Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Toxicol Sci. 2013 Jan;131(1):13-25. doi: 10.1093/toxsci/kfs268. Epub 2012 Sep 27.
Kidney injury molecule-1 (KIM-1) has been qualified by the Food and Drug Administration and European Medicines Agency as a urinary biomarker to monitor preclinical nephrotoxicity in rats and on a case-by-case basis for the translation of potentially nephrotoxic drugs into first-in human studies. Although mouse models are widely employed in preclinical studies, few urinary biomarker studies have been performed in mice due to limited urine availability and lack of sensitive assays. Here, we report the development and validation of two different assays for quantitative assessment of mouse urinary KIM-1 (uKIM-1) and compare the sensitivity of KIM-1 relative to other standard markers in ischemia reperfusion and aristolochic acid (AA)-induced kidney injury in mice. A sensitive, reproducible, and quantitative microbead-based KIM-1 ELISA was established, which requires only 10 μl urine for triplicate determination with an assay range of 12.21 pg/ml to 50 ng/ml. The second assay is a laminar flow dipstick assay, which has an assay range of 195 pg/ml to 50 ng/ml and provides quantitative assessment of KIM-1 in 15 min. uKIM-1 levels increased with increasing time of ischemia or time after AA administration. After only 10-min ischemia followed by 24-h reperfusion, uKIM-1 was significantly elevated by 13-fold, whereas serum creatinine (sCr), blood urea nitrogen, N-acetyl-β-glucosaminidase (NAG), and proteinuria levels did not change. After AA administration, uKIM-1 levels were significantly upregulated by greater than threefold within 12 h, whereas sCr and NAG levels were unchanged. Mouse KIM-1 was stable for multiple freeze-thaw cycles, for up to 5 days at room temperature and up to at least an year when stored at -80°C.
肾损伤分子 1（KIM-1）已被美国食品和药物管理局以及欧洲药品管理局认定为一种尿液生物标志物，可用于监测大鼠的临床前肾毒性，并在将具有潜在肾毒性的药物转化为首次人体研究时进行个案翻译。尽管小鼠模型在临床前研究中被广泛应用，但由于尿液可用性有限且缺乏敏感检测，很少在小鼠中进行尿液生物标志物研究。在这里，我们报告了两种不同的用于定量评估小鼠尿 KIM-1（uKIM-1）的检测方法的开发和验证，并比较了 KIM-1 相对于其他标准标志物在缺血再灌注和马兜铃酸（AA）诱导的小鼠肾损伤中的敏感性。建立了一种灵敏、重现性好且定量的基于微球的 KIM-1 ELISA，该方法仅需 10 μl 尿液，即可进行三次重复测定，检测范围为 12.21 pg/ml 至 50 ng/ml。第二种检测方法是层流试条检测法，其检测范围为 195 pg/ml 至 50 ng/ml，可在 15 分钟内定量评估 KIM-1。uKIM-1 水平随着缺血时间或 AA 给药后时间的增加而增加。仅 10 分钟缺血后再灌注 24 小时后，uKIM-1 水平显著升高 13 倍，而血清肌酐（sCr）、血尿素氮、N-乙酰-β-氨基葡萄糖苷酶（NAG）和蛋白尿水平没有变化。AA 给药后，uKIM-1 水平在 12 小时内显著上调 3 倍以上，而 sCr 和 NAG 水平不变。小鼠 KIM-1 在多次冻融循环中稳定，在室温下可稳定 5 天，在-80°C 下可至少稳定一年。
