CN116930340A - 犬尿喹啉酸作为肾小管OATs-MRP4通道内源性标志物的应用 - Google Patents
犬尿喹啉酸作为肾小管OATs-MRP4通道内源性标志物的应用 Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
本发明属于检测标志物技术领域,具体涉及犬尿喹啉酸作为肾小管OATs‑MRP4通道异常内源性标志物的应用和犬尿喹啉酸检测试剂作为指导有机阴离子类药物精准用药的标志物的应用。本发明发现犬尿喹啉酸为评价OATs‑MRP4通道异常功能的内源性标志物;且与头孢美唑等底物药物代谢存在相关性,能够用于评价OATs‑MRP4通道所有底物药物的代谢及排泄能力;同时也可作为急性肾损伤、慢性肾衰竭、尿毒症期、肾小球肾炎、糖尿病肾病和高血压肾病、原发性肾病综合征分型诊断和早期诊断的依据,实现上述疾病以及药物代谢排泄异常的早期诊断,并合理指导患者进行精准的药物治疗,提高治疗效果,具有显著的临床意义。
Description
技术领域
本发明属于检测标志物技术领域,具体涉及犬尿喹啉酸作为肾小管OATs-MRP4通道内源性标志物的应用和犬尿喹啉酸检测试剂作为指导有机阴离子类药物精准用药的标志物的应用。
背景技术
肾脏是机体重要的排泄器官之一,其排泄过程主要包括肾小球滤过、肾小管分泌和重吸收过程。研究显示,约32%的临床常见处方药经肾排泄,其中超过90%存在肾小管分泌。然而,目前国内外临床对肾功能评价的指标主要是评估肾小球滤过能力,如肌酐、胱抑素C、尿素氮、β2微球蛋白等,而缺乏评价肾小管分泌功能的标志物。因此,寻找评估肾小管分泌功能的特异性标志物对准确预测药物的肾排泄极其重要。
肾小管分泌主要借助于上皮细胞膜上的众多转运体来完成,这些摄取型转运体和外排型转运体形成了很多矢量转运的“通道”,不同的通道又有各自的特异性底物2,其中有机阴离子转运通道是由表达于基底侧膜的OAT1/3和表达于管腔侧膜的MRP2/4构成,介导了底物化合物的肾小管上皮细胞矢量转运过程,其底物药物临床应用十分广泛,如:NSAIDs、抗病毒药、抗生素类、甲氨蝶呤、5-氟尿嘧啶等。发明人意外地发现OATs-MRPs通道异常导致了上述相关药物的代谢异常,而准确诊断OATs-MRPs通道异常为药物代谢异常提供依据,可用于相关疾病及药物代谢异常的早期诊断。
因此,寻找肾小管OATs-MRPs通道功能评价的标志物对准确预测有机阴离子类药物的肾排泄至关重要。近年来,一些学者也致力于寻找评价OATs转运功能的内源性标志物,其中6β-hydroxycortisol是研究最为广泛的OAT1/3的特异性标志物。但是,遗憾的是,6β-hydroxycortisol经OAT1/3摄取后经MATE1和2-K介导外排进入尿液,并非由MRPs所介导。截至目前,国内外尚未见有关肾小管OATs-MRP4通道功能评价的内源性标志物的报道。
当肾脏受到损伤时,肾小球和肾小管的功能改变并非同步进行,因此,采用既被肾小球滤过又经肾小管分泌的内源性标志物是无法评估损伤发生在哪个部位,也就不能准确预测药物的肾脏排泄。而理想的肾小管OATs-MRPs通道功能评价的内源性标志物应具有不被肾小球滤过又特异性经该通道转运的特征,而这就给筛选肾小管OATs-MRPs通道功能评价的标志物带来了很大的困难。
发明人意外地发现犬尿喹啉酸具备能够不被肾小球滤过又特异性经该通道转运的特征,可以作为肾小管OATs-MRP4通道内源性标志物。
发明人还发现通过检测犬尿喹啉酸含量,能够实现NSAIDs、抗病毒药、抗生素类、甲氨蝶呤、5-氟尿嘧啶等的精准用药,具有重要的临床意义。
发明内容
针对上述技术问题,本发明的目的在于提供犬尿喹啉酸作为肾小管OATs-MRP4通道内源性标志物及其作为指导有机阴离子类药物精准用药标志物的应用。具体包括以下内容:
第一方面,本发明提供了犬尿喹啉酸作为检测肾小管OATs-MRP4通道异常的内源性标志物的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
第二方面,本发明提供了犬尿喹啉酸作为检测药物肾排泄异常的标志物的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述药物包括非甾体类消炎药、β-内酰胺类抗生素、抗病毒药、利尿剂、H2-受体拮抗剂、甲氨蝶呤、对氨基马尿酸中的一种或几种的组合。
优选地,所述药物为头孢菌素类抗生素。
优选地,所述头孢菌素类抗生素为头孢美唑。
第二方面,本发明提供了检测犬尿喹啉酸含量的试剂在制备检测药物肾排泄异常的试剂或试剂盒中的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述药物包括非甾体类消炎药、β-内酰胺类抗生素、抗病毒药、利尿剂、H2-受体拮抗剂、甲氨蝶呤、对氨基马尿酸中的一种或几种的组合。
优选地,所述药物为头孢菌素类抗生素。
优选地,所述头孢菌素类抗生素为头孢美唑。
第四方面,本发明提供了犬尿喹啉酸作为诊断肾损伤相关疾病的标志物的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述肾损伤相关疾病包括肾中毒、急性肾损伤、慢性肾衰竭、尿毒症期、肾小球肾炎、糖尿病肾病、高血压肾病、原发性肾病综合征。
第五方面,本发明提供了检测犬尿喹啉酸含量的试剂在制备诊断肾损伤相关疾病的试剂或试剂盒中的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述肾损伤相关疾病包括肾中毒、急性肾损伤、慢性肾衰竭、尿毒症期、肾小球肾炎、糖尿病肾病、高血压肾病、原发性肾病综合征。
第六方面,本发明提供了犬尿喹啉酸作为指导有机阴离子类药物精准用药的标志物的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述有机阴离子类药物包括非甾体类消炎药、β-内酰胺类抗生素、抗病毒药、利尿剂、H2-受体拮抗剂、甲氨蝶呤、对氨基马尿酸中的一种或几种的组合。
优选地,所述头孢菌素类抗生素为头孢菌素类抗生素。
优选地,所述头孢菌素类抗生素为头孢美唑。
第七方面,本发明提供了检测犬尿喹啉酸含量的试剂在制备指导有机阴离子类药物精准用药的试剂或试剂盒中的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述有机阴离子类药物包括非甾体类消炎药、β-内酰胺类抗生素、抗病毒药、利尿剂、H2-受体拮抗剂、甲氨蝶呤、对氨基马尿酸中的一种或几种的组合。
优选地,所述有机阴离子类药物为头孢菌素类抗生素。
优选地,所述头孢菌素类抗生素为头孢美唑。
第八方面,本发明提供了检测犬尿喹啉酸含量的试剂在制备筛选肾损伤相关疾病治疗药物的试剂或试剂盒中的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述肾损伤相关疾病包括肾中毒、急性肾损伤、慢性肾衰竭、尿毒症期、肾小球肾炎、糖尿病肾病、高血压肾病、原发性肾病综合征。
第九方面,本发明提供了犬尿喹啉酸作为诊断肾中毒的标志物的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述肾中毒包括急性肾中毒、药物性肾中毒。
第十方面,本发明提供了检测犬尿喹啉酸含量的试剂在制备诊断肾中毒的试剂或试剂盒中的应用,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
优选地,所述肾中毒包括急性肾中毒、药物性肾中毒。
第十一方面,本发明提供了一种检测有机阴离子类物品实现精准用药的方法,所述方法为:检测血浆犬尿喹啉酸含量,计算犬尿喹啉酸肾清除率,根据犬尿喹啉酸肾清除率调整有机阴离子类药物剂量,当所述患者的犬尿喹啉酸肾清除率低于正常水平时,减少药物剂量;当所述患者的犬尿喹啉酸肾清除率高于正常水平时,增加药物剂量。
优选地,所述有机阴离子类药物包括非甾体类消炎药、β-内酰胺类抗生素、抗病毒药、利尿剂、H2-受体拮抗剂、甲氨蝶呤、对氨基马尿酸中的一种或几种的组合。
优选地,所述有机阴离子类药物为头孢菌素类抗生素。
优选地,所述头孢菌素类抗生素为头孢美唑。
优选地,当所述犬尿喹啉酸血清水平增加时,降低原有给药剂量,当血清犬尿喹啉酸血清水平降低时,增加到原有给药剂量;药物剂量变化百分比与犬尿喹啉酸血清水平变化百分比相同。
优选地,当犬尿喹啉酸血清水平增加50%时,药物剂量降低到原有给药剂量的1/2;当血清犬尿喹啉酸血清水平降低50%时,药物剂量增加到原有给药剂量的2倍。
本发明的有益效果是:(1)发明人意外地发现犬尿喹啉酸具备能够不被肾小球滤过又特异性经该通道转运的特征,可以作为肾小管OATs-MRP4通道内源性标志物;(2)所述犬尿喹啉酸与头孢美唑等OATs-MRP4通道底物的AUC也存在明显的相关性,表明犬尿喹啉酸作为标志物能够用于评价OATs-MRP4通道底物的排泄能力;通过检测犬尿喹啉酸含量,能够实现NSAIDs、抗病毒药、抗生素类、甲氨蝶呤、5-氟尿嘧啶等的精准用药,具有重要的临床意义;例如,当犬尿喹啉酸血清水平增加50%时,药物剂量降低到原有给药剂量的1/2;当血清犬尿喹啉酸血清水平降低50%时,药物剂量增加到原有给药剂量的2倍;(3)所述犬尿喹啉酸在急性肾损伤、慢性肾衰竭、尿毒症期、肾小球肾炎、糖尿病肾病和高血压肾病患者显著增加,而在原发性肾病综合征显著降低,可作为上述疾病的诊断标志物,实现相关疾病的早期诊断。
附图说明
图1犬尿喹啉酸(kynurenic acid)在OAT1、OAT3和MRP4过表达细胞系中摄取(mean±S.D.,n=4;**p<0.01表示与HEK293T-MOCK相比;##p<0.01表示与未加HSA相比);
图2温度对犬尿喹啉酸在HEK293T-OAT1/3细胞摄取(a)和在HEK293T-MRP4细胞外排(b)的影响(mean±S.D.,n=4;**p<0.01表示与对照组相比;##p<0.01表示与4℃摄取相比);
图3温度对犬尿喹啉酸在double-Control和OAT1/3-MRP4细胞摄取的影响(a:4℃摄取;b:37℃摄取;mean±S.D.,n=4;**p<0.01表示与对照组相比);
图4OATs抑制剂丙磺舒(PROB)和MRP4抑制剂(MK-571)对犬尿喹啉酸细胞摄取(a和b)和外排(c和d)的影响(mean±S.D.,n=4;*p<0.05,**p<0.01表示与对照组相比;##p<0.01表示与未加抑制剂的MRP4过表达细胞系相比);
图5犬尿喹啉酸在双转染OAT1/3-MRP4细胞矢量转运评价(a和c:犬尿喹啉酸从顶侧膜(A)向基底侧膜转运(B),b和d:犬尿喹啉酸从基底侧膜(B)向顶侧膜(A)转运;mean±S.D.,n=4;**p<0.01表示与Double Control组相比;##p<0.01表示与未加抑制剂的双转染过表达细胞系相比);
图6犬尿喹啉酸在OCT2、MATE1、MATE2-K、OAT2、OATP4C1、P-gp、PEPT2、URAT1和OAT4过表达细胞系的摄取(mean±S.D.,n=6);
图7HEK293T-OAT1/3细胞中犬尿喹啉酸时间依赖性(a)和浓度依赖性摄取(b和c)(mean±S.D.,n=4);
图8d5-犬尿喹啉酸(d5-kynurenic acid)体内过程评价(a:d5-犬尿喹啉酸尿累积排泄量-时间曲线图和肾脏清除t1/2;b:d4-xanthurenic尿累积排泄量-时间曲线图和体内代谢率;c:犬尿喹啉酸尿累积排泄量-时间曲线图)(mean±S.D.,n=6;**p<0.01表示与对照组相比);
图9人体血清生化指标与犬尿喹啉酸相关性分析(ALB、GLO和TP,n=600;AST、ALT和TBIL,n=601;ALP和GGT,n=267;TBA,n=265;GLU,n=472;TC、TG和HDL-C,n=382;LDL-C,n=381;LDH和α-HBDH,n=216;CK,n=219,HCY,n=205;creatinine,n=678;Cys-C和β2-MG,n=77);
图10LC-MS/MS测定人血清中犬尿喹啉酸的色谱图和标准曲线图(a:标曲样本色谱图;b:CRF患者血清样本色谱图;c:人血清活性炭吸附后标准曲线);
图11rOAT1和rOAT3双基因敲除后犬尿喹啉酸血清水平、肾脏组织水平和清除率的影响(mean±S.D.,n=7;**p<0.01表示与野生型相比);
图12rOATs功能抑制后犬尿喹啉酸与头孢美唑相关性评价(a:头孢美唑血药浓度-时间曲线图;b:肌酐血药浓度-时间曲线图;c:犬尿喹啉酸血药浓度时间曲线图;d:犬尿喹啉酸/肌酐与头孢美唑AUC0-t的相关性;mean±S.D.,n=5;*p<0.05,**p<0.01表示与对照组相比);
图13健康人和不同肾损伤类型患者血清犬尿喹啉酸水平(健康人,n=50;急性肾损伤,n=5;慢性肾衰竭,n=73;尿毒症期,n=62;肾小球肾炎,n=9;糖尿病肾病,n=23;高血压肾病,n=10;原发性肾病综合征,n=30;**p<0.01表示与健康人相比显著增加,##p<0.01表示与健康人相比显著降低)。
具体实施方式
为使本发明更加容易理解,下面结合具体实施例进一步阐述本发明,但下述实施例仅用于说明本发明而不用于限制本发明的范围,下列实施例中未提及的具体实验方法,通常按照常规实验方法进行。
尿毒素和相关标准物质纯度均>90%。乙腈和甲醇为HPLC级(Sigma-Aldrich),其它试剂均为分析级。人血清白蛋白纯度为96%-98%(索莱宝,北京)。超滤管(截留分子>30kDa)购买于EMD Millipore Billerica(Billerica,MA,USA)。
SD雄性大鼠(180-250g)由中国农业科学院兰州兽医研究所提提供(合格证号:SCXK(甘)2020-0002),SPF级;所有动物饲养于兰州大学SPF动物房,于实验前12小时禁食,自由进水。
以下实施例所述方法具体包括:
体内蛋白结合率测定:取血清样本50μL+50μL内标+100μL乙腈,涡旋40s,14000rpm离心10min,取上清,用LC-MS/MS测定总浓度。取血清样本400μL加到超滤管中,于37℃,3500g,离心30min,取滤液50μL 加入50μL内标和100μL乙腈,涡旋40s,18000g离心10min,取上清,用LC-MS/MS测定游离浓度。
体外蛋白结合率测定:配犬尿喹啉酸溶液和4%HSA(PBS溶解,犬尿喹啉酸最终为浓度为100μM)。取标准品溶液40μL+360μL HSA,涡旋两分钟,于37℃摇床孵育2h。孵育结束后,取100μL结合液测定总浓度,另外300μL结合液加入到超滤管中,于37℃,3500g,离心30min后取滤液。总浓度和游离浓度的测定同体内蛋白结合率方法。蛋白结合率=(总浓度-游离浓度)/总浓度×100%。
转运体底物筛选:将HEK293T-MOCK和HEK293T-OAT1、HEK293T-OAT3和HEK293T-MRP4接种于12孔板(5×105个/mL,接种体积为1mL),接种24h后移除完全培养基,加入含有100μM的犬尿喹啉酸的buffer,MRP4摄取时同时加入4mM ATP,蛋白结合型尿毒素与4%HAS孵化成蛋白结合物,摄取30min,摄取结束后buffer冲洗2次,加入200μLH2O,收集细胞并超声破碎,BCA法蛋白定量,LC-MS/MS测定犬尿喹啉酸浓度。
温度对犬尿喹啉酸摄取的影响实验:将过表达细胞系接种于12孔板(5×105个/mL,接种体积为1mL),接种24h后,加入含有100μM的犬尿喹啉酸的buffer在37℃摄取30min,MRP4摄取时同时加入4mM ATP,摄取结束后buffer冲洗2次,加入200μL H2O,收集细胞并超声破碎,BCA法蛋白定量,LC-MS/MS测定犬尿喹啉酸浓度。4℃摄取时,将细胞置于4℃环境30min,随后加入4℃含有犬尿喹啉酸的buffer在4℃摄取30min,其它操作如上所述。
OATs-MRP4通道对犬尿喹啉酸转运的评价实验(Transweel转运实验):将双转染OAT1-MRP4和OAT3-MRP4的MOCK(5×105个/mL)500μL加入Transweel小室(6.5mm,孔径为0.4μM),下室中加入700μL完全培养基,培养3-5天后测定跨膜电阻确保单层细胞的完整性。A-B转运时,将500μL含有100μM的犬尿喹啉酸的buffer加入小室中,在下室中加入700μLbuffer,跨膜转运2h,收集下室buffer,LC-MS/MS测定犬尿喹啉酸浓度。B-A转运时,将700μL含有100μM的犬尿喹啉酸的buffer加入下室中,在小室中加入500μL buffer,跨膜转运2h,收集小室buffer,LC-MS/MS测定犬尿喹啉酸浓度。
抑制剂对摄取犬尿喹啉酸影响实验:OAT1/3过表达细胞系抑制摄取实验,将HEK293T-MOCK和HEK293T-OAT1和HEK293T-OAT3接种于12孔板(5×105个/mL,接种体积为1mL),接种24h后移除完全培养基,分别加入含有100μM的犬尿喹啉酸和不同浓度丙磺舒(0、0.5、1、5、10、50、100和500μM)的buffer,摄取30min后buffer冲洗2次,加入200μL H2O,收集细胞并超声破碎,BCA法蛋白定量,LC-MS/MS测定犬尿喹啉酸浓度,计算IC50。将HEK293T-OAT1/3或MOCK-OAT1/3-MRP4细胞接种于12孔板(5×105个/mL,接种体积为1mL),接种24h后移除完全培养基,分别加入含有100μM的犬尿喹啉酸和不同浓度MK-571(100和500μM)的buffer,摄取30min后buffer冲洗2次,加入200μL H2O,收集细胞并超声破碎,BCA法蛋白定量,LC-MS/MS测定犬尿喹啉酸浓度。
转运动力学实验:将HEK293T-MOCK和HEK293T-OAT1/3接种于12孔板(5×105个/mL,接种体积为1mL),接种24h后移除完全培养基,分别加入100μM犬尿喹啉酸的buffer,摄取2min、5min、10min、20min和30min,摄取完成后等体积buffer冲洗2次,加入200μL H2O,收集细胞并超声破碎,BCA法蛋白定量,LC-MS/MS测定犬尿喹啉酸浓度,分别确定犬尿喹啉酸的线性摄取时间。将HEK293T-MOCK和HEK293T-OAT1/3接种于12孔板(5×105个/mL,接种体积为1mL),接种24h后移除完全培养基,加入含有不同浓度犬尿喹啉酸的buffer,摄取时间为线性摄取时间内,摄取完成后buffer冲洗2次,加入200μL H2O,收集细胞并超声破碎,BCA法蛋白定量,LC-MS/MS测定犬尿喹啉酸浓度,根据米氏方程计算OAT1/3对犬尿喹啉酸的Km和Vmax。
犬尿喹啉酸清除半衰期的评价:SD大鼠随机分为Control、PROB和MK-571组,分别尾静脉给予d5-犬尿喹啉酸(2mg/kg)、d5-犬尿喹啉酸(2mg/kg)+PROB(60mg/kg)和d5-犬尿喹啉酸(2mg/kg)+MK-571(20mg/kg),给予后收集0-2、2-4、4-6、6-8、8-10和10-12h的尿液,测定犬尿喹啉酸、d5-犬尿喹啉酸、d4-xanthurenic acid的累计排泄量,并计算d5-犬尿喹啉酸的肾脏清除半衰期。
标志物影响因素的评价:收集2019年6月至2019年12月在兰州大学第一院肾病科住院患者的临床血液检测样本,共收集样本824例,评价血清中标志物与ALB(白蛋白)、GLO(球蛋白)、TP(总蛋白)、AST(谷草转氨酶)、ALT(谷丙转氨酶)、TBIL(总胆红素)、ALP(碱性磷酸酶)、GGT(谷氨酰转移酶)、TBA(总胆汁酸)、GLU(葡萄糖)、TC(总胆固醇)、TG(甘油三酯)、HDL-C(高密度脂蛋白)、LDL-C(低密度脂蛋白)、LDH(乳酸脱氢酶)、α-HNDH(α-羟基丁酸脱氢酶)、CK(肌酸激酶)、HCY(同型半胱氨酸)、creatinine(肌酐)、Cys-C(胱抑素-C)和β2-MG(β2-微球蛋白)水平的相关性,评价这些因素是与标志物的水平有关。所有程序均按照兰州大学第一医院人体实验伦理委员会的道德标准执行(NO.LDYYLL2018-32)。
标志物与头孢美唑相关性评价:大鼠随机分为Control、PROB(60mg/kg)和PROB(120mg/kg),分别口服给予生理盐水、60mg/kg丙磺舒和120mg/kg丙磺舒,每天1次,连续给予7天后收集尾静脉给予100mg/kg头孢美唑,股动脉收集5、10、15、30、60、120和240min的血液,HPLC测定头孢美唑的血药浓度,LC-MS/MS测定肌酐与标志物浓度,DAS2.0计算药动学参数。
实施例1OATs和MRP4底物的体外验证
转运体底物筛选实验结果显示,蛋白结合型和游离型犬尿喹啉酸在HEK293T-OAT1和HEK293T-OAT3摄取显著增加,而游离型在HEK293T-MRP4细胞中摄取量显著降低(见图1,其中每个小图从左至右与图标从上之下对应)。
温度对犬尿喹啉酸摄取的影响:在4℃时,HEK293T-OAT1、HEK293TOAT3、HEK293T-MPR4细胞与HEK293T-MOCK细胞对犬尿喹啉酸的摄取无明显差异,而在37℃时,犬尿喹啉酸在HEK293T-OAT1和HEK293T-OAT3细胞中显著增加(p<0.01),在HEK293T-MPR4细胞摄取量显著降低(p<0.01)(见图2,其中每个小图从左至右与图标从上之下对应)。与此一致,在4℃时,双转染OAT1-MRP4细胞和双转染OAT3-MRP4细胞的犬尿喹啉酸摄取量无明显差异(p>0.05),而在37℃时,OAT3-MRP4细胞中犬尿喹啉酸摄取量显著增加(p<0.01)。37℃时,OAT1-MRP4细胞中犬尿喹啉酸摄取量与HEK293T-MOCK细胞相比无明显差异,这是由于OAT1的摄取与MRP4的外排基本相等,使得细胞摄取量无明显变化(见图3)。这些结果表明犬尿喹啉酸的细胞摄取是OAT1/3依赖的,而其外排是MRP4依赖的。
抑制剂对摄取犬尿喹啉酸的影响:OATs特异性抑制剂丙磺舒能够显著抑制OAT1和OAT3介导的犬尿喹啉酸摄取,其IC50值分别为71.45μM和7.91μM。MRP4特异性抑制剂MK-571能够抑制MRP4介导的犬尿喹啉酸外排(p<0.01),同时,MK-571呈剂量依赖增加双转染细胞中犬尿喹啉酸摄取量(p<0.01)。这些结果证实OAT1/3和MRP4分别介导了犬尿喹啉酸的摄取和外排(见图4)。
OATs-MRP4通道对犬尿喹啉酸转运的评价:与对照细胞相比,在双转染OAT1/3-MPR4的细胞系中犬尿喹啉酸从顶侧膜(A)向基底侧膜(B)转运无明显差异(见图5中a和c,其中每个小图从左至右与图标从上之下对应)。相反,在双转染OAT1/3-MPR4的细胞系中犬尿喹啉酸从基底侧膜(B)向顶侧膜(A)转运显著增加(p<0.01),OATs抑制剂丙磺舒和MRP4抑制剂MK-571能够显著降低其转运量(p<0.01)(见图5中b和d,其中每个小图从左至右与图标从上之下对应)。这些结果表明犬尿喹啉酸是由摄取型转运体OATs和外排型转运体MRP4组成的矢量转运通道所介导。
实施例2OATs-MRP4通道标志物的特异性验证
在过表达人源不同肾小管转运体的细胞系中评价犬尿喹啉酸的摄取特异性,结果表明犬尿喹啉酸不经OCT2、MATE1、MATE2-K、OAT2、OATP4C1、P-gp、PEPT2、URAT1和OAT4介导摄取或外排(见图6),这些结果表明犬尿喹啉酸是OAT1/3-MRP4的特异性内源性标志物。
实施例3OATs-MRP4通道标志物的适宜性评价
适宜性评价包括Km、体内肾脏清除t1/2和检测方法三个方面。
Km的评价:动力学实验结果显示,犬尿喹啉酸在HEK293T-OAT1/3细胞中时间依赖性和浓度依赖性摄取见图7。犬尿喹啉酸的线性摄取实验为0-10min。浓度依赖摄取时间设定为5min,OAT1对犬尿喹啉酸的Km和Vmax分别为496.7μM和197.1pmol/mg protein/min;OAT3对犬尿喹啉酸的Km和Vmax分别为382.2μM和120.1pmol/mg protein/min,这些结果表明犬尿喹啉酸作为OATs-MRP4的标志物具有适宜的摄取亲和力。
犬尿喹啉酸肾脏清除t1/2的评价:d5-犬尿喹啉酸的肾脏清除t1/2为3.7±0.7h;d5-犬尿喹啉酸体内代谢为d4-xanthurenic acid,其代谢率为5.2-11.8%(见图8),这些结果表明犬尿喹啉酸在体内具有适宜的亲和力(0.5-8h),其代谢率较低。
LC-MS/MS检测犬尿喹啉酸具有检测时长短、前处理方法简单等,适用于临床检测(见图10)。同样,也可以采用ELISA、化学发光等对犬尿喹啉酸进行检测。
实施例4OATs-MRP4通道标志物影响因素评估
通过研究不同病生理指标与标志物的相关性,进一步验证标志物在体内的影响因素,为标志物的临床应用提供指导。标志物影响因素的评价实验结果显示,血清犬尿喹啉酸与ALB、GLO、TP、AST、ALT、TC、LDH、creatinine、Cys-C和β2-MG存在明显的相关性(p<0.01),但相关系数R2均小于0.3,属于弱相关性(见图9)。尽管肾损伤标志物creatinine、Cys-C和β2-MG与犬尿喹啉酸相关系数高于其它生化指标,但仍为弱相关,这是由于这些标志物并不能直接反应肾脏OATs-MRP4通道功能造成的。然而,血清犬尿喹啉酸和TBIL、ALP、GGT、TBA、GLU、TG、HDL-C、LDL-C、α-HBDH、CK和HCY并无相关性。由此可见,犬尿喹啉酸在体内几乎不受血清蛋白水平、肝功能、心功能、肾小球滤过功能等因素的影响,适合作为评价OATs-MRP4通道功能的内源性标志物。
实施例5OATs-MRP4通道标志物的应用
经体内验证、特异性和适宜性评价,最终获得OATs-MRP4通道的特异性良强和适宜的内源性标志物犬尿喹啉酸。
标志物的敏感性评价:在OATs-MRP4通道功能敲除后,犬尿喹啉酸血清水平增加3.9倍,其增加远远大于Cinhibition/Ccontrol>2倍。此外,犬尿喹啉酸的肾脏清除率显著降低(见图11,其中每个小图从左至右与图标从上之下对应)。这些结果表明犬尿喹啉酸在评价OATs-MRP4通道功能时敏感性强。
标志物与其通道底物药物肾排泄相关性评价:为了验证犬尿喹啉酸在评价OATs-MRP4通道功能的能力,大鼠给予OATs特异性抑制剂丙磺舒后,测定OATs-MRP4通道底物药物头孢美唑血药浓度,计算血浆犬尿喹啉酸与头孢美唑浓度的相关性。给予丙磺舒后,血浆犬尿喹啉酸和头孢美唑浓度呈剂量依赖性增加(见图12中a和c),而血浆肌酐浓度未发生明显变化。头孢美唑与犬尿喹啉酸AUC0-t存在明显的相关性,而与肌酐无明显相关性(见图12中d)。这些结果进一步证实在OATs功能抑制时,其底物药物的排泄显著降低,而肌酐水平无明显改变。
标志物在不同肾病人群中血清水平:与健康人相比,急性肾损伤、慢性肾衰竭、尿毒症、肾小球肾炎、糖尿病肾病和高血压肾病患者血清犬尿喹啉酸的水平显著增加(p<0.01),而原发性肾病综合征患者血清犬尿喹啉酸的水平显著降低(p<0.01)(见图13,其中每个小图从左至右与图标从上之下对应)。此外,该标志物的cutoff值为198.61±90.33nM。
本发明首先从欧洲尿毒素数据库和相关文献研究中筛选出26种候选的尿毒素,进行蛋白结合率评价,最终获得蛋白结合率100%的犬尿喹啉酸。
其次,将候选标志物进行体外和体内验证。体外结果表明,犬尿喹啉酸、为OAT1、OAT3和MRP4底物,同时经OATs-MRP4通道介导转运。
再次,将潜在标志物进行特异性和适宜性评价。在过表达肾小管分泌和重吸收转运体的细胞系验证潜在标志物的特异性,犬尿喹啉酸不经OCT2、MATE1、MATE2-K、OAT2、OATP4C1、P-gp、PEPT2、URAT1和OAT4介导摄取或外排,表明犬尿喹啉酸有良好的特异性。犬尿喹啉酸有适宜的OAT1和OAT3亲和力,犬尿喹啉酸在体内可能代谢为xanthurenic,但其代谢率低。犬尿喹啉酸具有适宜的半衰期,且检测方法灵敏、前处理简单。
综上所述:犬尿喹啉酸在评价OATs-MRP4通道功能时其特异性、敏感性、适宜性均良好,能够作为评价其通道功能的标志物。此外,犬尿喹啉酸在评价该通道底物药物肾排泄时具有良好的关联性,其显著强于目前临床使用的肌酐。而且犬尿喹啉酸也能够作为诊断肾损伤程度或类型的标志物,其cutoff值为198.61±90.33nM;通过检测犬尿喹啉酸含量及其肾清除率能够合理指导包括头孢美唑在内的有机阴离子类药物的精准用药,具有显著的临床指导意义,为病人合理用药提供依据,例如,当所述犬尿喹啉酸血清水平增加时,降低原有给药剂量,当血清犬尿喹啉酸血清水平降低时,增加到原有给药剂量;药物剂量变化百分比与犬尿喹啉酸血清水平变化百分比相同;优选地,当犬尿喹啉酸血清水平增加50%时,药物剂量降低到原有给药剂量的1/2;当血清犬尿喹啉酸血清水平降低50%时,药物剂量增加到原有给药剂量的2倍。
Claims (10)
1.犬尿喹啉酸作为检测肾小管OATs-MRP4通道异常的内源性标志物的应用,其特征在于,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
2.犬尿喹啉酸作为诊断肾损伤相关疾病的标志物的应用,其特征在于,所述肾损伤相关疾病包括肾中毒、急性肾损伤、慢性肾衰竭、尿毒症期、肾小球肾炎、糖尿病肾病、高血压肾病、原发性肾病综合征;所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
3.犬尿喹啉酸作为检测药物肾排泄异常的标志物的应用,其特征在于,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
4.检测犬尿喹啉酸含量的试剂在制备检测药物肾排泄异常的试剂或试剂盒中的应用,其特征在于,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
5.犬尿喹啉酸作为指导有机阴离子类药物精准用药的标志物的应用,其特征在于,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
6.检测犬尿喹啉酸含量的试剂在制备指导有机阴离子类药物精准用药的试剂或试剂盒中的应用,其特征在于,所述犬尿喹啉酸的结构式如下式(Ⅰ)所示:
7.如权利要求3-6任一所述的应用,其特征在于,所述有机阴离子类药物为非甾体类消炎药、β-内酰胺类抗生素、抗病毒药、利尿剂、H2-受体拮抗剂、甲氨蝶呤、对氨基马尿酸中的一种或几种的组合。
8.如权利要求7所述的应用,其特征在于,所述有机阴离子类药物为头孢菌素类抗生素。
9.如权利要求8所述的应用,其特征在于,所述头孢菌素类抗生素为头孢美唑。
10.一种检测有机阴离子类药物实现精准用药的方法,其特征在于,所述方法为:检测患者血浆犬尿喹啉酸含量,计算犬尿喹啉酸肾清除率,根据犬尿喹啉酸肾清除率调整有机阴离子类药物剂量,当所述患者的犬尿喹啉酸肾清除率低于正常水平时,减少药物剂量;当所述患者的犬尿喹啉酸肾清除率高于正常水平时,增加药物剂量;其中,所述有机阴离子类药物包括非甾体类消炎药、β-内酰胺类抗生素、抗病毒药、利尿剂、H2-受体拮抗剂、甲氨蝶呤、对氨基马尿酸中的一种或几种的组合。
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