Institute of Nephrology, Zhongda Hospital School of Medicine, Southeast Univ., No. 87 Ding JiaQiao Rd., Nanjing, China 210009.
Am J Physiol Renal Physiol. 2013 Oct 15;305(8):F1220-7. doi: 10.1152/ajprenal.00148.2013. Epub 2013 Aug 14.
Micro (mi)RNAs are frequently dysregulated in the development of renal fibrosis. Exosomes are small membrane vesicles that could be isolated from urine secreted from all nephron segments. Here we sought to observe for the first time whether miRNA in urine exosome could serve as a potential biomarker of renal fibrosis. Urine samples were collected from 32 chronic kidney disease (CKD) patients who underwent kidney biopsy and 7 controls. Exosome was isolated and confirmed by immunogold staining of exosome marker. Members of miR-29, miR-200, and RNU6B as endogenous control were detected by RT quantitative PCR. Electronic microscopy verified a typical shape of exosome with average size of 65.1 nm and labeled it with anti-CD9 and anti-aquaporin 2 antibody. Members of miR-29 and miR-200 are readily measured with reduced levels compared with controls (P < 0.05) and can robustly distinguish CKD from controls [area under the curve (AUC) varied from 0.902 to 1 by receiver operating characteristics analysis]. miR-29c correlated with both estimated glomerular filtration rate (r = 0.362; P < 0.05) and degree of tubulointerstitial fibrosis (r = -0.359; P < 0.05) for CKD patients. Moreover, miRNA in exosome was decreased in mild fibrosis group compared with moderated to severe group. miR-29a and miR-29c could predict degree of tubulointerstitial fibrosis with AUC of 0.883 and 0.738 (P < 0.05). The sensitivity and specificity for distinguishing mild from moderate to severe fibrosis were 93.8 and 81.3% with the use of miR-29a and 68.8 and 81.3% for miR-29c. Overall, miR-29c in urinary exosome correlates with both renal function and degree of histological fibrosis, suggesting it as a novel, noninvasive marker for renal fibrosis.
微小 RNA（miRNA）在肾纤维化的发展中经常失调。外泌体是可以从小鼠肾脏的尿液中分离出来的小膜泡。在这里，我们首次试图观察尿液外泌体中的 miRNA 是否可以作为肾纤维化的潜在生物标志物。收集了 32 名接受肾脏活检的慢性肾脏病（CKD）患者和 7 名对照者的尿液样本。通过对外泌体标志物的免疫金染色来分离和确认外泌体。通过 RT 定量 PCR 检测 miR-29、miR-200 和 RNU6B 作为内参。电子显微镜证实了外泌体的典型形状，平均尺寸为 65.1nm，并标记有抗 CD9 和抗水通道蛋白 2 抗体。与对照组相比，miR-29 和 miR-200 的成员易于测量，水平降低（P<0.05），并且可以可靠地区分 CKD 与对照组[通过接受者操作特征分析，曲线下面积（AUC）从 0.902 到 1 不等]。miR-29c 与 CKD 患者的估算肾小球滤过率（r=0.362；P<0.05）和肾小管间质纤维化程度（r=-0.359；P<0.05）相关。此外，与中度至重度纤维化组相比，轻度纤维化组中 exosome 的 miRNA 减少。miR-29a 和 miR-29c 可以预测肾小管间质纤维化的程度，AUC 分别为 0.883 和 0.738（P<0.05）。使用 miR-29a 区分轻度与中度至重度纤维化的敏感性和特异性分别为 93.8%和 81.3%，使用 miR-29c 分别为 68.8%和 81.3%。总体而言，尿外泌体中的 miR-29c 与肾功能和组织学纤维化程度相关，提示其为一种新的、非侵入性的肾纤维化标志物。
