糖尿病大鼠血糖控制前后肾小管上皮细胞VEGF、TGF-β1及Smad蛋白表达的动态研究

目的动态观察链脲佐菌素（STZ）诱导的糖尿病大鼠血糖控制前后肾小管上皮细胞（TEC）中血管内皮生长因子（VEGF）、转化生长因子β1（TGF-β1）、Smad2/3、Smad4的表达情况,探讨四者在糖尿病大鼠TEC表型转变和肾间质纤维化中可能发挥的作用及相互关系。方法实验动物随机分为5组,依病程长短分为①A组（2周组）,②B组（4周组）,③C组（8周组）,④D组（16周组）,⑤E组（24周组）,每组分别设有正常对照组（N组）和糖尿病组（a组）;另外,16周、24周两组加设胰岛素治疗组（b组）。采用尾静脉注射STZ法复制糖尿病大鼠模型;免疫组织化学方法检测肾小管VEGF、TGF-β1、Smad2/3、Smad4及α-平滑肌肌动蛋白（-αSMA）和纤连蛋白（FN）的表达;Western blot检测肾皮质VEGF和TGF-β1蛋白;PAS染色光镜观察肾小管基底膜变化及细胞外基质沉积情况等形态学改变;生化方法测定血糖、血肌酐及24小时尿蛋白量。结果正常对照组VEGF、TGF-β1及Smad2/3、Smad4在肾小管均有少量表达,-αSMA在肾小管无表达;糖尿病组肾小管前述四者的表达均显著高于正常对照组,且从16周开始肾小管上皮细胞可见α-SMA蛋白阳性表达;糖尿病16周时肾小管VEGF、TGF-β1、Smad2/3、Smad4两两之间呈正相关;随糖尿病进展,α-SMA及FN在肾小管表达增多,24h尿蛋白增多,肾脏肥大指数增大,而VEGF、TGF-β1二者都分别和-αSMA、FN、24h尿蛋白及肾脏肥大指数呈正相关性;胰岛素治疗后,VEGF、TGF-β1、Smad2/3、Smad4及FN的表达都比糖尿病组明显下降,且各指标之间的正相关性依然存在,-αSMA蛋白则呈阴性表达。结论糖尿病肾病大鼠肾小管上皮细胞表达的VEGF、TGF-β1及Smad2/3、Smad4参与了TEC表型转变和肾间质纤维化的发生,并且VEGF和TGF-β1相互作用,共同促进了肾脏损害。胰岛素对DN大鼠TEMT和肾间质纤维化的影响可能部分是通过间接阻断VEGF、TGF-β1和Smad2/3、Smad4在TEC中的合成来实现的。

DYNAMIC STUDY ON EXPRESSIONS OF VEGF, TGF-β1 AND SMAD PROTEINS IN RENAL TUBULES OF DIABETIC RATS BEFORE AND AFTER GLUCOSE CONTROL

Objective To investigate the expressions of vascular endothelial growth factor （VEGF）, transforming growth factor beta1 （TGF-β1）, Smad2/3 and Smad4 in the renal tubules of STZ-indueed diabetic rats before and after blood glucose is controlled, and explore their roles and relationship in the development of diabetic nephropathy. Methods 86 SD rats were randomly divided into 5 groups： group A （2 weeks）, group B （4 weeks）, group C （8 weeks）, group D （16 weeks） and group E （24 weeks）. Each group included a control subgroup and a diabetic model subgroup; in addition, group D and group E each still included a insuline-treated subgroup. The diabetic models were induced by injecting STZ through vena caudalis. The expressions of VEGF, TGF-β1, Stand2/3, Smad4, α-SMA and FN in renal tubules were assessed by immunohistochemistry and （or） Western blotting. The morphological changes of renal tissue, including those of the base membrane in renal tubules and ECM, were checked by microscopy and PAS staining. Blood glucose, blood creatinine and 24h urine protein were analysed by chemical methods. Results VEGF, TGF-β1, Smad2/3 and Smad4 were slightly expressed in the renal tubules of normal control rats, and α-SMA wasn＇t expressed in the renal tubules. The expression of the first 4 factors in renal tubules of diabetic rats was increased remarkably compared with that of the corresponding control groups, and α- SMA was found expressed in the renal tubules from week 16. In week 16 the expression of VEGF, TGF- β1, Smad2/3 and Smad4 were positively correlated respectively. With the development of diabetic nephropathy, the expression of α-SMA and FN in renal tubules, the 24h urine protein, and the kidney hy- pertrophy index were increased gradually, and had all positive relations with VEGF and TGF-β1 respectively. After insuline was injected, the expression of VEGF, TGF-β1, Smad2/3, Smad4 and FN in renal tubules of treated groups decreased remarkably compared with that of diabetic model groups, and α-SMA was negative. Conclusion VEGF, TGF-β1, Smad2/3 and Smad4 take part in the occurrence of tubular epithelial-mesenchymal transition （TEMT） and kidney mesenchyme fibrosis; VEGF and TGF-β1 interact and promote the development of diabetic nephropathy. The effects of insuline on TEMT and kidney mesenchyme fibrosis are partly achieved by indirectly breaking the synthesis of VEGF, TGF-β1, Smad2/3 and Smad4 in the tubular epithelial cell.