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浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (1): 41-50    DOI: 10.3785/j.issn.1008-9292.2018.02.06
    
Screening of mitochondrial DNA damage repair genes in rats with septic acute kidney injury
YANG Jingjuan(),WU Fengfeng,CHEN Jianghua,YANG Yi*()
Kidney Disease Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
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Abstract  

Objective: To screen genes involved in mitochondrial DNA (mtDNA) damage repair in rats with septic acute kidney injury (SAKI). Methods: Forty male C57BL/6J mice were randomly divided into SAKI group (n=28) and sham operation group (n=12). The SAKI mouse model was established by cecal ligation and puncture. Blood and kidney samples were collected at 8, 24, and 48 h after surgery. Serum creatinine and urea nitrogen were measured by a dry biochemical analyzer. Serum inflammatory cytokines were detected by ELISA. Histopathological changes were observed with HE staining. The mtDNA damage repair related genes were screened by RNA sequencing and bioinformatics analysis; the mRNA and protein expression levels of related genes were detected by real-time quantitative RT-PCR and immunohistochemisry, respectively. Results: Symptoms of sepsis were observed in SAKI group, and 16 out of 28 mice were died in the SAKI group; serum TNF-α, IL-6, creatinine and urea nitrogen levels were higher than those in the sham group (P < 0.05 or P < 0.01). Histopathological examination in SAKI group showed that renal tubular epithelial cells were swollen, inflammatory cells infiltrated, and a large number of cell vacuoles were seen, suggesting successful modeling. Mitochondrial DNA damage repair related genes Gadd45α, Bcl2l1, Cdkn1a, Jun, Rela, Nfkbia and Nfkb1 were screened out. The expression of these genes was detected by real-time RT-PCR, and the results were consistent with RNA sequencing trends. Immunohistochemical staining showed that Gadd45α was mainly expressed in the nucleus of renal tubular epithelial cells, and the positive rate of Gadd45α in SAKI group was higher than that in the sham operation group (P < 0.05). Conclusion: Gadd45α, Bcl2l1, Cdkn1a, Jun, Rela, Nfkbia and Nfkb1 genes are involved in mtDNA damage repair in rats with SAKI, indicating that these genes may be used as new targets for prevention and treatment of SAKI.

Key words： Sepsis      DNA damage      Genes, tumor suppressor      Mitochondria      Kidney/injuries      Kidney/pathology      Renal insufficiency, acute      Disease models, animal     
Received: 28 September 2017      Published: 12 June 2018
CLC:  R36  
  R692  
Corresponding Authors: YANG Yi     E-mail: jingjuyang@126.com;yangyixk@zju.edu.cn
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YANG Jingjuan
WU Fengfeng
CHEN Jianghua
YANG Yi
Cite this article:

YANG Jingjuan,WU Fengfeng,CHEN Jianghua,YANG Yi. Screening of mitochondrial DNA damage repair genes in rats with septic acute kidney injury. J Zhejiang Univ (Med Sci), 2018, 47(1): 41-50.

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http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2018.02.06     OR     http://www.zjujournals.com/med/Y2018/V47/I1/41


脓毒性急性肾损伤小鼠线粒体DNA损伤修复相关基因的筛选

目的: 筛选脓毒性急性肾损伤（SAKI）中参与线粒体DNA（mtDNA）损伤修复的相关基因。方法: 40只清洁级雄性C57BL/6J小鼠随机分为SAKI组（28只）和对照组（12只）。采用盲肠结扎穿刺术建立SAKI小鼠模型。分别于术后8、24、48 h采集两组的血液和肾脏标本，干式生化仪检测血清肌酐和尿素氮，ELISA法检测血清炎症因子表达水平；HE染色观察肾脏组织病理学变化。RNA测序和生物信息学分析筛选mtDNA损伤修复相关基因；实时定量RT-PCR法和免疫组织化学法检测相关基因的mRNA和蛋白表达水平。结果: SAKI组小鼠术后均出现脓毒症症状，死亡16只；血清炎症因子（TNF-α和IL-6）、肌酐和尿素氮水平均高于对照组（P < 0.05或P < 0.01）；肾小管上皮细胞肿胀，炎症细胞浸润，并可见大量细胞空泡形成，提示建模成功。生物信息学分析筛选出Gadd45α、Bcl2l1、Cdkn1a、Jun、Rela、Nfkbia和Nfkb1等线粒体DNA损伤修复相关基因，实时定量RT-PCR检测以上基因表达量结果与RNA测序趋势一致。SAKI组Gadd45α主要在肾小管上皮细胞核中表达，其阳性表达率高于对照组（P < 0.05）。结论: Gadd45α、Bcl2l1、Cdkn1a、Jun、Rela、Nfkbia和Nfkb1基因参与了SAKI中mtDNA损伤修复，可以作为SAKI防治的新靶点。


关键词： 脓毒症,  DNA损伤,  基因, 肿瘤抑制,  线粒体,  肾/损伤,  肾/病理学,  肾功能不全, 急性,  疾病模型, 动物 
引物名称 引物序列(5′→3′)
Gadd45α 正向：CTGCAGAGCAGAAGACCGAA
反向：GGGTCTACGTTGAGCAGCTT
Bcl2l1 正向：GGAGAGCGTTCAGTGATC
反向：AGGTGGTCATTCAGATAGGT
Jun 正向：ACGACCTTCTACGACGAT
反向：CATTGCTGGACTGGATGAT
Cdkn1a 正向：GTGATGTCCGACCTGTTC
反向：TCAAAGTTCCACCGTTCTC
Rela 正向：GGCATCTGTGGACAACTC
反向：CGCAATGGAGGAGAAGTC
Nfkbia 正向：GCCAGTGTAGCAGTCTTG
反向：CAGGTAGCCGTGGATAGA
Nfkb1 正向：CAAGAGTGATGACGAGGAG
反向：GTGGATGATGGCTAAGTGTA
Gapdh 正向：CATGGCCTTCCGTGTTCCTA
反向：CCTGCTTCACCACCTTCTTGAT
Tab 1 Primer sequences for real-time RT-PCR
Fig 1 Serum levels of inflammatory factors, creatinine and urea nitrogen
Fig 2 Histopathological examinations of kidneys in sham-operated and SAKI groups (H&E staining)
Fig 3 Volcano plot of differentially expressed genes in sham-operated and SAKI groups
序号 信号通路名称 基因数 富集得分
  均P < 0.05.
1 BIOCARTA_RELA_PATHWAY 16 0.731 220
2 BIOCARTA_AMI_PATHWAY 15 0.759 689
3 BIOCARTA_AKT_PATHWAY 17 0.652 274
4 BIOCARTA_ALK_PATHWAY 29 0.180 719
5 BIOCARTA_AT1R_PATHWAY 30 0.463 202
6 BIOCARTA_CHEMICAL_PATHWAY 20 0.338 688
7 BIOCARTA_SPPA_PATHWAY 19 0.295 740
8 BIOCARTA_ATM_PATHWAY 18 0.574 498
9 BIOCARTA_BCR_PATHWAY 33 0.370 569
10 BIOCARTA_BIOPEPTIDES_PATHWAY 38 0.264 800
11 BIOCARTA_CASPASE_PATHWAY 21 0.712 487
12 BIOCARTA_CCR3_PATHWAY 20 0.310 291
13 BIOCARTA_CD40_PATHWAY 15 0.626 474
14 BIOCARTA_G1_PATHWAY 24 0.302 741
15 BIOCARTA_G2_PATHWAY 21 0.404 999
16 BIOCARTA_CERAMIDE_PATHWAY 21 0.531 168
17 BIOCARTA_TID_PATHWAY 17 0.576 491
18 BIOCARTA_GCR_PATHWAY 17 0.583 920
19 BIOCARTA_CTCF_PATHWAY 20 0.472 224
20 BIOCARTA_CELLCYCLE_PATHWAY 20 0.348 596
Tab 2 Top20 signaling pathways in gene set enrichment analysis of differentially expressed genes
基因 排名得分 富集得分 核心富集
  ATM:毛细血管扩张共济失调突变.
Cdkn1a 3.694 151 0.169 178 是
Nfkbia 2.551 620 0.279 946 是
Gadd45α 2.287 872 0.388 357 是
Jun 1.479 635 0.421 320 是
Rela 1.376 330 0.483 236 是
Nfkb1 1.167 506 0.523 231 是
Rad51 0.947 872 0.533 983 是
Brca1 0.877 482 0.563 427 是
Rad50 0.682 282 0.546 523 是
Mre11a 0.619 685 0.558 859 是
Mdm2 0.579 492 0.574 498 是
Tab 3 Differentially expressed genes associated with ataxia-telangiectasia mutated(ATM) signaling pathway
基因 术后8 h 术后24 h 术后48 h
log2(倍数变化) P值 log2(倍数变化) P值 log2(倍数变化) P值
Ucp2 1.545 837 765 0.000 0 2.421 564 0.000 0 -0.232 888 755 0.000 1
Ucp3 2.005 799 517 0.019 1 1.877 483 0.023 9 -0.022 749 298 0.655 0
Bcl2l1 0.966 255 449 0.000 0 1.418 362 0.000 0 0.136 584 293 0.032 7
Txnrd1 0.633 360 128 0.000 0 1.394 716 0.000 0 0.226 891 292 0.000 0
Ung 0.420 837 016 0.164 8 1.362 909 0.000 0 0.392 288 202 0.283 3
Cry1 2.212 250 395 0.000 0 1.230 154 0.000 0 0.939 775 997 0.002 5
Ephx2 0.076 290 019 0.049 5 1.202 637 0.000 0 0.113 014 758 0.009 4
Trmt11 -0.232 064 313 0.178 3 1.038 919 0.000 0 0.676 802 335 0.000 1
Aifm3 -1.001 395 984 0.000 0 -1.084 27 0.000 0 -0.150 239 033 0.336 5
Cpt1c -2.203 653 849 0.000 0 -1.162 05 0.005 0 -0.910 274 568 0.043 5
Msrb2 -1.083 997 153 0.000 0 -1.186 08 0.000 0 -0.258 678 473 0.022 5
Alas2 1.274 288 353 0.000 0 -1.274 52 0.000 0 1.325 174 006 0.000 0
Sept4 -1.297 047 836 0.000 0 -1.365 54 0.000 0 -0.110 745 779 0.346 6
Gstk1 -0.254 009 677 0.000 3 -1.671 25 0.000 0 -0.197 267 995 0.009 8
Mpv17l 0.076 453 114 0.000 0 -2.021 58 0.000 0 -1.680 361 986 0.000 0
Idh1 -1.407 372 317 0.000 0 -2.546 52 0.000 0 0.104 786 086 0.000 0
Tab 4 Screening results of mitochondrial oxidative stress and DNA damage repair related genes
Fig 4 The protein interaction network of ataxia-telangiectasia mutated (ATM) signal pathway genes and mitochondria related genes
基因 术后8 h 术后24 h 术后48 h
log2倍数变化 P值 log2倍数变化 P值 log2倍数变化 P值
Gadd45α 1.825 227 0.000 0 2.287 872 0.000 0 -0.033 4 0.590 9
Bcl2l1 0.966 255 0.000 0 1.418 362 0.000 0 0.136 584 0.032 7
Cdkn1a 3.114 603 0.000 0 3.694 151 0.000 0 1.681 139 0.000 0
Jun 0.861 052 0.000 0 1.479 635 0.000 0 -0.805 31 0.000 0
Rela 0.823 518 0.000 0 1.376 33 0.000 0 0.093 765 0.000 0
Nfkbia 1.904 727 0.000 0 2.551 62 0.000 0 0.712 892 0.000 0
Nfkb1 0.149 964 0.000 0 1.167 506 0.000 0 -0.188 24 0.000 0
Tab 5 Sequencing results of protein interaction-related genes
Fig 5 Expression levels of mtDNA damage repair related genes in renal tissues of sham-operated and SAKI groups
Fig 6 Expression of Gadd45α protein in kidney tissues of sham-operated and SAKI groups
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