庆大霉素致大鼠急性肾损伤的尿液生物标志物

摘要

目的：探讨大鼠急性肾损伤后尿液新生物标志物时间和剂量依懒性，以寻找敏感和特异的新生物标志物。方法SD 大鼠分别 i m给予硫酸庆大霉素注射液0，5，20和80 mg·kg -1，每天1次，按照给药次数又分为给药1，3，7，14 d 和给药14 d 恢复期28 d 亚组，共计20组，给药后24 h，取血检测尿素氮（BUN）和肌酐（CRE），取尿用干化学法检测尿蛋白，用酶联免疫法（ELISA）检测肾损伤分子-1（KIM-1）、用电化学发光免疫法检测β2微球蛋白（β2-MG）；HE 染色观察肾组织病理改变。结果尿蛋白结果显示，给予庆大霉素80 mg·kg -17和14 d，个别雌性大鼠可见尿蛋白明显阳性（3＋），恢复期末有明显恢复趋势。给药80 mg·kg -17 d 组雌性大鼠 BUN 和 CRE 明显升高（P＜0．05），雄性大鼠 CRE 升高（P＜0．05），BUN有升高趋势；给药80 mg·kg -114 d 组雌雄大鼠 BUN 和 CRE 均明显升高（P ＜0．05），20 mg·kg -1组雌性大鼠 CRE 升高（P＜0．05），恢复期末，雌雄大鼠 BUN 和 CRE 恢复正常。肾组织病理学结果显示，给药20和80 mg·kg -13 d 组肾见局灶性肾小管扩张，给药20 mg·kg -17和14 d 组肾见炎细胞浸润和局灶性肾小管扩张，给药80 mg·kg -17和14 d 组肾见局灶性肾小管上皮细胞变性坏死、炎细胞浸润、局灶性管型（轻度），给药80 mg·kg -1恢复期28 d 组肾见嗜碱性小管、肾小管管型。给药20和80 mg·kg -13，7和14 d 组雌雄大鼠 KIM-1明显升高（P ＜0．05），给药80 mg·kg -1恢复期28 d 组雌雄大鼠和给药20 mg·kg -1的雄性大鼠KIM-1明显升高（P＜0．05），但有恢复趋势；给药80 mg·kg -13 d 组雌雄大鼠β2-MG 明显升高（P ＜0．05），给药20和80 mg·kg -17和14 d 组雌雄大鼠β2-MG 明显升高（P＜0．05或 P＜0．01），给药80 mg·kg -1雌雄大鼠和给药20 mg·kg -1的雌性大鼠恢复期28 d 组β2-MG 明显升高（P＜0．05），但也有恢复趋势。结论与传统的各项检测指标相比，KIM-1和β2-MG 在庆大霉素诱导的急性肾损伤模型中具有较好的敏感性和特异性。%OBJECTIVE To investigate the time and dose relation of new urine bio markers in rat model of acute kidney injury induced by genta mycin (GM)to search for more sensitive,noninvasive and specific markers than traditional approaches to monitor nephrotoxicity.METHODS SD Rats were im treated with GM5,20,80 mg·kg -1 or saline once daily.Rats were randomly divided into 20 subgroups:treated for 1 ,3,7,14 d and 14 d followed with 28 d recovery period.Ten rats per group (5 rats per sex)were scarified at 24 h after the last dosing or the end of recovery period.Blood sa mples were col-lected for blood urea nitrogen (BUN)and creatinine(CRE)analysis.Urine was collected at each nec-ropsy for urine protein by dry che mistry method,for kidney injury molecule-1 (KIM-1 )analysis by ELISA, and for β2-microglobulin (β2-MG)analysis by ELISA.Kidneys were obtained for histological exa mination after HE stains.RESULTS Positive protein(3 +)was noted for several fe male animals treated for 7 or 14 d at 80 mg·kg -1 and the tendency of recovery were noted at the end of recovery period.Co mpared with those in saline control group treated for 7 d,the seru m BUN and CRE levels for fe males and the CRE level for males were significantly increased at 80 mg·kg -1 (P 0.05).When treated for 14 d,the seru m BUN and CRE levels for fe males and males at 80 mg·kg -1 and the seru m CRE level for fe males at 20 mg·kg -1 were significantly increased when compared with those in saline control group(P <0.05). The seru m BUN and CRE recovered to base line for all animals treated for 14 d followed by a 28 d recov-ery period.Histopathological observation of kidney tissues indicated that focal tubule dilatation was noted for animals treated for 3 d at 20 and 80 mg·kg -1 ,infla mmatory cell infiltration and focal tubule dilatation were noted at 20 mg·kg -1 and focal renal tubular epithelial cell degeneration,infla mmatory cell infiltra-tion,focal casts (lightly)were noted at 80 mg·kg -1 for animals treated for 7 or 14 d.For animals treated for 14 d followed by a 28 d recovery period,only basophilic tubules and renal casts were noted at 80 mg·kg -1 .New urine bio markers determination indicated that KIM-1 level was significantly increased at 20 and 80 mg·kg -1 for animals treated for 3,7 or 14 d when compared with that in saline control group (P<0.05).For animals treated for 14 d followed by a 28 d recovery period,the KIM-1 level was still significantly higher than saline control group for males and fe males at 80 mg·kg -1 and males at 20 mg·kg -1 (P <0.05 ),but there was evidence for reversal.The β2-MG level was significantly increased at 80 mg·kg -1 for animals treated for 3 d(P<0.05),at 20 or 80 mg·kg -1 for animals treated for 7 or 14 d(P<0.05 or P<0.01 ),when compared with that in the saline control group.For animals treated for 14 d followed by a 28 d recovery period,the β2-MG level was still significantly higher than saline control group for males and fe males at 80 mg·kg -1 and fe males at 20 mg·kg -1 (P <0.05),but there was also evidence for reversal.CONCLUSION Urine KIM-1 and β2-GM are more sensitive and specific markers for early diagnosis of kidney injury induced by GM when compared with the traditional approaches to monitor nephrotoxicity.