Klotho蛋白对肾缺血再灌注大鼠急性肾损伤及纤维化的保护作用

doi:10.11958/20221063

天津医药 ›› 2023, Vol. 51 ›› Issue (4): 371-375.doi: 10.11958/20221063

Klotho蛋白对肾缺血再灌注大鼠急性肾损伤及纤维化的保护作用

刘竹枫(), 王文红(), 范树颖, 刘艳, 刘涛, 王欣, 吴瑕

天津市儿童医院（天津大学儿童医院）肾内科（邮编300134）

收稿日期:2022-07-05 修回日期:2022-11-25 出版日期:2023-04-15 发布日期:2023-04-20
通讯作者: 王文红 E-mail:liuzhufeng406@sina.cn;docwwh@163.com
作者简介:刘竹枫（1978），女，副主任医师，主要从事小儿肾脏疾病方面研究。E-mail：liuzhufeng406@sina.cn
基金资助:
天津市卫生健康委员会科技基金资助项目(ZC20088)

Protective effect of Klotho protein on acute renal injury and fibrosis in rats with ischemia-reperfusion

LIU Zhufeng(), WANG Wenhong(), FAN Shuying, LIU Yan, LIU Tao, WANG Xin, WU Xia

Department of Nephrology, Tianjin Children’s Hospital, Tianjin University Children′s Hospital, Tianjin 300134, China

Received:2022-07-05 Revised:2022-11-25 Published:2023-04-15 Online:2023-04-20
Contact: WANG Wenhong E-mail:liuzhufeng406@sina.cn;docwwh@163.com

刘竹枫, 王文红, 范树颖, 刘艳, 刘涛, 王欣, 吴瑕. Klotho蛋白对肾缺血再灌注大鼠急性肾损伤及纤维化的保护作用[J]. 天津医药, 2023, 51(4): 371-375.

LIU Zhufeng, WANG Wenhong, FAN Shuying, LIU Yan, LIU Tao, WANG Xin, WU Xia. Protective effect of Klotho protein on acute renal injury and fibrosis in rats with ischemia-reperfusion[J]. Tianjin Medical Journal, 2023, 51(4): 371-375.

摘要/Abstract

摘要：

目的探讨Klotho蛋白在缺血再灌注（I/R）大鼠引起的急性肾损伤及纤维化进程中的作用。方法 60只健康雄性SD大鼠按随机数字表法分为假手术组（Sham组）、肾脏缺血再灌注组（I/R组）和Klotho蛋白干预组（I/R+Kl组），每组20只。I/R组采用夹闭右侧肾蒂40 min再灌注法建立肾缺血再灌注急性肾损伤模型。Sham组游离出右侧肾动脉，但不夹闭右侧肾蒂，暴露40 min后缝合腹腔。I/R+Kl组手术方法同I/R组，造模成功后，腹腔注射Klotho蛋白（0.01 mg/kg）。3组分别于造模成功后第1、7、14、28天收集大鼠血、肾组织标本，分别检测大鼠血肌酐（Scr）、尿素氮（BUN）水平；HE染色观察肾组织损伤情况，Masson染色观察肾纤维化情况，酶联免疫吸附试验（ELISA）检测血清及肾组织Klotho蛋白水平，Western blot法检测肾脏组织Klotho蛋白的表达。结果与Sham组比较，I/R组及I/R+Kl组血Scr及BUN水平均升高（P<0.05），与I/R+Kl组比较，I/R组各时间点血Scr及BUN水平均升高（P<0.05）。HE染色结果显示，第1天时I/R组肾小管出现广泛急性坏死，I/R+Kl组损伤较轻微。Masson染色结果显示，I/R组第14天出现了轻度的纤维化，第28天时纤维化程度更为显著，I/R+Kl组纤维化程度较轻微（P<0.05）。ELISA结果显示，第1、14、28天时与Sham组比较，I/R+Kl组及I/R组血清及肾组织Klotho水平下降（P<0.05），I/R组血清及肾组织Klotho水平均低于I/R+Kl组（P<0.05）。Western blot结果显示，与Sham组比较，各时间点I/R组及I/R+Kl组Klotho水平均下降，而I/R+Kl组Klotho水平均高于I/R组（P<0.05）。结论肾缺血再灌注肾损伤时Klotho表达下调；外源性补充Klotho蛋白可缓解缺血再灌注肾损伤大鼠的急性肾损伤及纤维化，起到肾保护作用。

关键词: 急性肾损伤, 再灌注损伤, 纤维化, 肾疾病, Klotho蛋白

Abstract:

Objective To investigate the role of Klotho protein in acute renal injury and fibrosis induced by ischemia-reperfusion (I/R) in rats. Methods Sixty healthy SD rats were randomly divided into the sham operation group (sham group), the renal ischemia-reperfusion group (I/R group) and the Klotho protein intervention group (I/R+Kl group), with 20 rats in each group. In the I/R group, the acute renal injury model of I/R was established by clamping the right renal pedicle for 40 min. In the sham group, the right renal artery was dissociated, but the right renal pedicle was not clamped. After exposure for 40 minutes, the abdominal cavity was sutured. The operation method of the I/R+Kl group was the same as that of the I/R group. Kiotho protein (0.01 mg/kg) was injected intraperitoneally. The blood and renal tissue samples were collected on the 1st, 7th, 14th and 28th days after successful modeling in the three groups, and blood creatinine (Scr) and blood urea nitrogen (BUN) were measured respectively. HE staining was used to observe the damage of renal tissue. Masson staining was used to observe the renal fibrosis. ELISA was used to detect levels of Klotho protein in blood and renal tissue. Western blot assay was used to detect the expression of Klotho protein in renal tissue. Results Compared with the sham group, levels of SCR and BUN were increased in the I/R group and the I/R+Kl group (P<0.05). Compared with the I/R+Kl group, serum levels of SCR and BUN were increased in different time points in the I/R group (P<0.05). HE staining showed extensive acute necrosis of renal tubules in the I/R group, and the injury was mild in the I/R+Kl group. Masson staining showed that mild fibrosis occurred in the I/R group on the 14th day, and the degree of fibrosis was more significant on the 28th day, while the degree of fibrosis was very mild in the I/R+Kl group (P < 0.05). ELISA results showed that compared with the Sham group at day 1, 14 and 28, levels of Klotho in blood and renal tissue decreased significantly in the I/R+Kl group and the I/R group, and the level of Klotho decreased in the I/R group than that of the I/R+Kl group (P<0.05). Western blot results showed that compared with the Sham group, Klotho levels were decreased at all time points in the I/R group and the I/R+Kl group. while Klotho levels were higher in the I/R+Kl group than those in the I/R group (P<0.05). Conclusion Klotho expression is down-regulated during renal I/R injury. Exogenous supplementation of Klotho protein can alleviate acute renal injury and fibrosis in rats with renal I/R injury and play a role in renal protection.

Key words: acute kidney injury, reperfusion injury, fibrosis, kidney diseases, Klotho protein

中图分类号:

R726.92

图/表 8

图1 各组大鼠血肌酐水平比较 F第1天=52.521，F第7天=18.307，F第14天=148.194，F第28天=277.336，均P<0.05；a与Sham组相比，b与I/R组相比，P<0.05。

Fig.1 Comparison of serum creatinine levels between the three groups

图2 各组大鼠血尿素氮水平比较 F第1天=72.217，F第7天=92.919，F第14天=383.615，F第28天=219.687，均P<0.01；a与Sham组相比，b与I/R组相比，P<0.05。

Fig.2 Comparison of serum urea nitrogen levels between the three groups of rats

图3 缺血再灌注损伤后各组大鼠肾组织形态学改变（HE染色，×400）

Fig.3 Renal histomorphological changes of rats in each group after ischemia-reperfusion injury (HE staining, ×400)

图4 缺血再灌注损伤后各组大鼠肾组织纤维化改变（Masson染色，×400）

Fig.4 Changes of renal fibrosis in rats after ischemia-reperfusion injury (Masson staining, ×400)

图5 缺血再灌注损伤后各组大鼠肾组纤维化损伤评分 F第1天=951.278，F第7天=262.255，F第14天=374.990，F第28天=577.436，均P<0.01；a与Sham组相比，b与I/R组相比，P<0.05。

Fig.5 Renal fibrosis injury score of rats in each group after ischemia-reperfusion injury

图6 各组大鼠肾组织Klotho水平比较 F第1天=125.264**，F第7天=0.237，F第14天=107.545**，F第28天=145.164**，**P<0.01；a与Sham组相比，b与I/R组相比，P<0.05。

Fig.6 Comparison of Klotho protein levels in renal tissue of rats between the three groups

图7 各组大鼠血清Klotho蛋白水平比较 F第1天=126.533，F第7天=6.345，F第14天=48.208，F第28天=79.438，均P<0.01；a与Sham组相比，b与I/R组相比，P<0.05。

Fig.7 Comparison of serum Klotho protein levels of rats between the three groups

图8 Western blot法检测各组大鼠肾组织Klotho蛋白的表达 F第1天=113.096，F第7天=16.029，F第14天=58.570，F第28天=32.894，均P<0.01；a与Sham组相比，b与I/R组相比，P<0.05。

Fig.8 The expression levels of Klotho protein in renal tissue of rats detected by Western blot assay

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Klotho蛋白对肾缺血再灌注大鼠急性肾损伤及纤维化的保护作用

Protective effect of Klotho protein on acute renal injury and fibrosis in rats with ischemia-reperfusion

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