基于线粒体自噬探讨脂联素抑制肾小管上皮细胞程序性死亡的机制

Acute kidney injury (AKI) is caused by numerous factor such as anoxia and medications and is characterized by the damage of renal tubules. Necroptosis of renal tubular epithelial cells is one of the leading mechanism in tubule damage. Obesity decreases the level of plasma adiponectin, which may related to the high rate and severity of renal disease including AKI in obese group. Our previous study showed that obesity decreased the plasma adiponectin level and aggravated cisplatin-induced AKI and that adiponectin overexpressed rat had a lower degree of cisplatin-induced AKI and a high expression of autophagy related gene ATG16L1. Thus, it is hypothesized that adiponectin inhibits necroptosis and thereafter protects kidney from AKI via the induction of mitophagy. The models of renal tubular cells culture, ex vivo renal tubules culture and rodent animals will be adopted to observe the dynamic change of cisplatin/TNF-α induced necroptosis and mitophagy, as well as the effects of adiponectin. Moreover, this project puts an emphasis on illustrating whether the effects of adiponectin relate to mitophagy using the autophagy probes and autophagy inhibitor/inducer. At last, cisplatin induced AKI will be observed when the interference adeno associated virus are used to downregulate both plasma adiponectin and renal ATG16L1, respectively. Execution of this preject will provide the evidence for understanding the relation between mitophagy and necroptosis in AKI, as well as the novel idea for treating AKI.

急性肾损伤（AKI）由多种因素如缺氧及药物等导致，主要病理表现为肾小管损伤。肾小管上皮细胞程序性死亡是肾小管损伤的重要原因。肥胖状态下脂联素降低，AKI等肾脏疾病发生率及严重程度均增加。前期预实验发现，肥胖大鼠顺铂AKI加重而脂联素水平下降，脂联素过表达大鼠自噬基因ATG16L1表达升高，顺铂AKI减轻。课题组假设：脂联素通过促进线粒体自噬，抑制肾小管上皮细胞程序性死亡，减轻AKI。本课题拟采用肾小管上皮细胞、近端肾小管体外培养及动物模型，观察顺铂和TNF-α诱导的程序性死亡及线粒体自噬的动态变化，并探索脂联素的作用；重点以自噬和线粒体探针及自噬的诱导剂和抑制剂探明脂联素的作用是否与其调控线粒体自噬相关；通过干扰腺相关病毒同时下调大鼠血浆脂联素和肾脏ATG16L1，观察顺铂AKI的变化。本课题可为探明AKI进程中线粒体自噬与细胞程序性死亡的关系提供依据，并为AKI的防治提供新的思路。

庆大霉素(Gentamicin, GEN)是一种在我国广泛使用的氨基糖苷类抗生素，急性肾损伤(AKI)是GEN常见的毒性反应。肾小管上皮细胞的损伤是AKI发生的重要机制。本研究以NRK-52E、HK-2细胞和SD大鼠作为GEN诱导AKI的模型，研究细胞程序性坏死和线粒体自噬在GEN-AKI中的发生过程和功能。程序性坏死相关蛋白，包括RIPK1、RIPK3、MLKL和磷酸化的MLKL (p-MLKL)，均呈时间依赖性增加。通过使用RIPK1抑制剂NEC-1和RIPK3抑制剂CPD42，可以减轻GEN诱导的肾小管细胞死亡。此外，研究还证实，在体内和体外两种情况下，NEC-1干预后，GEN诱导的细胞凋亡和炎症均有所减弱。在MLKL敲低的背景下，NEC-1和CPD42的保护作用并不能提高。半胱天冬酶抑制剂Z-VAD能显著降低GEN诱导的细胞凋亡，但可增加坏死，轻度提高细胞的存活，且Z-VAD与NEC-1和CPD42无协同作用。线粒体自噬相关蛋白PINK-1和PARKIN在GEN刺激的早期即显著增加，荧光显微镜观察也确证了自噬小体的形成。使用自噬诱导剂雷帕霉素可以进一步诱导自噬的发生，并显著提高细胞的活力。综上所述，程序性死亡和线粒体自噬在GEN-AKI进程中发挥重要的作用，是潜在防治氨基糖苷类抗生素肾毒性的的干预靶点。此外，本项目还探究了caveolin-1对线粒体自噬影响及乙酰氨基酚导致的脂质积累的影响，综述了脓毒症AKI过程中肾脏细胞的程序性死亡的过程。