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目的观察重组腺相关病毒(recombinant adeno-associated virus,rAAV)介导的克老素(klotho,KL)基因表达对2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠肾脏纤维化的影响及其机制。方法将SD大鼠随机分为4组,随机选3组建立T2DM大鼠模型,分别经尾静脉注射rAAV.mKL(T2DM-mKL组)、rAAV.GFP(T2DM-GFP组)和PBS(T2DM-PBS组),正常对照(SD-PBS组)大鼠经尾静脉注射PBS,12周后,处死动物,收集肾脏组织标本,冰冻切片观察GFP,Masson染色观察肾脏组织病理变化及胶原纤维表达;免疫组化法检测各组大鼠肾脏组织中KL和波形蛋白(vimentin,VIM)的表达;RT-PCR法检测各组大鼠肾脏组织中Rho激酶(Rho associated coiled-coilforming protein kinase,ROCK)基因mRNA转录水平;Western blot法检测各组大鼠肾脏组织中ROCKⅠ蛋白活性。结果 T2DM-GFP组、T2DM-mKL组大鼠肾脏组织中GFP表达较强;T2DM-mKL组大鼠肾小球基底膜结构较清晰完整,肾小球系膜区及肾小管间质区胶原纤维明显较T2DM-PBS组和T2DM-GFP组减少;T2DM-mKL组KL蛋白的表达明显高于其他各组(P<0.01),VIM蛋白的表达明显低于其他各组,与T2DM-PBS组和T2DM-GFP组VIM蛋白的表达相比,差异有统计学意义(P<0.01);T2DM-mKL组ROCKⅠmRNA转录水平及其蛋白活性均明显低于T2DM-PBS组(P均<0.05)。结论 rAAV.mKL转染可明显增加T2DM大鼠肾脏中KL基因的表达,延缓糖尿病肾纤维化进程,其机制可能与KL抑制ROCK信号通路活性相关。
Objective To investigate the effect of recombinant adeno-associated virus (rAAV) -mediated klotho gene expression on renal fibrosis in type 2 diabetes mellitus (T2DM) rats and its mechanism . Methods The SD rats were randomly divided into 4 groups and randomly selected 3 groups to establish T2DM rat model. The rAAV.mKL (T2DM-mKL group), rAAV.GFP (T2DM-GFP group) and PBS (T2DM- PBS group). The rats in the normal control group (SD-PBS group) were injected with PBS through the caudal vein. After 12 weeks, the animals were sacrificed and the kidneys were collected. The frozen section was used to observe GFP and Masson staining to observe the histopathological changes and collagen fibers expression. The expression of KL and vimentin (VIM) in renal tissues of rats in each group were detected by histochemical method. The mRNA expression of Rho-associated coiled-coil forming protein kinase (ROCK) was detected by RT-PCR The level of ROCK Ⅰ protein in renal tissues of each group was detected by Western blot. Results The expression of GFP in T2DM-GFP group and T2DM-mKL group was stronger than that in T2DM-mKL group. The structure of glomerular basement membrane in T2DM-mKL group was more clear and complete. The glomerular mesangial area and tubulointerstitial collagen fibers The expression of KL protein in T2DM-mKL group was significantly higher than that in other groups (P <0.01), and the expression of VIM protein in T2DM-mKL group was significantly lower than that in other groups The expression of VIM protein in T2DM-GFP group was significantly lower than that in T2DM-PBS group (P <0.05). The mRNA and protein expression of ROCKⅠ in T2DM-mKL group were significantly lower than those in T2DM-PBS group. Conclusion Transfection of rAAV.mKL can significantly increase the expression of KL gene in the kidney of T2DM rats and delay the progression of diabetic nephropathy, which may be related to the inhibition of ROCK signaling pathway by KL.