Sun Qian, Zeng Cheng, Du Li, Dong Chong
Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China.
Organ Transplantation Center, Tianjin First Central Hospital, Tianjin 300192, P.R. China.
Exp Ther Med. 2021 Mar;21(3):190. doi: 10.3892/etm.2021.9622. Epub 2021 Jan 7.
The nuclear erythroid 2-related factor 2 (NRF2)/antioxidant response element (ARE) pathway has been shown to provide strong protection against oxidative stress injury induced by renal ischemia-reperfusion (IR). However, the endogenous regulatory mechanism of the NRF2/ARE pathway in renal IR injury is incompletely understood. A rat model of renal IR was established by occlusion of the bilateral renal pedicle for 45 min, followed by reperfusion for 24 h. Renal injury was assessed by light microscopy and levels of serum creatinine, blood urea nitrogen and neutrophil gelatinase-associated lipocalin was measured using enzyme-linked immunosorbent assay. Renal oxidative stress was also evaluated by measuring superoxide dismutase and malondialdehyde in renal tissues. Protein expression levels of brain and muscle ARNT-like 1 (BMAL1), nuclear factor erythroid 2-related factor 2 (NRF2), NAD(P)H dehydrogenase [quinone] 1 (NQO1), glutamate-cysteine ligase modifier (GCLM) and heme oxygenase 1 (HO1) in the kidney were determined by western blotting and immunohistochemistry. Reverse transcription-quantitative PCR was used to evaluate rhythmic transcription of the core clock genes (CLOCK and BMAL1) and the NRF2 gene. The nature of the binding of BMAL1 to the promoter regions in the NRF2 gene was assessed by chromatin immunoprecipitation assays in rat kidneys. BMAL1 was found to bind to the promoter of the NRF2 gene through an E-BOX element associated with strongly rhythmic activation of NRF2 in both the normal kidney and those exposed to IR. The ARE-regulated anti-oxidative stress protein was affected by the circadian rhythm of the NRF2 gene. As the NRF2 level was at a circadian nadir, the expression of the proteins NQO1, GCLM and HO1 was weakened, resulting in more serious renal oxidative stress injury and pathological and functional impairment induced by IR. It can be concluded that the circadian rhythm of the NRF2/ARE pathway controlled by the circadian clock is essential for regulating antioxidant stress in renal IR injury, which might prompt new therapeutic strategies associated with the diurnal variability of human kidney disease, including renal transplantation.
核红细胞2相关因子2（NRF2）/抗氧化反应元件（ARE）通路已被证明能为肾脏缺血再灌注（IR）诱导的氧化应激损伤提供强大保护。然而，NRF2/ARE通路在肾脏IR损伤中的内源性调节机制尚未完全明确。通过阻断双侧肾蒂45分钟，随后再灌注24小时，建立大鼠肾脏IR模型。通过光学显微镜评估肾脏损伤，并使用酶联免疫吸附测定法测量血清肌酐、血尿素氮和中性粒细胞明胶酶相关脂质运载蛋白水平。还通过测量肾组织中的超氧化物歧化酶和丙二醛来评估肾脏氧化应激。通过蛋白质印迹法和免疫组织化学法测定肾脏中脑和肌肉芳香烃受体核转运蛋白样蛋白1（BMAL1）、核因子红细胞2相关因子2（NRF2）、NAD（P）H脱氢酶[醌]1（NQO1）、谷氨酸-半胱氨酸连接酶修饰亚基（GCLM）和血红素加氧酶1（HO1）的蛋白表达水平。采用逆转录定量PCR评估核心生物钟基因（CLOCK和BMAL1）和NRF2基因的节律性转录。通过大鼠肾脏的染色质免疫沉淀试验评估BMAL1与NRF2基因启动子区域的结合性质。发现BMAL1通过与正常肾脏和IR暴露肾脏中NRF2强烈节律性激活相关的E-盒元件与NRF2基因的启动子结合。ARE调节的抗氧化应激蛋白受NRF2基因昼夜节律的影响。由于NRF2水平处于昼夜最低点，NQO1、GCLM和HO1蛋白的表达减弱，导致IR诱导的更严重的肾脏氧化应激损伤以及病理和功能损害。可以得出结论，由生物钟控制的NRF2/ARE通路的昼夜节律对于调节肾脏IR损伤中的抗氧化应激至关重要，这可能促使制定与人类肾脏疾病（包括肾移植）昼夜变化相关的新治疗策略。
