Klotho Reduces Necroptosis by Targeting Oxidative Stress Involved in Renal Ischemic-Reperfusion Injury
Klotho Reduces Necroptosis by Targeting Oxidative Stress Involved in Renal Ischemic-Reperfusion Injury
复制标题
Klotho 通过针对肾缺血再灌注损伤中涉及的氧化应激来减少坏死性凋亡
DOI：
10.1159/000488172
复制
发表时间：
2018-01-01
影响因子：
--
通讯作者：
Gu, Leyi
中科院分区：
文献类型：
--
作者：
Qian, Yingying;Guo, Xiangjiang;Gu, Leyi
背景/目的：Klotho是一种多功能蛋白，主要在肾小管上皮细胞中表达。在这里，我们研究了Klotho对肾缺血再灌注损伤（IRI）中坏死性凋亡的保护作用以及氧化应激在此过程中的作用。方法：采用双侧肾蒂钳夹法。将小鼠肾小管上皮细胞（TCMK-1）暴露于缺氧/复氧（H/R）或H2 O2。通过免疫荧光法检查急性肾损伤（阿基）患者和对照组的肾脏样本。Klotho蛋白和N-乙酰-L-半胱氨酸（NAC）被用来定义它们在介导坏死性凋亡中的作用。通过TUNEL染色、免疫印迹和实时PCR评估坏死性凋亡。通过ELISA、免疫印迹、比色法和硫代巴比妥酸反应物质测定研究氧化应激。结果：肾性IRI引起血清和肾脏Klotho缺乏，而尿中Klotho增加。IRI后坏死性凋亡标志物受体相互作用蛋白激酶（RIP）1、RIP 3、IL-1β和TUNEL阳性细胞的水平升高; Klotho可改善所有升高。在TCMK-1细胞中，Klotho和NAC减弱了由H/R或H2 O2诱导的RIP 1、RIP 3和LDH释放的升高。此外，Klotho在体内和体外实验中都降低了氧化应激生物标志物的水平，并提高了超氧化物歧化酶2的表达。对人体样本的研究进一步证实了Klotho缺乏症和阿基肾脏中RIP 3斑点的形成增加。结论：Klotho对肾小管上皮细胞的保护作用可能与抑制氧化应激有关。
Background/Aims: Klotho is a multifunctional protein expressed predominantly in kidney tubular epithelium. Here, we investigated the protective effects of Klotho on necroptosis in renal ischemic-reperfusion injury (IRI) and the role of oxidative stress in this process. Methods: Mice were subjected to bilateral renal pedicle clamping. Mouse renal tubular epithelial (TCMK-1) cells were exposed to hypoxia/reoxygenation (H/R) or H2O2. Kidney samples from acute kidney injury (AKI) patients and controls were examined by immunofluorescence. Klotho protein and N-acetyl-L-cysteine (NAC) were used to define their roles in mediating necroptosis. Necroptosis was assessed by TUNEL staining, immunoblotting, and real-time PCR. Oxidative stress was studied via ELISA, immunoblotting, colorimetric, and thiobarbituric acid reactive substances assays. Results: Renal IRI induced Klotho deficiency in the serum and kidney, but an increase in the urine. The levels of the necroptotic markers receptor-interacting protein kinase (RIP) 1, RIP3, IL-1β, and TUNEL-positive cells increased after IRI; all increases were ameliorated by Klotho. In TCMK-1 cells, Klotho and NAC attenuated the elevation in RIP1, RIP3, and LDH release induced by H/R or H2O2. Moreover, Klotho decreased the levels of oxidative stress biomarkers and elevated superoxide dismutase 2 expression in both in vivo and in vitro experiments. Studies in human samples further confirmed the Klotho deficiency and increased formation of RIP3 puncta in AKI kidneys. Conclusion: Klotho protects tubular epithelial cells from IRI and its anti-necroptotic role may be associated with oxidative stress inhibition.