Objective: To investigate the role and mechanism of trimethylamine N-oxide (TMAO), a uremic toxin, in renal fibrosis.
Methods: A total of 20 male BALB/c mice were randomly and evenly assigned to a Control group and a TMAO group. Mice in the Control group received intraperitoneal injection of normal saline, while mice in the TMAO group received intraperitoneal injection of TMAO (20 mg/[kg·d]). The injection was given once a day for 8 weeks. Histopathology and fibrosis of kidney were observed by H&E staining and Masson staining. Immunohistochemistry was performed to determine the levels of alpha smooth muscle actin (α-SMA), recombinant human fibronectin fragment (Fibronectin), and sterol-regulatory element binding protein 1 (SREBP1). Western blot was performed to determine α-SMA, SREBP1, phosphatidylinositol 3 kinase (PI3K), phospho-phosphatidylinositol 3 kinase (p-PI3K), protein kinase B (PKB, also known as AKT), and phospho-AKT (p-AKT) protein levels. HK2 cells were treated with SREBP1 small interfering RNA (siRNA) and PI3K/AKT inhibitor, respectively, and the reversal of the effects of TMAO was examined.
Results: Animal experiments showed that, compared with the Control group, the mice treated with TMAO experienced pathological damage and fibrosis of the kidney tissue and the expression levels of fibrosis markers, α-SMA and Fibronectin, in the kidney were increased (all P<0.05). According to the findings from further investigation, the TMAO-treatment group showed increased expression of SREBP1 and an up-regulation of PI3K phosphorylation ratio and AKT phosphorylation ratio compared with those of the Control group (all P<0.05). Cell experiments produced results similar to those of the animal experiment. After siRNA interference with SREBP1 expression, the expression levels of fibrosis marker proteins decreased (P<0.05). Besides, the high expression of SREBP1 caused by TMAO was inhibited after HK2 cells were incubated with LY294002, a PI3K-AKT pathway inhibitor (P<0.05).
Conclusion: TMAO may induce renal fibrosis by promoting the PI3K/AKT/SREBP1 pathway.
目的: 探讨尿毒症毒素氧化三甲胺（trimethylamine N-oxide, TMAO）在肾脏纤维化中的作用及其机制。
方法: 将20只雄性BALB/c小鼠随机平均分为对照组和TMAO组，每组10只。对照组腹腔注射生理盐水，TMAO组腹腔注射含TMAO的生理盐水〔20 mg/(kg·d) 〕，每日1次，持续8周；使用HE染色与Masson染色法观察分析小鼠肾脏切片病理及纤维化水平；采用免疫组化法检测肾脏组织中α-肌动蛋白（alpha smooth muscle actin, α-SMA）、重组人纤维连接蛋白片段（recombinant human fibronectin fragment, Fibronectin）、胆固醇调节元件结合蛋白-1（sterol-regulatory element binding protein 1, SREBP1）水平；采用Western blot检测肾脏组织中α-SMA、SREBP1、磷脂酰肌醇3激酶（phosphatidylinositol 3 kinase, PI3K）、磷酸化PI3K（phospho-PI3K, p-PI3K）、蛋白激酶B（protein kinase B, PKB，又称AKT）和磷酸化AKT（phospho-AKT, p-AKT）蛋白水平。分别用SREBP1 siRNA和PI3K/AKT抑制剂组处理HK2细胞，检测对TMAO效应的逆转作用。
结果: 动物实验显示，与对照组相比，腹腔注射TMAO后小鼠出现肾脏组织病理损伤和纤维化，纤维化标志物α-SMA、Fibronectin表达升高（P均<0.05）。相对于对照组，TMAO处理组小鼠肾脏中SREBP1表达升高（P<0.05），PI3K磷酸化比值、AKT磷酸化比值也发生了上调（P均<0.05）。细胞实验验证了上述结果。利用siRNA干扰肾小管上皮细胞中SREBP1表达后，纤维化指标蛋白表达下降（P<0.05)；利用PI3K-AKT通路抑制剂LY294002孵育HK2细胞后，TMAO导致的SREBP1高表达被抑制（P<0.05）。
结论: TMAO可能通过促进PI3K/AKT/SREBP1通路而诱导肾脏纤维化。
Keywords: PI3K/AKT pathway; Renal fibrosis; SREBP1; Trimethylamine N-oxide.
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