慢性铅染毒致雄性小鼠肾损伤的机制：基于环磷酸腺苷/蛋白激酶A信号通路
Mechanism of kidney damage induced by chronic lead exposure in mice: Based on cyclic adenosine monophosphate/protein kinase A signaling pathway
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摘要:
背景 铅染毒可引起肾脏损伤。研究表明环磷酸腺苷/蛋白激酶A（cAMP/PKA）信号通路可通过调节氧化酶活性介导活性氧过量产生，进而引起氧化应激和细胞凋亡。烟酰胺腺嘌呤二核苷酸磷酸（NADPH）途径是肾脏活性氧的主要来源之一。
目的 探讨铅是否通过cAMP/PKA途径调节肾脏中NADPH氧化酶4（NOX4）活性引起肾脏损伤。
方法 30只SPF级ICR雄性小鼠随机分为对照组和铅暴露组，每组15只。通过自由饮水的方式进行铅暴露，乙酸铅浓度分别为0、200mg·L-1。连续染毒90d后麻醉小鼠进行心脏采血，取肾脏、称重，计算脏器系数。采用石墨炉原子吸收分光光度仪检测血液和肾脏中铅含量，HE染色观察肾脏组织形态学改变，TUNEL法检测肾细胞凋亡水平；通过试剂盒检测肾组织中总超氧化物歧化酶（T-SOD）、铜锌超氧化物歧化酶（Cu/Zn-SOD）活力，谷胱甘肽（GSH）、丙二醛（MDA）和cAMP含量；采用实时荧光定量聚合酶链式反应法测定
PKA、NOX4 以及凋亡相关基因Bcl-2、Bax 和caspase3 mRNA相对表达水平。结果 两组小鼠体重和日饮水量差异无统计学意义。铅暴露组小鼠肾脏系数（0.89±0.02）%、血铅（100.13±17.23）μg·L-1和肾铅（2.58±0.21）μg·g-1水平均高于对照组（0.78±0.02）%、（1.88±0.50）μg·L-1、（0.04±0.01）μg·g-1（均
P < 0.05）。铅暴露组小鼠肾脏近曲小管和肾小球形态学损伤明显，细胞凋亡率高于对照组（0.013±0.003）%、（0.006±0.002）%；肾脏中GSH水平、T-SOD和Cu/Zn-SOD活性降低（P < 0.05），MDA含量升高（P < 0.05）；Bcl-2 mRNA相对表达量及Bcl-2/Bax值下降，caspase3 mRNA相对表达量则增高（P < 0.05）；肾组织中cAMP含量、PKA 和NOX4 mRNA相对表达量均升高，且NOX4 mRNA相对表达量与cAMP含量之间呈正相关（r =0.486，P < 0.05）。结论 铅可改变小鼠氧化酶NOX4活性，引起肾脏组织损伤，可能与cAMP/PKA信号通路有关。
Abstract:Background Lead can cause kidney damage. It has been shown that cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway can mediate reactive oxygen species (ROS) overproduction by regulating the activity of oxidase and then cause oxidative stress and apoptosis. Nicotinamide adenine dinucleotide phosphate (NADPH) pathway is one of the main sources of ROS in kidney.
Objective This experiment investigates whether lead cause kidney damage by regulating the activity of NADPH oxidase 4 (NOX4) through cAMP/PKA pathway.
Methods Thirty SPF ICR mice were randomly divided into a control group and a lead exposure group, with 15 mice in each group. The animals were given free drinking water containing 0 and 200 mg·L-1 lead acetate, respectively. After 90 days of continuous exposure, the mice were anesthetized to collect heart blood and kidney samples, weigh organs, and calculate organ coefficients. The lead levels in both blood and kidney were measured with graphite furnace atomic absorption spectrometer. Renal histomorphology was observed by HE staining and apoptosis was detected by TUNEL. The activities of total superoxide dismutase (T-SOD) and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), and the levels of glutathione (GSH), malondialdehyde (MDA), and cAMP were detected using corresponding kits. The relative mRNA expression levels of
PKA ,NOX4 ,Bcl-2 ,Bax , andcaspase3 were measured by quantitative real-time PCR.Results There were no significant differences in body weight and daily water intake between the two groups. The renal coefficient(0.89±0.02)%, blood lead level(100.13±17.23) μg·L-1, and renal lead level(2.58±0.21) μg·g-1 in the lead exposure group were higher than those in the control group(0.78±0.02)%, (1.88±0.50) μg·L-1, and (0.04±0.01) μg·g-1 (
P < 0.05). In the lead exposure group, the morphological damage of renal proximal convoluted tubules and glomeruli was obvious, and the apoptosis rate increased compared with the control group(0.013±0.003)% vs. (0.006±0.002)%; the GSH level, T-SOD activity, and Cu/Zn-SOD activity in kidney decreased (P < 0.05), while the MDA content increased (P < 0.05); the relative expression level ofBcl-2 mRNA and the ratio of Bcl-2/Bax decreased, and the relative expression level of caspase3 mRNA increased (P < 0.05); the level of cAMP and the relative expression levels ofPKA andNOX4 mRNA in renal tissues increased, and there was a positive correlation between the relative expression level ofNOX4 mRNA and the level of cAMP (r =0.486,P < 0.05).Conclusion Lead can change the activity of NOX4 and cause kidney damage, which is related to cAMP/PKA signaling pathway.
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