干预血栓调节蛋白表达对糖尿病肾病肾损伤的影响

Effect of modulating thrombomodulin expression on renal injury in rats with diabetic nephropathy

目的探讨干预血栓调节蛋白(thrombomodulin,TM)表达对糖尿病肾病(diabetic nephropathy,DN)肾损伤的影响及可能机制。方法①利用免疫组化检测25例DN患者肾组织以及6例正常肾组织中肝X受体-α(liver X receptor-α,LXR-α)、TM、凝血因子XⅢ-A(FXⅢ-A)和CD163表达;②在明确LXR-α激动剂T0901317上调db/db小鼠TM表达及TM干扰RNA(Ad-TM shRNA)下调TM表达作用后,将12周龄雄性db/db小鼠分为4组(n=6):DN生理盐水组、Ad-ctrl+DN组、T0901317+DN组、Ad-TM shRNA+DN组,C57BL6野生小鼠作为正常对照组。上述5组给药4周后,Western blot、免疫组化观察干预TM表达对肾组织TM、LXR-α、FXⅢ-A、CD163表达的影响,ELISA检测血炎症介质(TNF-α、IL-6)以及尿微量白蛋白的变化。结果DN患者肾组织TM、LXR-α表达量较对照组下降,且与肾小球滤过率正相关(P<0.05);而FXⅢ-A和CD163表达较对照组升高,且与肾小球滤过率负相关(P<0.05)。动物实验显示:DN生理盐水组与野生小鼠对照组相比、Ad-TM shRNA组与DN对照组相比,均表现为尿微量白蛋白和血TNF-α、IL-6水平上升、肾组织TM和LXR-α表达减少、FXⅢ-A和CD163表达增加(P<0.05),而T0901317+DN组较DN对照组,上述指标改善。结论表达上调的TM可能通过抑制肾组织FXⅢ-A、CD163表达以及下调血炎症介质水平而改善db/db小鼠尿蛋白水平。

Objective To investigate the effect of modulating thrombomodulin(TM)expression on renal injury in a rat model of diabetic nephropathy(DN)and explore the possible mechanism.Methods We examined the expression of liver X receptor-α(LXR-α),TM,coagulation factor XⅢ-A(FXⅢ-A)and CD163 in 25 renal biopsy samples from patients with DN and in 6 normal renal tissue specimens using immunohistochemistry.Twenty-four 12-week-old male db/db mice with DN were randomly assigned into 4 equal groups and treated daily with intragastric administration of normal saline or LXR-αagonist T0901317,or with intravenous injection of an adenoviral vector carrying TM shRNA or the control adenoviral vector for 7 consecutive days;6 wild-type C57BL6 mice with intragastric saline treatment served as the normal control group.Four weeks after the treatment,the expression of TM,LXR-α,FXⅢ-A and CD163 in the renal tissues of the mice were detected using Western blotting,the levels of tumor necrosis factor-α(TNF-α)and interleukine-6(IL-6)were detected by ELISA,and the urinary microalbumin level was determined.Results The renal expression levels of TM and LXR-αwere decreased(P<0.05)and those of FXⅢ-A and CD163 were increased significantly(P<0.05)in the kidney tissues of DN patients compared with normal kidney tissues.In db/db mouse model of DN,the expression levels of TM and LXR-αwere positively correlated while those of FXⅢ-A and CD163 were negatively correlated with glomerular filtration rate(P<0.05).Compared with the wild-type mice,the mouse model of DN showed significantly increased urinary microalbumin levels and serum levels of TNF-αand IL-6,lowered renal expressions of TM and LXR-α,and increased renal expression of FXⅢ-A and CD163;these changes were further exacerbated in the mice treated with Ad-TM shRNA(P<0.05)but significantly improved in the mouse models treated with T0901317.Conclusion Upregulating TM expression can reduce urinary microalbumin level in db/db mouse model of DN by inhibiting the renal expression of FXⅢ-A and CD163 and downregulating blood levels of inflammatory mediators.