en
×

分享给微信好友或者朋友圈

使用微信“扫一扫”功能。
通讯作者:

毛慧娟,E-mail:huijuanmao@126.com

中图分类号:R692.6

文献标识码:A

文章编号:1007-4368(2022)04-484-07

DOI:10.7655/NYDXBNS20220404

参考文献 1
BELLOMO R,KELLUM J A,RONCO C.Acute kidney in⁃jury[J].Lancet,2012,380(9843):756-766
参考文献 2
程小燕,邬步云,毛慧娟,等.住院患者急性肾损伤发生的流行病调查:一项单中心回顾性观察性研究[J].南京医科大学学报(自然科学版),2017,37(1):67-72
参考文献 3
BONVENTRE J V,YANG L.Cellular pathophysiology of ischemic acute kidney injury[J].J Clin Invest,2011,121(11):4210-4221
参考文献 4
PABLA N,DONG Z.Cisplatin nephrotoxicity:mecha⁃ nisms and renoprotective strategies[J].Kidney Int,2008,73(9):994-1007
参考文献 5
EDELING M,RAGI G,HUANG S,et al.Developmental signalling pathways in renal fibrosis:the roles of Notch,Wnt and Hedgehog[J].Nat Rev Nephrol,2016,12(7):426-439
参考文献 6
KAVVADAS P,KEUYLIAN Z,PRAKOURA N,et al.Notch3 orchestrates epithelial and inflammatory respons⁃ es to promote acute kidney injury[J].Kidney Int,2018,94(1):126-138
参考文献 7
TRIONFINI P,BENIGNI A,REMUZZI G.MicroRNAs in kidney physiology and disease[J].Nat Rev Nephrol,2015,11(1):23-33
参考文献 8
REN G L,ZHU J,LI J,et al.Noncoding RNAs in acute kidney injury[J].J Cell Physiol,2019,234(3):2266-2276
参考文献 9
BRANDENBURGER T,SALGADO SOMOZA A,DEVA⁃ UX Y,et al.Noncoding RNAs in acute kidney injury[J].Kidney Int,2018,94(5):870-881
参考文献 10
SONI H,MATTHEWS A T,PALLIKKUTH S,et al.Γ ⁃ secretase inhibitor DAPT mitigates cisplatin ⁃ induced acute kidney injury by suppressing Notch1 signaling[J].J Cell Mol Med,2019,23(1):260-270
参考文献 11
PAVKOVIC M,VAIDYA V S.MicroRNAs and drug ⁃in⁃ duced kidney injury[J].Pharmacol Ther,2016,163:48-57
参考文献 12
YANG Y,LIU H,LIU F,et al.Mitochondrial dysregula⁃ tion and protection in cisplatin nephrotoxicity[J].Arch Toxicol,2014,88(6):1249-1256
参考文献 13
KAUSHAL G P,KAUSHAL V,HERZOG C,et al.Au⁃ tophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity[J].Autophagy,2008,4(5):710-712
参考文献 14
BRAY S J.Notch signalling in context[J].Nat Rev Mol Cell Biol,2016,17(11):722-735
参考文献 15
DJUDJAJ S,CHATZIANTONIOU C,RAFFETSEDER U,et al.Notch⁃3 receptor activation drives inflammation and fibrosis following tubulointerstitial kidney injury[J].J Pathol,2012,228(3):286-299
参考文献 16
EL MACHHOUR F,KEUYLIAN Z,KAVVADAS P,et al.Activation of Notch3 in glomeruli promotes the develop⁃ ment of rapidly progressive renal disease[J].J Am Soc Nephrol,2015,26(7):1561-1575
参考文献 17
ZOU Y F,WEN D,ZHAO Q,et al.Urinary microRNA ⁃ 30c⁃5p and microRNA⁃192⁃5p as potential biomarkers of ischemia⁃reperfusion⁃induced kidney injury[J].Exp Biol Med(Maywood),2017,242(6):657-667
参考文献 18
KANKI M,MORIGUCHI A,SASAKI D,et al.Identifica⁃ tion of urinary miRNA biomarkers for detecting cisplatin⁃ induced proximal tubular injury in rats[J].Toxicology,2014,324:158-168
参考文献 19
ZOU D,GANUGULA R,ARORA M,et al.Oral delivery of nanoparticle urolithin A normalizes cellular stress and improves survival in mouse model of cisplatin ⁃ induced AKI[J].Am J Physiol Renal Physiol,2019,317(5):F1255-F1264
目录contents

    摘要

    目的:探讨 Notch3 在顺铂诱导的人肾小管上皮细胞(HK⁃2)损伤中的表达变化及其潜在的相关上游微小 RNA (microRNA,miRNA)调控机制。方法:体外培养HK⁃2细胞,Western blot及实时定量PCR检测Notch3在顺铂(20 μmol/L,24 h) 诱导HK⁃2细胞凋亡模型及正常细胞中的表达;Targetscan预测靶向Notch3的miRNA,实时定量PCR测定相关miRNA在顺铂诱导 HK⁃2细胞凋亡模型及正常细胞中的表达;然后,将HK⁃2细胞分别转染阴性对照模拟物(NC mimic)及miR⁃192⁃5p模拟物(miR⁃ 192⁃5p mimic),并依据给予或不给予顺铂处理(20 μmol/L,24 h),分为NC mimic组、NC mimic+Cisplatin组、miR⁃192⁃5p mimic组、 miR⁃192⁃5p mimic+Cisplatin 组。Western blot 及实时定量 PCR 检测 Notch3 的表达水平;流式细胞术检测细胞凋亡率,研究 miR⁃192⁃5p对顺铂所致肾小管上皮细胞凋亡及Notch3表达的影响;荧光素酶报告实验证实miR⁃192⁃5p与靶基因Notch3的靶向关系。结果:Notch3在顺铂诱导肾小管上皮细胞凋亡中的表达上调,同时miR⁃192⁃5p在顺铂诱导肾小管上皮细胞凋亡中的表达下调;实时定量 PCR 证实转染 miR⁃192⁃5p 模拟物后,miR⁃192⁃5p 的表达量升高;过表达 miR⁃192⁃5p 能够减轻顺铂诱导的肾小管上皮细胞凋亡,同时负向调控 Notch3 的表达;荧光素酶报告实验结果表明 miR⁃192⁃5p 直接靶向作用于 Notch3 的 3′⁃UTR,抑制Notch3的表达,证实Notch3是miR⁃192⁃5p的直接靶标。结论:miR⁃192⁃5p可通过直接抑制Notch3的表达而减轻顺铂所致的肾小管上皮细胞凋亡。

    Abstract

    Objective:To investigate the expression changes of Notch3 in cisplatin ⁃induced renal tubular epithelial cells(HK ⁃2) injury and the potential regulation mechanism related to upstream microRNA. Methods:Based on HK⁃2 cells apoptosis model induced by cisplatin(20 μmol/L,24 h),Western blot and RT⁃qPCR were used to analyze the expression of Notch3 in HK⁃2 cells. Targetscan was used to search potential miRNAs that bind the 3’⁃UTR of Notch3. RT⁃qPCR was used to detect the expression of miR⁃192⁃5p in HK ⁃2 cells after cisplatin treatment. Then,HK ⁃2 cells transfected with negative control mimic(NC mimic)and miR ⁃192⁃5p mimic respectively,and treated with or without cisplatin(20 μmol/L,24 h),were divided into four groups:NC mimic group(NC);NC mimic+ cisplatin group(NC+Cis);miR⁃192⁃5p mimic group(OE);miR⁃192⁃5p mimic+cisplatin group(OE+Cis). Western blot and RT⁃qPCR were used to demonstrate the effect of miR⁃192⁃5p on the expression of Notch3 after cisplatin treatment,while Western blot and flow cytometry were used to demonstrate the effect of miR⁃192⁃5p on apoptosis of HK⁃2 cells. Dual luciferase reporter assay was used to verified whether miR⁃192⁃5p targets Notch3. Results:Notch3 was up⁃regulated while miR⁃192⁃5p was down⁃regulated in HK⁃2 cells after cisplatin treatment compared with that in the normal controls. RT⁃qPCR showed that the expression of miR⁃192⁃5p in HK⁃2 cells was significantly increased after miR ⁃ 192 ⁃ 5p mimic transfection. The overexpression of miR ⁃ 192 ⁃ 5p alleviated cisplatin ⁃induced apoptosis of HK⁃2 cells and negatively regulated the expression of Notch3. The dual luciferase reporter assay results verified that miR⁃ 192⁃5p directly binds with 3’⁃UTR of Notch3. Conclusion:miR⁃192⁃5p can attenuate the apoptosis of renal tubular epithelial cells induced by cisplatin through directly inhibiting the expression of Notch3.

  • 急性肾损伤(acute kidney injury,AKI)是一种以肾功能突然下降为特征的临床综合征,其与住院患者的高病死率、远期慢性肾病的进展及其他类型的器官功能障碍密切相关[1-2]。AKI的主要特点是近端小管上皮细胞损伤和炎症,肾小管上皮细胞的功能障碍在缺血或毒性作用导致AKI的进展中起着关键作用,但其潜在机制尚未完全阐明[3-4]

  • Notch信号是一种高度保守的细胞间通信机制,可调节细胞发育、组织稳态及修复。近年来一系列研究表明Notch信号是肾脏发育、损伤和修复过程中的关键调节因子[5]。Kavvadas等[6] 在缺血再灌注肾损伤小鼠模型中发现Notch3异常高表达并证实Notch3参与了肾脏炎症反应并导致肾小管上皮细胞损伤,靶向Notch3或可为急性肾损伤提供一种新的治疗策略。然而Notch3在顺铂诱导的肾小管细胞损伤中的作用及其异常高表达的相关机制尚未阐明。

  • 微小RNA(microRNA,miRNA)是一类由内源基因编码的非编码单链RNA分子,其长度约为19~25个核苷酸,它们通过翻译抑制或促进RNA降解来介导靶基因的转录后调控,从而参与各种生理和病理过程[7]。在AKI的病理过程中,它们通过调节细胞凋亡、程序性死亡、炎症和细胞增殖等在肾脏的损伤和修复中发挥重要作用[8-9]。然而,目前还没有关于miRNA在顺铂诱导AKI过程中调控或靶向Notch3的相关研究报道。

  • 因此,本研究拟寻找靶向Nocth3的潜在miR⁃ NA,通过体外培养人肾小管上皮细胞,并转染相关miRNA类似物(mimics)以探讨相关miRNA调控及靶向Notch3在顺铂诱导肾小管上皮细胞凋亡中的作用,以期为防治肾小管坏死及急性肾损伤提供新的思路。

  • 1 材料和方法

  • 1.1 材料

  • 人近端肾小管上皮细胞株(HK⁃2细胞,中国典型培养物保藏中心);胎牛血清(FBS)、DMEM/F12培养基(Gibco公司,美国);顺铂(Cisplatin,Cis,Sigma公司,美国);miR⁃192⁃5p类似物(miR⁃192⁃5p mimic) 及其配对阴性miRNA类似物(negative control miRNA mimic,NC mimic)、miRNA相关PCR引物(茎环法)、miRNA转染试剂盒(广州锐博);TRIzol试剂 (Invitrogen公司,美国);PrimeScriptTM 反转录试剂盒、TB Green实时定量PCR试剂盒(TaKaRa公司,日本);miRNA反转录试剂盒(茎环法)、miRNA实时定量PCR试剂盒(南京诺唯赞);全蛋白提取试剂盒、 BCA法蛋白测定试盒(南京凯基);Notch3兔多克隆抗体(Abcam公司,英国);Caspase3和GAPDH抗体 (Proteintech公司,美国)。

  • 1.2 方法

  • 1.2.1 细胞培养与分组

  • HK⁃2细胞用含10%胎牛血清的DMEM/F12培养液于37℃ 5%CO2的细胞培养箱中培养。待细胞生长至70%~80%融合状态时用胰蛋白酶消化传代,接种于6孔板后待细胞生长至70%~80%融合状态用无血清培养基同步化培养24h。实验分为:①NC mimic组(NC);②NC mimic+Cisplatin组(NC+Cis);③miR⁃192⁃5p mimic组(OE);④miR⁃ 192⁃5p mimic+Cisplatin组(OE+Cis)。分别转染 (NC mimic、miR⁃192⁃5p mimic,加或不加顺铂20 μmol/L作用24h后,提取总RNA、总蛋白。

  • 1.2.2 实时荧光定量PCR

  • 按上述步骤干预细胞24h后用TRIzol氯仿⁃异丙醇法提取细胞总RNA,按照miRNA反转录说明书进行反转录,合成cDNA,反应体系10 μL,反转录条件:42℃ 2min,25℃ 5min,50℃ 15min, 85℃ 5min。实时荧光定量PCR反应体系为10 μL,反应条件:95℃ 5min,95℃ 10s,60℃ 30s,40个循环;95℃ 15s,60℃ 60s,95℃ 15s。mRNA实时定量PCR,按照mRNA反转录说明将RNA反转录成cDNA,反应条件:37℃ 15min,85℃ 5s。实时荧光定量PCR反应条件:95℃ 30s,95℃ 5s,60℃ 30s,40个循环。引物序列:内参β⁃ actin上游5′ ⁃AT⁃ GAAATCGCCGCACTG ⁃ 3′,下游5′ ⁃ TGAGCCTC⁃ GTCTCCCAC ⁃ 3′,Notch3上游5′ ⁃ TCAGGCTCT⁃ CACCCTTGG⁃3′,下游5′⁃AGTCACTGGCACGGTTG⁃ TAG⁃3′。采用LightCycler96实时荧光定量PCR仪 (瑞士罗氏公司)。miRNA以U6为内参,mRNA以 β⁃actin为内参,根据所得Ct值,运用2-ΔΔCT相对定量法分别计算miRNA及目的基因mRNA相对表达量。

  • 1.2.3 Western blot

  • 各组细胞按上述步骤干预24h后用预冷的RIPA裂解液、苯甲基磺酰氟(PMSF)和蛋白酶抑制剂裂解,提取总蛋白。测定蛋白浓度并配成统一浓度,加入上样缓冲液,沸水加热5~10min。制备5%浓缩胶和8%、10%分离胶,取30~50 μg总蛋白行十二烷基硫酸钠⁃聚丙烯酰胺凝胶电泳(SDS⁃PAGE) 分离蛋白分子,300mA恒流电转膜,室温下5%脱脂奶粉溶液封闭1h,分别加入一抗(Notch3抗体,1∶1 000;Caspase3抗体,1∶1 000;GAPDH抗体, 1∶2 000)后4℃过夜。TBST洗膜3次,每次10min,加入辣根过氧化物酶标记的二抗(1∶5 000)室温摇床孵育1~2h后,TBST洗膜3次,每次10min。电化学发光液(ECL)显色,凝胶成像系统图像扫描,Image J软件行结果分析。

  • 1.2.4 流式细胞仪分析

  • 用不含EDTA的胰酶消化收集细胞,PBS洗涤细胞2次(1 000r/min,离心5min),弃去上清,加入500 μL的Binding Buffer重悬细胞后加入5 μL Annexin V⁃FITC与5 μL碘化丙啶(propidium iodide, PI),混匀、室温避光反应5~15min,1h内行流式细胞仪检测。

  • 1.2.5 双荧光素酶报告基因检测

  • 带有miR⁃192⁃5p预测结合位点的Notch3定向缺失突变型(Notch3⁃MUT)和野生型(Notch3⁃WT) 3′⁃UTR的荧光素酶报告载体单独或与miR⁃ 192⁃5p mimic共转染至293T细胞中,细胞裂解后,按照双荧光素酶报告基因检测试剂盒说明书,采用酶标仪检测荧光素酶活性。

  • 1.3 统计学方法

  • 所有数据均使用SPSS22.0软件进行分析。每个实验重复3次,计量数据以均数±标准差(x- ± s)表示,两组间比较采用t检验,多组间比较采用方差分析(one ⁃way ANOVA),两两比较采用LSD ⁃t 检验。 P< 0.05为差异有统计学意义。

  • 2 结果

  • 2.1 顺铂处理后肾小管上皮细胞Notch3及凋亡相关蛋白cleaved⁃caspase3的表达及细胞凋亡率情况

  • 流式细胞分析示顺铂处理组细胞凋亡率明显升高(图1A);同时Western blot结果表明,与正常对照组比较,顺铂处理组Notch3及凋亡相关蛋白cleaved⁃caspase3表达均显著升高(P< 0.05,图1B); 实时荧光定量PCR表明,同正常对照组比较,顺铂处理组细胞Notch3mRNA表达显著上调(P< 0.001,图1C)。既往研究已报道药理性抑制Notch信号通路可以减轻顺铂诱导的HK⁃2细胞凋亡[10],综上提示Notch3或在顺铂所致的小管损伤中也起重要作用。

  • 2.2 靶向Notch3的潜在miRNA的筛选和检测

  • 使用公共预测平台Targetscan检索与Notch3的3′⁃UTR结合的潜在miRNA。然后,从既往的研究中选取了微阵列测量肾损伤样本中下调的miRNA[11]。两者取交集,从中选取1个miRNA(miR⁃ 192⁃5p)行进一步分析,实时荧光定量PCR表明,同正常对照组比较,顺铂处理组细胞miR⁃192⁃5p表达显著下调,差异有统计学意义(P< 0.01,图2)。

  • 2.3 miR⁃192⁃5p mimic的转染效率检测

  • 转染24h后,实时荧光定量PCR检测结果显示,与NC mimic组比较,miR⁃192⁃5p mimic组肾小管细胞内miR⁃192⁃5p明显升高,差异有统计学意义 (P< 0.001,图3A)。

  • 2.4 miR⁃ 192 ⁃ 5p对顺铂所致肾小管上皮细胞Notch3、凋亡相关蛋白cleaved⁃caspase3的表达及细胞凋亡率的影响

  • 将miR⁃192⁃5p mimic与NC mimic分别转染至HK⁃2细胞,加或不加顺铂处理24h后,实时荧光定量PCR检测及Western blot检测结果显示,同为顺铂处理诱导细胞凋亡情况下,与NC+Cis组比较,过表达miR⁃192⁃5p明显降低了Notch3mRNA及蛋白水平(P< 0.01,图3B、C)。同时,过表达miR⁃ 192⁃5p后肾小管上皮细胞凋亡相关蛋白cleaved⁃ caspase3表达及细胞凋亡率均明显降低,差异有统计学意义(图3D)。

  • 2.5 miR⁃192⁃5p与靶基因Notch3的靶向关系验证

  • 双荧光素酶报告实验检测发现过表达miR⁃ 192⁃5p可以降低pmiR⁃Notch3 3′⁃UTR⁃WT的荧光素酶活性,其活性下降39%(P< 0.01),而pmiR⁃ Notch3 3′⁃UTR⁃Mut与miR⁃192⁃5p mimic共同转染细胞的荧光素酶活性未见明显变化,以上结果证实了miR⁃192⁃5p与Notch3之间的直接作用关系 (图5)。

  • 图1 顺铂处理后HK⁃2细胞Notch3及cleaved⁃caspase3表达情况

  • Fig.1 The expression of Notch3and cleaved⁃caspase3in HK⁃2cells after cisplatin treatment

  • 图2 靶向Notch3的潜在miRNA的预测和检测

  • Fig.2 Gene prediction and verification of possible miRNAs targeting to Notch3

  • 图4 miR⁃192⁃5p对顺铂所致细胞凋亡和Notch3表达的影响

  • Fig.4 Effect of miR⁃192⁃5p on cisplatin⁃induced apoptosis and the expression of Notch3

  • 3 讨论

  • 凋亡以细胞自杀或程序性死亡的方式参与顺铂所致AKI的发展[4]。据报道,线粒体介导的凋亡信号通路是顺铂诱导的AKI和细胞损伤的关键机制[12]。作为凋亡的执行酶之一,caspase3可通过蛋白水解作用分解细胞内的组成蛋白成分,导致细胞最终形成凋亡小体。既往已有研究证实在肾损伤动物模型中,活化的caspase3表达明显增加[13]。本研究显示过表达miR⁃192⁃5p后,顺铂诱导的肾小管上皮细胞活化的caspase3(cleaved⁃caspase3)表达及细胞凋亡率降低,提示miR⁃192⁃5p可对顺铂所致的肾小管细胞凋亡起保护作用。

  • 图5 miR⁃192⁃5p与靶基因Notch3的靶向关系验证

  • Fig.5 Verification of the direct interaction between miR⁃192⁃5p and Notch3

  • Notch受体是高度保守的Ⅰ型跨膜糖蛋白,包括Notch1、Notch2、Notch3和Notch4,它们由细胞外结构域(NECD)和细胞内结构域(NICD)组成。 Notch受体的激活是由NECD与典型的Notch配体相互作用引起的,这些配体也是跨膜蛋白,包括Jagged1、Jagged2、dll1、dll3和dll4。与Notch配体的结合导致Notch的切割和NICD的释放,然后NICD转移到细胞核,与CSL转录因子相互作用,并启动Notch靶基因的转录从而发挥生物学效应[14]

  • 关于Notch信号在肾脏病理中的研究多集中于肾小球疾病及肾纤维化,已有研究大多集中于Notch1。Notch3是最近才研究增多的一种肾脏疾病介质。正常情况下,Notch3主要在血管平滑肌细胞中表达。单侧输尿管梗阻(unilateral ureteral obstruction,UUO)和肾毒性血清肾病动物模型中表明Notch3由受累肾实质细胞重新表达,并发挥了促进肾脏疾病进展的有害作用[15-16]。最近Kavvadas等[6] 发现在小鼠缺血再灌注肾损伤(ischemia⁃reper⁃ fusion injury,I/R)模型中,肾小管过表达Notch3,肾小管上皮细胞Notch3特异性敲除小鼠中则显示较轻的肾损伤及保留的肾功能,肾小管损伤及炎症浸润均有所改善。而Notch3信号激活的小鼠则在I/R和UUO后对肾脏损伤有较高的易感性,炎症反应及肾小管损伤加重[6]。本研究通过体外培养人肾小管上皮细胞证实,在顺铂毒性作用下,HK⁃2细胞中Notch3异常高表达,而抑制Notch3高表达则能够减轻肾小管上皮细胞凋亡。

  • 越来越多的证据表明,miRNA在AKI中起着重要的作用。miRNA水平异常可能是肾损伤发生和发展过程中蛋白质表达异常的原因之一[9]。多种miRNA及其相应靶基因在肾损伤或修复中发挥的作用已被研究所证实,这些发现表明miRNA是AKI的关键调节因子及潜在的治疗靶点[811]

  • 本研究先期预测了可能与Notch3的3′⁃UTR结合的miRNA,并与已报道的AKI中下调的miRNA取交集,从而得出11个潜在miRNA。进一步发现miR⁃192⁃5p在肾损伤活检样本中表达大幅下调(约6倍),此外也有研究报道在I/R和顺铂诱导的大鼠肾损伤模型尿液中miR⁃192⁃5p表达大幅增高[17-18]。近来Zou等[19] 也报道药物上调miR⁃192⁃5p的表达能够减轻顺铂诱导的小鼠肾损伤,但其具体机制尚未阐明。这些研究都提示miR⁃192⁃5p或在AKI过程中起着重要作用。本研究发现,顺铂处理的人近端肾小管上皮细胞miR⁃192⁃5p表达降低,而其靶基因Notch3表达升高。进一步发现过表达miR⁃192⁃5p后,其靶基因Notch3的表达被显著下调,同时cleaved ⁃caspase3表达及细胞凋亡率降低,这提示miR⁃192⁃5p可通过抑制其靶基因Notch3的表达,进而抑制线粒体凋亡途径,导致细胞凋亡相关蛋白cleaved⁃caspase3表达降低,减轻肾小管上皮细胞凋亡,但这其中的具体机制仍需进一步在动物实验中论证。

  • Notch3是AKI损伤的一种重要介质,本研究证实miR⁃192⁃5p可通过下调Notch3表达来减轻顺铂诱导的肾小管上皮细胞凋亡,这或能为急性肾损伤的防治提供一种新的研究思路和治疗策略。

  • 参考文献

    • [1] BELLOMO R,KELLUM J A,RONCO C.Acute kidney in⁃jury[J].Lancet,2012,380(9843):756-766

    • [2] 程小燕,邬步云,毛慧娟,等.住院患者急性肾损伤发生的流行病调查:一项单中心回顾性观察性研究[J].南京医科大学学报(自然科学版),2017,37(1):67-72

    • [3] BONVENTRE J V,YANG L.Cellular pathophysiology of ischemic acute kidney injury[J].J Clin Invest,2011,121(11):4210-4221

    • [4] PABLA N,DONG Z.Cisplatin nephrotoxicity:mecha⁃ nisms and renoprotective strategies[J].Kidney Int,2008,73(9):994-1007

    • [5] EDELING M,RAGI G,HUANG S,et al.Developmental signalling pathways in renal fibrosis:the roles of Notch,Wnt and Hedgehog[J].Nat Rev Nephrol,2016,12(7):426-439

    • [6] KAVVADAS P,KEUYLIAN Z,PRAKOURA N,et al.Notch3 orchestrates epithelial and inflammatory respons⁃ es to promote acute kidney injury[J].Kidney Int,2018,94(1):126-138

    • [7] TRIONFINI P,BENIGNI A,REMUZZI G.MicroRNAs in kidney physiology and disease[J].Nat Rev Nephrol,2015,11(1):23-33

    • [8] REN G L,ZHU J,LI J,et al.Noncoding RNAs in acute kidney injury[J].J Cell Physiol,2019,234(3):2266-2276

    • [9] BRANDENBURGER T,SALGADO SOMOZA A,DEVA⁃ UX Y,et al.Noncoding RNAs in acute kidney injury[J].Kidney Int,2018,94(5):870-881

    • [10] SONI H,MATTHEWS A T,PALLIKKUTH S,et al.Γ ⁃ secretase inhibitor DAPT mitigates cisplatin ⁃ induced acute kidney injury by suppressing Notch1 signaling[J].J Cell Mol Med,2019,23(1):260-270

    • [11] PAVKOVIC M,VAIDYA V S.MicroRNAs and drug ⁃in⁃ duced kidney injury[J].Pharmacol Ther,2016,163:48-57

    • [12] YANG Y,LIU H,LIU F,et al.Mitochondrial dysregula⁃ tion and protection in cisplatin nephrotoxicity[J].Arch Toxicol,2014,88(6):1249-1256

    • [13] KAUSHAL G P,KAUSHAL V,HERZOG C,et al.Au⁃ tophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity[J].Autophagy,2008,4(5):710-712

    • [14] BRAY S J.Notch signalling in context[J].Nat Rev Mol Cell Biol,2016,17(11):722-735

    • [15] DJUDJAJ S,CHATZIANTONIOU C,RAFFETSEDER U,et al.Notch⁃3 receptor activation drives inflammation and fibrosis following tubulointerstitial kidney injury[J].J Pathol,2012,228(3):286-299

    • [16] EL MACHHOUR F,KEUYLIAN Z,KAVVADAS P,et al.Activation of Notch3 in glomeruli promotes the develop⁃ ment of rapidly progressive renal disease[J].J Am Soc Nephrol,2015,26(7):1561-1575

    • [17] ZOU Y F,WEN D,ZHAO Q,et al.Urinary microRNA ⁃ 30c⁃5p and microRNA⁃192⁃5p as potential biomarkers of ischemia⁃reperfusion⁃induced kidney injury[J].Exp Biol Med(Maywood),2017,242(6):657-667

    • [18] KANKI M,MORIGUCHI A,SASAKI D,et al.Identifica⁃ tion of urinary miRNA biomarkers for detecting cisplatin⁃ induced proximal tubular injury in rats[J].Toxicology,2014,324:158-168

    • [19] ZOU D,GANUGULA R,ARORA M,et al.Oral delivery of nanoparticle urolithin A normalizes cellular stress and improves survival in mouse model of cisplatin ⁃ induced AKI[J].Am J Physiol Renal Physiol,2019,317(5):F1255-F1264

  • 参考文献

    • [1] BELLOMO R,KELLUM J A,RONCO C.Acute kidney in⁃jury[J].Lancet,2012,380(9843):756-766

    • [2] 程小燕,邬步云,毛慧娟,等.住院患者急性肾损伤发生的流行病调查:一项单中心回顾性观察性研究[J].南京医科大学学报(自然科学版),2017,37(1):67-72

    • [3] BONVENTRE J V,YANG L.Cellular pathophysiology of ischemic acute kidney injury[J].J Clin Invest,2011,121(11):4210-4221

    • [4] PABLA N,DONG Z.Cisplatin nephrotoxicity:mecha⁃ nisms and renoprotective strategies[J].Kidney Int,2008,73(9):994-1007

    • [5] EDELING M,RAGI G,HUANG S,et al.Developmental signalling pathways in renal fibrosis:the roles of Notch,Wnt and Hedgehog[J].Nat Rev Nephrol,2016,12(7):426-439

    • [6] KAVVADAS P,KEUYLIAN Z,PRAKOURA N,et al.Notch3 orchestrates epithelial and inflammatory respons⁃ es to promote acute kidney injury[J].Kidney Int,2018,94(1):126-138

    • [7] TRIONFINI P,BENIGNI A,REMUZZI G.MicroRNAs in kidney physiology and disease[J].Nat Rev Nephrol,2015,11(1):23-33

    • [8] REN G L,ZHU J,LI J,et al.Noncoding RNAs in acute kidney injury[J].J Cell Physiol,2019,234(3):2266-2276

    • [9] BRANDENBURGER T,SALGADO SOMOZA A,DEVA⁃ UX Y,et al.Noncoding RNAs in acute kidney injury[J].Kidney Int,2018,94(5):870-881

    • [10] SONI H,MATTHEWS A T,PALLIKKUTH S,et al.Γ ⁃ secretase inhibitor DAPT mitigates cisplatin ⁃ induced acute kidney injury by suppressing Notch1 signaling[J].J Cell Mol Med,2019,23(1):260-270

    • [11] PAVKOVIC M,VAIDYA V S.MicroRNAs and drug ⁃in⁃ duced kidney injury[J].Pharmacol Ther,2016,163:48-57

    • [12] YANG Y,LIU H,LIU F,et al.Mitochondrial dysregula⁃ tion and protection in cisplatin nephrotoxicity[J].Arch Toxicol,2014,88(6):1249-1256

    • [13] KAUSHAL G P,KAUSHAL V,HERZOG C,et al.Au⁃ tophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity[J].Autophagy,2008,4(5):710-712

    • [14] BRAY S J.Notch signalling in context[J].Nat Rev Mol Cell Biol,2016,17(11):722-735

    • [15] DJUDJAJ S,CHATZIANTONIOU C,RAFFETSEDER U,et al.Notch⁃3 receptor activation drives inflammation and fibrosis following tubulointerstitial kidney injury[J].J Pathol,2012,228(3):286-299

    • [16] EL MACHHOUR F,KEUYLIAN Z,KAVVADAS P,et al.Activation of Notch3 in glomeruli promotes the develop⁃ ment of rapidly progressive renal disease[J].J Am Soc Nephrol,2015,26(7):1561-1575

    • [17] ZOU Y F,WEN D,ZHAO Q,et al.Urinary microRNA ⁃ 30c⁃5p and microRNA⁃192⁃5p as potential biomarkers of ischemia⁃reperfusion⁃induced kidney injury[J].Exp Biol Med(Maywood),2017,242(6):657-667

    • [18] KANKI M,MORIGUCHI A,SASAKI D,et al.Identifica⁃ tion of urinary miRNA biomarkers for detecting cisplatin⁃ induced proximal tubular injury in rats[J].Toxicology,2014,324:158-168

    • [19] ZOU D,GANUGULA R,ARORA M,et al.Oral delivery of nanoparticle urolithin A normalizes cellular stress and improves survival in mouse model of cisplatin ⁃ induced AKI[J].Am J Physiol Renal Physiol,2019,317(5):F1255-F1264