脓毒血症肾小管上皮细胞PGC-1α/TFAM信号通路改变及线粒体功能障碍

    Changes of PGC-1α/TFAM signaling pathway and mitochondrial dysfunction of renal tubular epithelial cells during sepsis-associated acute kidney injury

      摘要
    • 摘要: 目的 探讨脓毒血症急性肾损伤时肾小管上皮细胞过氧化物酶体增殖物激活受体γ辅激活因子1α（peroxisome proliferatoractivated receptor γ coactivator-1α，PGC-1α）和线粒体转录因子A （mitochondrial transcription factor A，TFAM）的信号通路改变及线粒体损伤。方法 8~10周雄性C57BL/6小鼠腹腔内注射脂多糖（lipopolysaccharide，LPS，10 mg/kg）诱导脓毒血症急性肾损伤（acute kidney injury，AKI）模型，检测血清尿素氮、肌酐和肾损伤分子1和肾脏组织病理学改变，电镜下观察小鼠近端肾小管上皮细胞的自噬现象和线粒体的形态改变。利用Western blot、实时荧光定量PCR法和免疫荧光、免疫组化等方法检测小鼠肾组织内LC3Ⅱ、p62、PGC-1α、TFAM和线粒体DNA与核DNA比值的表达变化。体外培养人近端肾小管上皮细胞株（HK-2细胞），LPS诱导脓毒血症AKI肾小管上皮细胞模型，检测PGC-1α和TFAM的表达变化，MTT法检测细胞活力；JC-1检测线粒体膜电位变化，MitoTracker标记线粒体形态学变化；Mito-SOXTM检测线粒体超氧化物含量改变。结果 与对照组相比，模型组小鼠血清尿素氮、肌酐和肾损伤分子1明显升高，白细胞介素（interleukin，IL）-1β、IL-6及高迁移率族蛋白B1（high mobility group box 1 protein，HMGB1）等炎症及氧化应激因子转录增加。光镜下模型组近端肾小管上皮细胞未见明显坏死或凋亡，但电镜下可见线粒体明显肿胀、变圆，嵴排列紊乱或消失，自噬体增加，肾组织中自噬标记蛋白LC3Ⅱ及p62表达增加（P<0.05），PGC-1α和TFAM mRNA及蛋白表达量显著下降（P<0.05），线粒体DNA与核DNA比值下降（P<0.05）。体外LPS刺激可引起HK-2细胞活力下降，线粒体膜电位下降，线粒体片段化改变以及线粒体超氧化物含量升高。HK-2细胞PGC-1α和TFAM蛋白表达量随LPS处理浓度增加而减少（P<0.05）。结论 PGC-1α/TFAM信号通路参与脓毒血症时近端肾小管上皮细胞的线粒体功能障碍，可能是脓毒血症AKI新的干预靶点。

       

      Abstract: Objective To explore the changes of signaling pathways of peroxisome proliferator-activated receptor γ coactivator 1α and mitochondrial transcription factor A and mitochondrial damage in renal tubular epithelial cells during sepsis-associated acute kidney injury(AKI).Methods A murine model of septic AKI model was established after 8-10-week-old male C57BL/6 mice received an intraperitoneal injection of lipopolysaccharide(LPS;10 mg/kg).Serum levels of urea nitrogen,creatinine and kidney injury molecule 1 were measured at 24h post-injection.Renal pathological damage was observed after periodic acid-Schiff(PAS)stain.Electron microscope was employed for observing the changes of mitochondrial morphology and count in proximal renal tubular epithelial cells.Western blot and real-time fluorescent quantitative polymerase chain reaction(PCR)were employed for detecting the mRNA/protein expression levels of LC3II,p62,PGC-1α,TFAM and the ratio of mitochondrial DNA to nuclear DNA.In LPS-induced endotoxemia AKI model of renal tubular epithelial cell,PGC-1α and TFAM were measured by Western blot,RT-qPCR and immunofluorescence.MTT was utilized for detecting cell viability;mitochondrial membrane potential observed by JC-1;mitochondrial morphological change detected by MitoTracker and mitochondrial superoxide content assessed by Mito-SOXTM.Results As compared with control group,blood urea nitrogen,serum creatinine and kidney injury molecule 1 spiked markedly in model group.And proximal renal tubular epithelial cells had mild vacuolar degeneration and a greater transcription of inflammation and oxidative stress factors such as IL-1β,IL-6 and HMGB1.Under electron microscope,mitochondria in proximal renal tubular epithelial cells in model group became obviously swollen and rounded with broken ridges and more autophagosomes and the expressions of autophagy marker proteins LC3II and p62 were up-regulated in kidney tissue(P<0.05)and the mRNA/protein expressions of PGC-1α and TFAM declined markedly(P<0.05).The ratio of mitochondrial DNA to nuclear DNA(mtDNA:nDNA)declined in renal cortex tissues of LPS-injected mice as compared with control mice(P<0.05).In vitro LPS stimulation lowered HK-2 cell viability and boosted mitochondrial dysfunction.The protein expressions of PGC-1α and TFAM declined with the rising concentration of LPS in HK-2 cells(P<0.05).Conclusion The signaling pathway of PGC-1α/TFAM may be a novel intervention target for septic AKI.

       

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