Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome
Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome
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DOI：
10.1038/ki.2010.262
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发表时间：
2010-10-01
影响因子：
19.6
通讯作者：
Klein, Jon B.
中科院分区：
文献类型：
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作者：
Rood, Ilse M.;Deegens, Jeroen K. J.;Klein, Jon B.
尿微泡，如40-100 nm的外来体和100- 1000 nm的微粒，含有许多蛋白质，可作为肾脏疾病的生物标志物。微泡已被从正常尿液中分离出超离心或纳米膜超滤，然而，这些方法在分离肾病范围蛋白尿患者的微泡中的效率知之甚少。在这里，我们比较了三种技术，从肾病尿中分离微泡：纳米膜超滤，超离心，超离心后的尺寸排阻色谱法（UC-SEC）。超滤或超浓缩后，高丰度的尿蛋白仍以足够的量存在，从而使MALDI-TOF-TOF质谱法检测到的丰度较低的微泡蛋白钝化。微泡标志物脑啡肽酶，水通道蛋白-2，podocalyxin高度富集后UC-SEC相比，通过超滤或ultracentravation单独的制剂。UC-SEC级分的电子显微镜发现不同大小的微泡，与外来体和微粒的存在相容。因此，UC-SEC超离心后进一步富集和纯化微粒，有助于寻找可用于预测肾病综合征临床病程的预后生物标志物。Kidney International（2010）78，810-816; doi：10.1038/ki.2010.262; 2010年8月4日在线发表
Urinary microvesicles, such as 40-100 nm exosomes and 100-1000nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by sizeexclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome. Kidney International (2010) 78, 810-816; doi: 10.1038/ki.2010.262; published online 4 August 2010