大鼠肾缺血再灌注过程中流体剪切力的缺失降低腺苷酸活化蛋白激酶的活性

The absence of fluid shear stress reduces AMPK activity in the renal I/R process

目的 探讨大鼠肾缺血再灌注（IR）过程中流体剪切力（FSS）改变引起肾损伤的机制.方法 1.细胞实验：将人脐静脉内皮细胞株（HUVEC）分为4组,（1）应用平板流体小室系统对HU-VEC加载12 dyn/cm2的FSS 30、45、90 min;（2）将HUVEC加载FSS2 h后停止加力,并分别培养1、3、8、12 h;（3）将0、1、2、4、8 mmol二甲双胍分别对HUVEC预处理培养24 h;（4）对照组为正常培养HUVEC.采用蛋白质印迹法检测各组细胞内p-AMPK/AMPK蛋白表达水平.2.体内实验：取成功建立IR模型的SD大鼠16只,采用随机数字表法分4组,每组4只：（1）低温静态保存（CS）组：将离体大鼠双肾冷保存4h;（2）低温机械灌注保存（HMP）组：将离体大鼠双肾持续灌注0℃乳酸林格液4h.（3）二甲双胍处理组（Met-CS组）：术前连续3d腹腔注射二甲双胍,获取离体大鼠双肾后冷保存4h;（4）对照组离体大鼠双肾热缺血30min后不做任何处理.采用原位末端标记法、HE染色等观察各组大鼠肾组织损伤情况,采用免疫组织化学法检测肾组织内p-AMPK蛋白表达和分布规律.分析FSS缺失与肾组织AMPK表达间的相关性.结果 FSS可上调HUVEC内p-AMPK表达水平,并随作用时间延长表达升高;停力后,HUVEC内p-AMPK蛋白表达随时间延长逐渐降低（P＜0.05）;二甲双胍可激活AMPK活性,并随浓度增加而升高（P＜0.05）.HMP组肾组织内p-AMPK含量明显高于CS组（P＜0.05）,HMP组肾组织内p-AMPK表达主要分布于肾小管处,少数于肾小球内皮细胞和血管处.HMP组肾组织细胞凋亡率明显低于CS组（P＜0.05）.HMP组大鼠肾组织损伤轻,无水肿,肾小管轻度扩张;而CS组肾组织损伤严重,肾小管明显水肿.结论 大鼠肾IR过程中,FSS改变可能通过AMPK途径影响肾组织损伤.

Objective To investigate the mechanism of renal injury induced by changes in flow shear stress （FSS） during renal ischemia/reperfusion （I/R）.Methods 1.In vitro,HUVECs were divided into 4 groups：（1） HUVECs were loaded with 12 dyn/cm2 force for 30,45,and 90 min by using plate fluid chamber system.（2） Cells were loaded with FSS for 2 h,and then cultured for 1,3,8 and 12 h respectively;（3） HUVECs were pretreated with 0,1,2,4 and 8 mmol metformin and cultured for 24 h.（4） HUVECs in control group were cultured normally.The expression of p-AMPK/AMPK protein was detected by Western blotting in each group.2.In vivo,16 SD rats with successful establishment of IR model were randomly divided into 4 groups （n =4 in each group）：（1） static cold storage （CS） group：isolated kidneys were stored for 4 h;（2） hypothermia machine perfusion （HMP） group：isolated kidneys were continuously perfused with 0 ℃ lactated Ringer＇s solution for 4 h;（3）metformin treatment group （Met-CS）：metformin was intraperitoneally injected 3 days before surgery,and the isolated kidneys were obtained after cold preservation for 4 h;（4）rat kidneys of control group were just subjected to thermal ischemia for 30 min.The injury of renal tissue in each group was observed by TUNEL and HE staining.The expression and distribution of p-AMPK protein in renal tissues were detected by immunohistochemistry.The correlation between FSS loss and AMPK expression in kidney tissue was analyzed.Results The expression of p-AMPK in HUVECs could be up-regulated by FSS,and the expression of p-AMPK protein increased with the prolongation of time.After stopping FSS,the expression of p-AMPK protein in HUVECs gradually decreased with time （P〈0.05）.Metformin could activate AMPK activity in a concentration-dependent manner （P〈0.05）.The content of p-AMPK in renal tissue of HMP group was significantly higher than that of CS group （P〈0.05）.The expression of p-AMPK in renal tissue of HMP group mainly distributed in the renal tubules,and few in glomerular endothelial cells and blood vessels.The apoptosis rate of renal tissue in HMP group was significantly lower than that in CS group （P〈0.05）.In the HMP group,the damage of the renal tissue was mild,there was no swelling,and the renal tubules were slightly expanded.In the CS group,the renal tissue was severely damaged and the renal tubules were markedly swollen.Conclusion During the course of renal IR in rats,changes in FSS may affect renal tissue damage through the AMPK pathway.