Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator
Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator
复制标题
DOI：
10.1152/ajprenal.00434.2006
复制
发表时间：
2007-05-01
影响因子：
4.2
通讯作者：
Star, Robert A.
中科院分区：
文献类型：
--
作者：
Cheruvanky, Anita;Zhou, Hua;Star, Robert A.
尿液外泌体从所有肾单位段中排出，可作为肾脏疾病分类的生物标志物。通过已建立的超速离心方法分离尿液外泌体对于临床实验室的使用有一些限制。我们寻求一种快速而简单的方法来获取尿液外泌体。我们使用市售的纳米膜浓缩器，通过在 3,000 g 下离心 10 - 30 分钟来富集尿液中的外泌体。从纳米膜浓缩器中回收尿外泌体标志物肿瘤易感基因101、水通道蛋白-2、神经元特异性烯醇化酶、膜联蛋白V、血管紧张素转换酶和足萼蛋白（PODXL）并通过Western blotting检测，并通过电镜观察发现尿囊泡的典型特征。仅在 0.5 毫升尿液中即可检测到外泌体标记物。通过纳米膜方法，可以从-80℃冷冻或4℃冷藏过夜然后在-80℃保存的尿液样本中回收外泌体蛋白。通过富集外泌体，我们可以检测到足细胞标记物PODXL，在5名患有局灶节段性肾小球硬化症和大量蛋白尿的男性患者中，足细胞标记物PODXL下降了71%。我们的结论是：1）纳米膜浓缩器的使用简化并加速了尿液外泌体的富集； 2）纳米膜浓缩器可以浓缩临床尿样中的外泌体蛋白。这种增强的方法可以加速尿液外泌体生物标志物从实验室到临床的转化，用于肾脏疾病的诊断、分类和预测。
Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10 - 30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin- 2, neuron- specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin ( PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at -80 degrees C or refrigerated overnight at 4 degrees C then stored at -80 degrees C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases.