瞬时受体电位通道M7和程序性坏死标志物RIP1在大鼠肾脏缺血再灌注损伤中的表达及意义

Expression and significance of TRPM7 and necroptosis marker RIP1 in rat kidney ischemia /reperfusion injury

目的观察大鼠缺血再灌注损伤模型中,瞬时受体电位通道M7(trallsient receptor potential melastatiIl related7,TRPM7)的表达变化及其与肾组织病理损伤、程序性坏死标志物RIP1、聚腺苷酸二磷酸核糖转移酶-1(PARP-1)表达变化的关系,初步探讨TRPM7在肾脏缺血再灌注损伤细胞程序性坏死中的作用及其可能的机制。方法 55只雄性SD大鼠建立缺血再灌注模型后,于再灌注后24、48 h、5、7 d取动脉血检测大鼠血肌酐、尿素氮水平;肾组织标本HE染色观察肾组织病理损伤;免疫组化和Western blot检测肾组织TRPM7、PARP-1、RIP1定位及表达变化。结果对照组和假手术组相比,血肌酐尿素氮水平、肾脏组织形态无明显变化;与对照组、假手术组相比,缺血再灌注后大鼠血肌酐尿素氮水平明显升高,再灌注后24 h达到最高值,随后逐渐下降,7 d左右降至接近正常。HE染色显示肾组织在再灌注24、48 h均出现明显急性肾小管损伤,5 d时开始修复,至第7天基本修复。免疫组化及Western blot显示,TRPM7主要在肾小管上皮细胞表达,再灌注后TRPM7表达显著升高,其中24 h达高峰,随后逐渐下降。PARP-1、RIP1的表达变化有相似的趋势。相关性分析显示TRPM7表达变化与肾小管急性损伤评分呈正相关(r=0.71,P<0.05)。结论瞬时受体电位通道M7可能参与了肾脏缺血再灌注损伤,通过下游钙依赖的信号传导途径调控PARP-1、RIP1影响程序性坏死发挥作用。

Objective To investigate the expression changes of transient receptor potential melastatin related 7 （TRPM7）, poly（ADP-ribose） polymerase 1 （PARP-1）, and necroptosis marker receptor interacting protein-1 （RIP1） in rat kidneys after ischemia/reperfusion （I/R） injury and their relationship with nephridial tissue injury so as to preliminarily explore the role of TRPM7 in the kidney I/R injury of programmed cell necrosis and its possible mechanism. Methods Renal I/R injury models of 55 male SD rats were established. Serum creatinine （SCr） and urea nitrogen （BUN） were detected on days 1, 2, 5 and 7 after kidney I/R. Renal injury was observed under a light microscope with PAS staining. TRPM7, PARP-1, and RIP1 were tested by immunohistochemistry and Western blotting. Results Comparing the control group with the sham operation group, the levels of SCr and BUN and the renal morphology had no significant difference. The levels of SCr and BUN were significantly higher in the I/R group than in the sham operation group. The levels of SCr and BUN in the I/R group reached the peak at the time point of 24 h after reperfusion, and then gradually declined, falling to be nearly normal levels about 7 d. HE staining results showed that nephridial injury appeared obviously at the time points of 24 and 48 h after reperfusion, and started to repair on day 5 and recovered on day 7. Immunohistochemistry and Western blot results showed that TRPM7 was expressed mainly in the renal tubular epithelial cells. In the I/R group, TRPM7 expression in kidney tissue started to rise significantly after reperfusion, reached the peak at the time point of 24 h after reperfusion, and then declined gradually. The changes of PARP-1 and RIP1 expression were similar to TRPM7 expression. Correlation analysis showed that there was a positive relation between the changes of TRPM7 expression and acute renal tubular injury grading （r=0.71, P〈0.05）. Conclusion TRPM7 expression rises in the early period of kidney I/R injury, and then returns to the normal level gradually. The expression of PARP-1 and RIP1 and nephridial tissue injury have similar change trends. TRPM7 may participate in the kidney I/R injury through the downstream calcium-dependent signal pathways regulating PARP 1 and RIP1 to influence necroptosis.