Urine extracelluar vesicle determination and its research progress

doi:10.3969/j.issn.1673-8640.2017.010.022

Abstract

Abstract:

Extracelluar vesicles（EV） are vesicles which are released from most cell types during budding and cell membrane fission. EV specific molecules,such as proteins,nucleic acids and lipids,are expected as novel biomarkers. EV exists in the body fluid of human body. Urine is a good target for the research of EV,with the advantages of sufficient volume,obtaining easily and non-invasive operation. The component urine EV may be potential as biomarkers of various diseases. This review mainly focuses on the determination of urine EV and its clinical application.

Key words: Extracelluar vesicle, Urine, Novel biomarker

CLC Number:

R446.1

XU Yong, ZHENG Lei. Urine extracelluar vesicle determination and its research progress[J]. Laboratory Medicine, 2017, 32(10): 933-940.

Figures/Tables 2

References 58

[1]	GÁMEZ-VALERO A,LOZANO-RAMOS S I,BANCU I,et al. Urinary extracellular vesicles as source of biomarkers in kidney diseases[J]. Front Immunol,2015,6:6.
[2]	WITWER K W,BUZÁS E I,BEMIS L T,et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research[J]. J Extracell Vesicles,2013,2.
[3]	MITCHELL P J,WELTON J,STAFFURTH J,et al.Can urinary exosomes act as treatment response markers in prostate cancer?[J]. J Transl Med,2009,7:4.
[4]	PISITKUN T,JOHNSTONE R,KNEPPER M A.Discovery of urinary biomarkers[J]. Mol Cell Proteomics,2006,5(10):1760-1771.
[5]	ZHOU H,YUEN P S,PISITKUN T,et al.Collection,storage,preservation,and normalization of human urinary exosomes for biomarker discovery[J]. Kidney Int,2006,69(8):1471-1476.
[6]	LV L L,CAO Y,LIU D,et al.Isolation and quantification of microRNAs from urinary exosomes/microvesicles for biomarker discovery[J]. Int J Biol Sci,2013,9(10):1021-1031.
[7]	YUANA Y,BÖING A N,GROOTEMAAT A E,et al. Handling and storage of human body fluids for analysis of extracellular vesicles[J]. J Extracell Vesicles,2015,4:29260.
[8]	ALVAREZ M L,KHOSROHEIDARI M,KANCHI RAVI R,et al.Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers[J]. Kidney Int,2012,82(9):1024-1032.
[9]	LIN H,LIU X,XU X,et al.Quantification and size distribution of 24-hour urinary extracellular vesicles from healthy adults[J]. Nan Fang Yi Ke Da Xue Xue Bao,2015,35(11):1530-1534.
[10]	GONZALES P A,ZHOU H,PISITKUN T,et al.Isolation and purification of exosomes in urine[J]. Methods Mol Biol,2010,641:89-99.
[11]	MUSANTE L,SARASWAT M,RAVIDA A,et al.Recovery of urinary nanovesicles from ultracentrifugation supernatants[J]. Nephrol Dial Transplant,2013,28(6):1425-1433.
[12]	罗伊,王从容. 影响尿液储存稳定性的常见因素[J]. 检验医学,2014,29(1):86-90.
[13]	ROOD I M,DEEGENS J K,MERCHANT M L,et al.Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome[J]. Kidney Int,2010,78(8):810-816.
[14]	CHERUVANKY A,ZHOU H,PISITKUN T,et al.Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator[J]. Am J Physiol:Renal Physiol,2007,292(5):F1657-F1661.
[15]	MUSANTE L,TATARUCH D E,HOLTHOFER H.Use and isolation of urinary exosomes as biomarkers for diabetic nephropathy[J]. Front Endocrinol(Lausanne),2014,5:149.
[16]	TAYLOR D D,ZACHARIAS W,GERCEL-TAYLOR C.Exosome isolation for proteomic analyses and RNA profiling[J]. Methods Mol Biol,2011,728:235-246.
[17]	SANTANA S M,ANTONYAK M A,CERIONE R A,et al.Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations[J]. Biomed Microdevices,2014,16(6):869-877.
[18]	LIGA A,VLIEGENTHART A D,OOSTHUYZEN W,et al.Exosome isolation:a microfluidic road-map[J]. Lab Chip,2015,15(11):2388-2394.
[19]	MUSANTE L,TATARUCH D,GU D,et al.A simplified method to recover urinary vesicles for clinical applications,and sample banking[J]. Sci Rep,2014,4:7532.
[20]	LIU X,CHINELLO C,MUSANTE L,et al.Intraluminal proteome and peptidome of human urinary extracellular vesicles[J]. Proteomics-Clin Appl,2015,9(5-6):568-573.
[21]	HUANG X,YUAN T,LIANG M,et al.Exosomal miR-1290 and miR-375 as prognostic markers in castration-resistant prostate cancer[J]. Eur Urol,2015,67(1):33-41.
[22]	SAMSONOV R,SHTAM T,BURDAKOV V,et al.Lectin-induced agglutination method of urinary exosomes isolation followed by mi-RNA analysis:application for prostate cancer diagnostic[J]. Prostate,2016,76(1):68-79.
[23]	WANG D,SUN W.Urinary extracellular microvesicles:isolation methods and prospects for urinary proteome[J]. Proteomics,2014,14(16):1922-1932.
[24]	BIJNSDORP I V,GELDOF A A,LAVAEI M,et al.Exosomal ITGA3 interferes with non-cancerous prostate cell functions and is increased in urine exosomes of metastatic prostate cancer patients[J]. J Extracell Vesicles,2013,2.
[25]	林铖,姜傥. 液体活检技术在非小细胞肺癌患者EGFR-TKI继发耐药中的应用[J]. 检验医学,2016,31(10):835-843.
[26]	ZHOU H,PISITKUN T,APONTE A,et al.Exosomal fetuin-a identified by proteomics:a novel urinary biomarker for detecting acute kidney injury[J]. Kidney Int,2006,70(10):1847-1857.
[27]	ZHOU H,CHERUVANKY A,HU X,et al.Urinary exosomal transcription factors,a new class of biomarkers for renal disease[J]. Kidney Int,2008,74(5):613-621.
[28]	DU CHEYRON D,DAUBIN C,POGGIOLI J,et al.Urinary measurement of Na+/H+ exchanger isoform 3 (NHE3) protein as new marker of tubule injury in critically ill patients with ARF[J]. Am J Kidney Dis,2003,42(3):497-506.
[29]	SONODA H,YOKOTA-IKEDA N,OSHIKAWA S,et al.Decreased abundance of urinary exosomal aquaporin-1 in renal ischemia-reperfusion injury[J]. Am J Physiol Renal Physiol,2009,297(4):F1006-F1016.
[30]	ZHOU H,KAJIYAMA H,TSUJI T,et al.Urinary exosomal Wilms' tumor-1 as a potential biomarker for podocyte injury[J]. Am J Physiol Renal Physiol,2013,305(4):F553-F559.
[31]	MOON P,LEE J,YOU S,et al.Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy[J]. Proteomics,2011,11(12):2459-2475.
[32]	BARUTTA F,TRICARICO M,CORBELLI A,et al.Urinary exosomal microRNAs in incipient diabetic nephropathy[J]. PLoS One,2013,8(11):e73798.
[33]	GUDEHITHLU K P,GARCIA-GOMEZ I,VERNIK J,et al.In diabetic kidney disease urinary exosomes better represent kidney specific protein alterations than whole urine[J]. Am J Nephrol,2016,42(6):418-424.
[34]	SUN A L,DENG J T,GUAN G J,et al.Dipeptidyl peptidase-Ⅳ is a potential molecular biomarker in diabetic kidney disease[J]. Diab Vasc Dis Res,2012,9(4):301-308.
[35]	ZUBIRI I,POSADA-AYALA M,SANZ-MAROTO A,et al.Diabetic nephropathy induces changes in the proteome of human urinary exosomes as revealed by label-free comparative analysis[J]. J Proteomics, 2014,96:92-102.
[36]	GUTWEIN P,SCHRAMME A,ABDEL-BAKKY M S,et al. ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases[J]. J Biomed Sci,2010,17:3.
[37]	LV L L,CAO Y H,NI H F,et al.MicroRNA-29c in urinary exosome/microvesicle as a biomarker of renal fibrosis[J]. Am J Physiol Renal Physiol,2013,305(8):F1220-F1227.
[38]	LV L L,CAO Y H,PAN M M,et al.CD2AP mRNA in urinary exosome as biomarker of kidney disease[J]. Clin Chim Acta,2014,428:26-31.
[39]	PEAKE P W,PIANTA T J,SUCCAR L,et al.A comparison of the ability of levels of urinary biomarker proteins and exosomal mRNA to predict outcomes after renal transplantation[J]. PLoS One,2014,9(2):e98644.
[40]	ALVAREZ S,SUAZO C,BOLTANSKY A,et al.Urinary exosomes as a source of kidney dysfunction biomarker in renal transplantation[J]. Transplant Proc,2013,45(10):3719-3723.
[41]	DIMUCCIO V,RANGHINO A,PRATICÒ BARBATO L,et al.Urinary CD133+ extracellular vesicles are decreased in kidney transplanted patients with slow graft function and vascular damage[J]. PLoS One,2014,9(8):e104490.
[42]	HOGAN M C,MANGANELLI L,WOOLLARD J R,et al.Characterization of PKD protein-positive exosome-like vesicles[J]. J Am Soc Nephrol,2009,20(2):278-288.
[43]	CORCORAN C,RANI S,O'DRISCOLL L. MiR-34a is an intracellular and exosomal predictive biomarker for response to docetaxel with clinical relevance to prostate cancer progression[J]. Prostate,2014,74(13):1320-1334.
[44]	ISIN M,UYSALER E,ÖZGÜR E,et al. Exosomal lncRNA-p21 levels may help to distinguish prostate cancer from benign disease[J]. Front Genet,2015,6:168.
[45]	NILSSON J,SKOG J,NORDSTRAND A,et al.Prostate cancer-derived urine exosomes:a novel approach to biomarkers for prostate cancer[J]. Br J Cancer,2009,100(10):1603-1607.
[46]	OVERBYE A,SKOTLAND T,KOEHLER C J,et al.Identification of prostate cancer biomarkers in urinary exosomes[J]. Oncotarget,2015,6(30):30357-30376.
[47]	ROYO F,ZUNIGA-GARCIA P,TORRANO V,et al.Transcriptomic profiling of urine extracellular vesicles reveals alterations of CDH3 in prostate cancer[J]. Oncotarget,2016,7(6):6835-6846.
[48]	BECKHAM C J,OLSEN J,YIN P N,et al.Bladder cancer exosomes contain edil-3/del1 and facilitate cancer progression[J]. J Urol,2014,192(2):583-592.
[49]	PEREZ A,LOIZAGA A,ARCEO R,et al.A pilot study on the potential of RNA-associated to urinary vesicles as a suitable non-invasive source for diagnostic purposes in bladder cancer[J]. Cancers(Basel),2014,6(1):179-192.
[50]	CHEN C L,LAI Y F,TANG P,et al.Comparative and targeted proteomic analyses of urinary microparticles from bladder cancer and hernia patients[J]. J Proteome Res,2012,11(12):5611-5629.
[51]	RAIMONDO F,MOROSI L,CORBETTA S,et al.Differential protein profiling of renal cell carcinoma urinary exosomes[J]. Mol Biosyst,2013,9(6):1220-1233.
[52]	VIÑUELA-BERNI V,DONÍZ-PADILLA L,FIGUEROA-VEGA N,et al. Proportions of several types of plasma and urine microparticles are increased in patients with rheumatoid arthritis with active disease[J]. Clin Exp Immunol,2015,180(3):442-451.
[53]	FRASER K B,RAWLINS A B,CLARK R G,et al.Ser(P)-1292 LRRK2 in urinary exosomes is elevated in idiopathic Parkinson's disease[J]. Mov Disord,2016, 31(10):1543-1550.
[54]	BARRERA-CHIMAL J,PEREZ-VILLALVA R,CORTES-GONZALEZ C,et al.Hsp72 is an early and sensitive biomarker to detect acute kidney injury[J]. EMBO Mol Med,2011,3(1):5-20.
[55]	HERR P,KORNIYCHUK G,YAMAMOTO Y,et al.Regulation of TGF-(beta) signalling by N-
	acetylgalactosaminyltransferase-like 1[J]. Development,2008,135(10):1813-1822.
[56]	MESICEK J,LEE H,FELDMAN T,et al.Ceramide synthases 2,5,and 6 confer distinct roles in radiation-induced apoptosis in HeLa cells[J]. Cell Signal,2010,22(9):1300-1307.
[57]	SKOTLAND T,EKROOS K,KAUHANEN D,et al.Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers[J]. Eur J Cancer,2017,70:122-132.
[58]	LI Y,ZHANG Y,QIU F,et al.Proteomic identification of exosomal LRG1:a potential urinary biomarker for detecting NSCLC[J]. Electrophoresis,2011,32(15):1976-1983.

方法	原理	优点	局限性
超高速离心^[1,11]	基于EV大小和密度不同沉淀	保持EV完整性,利于RNA、蛋白质分析	耗时长、工作量大、需特殊设备
超高速离心+蔗糖梯度^[3,8]	在超高速离心基础上,据密度不同EV悬浮于不同浓度蔗糖溶液中	去除高丰度蛋白,利于蛋白质分析,可将EV分成不同的群	耗时长、步骤复杂、不适宜大批量标本
超滤法^[13-15]	基于EV大小不同分离	简单、快速、标本量小	有蛋白污染、不适宜 RNA、蛋白质分析
离心-尺寸排阻法^[13]	基于EV大小不同分离	去除高丰度蛋白,利于蛋白质分析	耗时长、工作量大、提取效率较低
沉淀法^[8]	基于聚乙二醇分离	简单、快速、操作标准化、利于miRNA分析	有蛋白污染,不适宜蛋白质分析
免疫亲和力法^[16]	基于EV标志物与其抗体特异性结合分离	能得到某一特定类型EV	非特异性EV
微流控法^[17-18]	基于液流力学分离	简便、快速、可将EV分成不同的群	提取效率不高、不适宜大标本量标本
液压透析法^[15,19-20]	基于EV大小不同分离	可处理大量标本、保持EV完整性、无需特殊设备	需后续处理高丰度蛋白

方法	原理	优点	局限性
超高速离心^[1,11]	基于EV大小和密度不同沉淀	保持EV完整性,利于RNA、蛋白质分析	耗时长、工作量大、需特殊设备
超高速离心+蔗糖梯度^[3,8]	在超高速离心基础上,据密度不同EV悬浮于不同浓度蔗糖溶液中	去除高丰度蛋白,利于蛋白质分析,可将EV分成不同的群	耗时长、步骤复杂、不适宜大批量标本
超滤法^[13-15]	基于EV大小不同分离	简单、快速、标本量小	有蛋白污染、不适宜 RNA、蛋白质分析
离心-尺寸排阻法^[13]	基于EV大小不同分离	去除高丰度蛋白,利于蛋白质分析	耗时长、工作量大、提取效率较低
沉淀法^[8]	基于聚乙二醇分离	简单、快速、操作标准化、利于miRNA分析	有蛋白污染,不适宜蛋白质分析
免疫亲和力法^[16]	基于EV标志物与其抗体特异性结合分离	能得到某一特定类型EV	非特异性EV
微流控法^[17-18]	基于液流力学分离	简便、快速、可将EV分成不同的群	提取效率不高、不适宜大标本量标本
液压透析法^[15,19-20]	基于EV大小不同分离	可处理大量标本、保持EV完整性、无需特殊设备	需后续处理高丰度蛋白

疾病	标志物	分离方法	参考文献
急性肾损伤	Fetuin A	超高速离心法	[26]
	ATF3	超高速离心法	[27]
	NHE3	超高速离心法	[28]
肾脏缺血再灌注	水通道蛋白-1	超高速离心法	[29]
局灶节段性肾小球硬化	Wilm's tumor 1	超高速离心法	[30]
IgA肾病	α1-抗胰酶蛋白、氨肽酶N、vasorin前体、血浆铜蓝蛋白	超高速离心联合蔗糖梯度法	[31]
糖尿病肾病	miR-130、 miR-145、 miR-155 和 miR-424	超高速离心法	[32]
糖尿病肾病	明胶酶、血浆铜蓝蛋白	超高速离心法	[33]
肾小球疾病	二肽基肽酶4	滤器-离心法	[34]
	AMBP、MLL3和 VDAC1	超高速离心法	[35]
	ADAM10	超高速离心法	[36]
肾纤维化	miR-29c和miR-200	超高速离心法	[37]
肾纤维化	CD2AP	超高速离心法	[38]
慢性肾病	IL-18和 NGAL	超滤法	[39]
肾脏移植	NGAL	超高速离心法	[40]
肾脏移植	CD133	超高速离心法	[41]
多囊肾	PKD1、PKD2和PKHD1	超高速离心法	[42]
前列腺癌	ITGA3和ITGB1	超高速离心法	[24]
	miR-34a	超高速离心法	[43]
	LncRNA-p21	尿液外泌体RNA分离试剂盒	[44]
	TMPRSS2:ERG融合基因和PCA-3	超高速离心法	[45]
	TM256和LAMTOR1	超高速离心法	[46]
	钙黏蛋白3	超高速离心法	[47]
膀胱癌	EDIL-3	超高速离心联合蔗糖梯度法	[48]
	LASS2和GALNT1	超高速离心法	[49]
	TACSTD2	超高速离心法	[50]
肾癌	MMP-9、DKK4、EMMPRIN和PODXL	超高速离心法	[51]
类风湿关节炎	CD14、CD3和CD19	超高速离心法	[52]
帕金森病	Ser（P）-1292 LRRK2	超高速离心法	[53]

疾病	标志物	分离方法	参考文献
急性肾损伤	Fetuin A	超高速离心法	[26]
	ATF3	超高速离心法	[27]
	NHE3	超高速离心法	[28]
肾脏缺血再灌注	水通道蛋白-1	超高速离心法	[29]
局灶节段性肾小球硬化	Wilm's tumor 1	超高速离心法	[30]
IgA肾病	α1-抗胰酶蛋白、氨肽酶N、vasorin前体、血浆铜蓝蛋白	超高速离心联合蔗糖梯度法	[31]
糖尿病肾病	miR-130、 miR-145、 miR-155 和 miR-424	超高速离心法	[32]
糖尿病肾病	明胶酶、血浆铜蓝蛋白	超高速离心法	[33]
肾小球疾病	二肽基肽酶4	滤器-离心法	[34]
	AMBP、MLL3和 VDAC1	超高速离心法	[35]
	ADAM10	超高速离心法	[36]
肾纤维化	miR-29c和miR-200	超高速离心法	[37]
肾纤维化	CD2AP	超高速离心法	[38]
慢性肾病	IL-18和 NGAL	超滤法	[39]
肾脏移植	NGAL	超高速离心法	[40]
肾脏移植	CD133	超高速离心法	[41]
多囊肾	PKD1、PKD2和PKHD1	超高速离心法	[42]
前列腺癌	ITGA3和ITGB1	超高速离心法	[24]
	miR-34a	超高速离心法	[43]
	LncRNA-p21	尿液外泌体RNA分离试剂盒	[44]
	TMPRSS2:ERG融合基因和PCA-3	超高速离心法	[45]
	TM256和LAMTOR1	超高速离心法	[46]
	钙黏蛋白3	超高速离心法	[47]
膀胱癌	EDIL-3	超高速离心联合蔗糖梯度法	[48]
	LASS2和GALNT1	超高速离心法	[49]
	TACSTD2	超高速离心法	[50]
肾癌	MMP-9、DKK4、EMMPRIN和PODXL	超高速离心法	[51]
类风湿关节炎	CD14、CD3和CD19	超高速离心法	[52]
帕金森病	Ser（P）-1292 LRRK2	超高速离心法	[53]