Zhou H, Yuen P S T, Pisitkun T, Gonzales P A, Yasuda H, Dear J W, Gross P, Knepper M A, Star R A
Renal Diagnostics and Therapeutics Unit, NIDDK, Bethesda, MD 20892-1268, USA.
Kidney Int. 2006 Apr;69(8):1471-6. doi: 10.1038/sj.ki.5000273.
Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.
含有顶端膜和细胞内液的尿液外泌体通常从所有肾单位段分泌到尿液中，并可能携带肾功能障碍和结构损伤的蛋白质标志物。我们研究了尿液外泌体蛋白质的收集、储存和保存方法。我们从健康志愿者收集尿液，添加蛋白酶抑制剂，并将尿液样本分别在4℃、-20℃和-80℃储存1周或7个月。样本在有或没有剧烈涡旋的情况下解冻，然后分离出三个部分：尿沉渣、上清液和外泌体部分。测量了蛋白质浓度、电泳图谱以及七种外泌体相关蛋白的丰度。在尿沉渣或上清液部分未检测到外泌体相关蛋白。蛋白酶抑制剂可防止外泌体相关蛋白的降解。与新鲜尿液相比，在-20℃冷冻导致外泌体大量损失。相比之下，在-80℃冷冻后的回收率几乎是完整的。解冻后剧烈涡旋显著提高了在-20℃或-80℃冷冻尿液中的外泌体回收率，即使冷冻7个月也是如此。第一次晨尿和第二次晨尿的回收率相似。胞质外泌体相关蛋白的丰度在长期储存期间没有下降。我们得出结论：（1）蛋白酶抑制剂对于保存至关重要；（2）在-80℃储存并在解冻后剧烈涡旋可使尿液外泌体的回收率最大化；（3）第一次晨尿和第二次晨尿中外泌体相关蛋白的差异很小，表明尿路/膀胱中的蛋白质降解极少；（4）尿液外泌体在长期储存期间保持完整。这些尿液收集、储存和处理条件可能对未来的生物标志物发现工作有用。
