CN108088987A - 以尿液生物标记筛检糖尿病肾病变高危险性的用途与方法 - Google Patents
以尿液生物标记筛检糖尿病肾病变高危险性的用途与方法 Download PDFInfo
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Abstract
本发明涉及以尿液生物标记筛检糖尿病肾病变高危险性的用途与方法。本发明提供一种尿液生物标记,尤其是关于一种能够用以筛检糖尿病肾病变高危险性族群的尿液生物标记及其筛检方法。藉由本发明的尿液生物标记‑糖化尿调理素,相较于一般PCR或ACR检测,能够在糖尿病患者族群中,更准确地早期发现具有发生糖尿病肾病变高危险性的病人,而能对其进一步的检测并提早进行治疗,以避免慢性肾脏病进程持续进行,而威胁到病人的生命。
Description
技术领域
本发明是关于一种尿液生物标记,尤其是关于一种能够用以筛检糖尿病肾病变高危险性族群的尿液生物标记及其筛检方法。
背景技术
在医学发达的二十一世纪,世界各地民众罹患糖尿病与糖尿病前期的盛行率依然持续攀升,2013年时的全球糖尿病人口约3亿8,200万人,预估到2035年时将高达5亿9,200万人,相当于全球20~79岁成年人口的10.1%。发生慢性肾脏病(Chronic KidneyDisease,CKD)最常见的危险因子之一就是糖尿病,而由糖尿病所引起的并发症最主要的即为糖尿病肾病变(Diabetic nephropathy,DN)而导致的末期肾脏病(End-Stage RenalDiseases,ESRD),该并发症使患者苦于血液透析或必须进行肾脏移植,并面临死亡的威胁。根据美国NHANES(National Health and Nutrition Examination Survey)对20岁以上成年人持续10年以上的追踪调查后发现,糖尿病患者的全死因死亡率增加了3.4倍,而CKD病人的全死因死亡率则增加至9倍,然而,当糖尿病合并CKD时,全死因死亡率即大幅增加至23.4倍(请参Afkarian M,Sachs MC,Kestenbaum B,et al.Kidney disease andincreased mortality risk in type 2diabetes.J Am Soc Nephrol.2013;24(2):302-308),显见糖尿病肾病变已成为糖尿病患者最严重的头号威胁。
即便知晓上述情况,需要进行肾脏移植的末期肾脏病病患依然不断增加,可知现今对于糖尿病合并症的发生仍无法有效预防,也反应出医学上对于糖尿病肾病变致病机制的了解不足,以及对末期肾脏升危险性的筛检或预测的欠缺。目前临床上有几种方法可筛检糖尿病肾病变,例如检测血糖异常病人尿液中的白蛋白-肌酸酐比值(urine albumin tocreatinine ratio,ACR)或蛋白质-肌酸酐比值(urine protein to creatinine ratio,PCR),当ACR或PCR愈高,病人日后肾功能衰退的状况就愈显著。而其中,是否具有微白蛋白尿(microalbuminuria)已是诊断早期糖尿病肾病变的标准方法。然而,许多病人发现有微白蛋白尿(microalbuminuria)时,其肾脏已出现晚期的病理改变,因此以微白蛋白尿做为糖尿病患者发生肾病变危险性的早期筛检或预测标记,并非一适当且准确的检测方式,其尚必须搭配其他的检测数据。尚且,许多第二型糖尿病患者,经肾脏切片分析后,却未发现合并白蛋白尿异常,亦即,部分糖尿病人的病理为非典型糖尿病肾病变,而其中更有些病人同时存有非糖尿病肾病变与糖尿病肾病变,因此,白蛋白尿的检测有其局限性;此外,与白种人相较,亚洲糖尿病患者的白蛋白尿的盛行率与发生率都较高,肾脏病恶化的速度也比白种人快,因此,找出一种能够精确检测早期糖尿病肾脏病及其进程的生物标记,提早筛检出这些可能造成肾病变的糖尿病患者,以便早期治疗,实为一迫切的需求。
如前所述,糖尿病肾病变的致病机制并不清楚,但从多种研究显示,血糖异常扮演了重要的角色。以第一型糖尿病患者为例,严格的血糖控制可减少其白蛋白尿出现及恶化的机会;除了血糖异常外,目前认为最相关的影响因素之一即为过度糖化终产物(advancedglycation end product,AGEs)。过度糖化终产物是由一些还原糖,例如葡萄糖,与蛋白质、脂质或核酸中的胺基,经过一系列的梅纳反应(Maillard reaction)后形成希夫碱(Schiffbases)与阿玛得利产物(Amadori products)而生成。过度糖化终产物持续在人体内生成,即便是血糖值正常的个体中,但在糖尿病患者体内却会加速生成,其最后可由肾脏所排除或代谢,但在末期肾脏病的血清或组织中却会大量累积,相较于一般无肾脏病的糖尿病患者,末期肾脏病糖尿病患者体组织中的AGEs含量高达2倍之多。
在健康人体的尿液中所存在最丰富的尿蛋白称之为尿调理素(uromodulin,也称之为Tamm-Horsfall protein),一般是由粗升肢亨氏环(TALH)与远曲小管前端的上皮细胞所表现。尿调理素可避免第一型纤毛表现大肠杆菌对上尿道的感染,并能调升免疫反应以及肾小管的运送功能。在一些研究中,尿调理素并被认为参与了慢性肾脏病的致病机制。当尿调理素失去具保护性的活性时,尿调理素可能损害肾小管的复原,并造成间质纤维病(interstitial fibrosis)与不可逆的肾元死亡(请参Allison A Eddy.Scrapingfibrosis:UMODulating renal fibrosis.Nat Med.2011;17:553-5)。此外,尿调理素也被确认与肾丝球过滤率(eGFR)与糖尿病肾病变有所关联(请参Ahluwalia TS,Lindholm E,Groop L,Melander O.Uromodulin gene variant is associated with type 2diabeticnephropathy.JHypertens 2011;29:1731–1734)。
发明内容
本发明目的之一在于提供一种能够早期预测发生糖尿病肾病变高危险性的生物标记,藉以对罹患糖尿病的患者进行筛检,以能早期发现肾脏病而早期治疗,避免未能及时发现而导致的末期肾脏病乃至于死亡,以减少糖尿病患者严重合并症发生的机率
为了达成前述的目的,本发明提供一种尿液生物标记,其可做为筛检糖尿病肾病变高危险性的用途,其中该尿液生物标记是糖化尿调理素(glycateduromodulin)。
为了达成前述的目的,本发明同时提供一种以尿液生物标记筛检糖尿病肾病变高危险性的方法,包括以下步骤:提供一待测者的尿液检体;以一检测方法检测该尿液检体中是否存有糖化尿调理素;以及当该尿液检体中检测出存有糖化尿调理素时,判断该待测者具有糖尿病肾病变发生的高危险性。
在本发明的一实施例中,所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其中该待测者是糖尿病患者。在本实施例的一态样中,该待测者的年龄是约小于65岁。
在本发明前述实施例的一态样中,所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其中该待测者是慢性肾脏病第1至3a期的糖尿病患者。
在本发明的一实施例中,所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其中该尿液检体是进一步将尿液离心后的上清液。
在本发明的一实施例中,所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其中该离心步骤可在16,000xg~20,000xg下进行,较佳为18,000xg,之后回收其上清液。前述离心步骤亦可利用连续式离心方式,进一步以100,000xg~120,000xg进行离心,较佳为110,000xg,再回收其上清液。
在本发明的一实施例中,所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其中该检测方法可为西方墨点法、质谱法、免疫侦测法、或层析法,但并不以此为限。
在本发明的一实施例中,所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其中该糖化尿调理素的值是8,000a.u.(arbitrary unit,任意单位)以上,较佳为9,000a.u.以上。
藉由本发明尿液生物标记的检测,相较于一般PCR或ACR检测,更能够在糖尿病患者族群中,准确地早期发现具有发生糖尿病肾病变高危险性的病人,而能对其进一步的检测并提早进行治疗,以避免慢性肾脏病进程持续进行,让病人苦于血液透析,或进而威胁其生命。
此外,在本发明的一实施例中,所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其中当该尿液检体中检测出存有糖化尿调理素时,可进一步结合该待检测者所测量的一白蛋白-肌酸酐比值(ACR)或蛋白质-肌酸酐比值(PCR),判断该待测者具有糖尿病肾病变发生的高危险性,以提高传统ACR或PCR检测的预测准确率。
以下将进一步说明本发明的实施方式,下述所列举的实施例是用以阐明本发明,并非用以限定本发明的范围,任何熟习此技艺者,在不脱离本发明的精神和范围内,当可做些许更动与润饰,因此本发明的保护范围当视后附的申请专利范围所界定者为准。
附图说明
图1是本发明实施例于病人尿液中所分离并确认的糖化尿调理素的西方墨点法分析结果图;
图2是本发明实施例于病人尿液中所分离并确认的糖化尿调理素的西方墨点法分析结果图与检测出具糖化尿调理素的病人数统计表;
图3是本发明实施例于非糖尿病与糖尿病患者的尿液中所发现糖化尿调理素的中位数比较图;
图4是本发明实施例于非糖尿病与糖尿病患者的尿液中所发现糖化尿调理素与慢性肾脏病(CKD)分期的关系图;
图5是本发明实施例中糖化尿调理素含量与糖尿病所致慢性肾脏病(CKD)机率的关系图;
图6是本发明实施例中糖化尿调理素与PCR、ACR于糖尿病肾病变以及非糖尿病肾病变间关于AUC-ROC的分析结果图。
具体实施方式
本发明将对糖尿病肾病变与非糖尿病肾病变病人的尿液分离出过度糖化终产物,并以LC-MS/MS以及西方墨点法分析,确认糖化尿调理素主要存在于发生肾脏病的糖尿病患者尿液中。
实施例1糖化尿调理素的分离与分析
首先由彰化基督教医院肾脏病门诊病人中,排除检验前3个月曾发烧、受感染、具肝脏、心脏病史、内分泌失调、经手术、外伤或住院的人员后,选定84位肾脏病患者进行检测,其中35位为糖尿病患者,另外49位为非糖尿病患者。该些病人经过8小时禁食后,收集其静脉血以及早晨第一次排放的尿液,尿液于分装后即冷冻于-80℃中备用。
为知悉糖化尿调理素分泌分布的情形,首先将84位待测病人的尿液解冻后进行连续式离心,于4℃,18,000xg下离心3小时(收集第一次沉淀物),再于4℃,110,000xg下离心3小时后,收集第二次沉淀物与上清液,将前后二次离心沉淀物回溶后与最后的上清液,分别以抗尿调理素抗体进行免疫沉淀,再以LC-MS/MS纯化、分析,并以西方墨点法(分别利用抗尿调理素抗体与抗过度糖化终产物抗体)加以确认,其结果如图1所示。另一方面,同样于4℃,18,000xg下离心3小时后,取其上清液,以抗尿调理素抗体进行免疫沉淀,再以西方墨点法(利用抗过度糖化终产物抗体)加以确认,即可确认糖化尿调理素是否出现在糖尿病或非糖尿病尿液中,其结果如图2所示。前述离心方式与条件仅为例示,而检测尿调理素的方法,除西方墨点法外,亦可利用其他免疫分析法(例如ELISA)或是层析法、质谱法等该技术领域所知悉的方法,其并未设有特别的限制。
请同时参见图1与图2,该二图是关于本发明实施例于病人尿液中所分离并确认的糖化尿调理素的西方墨点法分析结果图。由该二图可知,糖化尿调理素主要发现在发生肾脏病的糖尿病患者的尿液中,其比例为54.28%,而在非糖尿病患者则仅有16.33%。且特别得的是,糖化尿调理素主要存在于尿液离心后的上清液中。藉此,本发明发现了在糖尿病肾病变与非糖尿病肾病变病人尿液中一重要的成分差异,相较于一般人尿液中普遍可以获得的尿调理素,本发明发现「糖化尿调理素」在糖尿病肾病变与非糖尿病肾病变病人尿液中存在着差异,因此可利用于预测糖尿病肾病变的发生。
此外,尿液或血清中的肌酸酐浓度则基于杰弗反应(Jaffe reaction)以反应动力学方法加以计算,每一检体进行二次测试,使组内变异系数低于5%。尿液中的蛋白质、白蛋白则以免疫比酌法(immunoturbidmetric method,Roche Diagnostics GmbH)或其他临床上常见的测量法计算浓度,其后与前述所测得的肌酸酐浓度进行PCR与ACR的比值分析。另外,血清中的肌酸酐浓度值则利用于肾丝球过滤率(eGFR)的计算。
关于测得的数值,是以中位数(四分位数)或百分比N(%)表示,而关于糖尿病或非糖尿病患者血清中糖化尿调理素浓度的类别变异统计分析,则是利用卡方检定(Chi-Square test)或费雪尔正确性检定(Fisher’s Exact test)比较分析后获得。至于糖尿病或非糖尿病患者血清中糖化尿调理素浓度的连续变异统计分析,是以无母数的魏克生等级和(Nonparametric Wilcoxon rank-sum test)检定进行。另一方面,关于不同糖化尿调理素浓度与糖尿病肾脏病的机率关系则是以逻辑回归模型进行预测。以上统计分析是以19版SPSS统计软件(IBM,USA)进行,当P<0.05时表示具有统计显著性。
请参见表1,表1除病人临床特征的统计外,还包括糖化尿调理素浓度与肾丝球过滤率(eGFR)的比较。慢性肾脏病(CKD)可利用以下eGFR来分期:(1)第一期:≧90mL/min/1.73m2,肾丝球过滤率正常或增加,但有蛋白尿、血尿等肾脏损伤状况;(2)第二期:60~89mL/min/1.73m2,肾丝球过滤率轻微下降,并有蛋白尿、血尿等状况。患病后2~3年起发生,没有症状;(3)第三期:30~59mL/min/1.73m2(3a期:45~59;3b期:30~44),肾丝球过滤率中度下降,患病后7~15年起发生;(4)第四期:15~29mL/min/1.73m2,肾丝球过滤率严重下降,患病后10~30年发生,尿液白蛋白每天超过300mg;(5)第五期:<15mL/min/1.73m2,即末期肾脏病(End-Stage Renal Disease,ESRD),患病后20~40年发生,血液检查肾功能异常,大部份病人肾功能会逐渐恶化,出现尿毒症,需进行肾脏替代疗法。
表1
请同时参见图3,该图是本发明实施例于非糖尿病与糖尿病患者的尿液中所发现糖化尿调理素的中位数比较图。从表1与图3可以发现,在糖尿病患者组中,其尿液中所存有的糖化尿调理素中位数高达6027a.u.,具有显著相关性,而在非糖尿病患者组中则为0。此外,以皮尔森相关系数分析法(Person correlation analysis)后发现(表中未示),糖化尿调理素与年龄间的显著水平为0.773(双尾),而糖化尿调理素与身体质量指数(BMI)间的显著水平为0.773(双尾),二组相互间都不具统计显著性。
然而,请同时参见以下表2与图4,表2与图4是非糖尿病与糖尿病患者的尿液中糖化尿调理素浓度与慢性肾脏病(CKD)分期的关系图表。从表2与图4(前期、晚期中的右侧柱状图)可知,糖化尿调理素浓度于糖尿病患者与CKD前期、晚期间皆具有显著相关性,特别是当糖化尿调理素浓度值大于9000a.u.时有较高比例是糖尿病患者(约有60%的阳性预测值)。
表2
此外,请参见以下表3,其是非糖尿病与糖尿病患者的尿液中糖化尿调理素浓度与年龄的关系表。从表3可知,糖化尿调理素浓度于糖尿病患者与年龄间具有显著相关性,特别是当糖尿病患者的年龄小于65岁时有较高的显著性。
表3
请参见图5,该图是糖化尿调理素含量与糖尿病所致慢性肾脏病(CKD)机率的关系图。由图5可知,当糖化尿调理素含量愈高,罹患糖尿病慢性肾脏病的机率也愈高。例如当糖化尿调理素含量为8,959a.u.时,糖尿病慢性肾脏病的机率为57%,而当糖化尿调理素含量为16,821a.u.时,糖尿病慢性肾脏病的机率则提高至79%。
为进一步确认以糖化尿调理素作为生物标记的准确性,本发明实施例同时以一般常见的PCR、ACR生物标记与具有糖尿病肾病变与非糖尿病肾病变病人进行相关性分析,比较二者间的差异。所采用的方法是接收者操作特征曲线(receiver operatingcharacteristic curve,ROC曲线)与曲面下面积(Area under the Curve of ROC,AUC-ROC)分析,其结果如图6所示。由图6可知,糖化尿调理素的AUC-ROC为0.715(95%CI:0.597-0.834,p值=0.001),ACR的AUC-ROC则为0.799(95%CI:0.696–0.903,p值=0.001),而PCR的AUC-ROC为0.480(95%CI:0.341–0.619,p值=0.754)。因此,利用糖化尿调理素作为生物标记,其正确率与ACR相当接近,是一优秀的预测方式。
实施例2糖尿病肾病变危险性预测模型
糖尿病肾病变危险性预测模型是利用多变异逻辑回归进行分析,而预测能力则是利用一致性统计量(c-statistics)、净分类改善度(category-free netreclassification improvement,cfNRI)与综合区分改善度(integrated discriminationimprovement,IDI)进行分析,其结果如表4。
表4
由表4可知,从模型1a的ACR以及模型1b的ACR与糖化尿调理素调整,糖化尿调理素对于糖尿病患者发生慢性肾脏病具有良好的预测性(odds ratio1.14(95%CI:1.01–1.29),P值=0.028);相较之下,模型2a的PCR以及模型2b的PCR与糖化尿调理素调整,糖化尿调理素对于糖尿病患者发生慢性肾脏病具有更佳的预测性(odds ratio 1.23(95%CI:1.11–1.38),P值<0.0001)。然而,观察其变异数膨胀因子,仅分别为1.105与1.022,因此,ACR与糖化尿调理素以及PCR与糖化尿调理素间并不具共线性,为彼此独立的变量。
为进一步分析本发明糖化尿调理素做为生物标记,用以评估糖尿病患者不同层次的危险性,将进行一致性统计量、净分类改善度与综合区分改善度的计算,其结果如下表5所示。
表5
综合区分改善度(IDI),是用以量化原生物标记加入糖化尿调理素后所提高预测概率的能力。由表5可知,当尿液中的糖化尿调理素结合ACR时,IDI为0.046(95%CI:0.002-0.09,P值=0.048),其预测概率显著增加了4.6%,而当糖化尿调理素结合PCR时,IDI为0.19(95%CI:0.103-0.277,P值<0.0001),预测概率则显著增加了19%。
净分类改善度(cfNRI),则是用以量化原生物标记加入糖化尿调理素后是否能提升正确分类个数的能力。如表5所示,当尿液中的糖化尿调理素结合ACR时,cfNRI正确分类的比例增加75.92%(95%CI:36.96-114.88,P值<0.0001),而当糖化尿调理素结合PCR时,cfNRI正确分类的比例亦是增加75.92%(95%CI:36.96-114.88,P值<0.0001)。因此,若利用本发明所发现的糖化尿调理素的检测结果,结合传统ACR或PCR的检测结果,将更能进一步提升传统ACR或PCR的检测预测率。
藉由上述检测试验可知,藉由本发明所发现的糖化尿调理素,以其作为尿液生物标记,可针对糖尿病人进行早期的筛检,以找出具有发生糖尿病肾病变的个体,而能对其进行进一步的检查,并可提早治疗,避免慢性肾脏病进程的进行,以能提高生活质量并降低死亡率。
Claims (9)
1.一种以尿液生物标记筛检糖尿病肾病变高危险性的用途,其中该尿液生物标记是糖化尿调理素(glycated uromodulin)。
2.一种以尿液生物标记筛检糖尿病肾病变高危险性的方法,包括以下步骤:
提供一待测者的尿液检体;
以一检测方法检测该尿液检体中是否存有糖化尿调理素;以及
当该尿液检体中检测出存有糖化尿调理素时,判断该待测者具有糖尿病肾病变发生的高危险性。
3.根据权利要求2所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其特征在于,所述待测者是糖尿病患者。
4.根据权利要求3所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其特征在于,所述待测者的年龄是小于65岁。
5.根据权利要求2所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其特征在于,所述尿液检体是进一步将尿液离心后的上清液。
6.根据权利要求5所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其特征在于,所述离心步骤是在16,000xg~120,000xg下进行。
7.根据权利要求2所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其特征在于,所述检测方法是西方墨点法、质谱法、免疫侦测法、或层析法。
8.根据权利要求2至7中任一项所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其特征在于,所述糖化尿调理素的值是9,000a.u.以上。
9.根据权利要求2所述的以尿液生物标记筛检糖尿病肾病变高危险性的方法,其特征在于,当该尿液检体中检测出存有糖化尿调理素时,进一步结合该待检测者所测量的一白蛋白-肌酸酐比值(ACR)或蛋白质-肌酸酐比值(PCR),判断该待测者具有糖尿病肾病变发生的高危险性。
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WO2023056924A1 (zh) * | 2021-10-09 | 2023-04-13 | 北京大学第一医院 | 尿调蛋白Sda抗原糖基化检测试剂盒及其在预测早期肾损伤中的应用 |
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WO2023056924A1 (zh) * | 2021-10-09 | 2023-04-13 | 北京大学第一医院 | 尿调蛋白Sda抗原糖基化检测试剂盒及其在预测早期肾损伤中的应用 |
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