【作者】 彭亮；

【导师】 刘虹；

【作者基本信息】 中南大学 ， 内科学， 2022， 博士

【摘要】 背景:急性肾损伤(acute kidney injury,AKI)是一种主要表现为急性肾脏排泄功能障碍的综合征,肾小管细胞损伤和死亡是AKI最为关键的病理特征。蛙皮素受体激活蛋白(bombesin receptor-activated protein,BRAP,C6ORF89蛋白)最初是作为一种G蛋白偶联受体-蛙皮素受体亚型3(bombesin-like receptor-3,BRS-3)的配体被发现的。目前发现的BRAP的生物学作用包括促进细胞周期进程、创伤修复和组蛋白去乙酰化酶(histone deacetylase,HDAC)的激活。BRAP在肾小管上皮细胞中有表达,但其在AKI中的作用尚不清楚。目的:本研究旨在探索BRAP在AKI中的表达变化及作用,并进一步在顺铂诱导的AKI中进行机制研究。方法:1.使用C57/bl6J小鼠,腹腔注射顺铂诱导AKI,实时荧光定量PCR(real-time fluorescent quantitative PCR,Real-time PCR)及Western blot检测不同时间点BRAP m RNA及蛋白表达水平。使用顺铂孵育人近端肾小管上皮细胞株(human kidney proximal tubular epithelial cells,HK2细胞),观察其对BRAP表达的时间及浓度依赖性作用。使用AKI患者肾脏组织及肾癌患者切除的癌旁健康肾脏组织标本、顺铂诱导的急性肾损伤小鼠肾组织标本或对照健康小鼠肾组织标本进行BRAP免疫染色分析。使用人或小鼠急性肾损伤相关的RNA测序(RNA-Sequencing,RNA-seq)数据集,分析BRAP、KIM-1等表达情况。2.使用8-10周雄性全身敲除BRAP的小鼠及同窝野生型小鼠,在伴或并不伴有泛天冬氨酸特异性半胱氨酸蛋白酶(caspase)抑制剂Z-VAD预处理的情况下,腹腔注射顺铂构建AKI模型。进行血肌酐检测及苏木素-伊红(hematoxylin-eosin,HE)染色评价肾脏损伤程度。肾组织脱氧核糖核苷酸末端转移酶标记(Terminaldeoxynucleotidyl transferase d UTP nick end labeling,TUNEL)染色及活性caspase3(cleaved-caspase3)和磷酸化混合谱系酶域样蛋白(phosphomixedlineage kinase domainlike,p-MLKL)的Western blot和免疫组化染色检测细胞死亡。在4-5周BRAP敲除鼠中行双侧肾脏原位注携带BRAP质粒的腺相关病毒(AAV9-BRAP-FLAG-Zs Green)或相应对照(AAV9-NC-Zs Green),30天后再予顺铂腹腔注射构建AKI模型。分为四组:AAV9-BRAP+生理盐水,AAV9-BRAP+顺铂,AAV9-NC+生理盐水,AAV9-NC+顺铂组。检测各组小鼠上述指标的变化。3.在8-10周雄性BRAP敲除小鼠及同窝野生型小鼠,构建顺铂诱导的AKI模型。使用野生型小鼠,气管内滴注BRAP-AAV9构建肺部过表达BRAP小鼠。10天后,这些小鼠被腹腔注射顺铂诱导AKI。对BRAP敲除小鼠、过表达BRAP小鼠及相应的对照组小鼠进行肺组织中性粒细胞弹性蛋白酶免疫荧光染色、TUNEL染色及Evans blue染色。4.在HK2细胞中,通过基因干预上调或下调BRAP表达,继以顺铂孵育构建急性损伤模型。流式细胞术观察细胞凋亡及坏死比例,Western blot及免疫荧光检测cleaved-caspase3和p-MLKL的表达。在转染BRAP或空载质粒的HK2细胞中,在顺铂干预前使用Z-VAD或坏死性凋亡抑制剂Necrostatin-1(Nec-1)预处理,Hochest染色及细胞计数试剂盒(Cell Counting Kit-8,CCK8)检测细胞凋亡及存活率。5.使用8-10周雄性全身敲除BRAP的小鼠及同窝野生型小鼠,脂多糖(lipopolysaccharide,LPS)10mg/kg腹腔注射构建炎症诱导的AKI模型,双侧肾蒂夹闭建立缺血再灌注AKI模型。检测各组小鼠血肌酐水平及肾脏组织中BRAP、cleaved-caspase3蛋白水平并进行TUNEL染色。在LPS诱导的AKI小鼠肾组织中进行F4/80染色。Real-time PCR检测炎症因子白细胞介素1β(Interleukin-1β,IL-1β)、IL-6、IL-10及人单核细胞趋化蛋白(Monocyte chemotactic protein-1,MCP-1)m RNA表达。对缺血再灌注AKI小鼠进行cleaved-caspase3及p-MLKL的Western blot及免疫组化染色。6.通过RNA-seq数据集分析,我们发现在AKI中,脱乙酰化酶sirtuin-2(SIRT2)可能是与BRAP作用相关的HDAC。通过Real-time PCR、Western blot及免疫荧光染色等方法在体内及体外揭示BRAP对SIRT2的调节作用。为了寻找BRAP影响SIRT2蛋白水平的机制,我们使用了MG132(一种泛素蛋白酶体抑制剂)来阻断蛋白酶体降解,使用Ubi Browser软件寻找能够介导BRAP和SIRT2降解的E3泛素连接酶。然后在HK2细胞中上调或下调BRAP表达,接下来进一步检测SIRT2的蛋白水平、泛素化SIRT2及SIRT2与E3泛素连接酶的结合水平。7.使用染色质免疫共沉淀—高通量测序(Chromatin Immunoprecipitation-Sequencing,Ch IP-Seq)数据集分析P65可能为调节BRAP表达的转录因子。在顺铂干预的小鼠及HK2细胞中,使用Western blot及免疫组化检测P65的表达及激活。在HK2细胞中过表达P65或使用P65抑制剂SC75741,观察BRAP的m RNA及蛋白表达变化。通过双荧光素酶报告实验和Ch IP-PCR检测P65与BRAP基因的启动子区域的结合。结果:1.免疫组化显示伴有AKI的患者和小鼠肾组织中BRAP在肾小管上皮细胞中表达降低。顺铂诱导HK2细胞BRAP下调,并呈时间和剂量依赖性。RNA-seq分析显示肾移植患者供肾活检标本中BRAP表达水平较移植前显著降低,且移植后供肾组织中BRAP的m RNA表达水平与KIM-1 m RNA水平呈正相关。小鼠RNA-seq数据也显示AKI小鼠中同样存在BRAP表达降低,且其表达水平与KIM-1正相关。2.在顺铂诱导的AKI中,与BRAP敲除小鼠相比,野生型小鼠肾小管损伤更重。肾组织中TUNEL阳性、p-MLKL阳性、cleaved-caspase3阳性细胞的数量和血清肌酐浓度在野生型小鼠中高于BRAP敲除鼠。在顺铂诱导的AKI中,在BRAP敲除鼠肾脏中重新表达BRAP导致上述指标加重。在顺铂诱导的AKI小鼠中,Z-VAD能够减轻野生型小鼠肾功能减退和组织损伤,但在BRAP敲除鼠中,这种保护作用消失。Z-VAD能够增加顺铂诱导的AKI小鼠中MLKL的激活,但是这种效果在BRAP敲除小鼠中更明显。3.顺铂诱导的AKI小鼠肺部组织中性粒细胞及TUNEL阳性细胞增多,Evans blue沉积增加。上述指标在BRAP敲除鼠中降低,而在肺部过表达BRAP的小鼠中升高。4.在顺铂处理的HK2细胞中,BRAP敲减降低了顺铂诱导的细胞凋亡和坏死性凋亡,而过表达BRAP则增加了凋亡和坏死性凋亡。在HK2细胞中,Z-VAD对细胞的保护作用比Nec-1更强。而且Nec-1预处理仅降低了在BRAP过表达的HK2细胞中顺铂诱导的细胞死亡,对BRAP正常水平的细胞不起作用。5.在LPS及缺血再灌注诱导的急性肾损伤小鼠模型中,BRAP的蛋白表达均降低。在BRAP敲除鼠中,TUNEL染色、HE染色及血肌酐检测证实LPS及缺血再灌注诱导的急性肾组织损伤及肾功能衰退、细胞死亡均较野生型小鼠轻。在LPS诱导的AKI中,BRAP敲除小鼠肾脏组织中F4/80阳性细胞数量及炎症因子IL-1β、IL-6、MCP-1表达水平均显著低于野生型小鼠,cleaved-caspase3蛋白水平也较低。在缺血再灌注诱导的AKI小鼠中,BRAP敲除小鼠肾脏组织中cleaved-caspase3及p-MLKL表达均较野生型小鼠少。6.根据RNA-seq数据及文献资料,顺铂诱导的AKI中SIRT2表达降低,与肾脏保护作用相关。Western blot和免疫染色结果证实在顺铂诱导的AKI小鼠和细胞模型中SIRT2降低。体内及体外实验均证明过表达BRAP导致SIRT2蛋白水平增高,而敲减BRAP降低SIRT2蛋白水平。但是BRAP对SIRT2 m RNA表达水平无影响。通过MG132阻断蛋白酶体降解,顺铂诱导的BRAP和SIRT2降低能被部分或完全逆转。过表达BRAP减少SIRT2的泛素化,而BRAP敲减增加了SIRT2的泛素化水平。我们使用Ubi Browser预测羟甲基戊二酰辅酶A还原酶降解蛋白1(3-hydroxy-3-methylglutaryl coenzyme A(HMG-Co A)reductase degradation 1,HRD1)可能是能够介导BRAP及SIRT2泛素化降解的E3泛素连接酶。通过使用免疫共沉淀发现,过表达BRAP减少了SIRT2与HRD1结合,敲减BRAP则增加了SIRT2与HRD1的结合。7.我们通过分析Ch IP-seq数据库Ch Ip-atlas(http://chip-atlas.org)发现在C6ORF89启动子区域存在P65峰富集。顺铂活化NF-κB亚基P65,过表达P65减少BRAP m RNA表达,抑制P65转录可增加HK2细胞中BRAP的m RNA水平。双荧光素酶报告基因实验证实P65能与C6ORF89启动子结合并抑制其转录。Ch IP-PCR检测发现P65能够结合于C6ORF89转录起始位点上游-834至-1150区域,在顺铂处理的情况下,这种结合是增强的。结论:1、AKI中肾小管上皮细胞BRAP表达降低是一种自身保护性调节机制。BRAP敲减缓解AKI小鼠急性肾功能衰退、肾脏及肺部组织损伤。因此,抑制BRAP具有AKI治疗潜力。2、BRAP敲除缓解急性肾损伤中细胞凋亡和坏死性凋亡、炎症。3、SIRT2在顺铂诱导的AKI中下调,BRAP能够通过影响SIRT2与E3泛素连接酶HRD1结合和后续的泛素化降解,调节SIRT2蛋白水平。4、在顺铂诱导的AKI中,P65结合于BRAP基因启动子,负调控BRAP的转录。图28幅,表15个,参考文献110篇更多还原

【Abstract】 Background:Acute kidney injury(AKI)is a syndrome manifested by the rapid loss of the kidney excretory function.A key pathological feature of AKI is the injury and death of tubular epithelial cells.Bombesin receptor-activated protein(BRAP,C6ORF89 protein)was initially discovered as a ligand for a previously orphan G protein-coupled receptor bombesin-like receptor-3.At present,accepted biological effects of BRAP include cell cycle progression,wound repair,and the activation of histone deacetylases(HDACs).BRAP is expressed in renal tubular epithelial cells,however,its role in kidney disease is unknown.Objective:The present study aimed to investigate the expression and functional role of BRAP in AKI,and further explore the molecular mechanisms involved in cisplatin(CP)-induced AKI.Methods:1.C57/bl6 J mice were injected with CP to induce AKI.Real-time PCR and Western blot were used to detect the mRNA and protein expression of BRAP at different time points separately.Human kidney proximal tubular epithelial(HK2)cells were incubated with CP,then time and concentration-dependent effects of CP on BRAP expression were analyzed.Immunohistochemical staining of BRAP was performed in renal biopsy sections from patients with acute kidney injury,para carcinoma kidney tissues,and renal sections from CP-induced AKI or healthy mice.The RNA-Sequencing(RNA-seq)datasets of AKI patients and mice were searched in the GEO database.The expression of BRAP and kidney injury marker KIM-1 was analyzed.2.8-10 weeks of male BRAP whole-body knockout mice(BRAP-/-)and their wild-type(WT)littermates were received intraperitoneal injectionsof CP with or without Z-VAD(a pan-caspase inhibitor)pretreatment.We assessed kidney injury by evaluating histology via hematoxylin-eosin(HE)staining and measuring serum creatinine levels.Terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL)staining,Western blot,and immunostaining of cleaved-caspase3 and phospho-mixedlineage kinase domainlike(p-MLKL)were performed in kidney tissues to evaluate cell death.Adenoviruses expressing BRAP(AAV9-BRAP-FLAG-Zs Green)or control(AAV9-Zs Green)were orthotopic bilateral injected into the kidneys of 4-5 weeks of BRAP-/-mice.CP was injected intraperitoneally to induce AKI one month later.The mice were divided into 4 groups:AAV9-BRAP + NS,AAV9-BRAP + CP,AAV9-NC+NS,and AAV9-NC + CP.All the above indices were examined in each group of mice.3.8-10 weeks of male BRAP-/-mice and their WT littermates were abdominally injected with CP to induce AKI.Intratracheal administration of adenoviruses expressing BRAP induced BRAP expression in the lungs of WT mice.After ten days,the mice were administered CP intraperitoneally.TUNEL staining,Evans blue dye,and immunofluorescent staining of neutrophil elastase were performed in lungs from AKI mice and their corresponding control mice.4.BRAP were up-regulated or down-regulated in HK2 cells,followed by incubation of these cells with CP for 24 h.Cell apoptosis and necrosis were determined by flow cytometry.The expression of cleaved-caspase3 and p-MLKL was assessed by Western blot and immunofluorescent staining.HK2 cells transfected with BRAP or negative control plasmid were pretreated with Z-VAD or necrostatin-1(Nec-1,a necroptosis inhibitor)and then exposed to CP.Hochest staining and Cell Counting Kit-8(CCK8)assay were used to assess cell apoptosis and survival separately.5.8-10 week male WT or BRAP-/-mice were intraperitoneally injected with lipopolysaccharide(LPS)to establish the sepsis-induced AKI models.Models of kidney ischemia/reperfusion injury(IRI)were carried out by clamping bilateral renal pedicles.We examined the level of serum creatinine,performed HE staining,TUNEL staining,and Western blot for BRAP and cleaved-caspase3 in renal tissues of mice.We performed immunostaining for F4/80 and checked the mRNA expression of IL-1β,IL-6,IL-10,and Monocyte chemotactic protein-1(MCP-1)in LPS-induced AKI mice.Immunostaining and Western blot for p-MLKL were performed in IRI-induced AKI mice.6.We found that deacetylase sirtuin-2(SIRT2)may be associated with BRAP in AKI by analyzing RNA-seq datasets.The regulation of BRAP on SIRT2 was examined by Real-time PCR,Western blot,and immunofluorescence assay in vitro and vivo.To explore the regulation of BRAP on the protein levels of SIRT2,we used MG132(a ubiquitin-proteasome inhibitor)to block proteasomal degradation.Ubi Browser software was used to search the E3 ubiquitin ligase that mediates ubiquitination and degradation of BRAP and SIRT2.After that,we up-regulated or down-regulated BRAP in HK2 cells,then assessed the expression of SIRT2,the ubiquitination level of SIRT2,and the levels of binding between BRAP and the E3 ubiquitin ligase.7.Chromatin Immunoprecipitation-Sequencing(Ch IP-seq)analysis revealed that p65 might serve as a transcriptional factor to regulate BRAP expression.The expression and activation of P65 were examined by Western blot and immunohistochemical method in CP-treated mice and HK2 cells.Following overexpression of P65 or treatment with SC75741(a P65 inhibitor),HK2 cells were lysed to detect the mRNA and protein levels of BRAP.Dual-luciferase reporter and Ch IP-PCR assays were used to detect the interaction between P65 and C6ORF89 promoter region.Results:1.Immunohistochemical analysis demonstrated that the expression of BRAP was decreased in renal tubular epithelial cells of AKI patients and mice.In HK2 cells,CP treatment induced downregulation of BRAP expression in a dose-and time-dependent manner.The BRAP levels in kidney allograft recipients were decreased and positively related to the KIM-1 mRNA levels after kidney transplantation in RNA-seq data.The same results were obtained in AKI mice from RNA-seq datasets.2.After CP treatment,WT mice exhibited exacerbated tubular injury compared to BRAP-/-mice.The number of TUNEL-positive,p-MLKL-positive,cleaved-caspase3-positive cells in kidney sections and the serum creatinine concentrations were higher in WT mice than those in BRAP-/-mice.In BRAP-/-mice,re-expression of BRAP in the kidneys exacerbated the indexes mentioned above during CP-induced AKI.Z-VAD pretreatments attenuated CP-induced blood creatinine increase and kidney injury in WT mice,but not in BRAP-/-mice.The activation of MLKL was magnified by Z-VAD in CP-treated mice,especially in BRAP-/-mice.3.In CP-induced AKI mice,Evans Blue dye extravasation and the number of neutrophils and TUNEL-positive cells were increased in the lung tissues.All above indexes were augmented by BRAP overexpression and attenuated by BRAP knockout in the lung tissues.4.BRAP knockdown alleviated CP-induced tubular cell apoptosis and necroptosis in HK2 cells,whereas overexpression of BRAP aggravated them.The cytoprotective effect of Z-VAD was more substantial than Nec-1 in CP-treated HK2 cells.Furthermore,Nec-1pretreatment reduced the CP-induced cell death in BRAP overexpression HK2 cells but did not work in cells with normal BRAP levels.5.BRAP expression was also dramatically decreased in IRI-or LPS-induced AKI.BRAP knockout notably improved IRI-and LPS-induced renal function decline,tubular damage,and cell death,as demonstrated by the level of serum creatinine,HE staining,and TUNEL staining.In LPS-induced AKI mice,there were fewer F4/80 positive cells,lower levels of cleaved-caspase3,and inflammatory factors(IL-1β、IL-6、MCP-1)in renal tissues.In IRI-induced AKI mice,the expression of cleaved-caspase3 and p-MLKL were lower in BRAP-/-mice than in WT mice.6.According to RNA-Seq data and the literature,SIRT2 was down-regulated in CP-induced AKI,associated with renoprotective function.Western blot and immunofluorescence stainig results proved that SIRT2 was down-regulated in CP treated mice and cells.SIRT2 protein levels were up-regulated by BRAP overexpression and down-regulated by knocking down BRAP in vivo and vitro.BRAP did not affect SIRT2 mRNA levels.The decreased protein levels of BRAP and SIRT2 after CP treatment was partially or wholly reversed by MG132.Overexpressed BRAP decreased,and knockdown BRAP increased the polyubiquitinated SIRT2 proteins.We used Ubi Browser software and predicted that the E3 ubiquitin ligase 3-hydroxy-3-methylglutaryl coenzyme A(HMG-Co A)reductase degradation 1(HRD1)could bind to BRAP and SIRT2,which may contribute to their degradation by ubiquitination.Immunoprecipitation(IP)-based pull-down revealed that BRAP knockdown significantly enhanced the binding between SIRT2 and HRD1,whereas ectopic BRAP expression reduced their interaction.7.We analyzed Ch IP-seq data using Ch Ip-atlas(http://chip-atlas.org)and found a significant enrichment of P65 in the C6ORF89 promoter region.CP treatment activated NF-κB subunit P65.P65 overexpressing decreased,and inhibition of P65 increased the mRNA levels of BRAP in HK2 cells.Dual-luciferase reporter gene assay verified P65 binding to the C6ORF89 promoter and reduced its mRNA expression.Ch IP assay determined that P65 could bind to the binding site 1(-1150 to-834)in the C6ORF89 promoter,and CP treatment enhanced this binding.Conclusion:1.The downregulated BRAP expression during AKI is an adaptive mechanism employed by tubular cells.BRAP knock-out ameliorated acute renal failure,tubular injury,and remote organ injury(lung)in CP-induced AKI.Hence,inhibition of BRAP may have therapeutic potential in treating AKI.2.BRAP knockout attenuated cell apoptosis,necroptosis,and inflammation during AKI.3.SIRT2 was down-regulated in CP-induced AKI,and BRAP regulated the SIRT2 protein levels by affecting SIRT2’s interactions with E3 ubiquitin ligase HRD1 and subsequent proteasomal degradation.4.P65 served as a transcriptional factor to repress BRAP expression during CP-induced AKI.更多还原