上调intermedin表达对大鼠肾脏缺血再灌注的保护作用

目的 观察上调肾脏intermedin( IMD)表达对大鼠肾脏缺血再灌注(I/R)的影响.方法 24只健康雄性Wistar大鼠随机分为假手术组、肾脏I/R组、IMD+I/R组、空质粒+I/R组,每组6只.所有动物于I/R术24h后杀检,取肾组织进行光镜检查,留取血清测定尿素氮(BUN)和血清肌酐(Scr)的浓度.免疫组织化学方法、半定量RT-PCR、Western印迹检测肾组织IMD表达及部位.Western印迹测定内皮素1(ET-1)、肿瘤坏死因子α(TNF-α)蛋白的表达.结果 HE、PAS染色结果显示,I/R组肾小管及间质病理损伤显著重于假手术组,IMD+I/R组小管间质损伤程度较肾脏I/R模型组及空质粒+I/R模型组明显减轻(1.5±0.8比7.6±2.3和7.0±1.8,均P＜0.05].与假手术组[(BUN 3.85±0.21 mmol/L,Scr(48.67±3.61) μmol/L相比,I/R组、IMD+I/R组以及空质粒+I/R组BUN(10.13±2.14) mmol/L,( 7.73±1.03) mmol/L,( 9.77±1.92) mmol/L和Scr(80.33±7.15) μmol/L,(58.50±:3.27)μmol/L,(75.67±7.58) μmol/L均明显升高(均P＜ 0.05),其中IMD+I/R组较I/R组以及空质粒+I/R组BUN和Scr水平显著降低(均P＜ 0.05).免疫组化结果显示,假手术组IMD呈弱阳性表达,主要位于肾小管间质细胞胞质内,I/R组肾组织IMD在肾小管上皮细胞和间质表达较假手术组上调；IMD+I/R组肾组织中IMD表达较I/R组明显上调(P＜0.01).与I/R组及空质粒+I/R组相比IMD+I/R组肾组织IMD mRNA,相对含量分别增加了60.7％、66.1％,蛋白相对含量分别增加了51.4％、55.9％.此外,与I/R组相比,IMD+I/R组肾组织ET-1、TNF-α蛋白表达显著降低(ET-1:0.17±0.02比0.08±0.02；TNF-α:0.35±0.02比0.21±0.04,均P＜0.05).结论 在大鼠肾脏I/R前上调IMD表达可能通过抑制ET-1、TNF-α表达保护肾脏结构及功能.

Up-regulation of intermedin protects kidney from ischemia/reperfusion injury

Objective To investigate the effect of intermedin (IMD) on renal ischemia/reperfusion(I/R) injury after the up-regulation of IMD. Methods A total of 24 healthy Wistar male rats were randomly divided into four groups, sham-operated group, I/R group, IMD gene transfection +I/R group and empty plamid +I/R group. All the animals were killed at the end of 24 h of reperfusion. Histological changes and renal function were estimated. The expression and site of IMD were determined by Immunohistochemistry method, semi-quantitative RT-PCR and Western blotting. The protein expressions of endothelin 1 (ET-1), tumor necrosis factor α (TNF-α) were detected by Western blotting. Results Compared with sham-operated group, tubulointerstitial pathological injury was significant aggravated in I/R group (7.6±2.3) and empty plamid +I/R group(7.0±1.8), and such injury was improved in IMD+I/R group (1.5±0.8) (P<0.05). Compared with I/R group and empty plamid +I/R group, the renal dysfunction of IMD +I/R group was obviously lessened [BUN：(7.73±1.03) mmol/L vs (10.13±2.14) mmol/L， (9.77±1.92) mmol/L; Scr: (58.50±3.27) μmol/L vs (80.33±7.15) μmol/L, (75.67±7.58) μmol/L, all P<0.05]. IMD expression was weak in the plasma of tubulointerstitial cells in sham-operated group, and was up-regulated in I/R group. Compared with I/R group, immunohistochemical IMD expression increased obviously (262.03±67.89 vs 175.57±48.06, P＜0.01). The mRNA expression of IMD in IMD+I/R group was up-regulated significantly by 60.7% , 66.1% and the protein expression of IMD in IMD+I/R group increased significantly by 51.4%, 55.9% as compared to I/R and empty plasmid +I/R group. Meanwhile, the protein expressions of ET-1 and TNF-α in IMD+I/R group were obviously lower compared with those in I/R group (ET-1: 0.08±0.02 vs 0.17±0.02; TNF-α: 0.21±0.04 vs 0.35±0.02, all P<0.05). Conclusion IMD gene transfected into kidneys of rats prior to I/R surgery can attenuate the over-expressions of both ET-1 and TNF-α in I/R injured rat kidneys as well as the damages to the structure and function of the kidneys.