatory reaction and other pathophysiological processes through p53 dependent and independent signaling pathways. Our preliminary data shown that in patients accompanying with TIF and mice model of Unilateral Ureteral Obstruction (UUO) the expression of MDM2 was upregulated. Nevertheless, pharmacological inhibition of the association between MDM2 and p53 cannot reverse the severity of TIF as well as the transdifferentiation and ECM accumulation in NRK-49F cells (Normal Rat Kidney Fibroblast) exposed to TGF-β1. Thus, it is proposed that MDM2 mediates fibroblast activation and TIF via p53 independent pathway.. Interestingly, we found the abundance of Notch1 in renal cortex was elevated markedly in UUO mice. Notch1 is a transmembrane protein and involves in various biological processes, such as cell proliferation, transdifferentiation and collagen production. In cancer cells and myoblasts MDM2 can activate Notch1. Moreover, our preliminary data indicated the overexpressed Notch1 co-localized with MDM2 in the interstitial fibroblast. It is suggested that Notch1 may be the downstream target of MDM2 during renal fibroblast activation.. Taken together, we hypothesize that MDM2-Notch1 signaling pathway is implicated in fibroblast activation and TIF development. On the basis of our previous experiments we are going to investigate the significance of MDM2-Notch1 signaling pathway in TIF patients and MDM2 fibroblast conditional knockout UUO mice, as well as in vitro cultured fibroblast isolated from above conditional knockout mice and NRK-49F exposed TGF-β. By this investigation we wish to clarify the molecular mechanisms of fibroblast activation and TIF and shed light on the exploration of its effective therapies.

肾间质成纤维细胞活化是肾小管间质纤维化(TIF)的关键环节，但其发生机制未明。MDM2是一E3泛素连接酶，可调控细胞周期、分化、炎症反应等病理生理过程。我们的前期研究发现，MDM2在TIF患者与UUO小鼠肾间质的成纤维细胞中表达增强，并伴随成纤维细胞活化；然而阻断经典的MDM2-p53通路未能抑制上述病变。有趣的是，我们发现TIF病变中Notch1表达亦显著增强，且与MDM2共定位于肾间质成纤维细胞。上述结果提示，MDM2-Notch1信号通路可能参与成纤维细胞活化与TIF进程。本项目拟在前期研究基础上，以TIF患者、成纤维细胞特异性MDM2基因敲除小鼠和体外培养的肾脏成纤维细胞为研究对象，通过表征与调控MDM2、Notch1的表达，探讨MDM2-Notch1信号通路在成纤维细胞活化和TIF进程中的关键作用，以期阐明成纤维细胞活化的分子机制，并为TIF的防治提供新的理论依据。

慢性肾脏病（Chronic Kidney Disease, CKD）是危胁人类健康的重要疾病之一，也是导致患者发展至终末期肾脏病的主要原因。鼠双微体2（Murine Double Minute 2, MDM2）属于E3泛素化连接酶，其在肿瘤细胞增殖以及炎症反应过程中发挥着重要作用。在肾小球中MDM2过表达参与了足细胞损伤。然而在肾脏纤维化的发生与进展中，MDM2的作用尚不清楚。在本研究中，我们通过患者肾活检标本、单侧输尿管结扎小鼠模型(UUO)与体外培养大鼠成纤维细胞（NRK-49F），探索与揭示了MDM2在肾小管间质纤维化（TIF）中的作用与机制。我们研究结论如下：在TIF患者、UUO小鼠小管间质区域以及活化的成纤维细胞中MDM2表达上调，并且MDM2介导的Notch1泛素化修饰-蛋白酶体降解是其导致纤维化作用的重要机制，而经典的MDM2-p53通路并未参与此病理生理过程。在上述研究的基础上，我们进一步探索MDM2在急性肾脏病（Acute Kidney Disease, AKI）向CKD进展中的作用与机制。通过患者肾活检标本、多次小剂量顺铂（Cisplatin, CP）诱导的AKI向CKD进展（AKI-CKD）小鼠模型与体外培养大鼠肾小管上皮细胞（NRK-52E），探讨了近端肾小管上皮细胞（Proximal Tubular Epithelium, PTE）中MDM2在AKI-CKD进展中的作用及机制。我们得出以下结论：1.在体内和体外多次小剂量顺铂（CP）诱导的AKI-CKD模型中，PTE中MDM2的表达定位从细胞核转移至细胞膜，且此转位现象参与并促进AKI向CKD进展；2.多次小剂量顺铂（CP）诱导细胞核MDM2表达减少，伴有p53表达上调及G2/M阻滞；3.多次小剂量顺铂（CP）诱导细胞膜MDM2集聚，并且促进整合素β8的降解，从而减弱整合素β8对TGF-β1的抑制效应，最终导致TGF-β1信号通路激活，促进肾脏纤维化的发生与进展。