法尼醇 X 受体基因敲除加重顺铂诱导的肾损伤。
Knockout of farnesoid X receptor gene aggravates cisplatin-induced kidney injury.
机构信息
Department of Nephrology, Third Xiangya Hospital, Central South University, Changsha 410013.
Medical College of Hunan Traditional Chinese Medical College, Zhuzhou Hunan 412012.
出版信息
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2022 Feb 28;47(2):174-182. doi: 10.11817/j.issn.1672-7347.2022.210423.
OBJECTIVES
Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand activated transcription factors and belongs to bile acid receptor. Studies have shown that the expression of FXR in renal tissue can reduce renal injury via regulation of glucose and lipid metabolism, inhibition of inflammatory response, reduction of oxidative stress and renal fibrosis. However, it is unclear whether FXR is involved in autophagy in renal diseases. This study aims to investigate the role of FXR in cisplatin-induced acute renal injury and whether its mechanism is related to autophagy regulation.
METHODS
Twelve male WT or FXR-KO mice at 12 weeks were randomly divided into a WT group, a WT+cisplatin group, a FXR-KO group, and a FXR-KO+cisplatin group, with 6 mice in each group. The WT+cisplatin group and the FXR-KO+cisplatin group were intraperitoneally injected with cisplatin (20 mg/kg), and the WT group and the FXR-KO group were intraperitoneally injected with equal volume of cisplatin solvent. Seventy-two hours later, the mice were killed and blood and renal tissue samples were collected. The levels of SCr and BUN were detected by immunoturbidimetry. After the staining, the pathological changes of renal tissue were observed under optical microscope. The protein levels of LC3 and p62 were detected by Western blotting and immunohistochemistry. The clearance of damaged mitochondria and the accumulation of lysosomal substrate were observed under electron microscope. The apoptosis of renal tubular epithelial cells was detected by TUNEL.
RESULTS
Compared with the WT group or the FXR-KO group, both SCr and BUN levels in the WT+cisplatin group or the FXR-KO+cisplatin group were significantly increased (<0.01 or <0.001), and SCr and BUN levels in the FXR-KO+cisplatin group were significantly higher than those in the WT+cisplatin group (both <0.05). Under the light microscope, there were no obvious pathological changes in the renal tissue of mice in the WT group and the FXR-KO group. Both the WT+cisplatin group and the FXR-KO+cisplatin group had vacuolar or granular degeneration of renal tubular epithelial cells, flat cells, lumen expansion, brush edge falling off, and even exposed basement membrane and tubular formation. The scores of renal tubular injury in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both <0.001), and the score in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (<0.05). Under the transmission electron microscope, the mitochondria of mouse tubular epithelial cell in the WT+cisplatin group and the FXR-KO+cisplatin group was swollen, round, vacuolated, cristae broken or disappeared; the lysosome was uneven and high-density clumps, and the change was more obvious in the FXR-KO+cisplatin group. Western blotting showed that the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased in the WT+cisplatin group compared with the WT group and the FXR-KO+cisplatin group compared with FXR-KO group (<0.05 or <0.01); compared with the FXR-KO group, the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased significantly in the FXR-KO+cisplatin group (both <0.05). Immunohistochemistry results showed that the expression of total LC3 and p62 in renal cortex of the WT+cisplatin group and the FXR-KO+cisplatin group was increased significantly, especially in the FXR-KO+cisplatin group. TUNEL results showed that the mice in the WT group and the FXR-KO group had negative staining or only a few apoptotic tubular epithelial cells, and the number of apoptotic cells in the WT+cisplatin group and the FXR-KO+cisplatin group were increased. The apoptosis rates of renal tubular epithelial cells in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both <0.001), and the apoptosis rate in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (<0.05).
CONCLUSIONS
Knockout of gene aggravates cisplatin induced acute renal injury, and its mechanism may be related to inhibiting autophagy and promoting apoptosis.
目的
法尼醇 X 受体（FXR）是配体激活转录因子核受体超家族的成员，属于胆汁酸受体。研究表明，肾组织中 FXR 的表达可以通过调节葡萄糖和脂质代谢、抑制炎症反应、减少氧化应激和肾纤维化来减轻肾损伤。然而，FXR 是否参与肾脏疾病中的自噬尚不清楚。本研究旨在探讨 FXR 在顺铂诱导的急性肾损伤中的作用及其机制是否与自噬调节有关。
方法
12 只 12 周龄雄性 WT 或 FXR-KO 小鼠随机分为 WT 组、WT+顺铂组、FXR-KO 组和 FXR-KO+顺铂组，每组 6 只。WT+顺铂组和 FXR-KO+顺铂组腹腔注射顺铂（20mg/kg），WT 组和 FXR-KO 组腹腔注射等体积顺铂溶剂。72 小时后处死小鼠，采集血样和肾组织样本。采用免疫比浊法检测 SCr 和 BUN 水平。染色后，在光学显微镜下观察肾组织的病理变化。采用 Western blot 和免疫组化法检测 LC3 和 p62 的蛋白水平。在电子显微镜下观察受损线粒体的清除和溶酶体底物的积累。采用 TUNEL 法检测肾小管上皮细胞的凋亡。
结果
与 WT 组或 FXR-KO 组相比，WT+顺铂组或 FXR-KO+顺铂组的 SCr 和 BUN 水平均明显升高（均<0.01 或<0.001），且 FXR-KO+顺铂组的 SCr 和 BUN 水平明显高于 WT+顺铂组（均<0.05）。WT 组和 FXR-KO 组小鼠的肾组织在光镜下无明显病理改变。WT+顺铂组和 FXR-KO+顺铂组肾小管上皮细胞有空泡或颗粒状变性、扁平细胞、管腔扩张、刷缘脱落，甚至暴露基底膜和管型形成。WT+顺铂组和 FXR-KO+顺铂组肾小管损伤评分均明显高于 WT 组和 FXR-KO 组（均<0.001），且 FXR-KO+顺铂组评分明显高于 WT+顺铂组（<0.05）。在透射电镜下，WT+顺铂组和 FXR-KO+顺铂组小鼠肾小管上皮细胞的线粒体肿胀、呈圆形、有空泡、嵴断裂或消失；溶酶体不均匀，高密度聚集，FXR-KO+顺铂组变化更明显。Western blot 结果显示，与 WT 组相比，WT+顺铂组 LC3-II/LC3-I 比值降低，p62 表达增加（均<0.05 或<0.01）；与 FXR-KO 组相比，FXR-KO+顺铂组 LC3-II/LC3-I 比值降低，p62 表达明显增加（均<0.05）。免疫组化结果显示，WT+顺铂组和 FXR-KO+顺铂组肾皮质总 LC3 和 p62 的表达明显增加，尤其是 FXR-KO+顺铂组。TUNEL 结果显示，WT 组和 FXR-KO 组小鼠肾小管上皮细胞染色阴性或仅有少数凋亡细胞，WT+顺铂组和 FXR-KO+顺铂组凋亡细胞数增加。WT+顺铂组和 FXR-KO+顺铂组肾小管上皮细胞的凋亡率明显高于 WT 组和 FXR-KO 组（均<0.001），FXR-KO+顺铂组的凋亡率明显高于 WT+顺铂组（<0.05）。
结论
基因敲除加重顺铂诱导的急性肾损伤，其机制可能与抑制自噬和促进凋亡有关。
Zotero 插件