热缺血时间对小鼠肾脏缺血再灌注损伤后肾功能的影响

Effect of warm ischemia time on renal function after renal ischemia-reperfusion injury in mice

目的探讨热缺d血t时间对小鼠肾脏缺血再灌注损伤(IRI)后肾功能的影响及与肾脏损伤分子1(KIM-1)的关系。方法选取雄性C57BL/6小鼠36只,异氟烷吸入麻醉下阻断双侧肾蒂血管,建立双侧肾脏IRI模型。根据肾缺血时间将小鼠分为四组,即假手术组(Sham组)、阻断28min组(28Min组),30min组(30Min组)和32min组(32Min组)。术后动态监测各组fu清肌酐(Ser)和血尿素氮(BUN)的变化。术后48 h取肾脏组织行Western blot和免疫荧光染色法检测近端肾小管细胞(P'TCs)中KIM-1的表达。术后6周取肾脏组织行苦味酸-天狼星红染色法检测小鼠肾纤维化程度。结果除Sham组外,其余各组小鼠均出现术后Ser及BUN升高,Ser和BUN的达峰时间为24 h,随后缓慢下降,至术后2周达稳定水平。IRI急性期评估提示,除Sham组外,其余各组小鼠术后48 h PTCs表达KIM-1明显上调,且KIM-1的表达量与肾损伤程度及热缺血时间成正比。慢性期评估表明,Sham组和28Min组术后6周无明显肾纤维化,而30Min组和32Min组出现明显肾脏纤维化,且纤维化程度随热缺血时间的延长而加重。术后6周观察期内,Sham组.28Min组和30Min组均无小鼠死亡,而32Min组有50%小鼠死亡。结论小鼠肾脏IRI的损伤程度随热缺血时间的延长而加重。IRI导致的轻微肾损伤是可逆的,急性期后肾功能可逐渐恢复正常。而严重的肾损伤会明显增加小鼠病死率,且会长期影响肾功能。KIM-I可用于监测IRI引起的肾损伤,并评价肾脏损伤的严重程度。

Objective To investigate the influence of different warm ischemia time on renal function after renal ischemia-reperfusion injuiy in mice and its correlation with kidney injuiy molecule-1(KIM-1),so as to assisting urologists lo optimize the time of renal arteiy occlusion during the surgery of partial nephrectomy.Methods A total of 36 male C57BIV6 mice were divided randomly into 4 groups(Sham,28 Min,30 Min and 32 Min)according to different renal pedicle occlusion duration.Serum creatinine(S cr)and blood urea nitrogen(BUN)were monitored before surgery,and at each time-point of 24 hours,72 hours and weekly from 1 week to 6 weeks after the surgeiy.Western blot and ininiunonuoreseence staining were used to detect the KIM-1 expression in kidney proximal tubular cells(PTCs)in the acute phase of 48 hours post the procedure.Picrosirius red staining was used to evaluate the kidney fibrosis severity as the indii-ator of chronic phase of kidney injury 6 weeks post the surgery.Results A half of mice in the group of 32 Min died within 1 week after the surgeiy,while mice in the other groups survived until euthanasia.Phe mice receiving renal pedicle occlusion exhibited incTeased Scr and BUN after the surgen while the sham-operated mice did not.The peak concentration of Scr and BUN both appeared at 24 hours post the surgen-,then decreased gradually till 2 weeks after the surgery and sustained stably thereafter.The peak and steady concentration of Scr and BUN showed a positive correlation with warm ischemia duration.Western blot and immunofluorescence staining showed FrCs presenting KIM-1 promptly and dramatically increased 48 hours post the surgeiy and indicated a positive correlation between KIM-1 expression and the warm ischemia duration.Moreover,picrosirius red staining implicated that the kidney librosis also deteriorated with the extended warm ischemia time.Conclusions The severity of kidney injuiy deteriorates with the extended warm ischemia time.Hie mice surviving the episode of acute kidney injury(AKI)could make a full renal recove r'when the injury in the acute phase is mild.However,AKI is appreciated to he markedly associated with increased risk of future chronic kidney disease and end-stage renal disease wiien the injury is severe.KIM-1 could be used as a biomarker to indicate the ischemia-repe^rfusion injury inducing kidney injury and to evaluate the severity.