Klotho基因高表达对糖尿病肾病大鼠肾脏的保护作用

Protective effect of high expression of Klotho gene on kidney of rats with diabetic nephropathy

目的观察Klotho基因高表达对糖尿病肾病(diabeticnephropathy,DN)大鼠肾脏的保护作用。方法采用RT-PCR法从健康Wistar大鼠肾脏中分段扩增Klotho基因,将两段基因连接后,亚克隆至腺病毒载体shuttle中,构建重组质粒shuttle/Klotho,与腺病毒骨架质粒共转染AD293细胞进行包装后,检测重组腺病毒的滴度。经Wistar大鼠腹腔注射链脲佐菌素(streptozotocin,STZ)复制DN模型,将DN模型大鼠随机分成DN组、Ad组和Klotho组,从糖尿病(diabetes mellitus,DM)模型建立成功后2周开始,Ad组每只经尾静脉注射3×108 PFU不含Klotho基因的腺病毒,Klotho组每只经尾静脉注射3×108 PFU含Klotho基因的重组腺病毒,DN组每只经尾静脉注射等体积生理盐水;并设对照组(即Con组,制备糖尿病模型时,经腹腔注射等量柠檬酸缓冲液,治疗时经尾静脉注射等体积生理盐水)。各组均每2周注射1次。实验期间,每天观察各组大鼠一般情况。分别于糖尿病模型建立成功后第4、8、16周,称量大鼠体重并处死,取左侧肾组织称重,计算肾脏指数(renal index,RI);显微镜下观察肾脏的病理学改变;RT-PCR法检测大鼠肾脏组织中Klotho基因mRNA的转录水平;ELISA法检测尿微量白蛋白水平,比浊法检测血尿素氮和血肌酐水平。结果酶切及测序鉴定证实克隆的Klotho基因正确。含Klotho基因的重组腺病毒的滴度为4.2×108 PFU/ml。DN、Ad和Klotho组大鼠从DM模型建立成功后第2周开始毛色逐渐无光泽,体重降低,活动能力减弱,食量、饮水量增加,粪便偏稀,精神状态差;从第6周开始,Klotho组大鼠的上述症状均有所改善。DM模型建立成功后第4、8、16周,与Con组相比,DN和Ad组大鼠的RI、尿微量白蛋白、血肌酐和血尿素氮水平均显著增加(P<0.01),而Klotho组RI和尿微量白蛋白水平显著低于DN和Ad组(P<0.05或P<0.01),血肌酐和血尿素氮水平也下降,且第8、16周DN和Ad组大鼠血肌酐和血尿素氮水平高于第4周,而Klotho组无此现象;DN和Ad组大鼠的肾小球体积明显增大,肾小球系膜细胞增生,系膜区明显增宽,基膜增厚、基质大量堆积,部分肾小球出现硬化,肾小管上皮细胞肿胀,而Klotho组大鼠肾脏上述病变较DN和Ad组减轻;DN和Ad组大鼠肾脏组织中Klotho基因mRNA的转录水平明显减少(P<0.05或P<0.01),Klotho组变化不明显,但高于DN和Ad组(P<0.01)。结论 Klotho基因高表达可减轻DN大鼠的肾脏损伤,对肾脏具有保护作用。

Objective To observe the protective effect of high expression of Klotho gene on kidney of rats with diabetic nephropathy （DN）. Methods Klotho gene was amplified fractionally from the kidney of healthy Wistar rats by RT-PCR, then linked and subcloned into adenoviral shuttle vector. The constructed recombinant plasmid shuttle/Klotho was cotransfected to AD293 cells with adenovirus backbone plasmid for packaging, and the obtained recombinant adenovirus was determined for titer. Wistar rats were injected i.p. with streptozotocin （STZ） to copy the model of DN, using those injected with citrated buffer as control （Con）. The model rats were randomly divided into DN, Ad and Klotho groups. The rats in Ad group were injected i.v. with 3 × 10^8 PFU adenovirus containing no Klotho gene 2 weeks after successful copy of diabetes mellitus （DM）model, once 2 weeks for 8 times, while those in Klotho group with 3 × 10^8 PFU adenovirus containing Klotho gene, and those in DM and Con groups with physiological saline at an equal volume. The rats were observed for general status daily, then weighed and killed 4, 8 and 16 weeks after copy of DM model respectively, of which the left kidneys were weighed to calculate the renal index （RI）. The pathological change of kidney was observed by microscopy. The transcription level of Klotho mRNA in rat kidney was determined by RT-PCR, while the microalbuminuria （MAU） by ELISA, and the urea nitrogen and serum creatinine levels by turbidimetry. Results Restriction analysis and sequencing proved that Klotho gene was cloned correctly. The titer of recombinant adenovirus containing Klotho gene was 4. 2 × 10^8 PFU/ml. Dull hair color, decreased bodyweight, abated activity ability, increased food and water intakes, loose feces and poor mental state were observed in the rats in DM, Ad and Klotho groups starting from week 2 after copy of DM model. All the symptoms of rats in Klotho group were improved from week 6. Compared with those in Con group, the RIs, MAU as well as urea nitrogen and serum creatinine levels in DN and AD groups at weeks 4, 8 and 16 increased significantly （P 〈 0. 01）. However, the RI and MAU in Klotho group were significantly lower than those in DN and Ad groups （P 〈 0. 05 or P 〈 0. 01 ）, while the urea nitrogen and serum creatinine levels decreased. Both the urea nitrogen and serum creatinine levels in DN and Ad groups, but not in KJlotho group, at weeks 8 and 16 were higher than those at week 4. Obviously increased glomerular volume, mesangial cell proliferation, increased width of mesangial region, increased thickness of basement membrane, a large quantity of accumulated matrix, partial glomerular sclerosis and swelling of renal tubular epithelial ceils were observed in DN and Ad groups, while were alleviated in Klotho group. The transcription levels of Klotho mRNA in kidney tissues of rats in DN and Ad groups decreased significantly （P 〈 0. 05 or P 〈 0. 01 ）. However, the transcription level showed no significant change in Klotho group, which was higher than those in DN and Ad groups （P 〈 0. 01）. Conclusion High expression of Klotho gene relieved the injury of kidney of rats with DN and showed a protective effect on kidney.