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中华肾病研究电子杂志

中华肾病研究电子杂志 ›› 2026, Vol. 15 ›› Issue (05) : 253 -259. doi: 10.3877/cma.j.issn.2095-3216.2026.05.002

论著

小鼠肾小管上皮细胞特异性c-kit基因敲除减轻单侧肾缺血-再灌注损伤诱导的肾间质纤维化
邢增辉1,2, 国文凯3, 刘超1, 王洪峰1,2, 郑升春1,2, 陈艳1,2, 韩冰4, 张一绚1, 王桂秀5, 陈香美1,†(), 孙雪峰1,†()   
  1. 1100853　北京，解放军总医院第一医学中心肾脏病医学部、肾脏疾病全国重点实验室、肾病与泌尿系统疾病国家临床医学研究中心、重症肾脏疾病器械与中西医药物研发北京市重点实验室、数智中医泛血管疾病防治北京市重点实验室、国家中医药管理局高水平中医药重点学科(zyyzdxk-2023310)、国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-D-202402)
    2100853　北京，解放军医学院
    3250033　济南，山东大学齐鲁第二医院肾脏多学科创新医学中心
    4300121　天津市人民医院（南开大学第一附属医院）肾脏病科
    5266200　青岛市即墨区中医医院小儿输液室
  • 收稿日期:2026-01-07 出版日期:2026-10-28
  • 通信作者: 陈香美, 孙雪峰
  • 基金资助:
    国家自然科学基金(82200780)

Renal tubular epithelial cell-specific c-kit gene knockout alleviates renal interstitial fibrosis induced by unilateral ischemia-reperfusion injury in mice

Zenghui Xing1,2, Wenkai Guo3, Chao Liu1, Hongfeng Wang1,2, Shengchun Zheng1,2, Yan Chen1,2, Bing Han4, Yixuan Zhang1, Guixiu Wang5, Xiangmei Chen1,†(), Xuefeng Sun1,†()   

  1. 1Senior Department of Nephrology, First Medical Center of Chinese PLA General Hospital, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney and Urological Diseases, Beijing Key Laboratory of Medical Devices and Integrated Traditional Chinese and Western Drug Development for Severe Kidney Diseases, Beijing Key Laboratory of Digital Intelligent TCM for the Preventionand Treatment of Pan-vascular Diseases, Key Disciplines of National Administration of Traditional Chinese Medicine(zyyzdxk-2023310), Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-D-202402), Beijing 100853, China
    2Chinese PLA Medical School, Beijing 100853
    3Multidisciplinary Innovation Center of Nephrology, Qilu Second Hospital of Shandong University, Jinan 250033, Shandong Province
    4Department of Nephrology, Tianjin People′s Hospital (First Affiliated Hospital of Nankai University), Tianjin 300121
    5Pediatric Infusion Room, Jimo District Hospital of Traditional Chinese Medicine, Qingdao 266200, Shandong Province; China
  • Received:2026-01-07 Published:2026-10-28
  • Corresponding author: Xiangmei Chen, Xuefeng Sun
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引用本文：

邢增辉, 国文凯, 刘超, 王洪峰, 郑升春, 陈艳, 韩冰, 张一绚, 王桂秀, 陈香美, 孙雪峰. 小鼠肾小管上皮细胞特异性c-kit基因敲除减轻单侧肾缺血-再灌注损伤诱导的肾间质纤维化[J/OL]. 中华肾病研究电子杂志, 2026, 15(05): 253-259.

Zenghui Xing, Wenkai Guo, Chao Liu, Hongfeng Wang, Shengchun Zheng, Yan Chen, Bing Han, Yixuan Zhang, Guixiu Wang, Xiangmei Chen, Xuefeng Sun. Renal tubular epithelial cell-specific c-kit gene knockout alleviates renal interstitial fibrosis induced by unilateral ischemia-reperfusion injury in mice[J/OL]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2026, 15(05): 253-259.

目的

探究小鼠肾小管上皮细胞特异性c-kit基因敲除对肾单侧缺血再灌注损伤(unilateral ischemia-reperfusion injury，uIRI)后巨噬细胞浸润及肾间质纤维化的影响。

方法

选取肾小管上皮细胞c-kit基因特异性敲除(Ggt1-Cre c-kit－/－)小鼠、干细胞因子受体LoxP纯合(c-kitloxp/loxp)小鼠、野生型(wild type, WT)8周龄雄性C57BL/6J小鼠构建uIRI模型。实验小鼠分为6组：Ggt1-Cre c-kit －/－ uIRI组、c-kitloxp/loxp uIRI组、C57BL/6J uIRI组(WT uIRI组)、Ggt1-Cre c-kit －/－假手术组、c-kitloxp/loxp假手术组、C57BL/6J假手术组(WT假手术组)，12只/组。分别于术后1 d、3 d、14 d和28 d取材，检测血清肌酐、血尿素氮水平和肾脏组织病理学改变，天狼星红染色评估肾间质纤维化；免疫组化观察巨噬细胞浸润情况；Western印迹和RT-qPCR法检测α-平滑肌肌动蛋白、波形蛋白、Ⅰ型胶原的蛋白及mRNA表达。组间比较采用方差分析。

结果

与WT uIRI组及c-kitloxp/loxp uIRI组相比，Ggt1-Cre c-kit －/－ uIRI组小鼠在uIRI术后各时间点的血清肌酐与血尿素氮水平均较低(P均<0.05)；uIRI术后3 d、14 d，Ggt1-Cre c-kit －/－ uIRI组巨噬细胞浸润数量亦较其他两组减少；uIRI术后28 d，Ggt1-Cre c-kit －/－ uIRI组肾间质纤维化评分以及纤维化标志物α-平滑肌肌动蛋白、波形蛋白、Ⅰ型胶原的mRNA及蛋白水平均低于其他两组(P均<0.05)。

结论

肾小管上皮细胞c-kit基因敲除可显著减轻肾间质纤维化，并抑制巨噬细胞聚集，提示c-kit基因有可能成为肾脏疾病治疗的新靶点。

关键词: 肾小管上皮细胞, c-kit基因, 基因敲除, 缺血再灌注损伤, 巨噬细胞, 肾间质纤维化
Objective

To investigate the effects of renal tubular epithelial cell-specific c-kit gene knockout on macrophage infiltration and renal interstitial fibrosis following unilateral ischemia-reperfusion injury (uIRI) in mice.

Methods

Eight-week-old male renal tubular epithelial cell-specific c-kit knockout mice (Ggt1-Cre c-kit-/-), c-kitloxp/loxp mice, and wild-type (WT) C57BL/6J mice were used to establish the uIRI model. Experimental mice were divided into six groups (n= 12 per group): Ggt1-Cre c-kit-/- uIRI group, c-kitloxp/loxp uIRI group, C57BL/6J uIRI group (WT uIRI group), Ggt1-Cre c-kit-/- sham group, c-kitloxp/loxp sham group, and C57BL/6J sham group (WT sham group). Kidney tissue and blood samples were collected at 1, 3, 14, and 28 days post-operation. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured, and renal histopathological changes were evaluated. Sirius red staining was used to assess renal interstitial fibrosis. Macrophage infiltration was examined by immunohistochemistry. The protein and mRNA expression levels of α-smooth muscle actin (α-SMA), vimentin, and collagen I were detected by Western blotting and RT-qPCR, respectively. ANOVA software was used for statistical analysis.

Results

Compared with the WT uIRI group and c-kitloxp/loxp uIRI group, the Ggt1-Cre c-kit-/- uIRI group exhibited significantly lower SCr and BUN levels at all time points after uIRI operation (all P<0.05). On 3 and 14 days post-operation, the number of infiltrating macrophages in the Ggt1-Cre c-kit-/- uIRI group was significantly reduced compared with the other two uIRI groups. On postoperative day 28, the renal interstitial fibrosis score and the mRNA and protein expression levels of fibrosis markers (α-SMA, vimentin, and collagen I) in the Ggt1-Cre c-kit-/- uIRI group were significantly lower than those in the other two uIRI groups (all P<0.05).

Conclusion

Renal tubular epithelial cell-specific c-kit gene knockout significantly attenuates renal interstitial fibrosis and inhibits macrophage infiltration, suggesting that c-kit gene may serve as a new therapeutic target for kidney diseases.

Key words: Renal tubular epithelial cell, c-kit gene, Gene knockout, Ischemia-reperfusion injury, Macrophage, Renal interstitial fibrosis
表1 鼠尾鉴定基因引物序列
引物组 扩增子长度(bp) 基因型 序列(5′→3′)
c-kitloxp正向引物 188/247 WT/c-kitloxp AATGATGTTCATCGAGCATGAGAG
c-kitloxp反向引物     GAGACATGAACATTAAGGGATGCTG
Ggt1-Cre正向引物 428 Ggt1-Cre CATCACATCAG GCACCCCAGAA
Ggt1-Cre反向引物     GAACATCTTCAGGTTCTGCGGGA
表2 实时荧光定量逆转录聚合酶链式反应引物序列
检测指标 种属 上游引物序列(5′-3′) 下游引物序列(5′-3′)
18S 小鼠 CCTCTATGCCAACACAGTGCTGTCT GCTCAGGAGGAGCAATGATCTTGA
α-平滑肌肌动蛋白 小鼠 AGCCATCTTTCATTGGGATGG CCCCTGACAGGACGTTGTTA
波形蛋白 小鼠 GGCTGCGAGAGAAATTGCAGG AAGACGTGCCAGAGAAGCATT
Ⅰ型胶原 小鼠 ACACGCAAGGCAATGAGACT TGGTCACTTGCACTGGTTGA
表3 各组小鼠肾单侧缺血再灌注损伤后肾功能动态变化
肾功能指标 分组 假手术 uIRI 1 d uIRI 3 d uIRI 14 d uIRI 28 d
血清肌酐(μmol/L) WT组 12.301±0.466 179.094±9.407ab 50.420±8.807ab 28.335±2.880ab 20.917±0.798b
  c-kitloxp/loxp组 12.949±2.137 175.612±5.704ac 52.197±4.542ac 28.073±0.324ac 20.077±2.196b
  Ggt1-Cre c-kit－/－组 12.253±3.031 114.227±9.124a 35.710±1.040a 23.189±6.712a 16.907±1.642
血尿素氮(mmol/L) WT组 6.923±1.714 51.143±4.493ab 27.472±2.560ab 20.672±2.560ab 12.552±0.756ab
  c-kitloxp/loxp组 6.610±1.286 51.326±1.248ab 27.400±0.544ab 20.637±0.574ab 12.947±0.962ab
  Ggt1-Cre c-kit－/－组 6.886±0.792 36.162±2.876a 22.706±0.419a 17.490±1.325a 10.482±0.270
图1 肾单侧缺血再灌注损伤术后各组小鼠肾组织病理学变化注：WT，野生型C57BL/6J小鼠组；c-kitloxp/loxp，干细胞因子受体基因c-kit LoxP纯合小鼠组；Ggt1-Cre c-kit －/－，肾小管上皮细胞特异性c-kit基因敲除小鼠组；Sham，假手术组；uIRI，unilateral ischemia-reperfusion injury，单侧缺血再灌注损伤；比例尺＝20 μm，n＝3；组间差异采用双因素方差分析；与Ggt1-Cre c-kit－/－uIRI组28 d相比，aP<0.05
图2 肾单侧缺血再灌注损伤术后28 d各组小鼠肾脏纤维化相关蛋白mRNA水平与半定量分析注：WT：野生型C57BL/6J小鼠组；c-kitloxp/loxp：干细胞因子受体基因c-kit LoxP纯合小鼠组；Ggt1-Cre c-kit －/－：肾小管上皮细胞特异性c-kit基因敲除小鼠组；Sham：假手术组；uIRI，unilateral ischemia-reperfusion injury，单侧缺血再灌注损伤；α-SMA：α-smooth muscle actin，α-平滑肌肌动蛋白；Vimentin：波形蛋白、Collagen I：I型胶原；组间差异采用双因素方差分析；与Ggt1-Cre c-kit －/－uIRI组28 d相比，aP<0.05
图3 肾单侧缺血再灌注损伤术后28 d各组小鼠肾脏纤维化相关蛋白表达与半定量分析注：WT：野生型C57BL/6J小鼠组；c-kitloxp/loxp：干细胞因子受体基因c-kit LoxP纯合小鼠组；Ggt1-Cre c-kit －/－：肾小管上皮细胞特异性c-kit基因敲除小鼠组；Sham：假手术组；uIRI，unilateral ischemia-reperfusion injury，单侧缺血再灌注损伤；α-SMA：α-smooth muscle actin，α-平滑肌肌动蛋白；Vimentin：波形蛋白、Collagen Ⅰ：Ⅰ型胶原；GAPDH：glyceraldehyde-3-phosphate dehydrogenase，甘油醛-3-磷酸脱氢酶；组间差异采用双因素方差分析；与Ggt1-Cre c-kit －/－uIRI组28 d相比，aP<0.05
图4 肾单侧缺血再灌注损伤术后各组巨噬细胞标志物F4/80免疫组织化学染色及半定量分析注：WT：野生型C57BL/6J小鼠组；c-kitloxp/loxp：干细胞因子受体基因c-kit LoxP纯合小鼠组；Ggt1-Cre c-kit －/－：肾小管上皮细胞特异性c-kit基因敲除小鼠组；Sham：假手术组；uIRI，unilateral ischemia-reperfusion injury，单侧缺血再灌注损伤；比例尺＝20 μm，n＝3；组间差异采用双因素方差分析；与Ggt1-Cre c-kit －/－uIRI组3 d相比，aP<0.05；与Ggt1-Cre c-kit －/－uIRI组14 d相比，bP<0.05；Positive area ratio，阳性面积比
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