Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603, USA.
Kidney Int. 2010 Apr;77(8):736-42. doi: 10.1038/ki.2009.550. Epub 2010 Feb 3.
Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidney disease. Current protocols for their isolation use a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high speed (200,000 x g). Analysis using western blot and electron microscopy found that urinary exosomes are also present in the low-speed pellet entrapped by polymeric Tamm-Horsfall protein, thus diminishing the procedure's reproducibility. Here we show that addition of dithiothreitol to the low-speed pellet disrupted the polymeric network, presumably by reduction of disulfide bonds linking the monomers. This modification shifted the exosomal proteins from the low- to the high-speed pellet. Also, by shifting the Tamm-Horsfall protein to the high-speed pellet, the use of dithiothreitol makes it feasible to use Tamm-Horsfall protein to normalize excretion rates of exosomal proteins in spot urines. We tested this by western blot, and found that there was a high degree of correlation between exosomal proteins and Tamm-Horsfall protein in the high-speed pellet. Since the yield of exosomes by differential centrifugation can be increased by chemical reduction, Tamm-Horsfall protein may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical.
尿液外泌体被提议作为发现肾脏疾病蛋白生物标志物的起始材料。目前用于分离外泌体的方案使用两步差速离心法。由于其密度较低，外泌体预计将留在低速（17,000 x g）上清液中，只有当样品在高速（200,000 x g）下旋转时才会沉降。使用 Western blot 和电子显微镜分析发现，尿液外泌体也存在于被聚合物 Tamm-Horsfall 蛋白截留的低速沉淀中，从而降低了该程序的重现性。在这里，我们表明，向低速沉淀中添加二硫苏糖醇会破坏聚合物网络，可能是通过还原连接单体的二硫键。这种修饰将外泌体蛋白从低速沉淀转移到高速沉淀中。此外，通过将 Tamm-Horsfall 蛋白转移到高速沉淀中，二硫苏糖醇的使用使得使用 Tamm-Horsfall 蛋白来标准化斑点尿液中外泌体蛋白的排泄率成为可能。我们通过 Western blot 对此进行了测试，发现高速沉淀中外泌体蛋白和 Tamm-Horsfall 蛋白之间存在高度相关性。由于差速离心法中通过化学还原增加了外泌体的产量，因此当定量尿液收集不可行时，Tamm-Horsfall 蛋白可能是尿液外泌体研究的合适归一化变量。
