血管生成素及其受体、血管内皮生长因子与糖尿病肾脏微血管病变的关系

目的 探讨血管生成素(Ang)及其受体(Tie-2)和血管内皮生长因子(VEGF)在糖尿病肾脏中的表达变化规律，研究其与肾脏微血管结构变化的关系。 方法 将雄性成年SD大鼠分成糖尿病组和对照组，糖尿病组采用链脲佐菌素(STZ)腹腔注射造模。采用RT-PCR以及免疫组化技术，连续多时点观察肾脏Ang-1、Ang-2、Tie-2、VEGF以及血栓调节蛋白(TM-1) mRNA和蛋白表达变化规律，并分析其相关性。 结果 (1)糖尿病组Ang-1 mRNA于第4、8周时显著上调(吸光度A，83.58±10.23、88.59±6.97)， 第24周时低于对照组 (47.13±8.02比64.53±8.77，P < 0.05)。免疫组化显示Ang-1突出表达于肾小球，糖尿病组第4周后肾小球阳性染色明显强于对照组(A对数值，4周1.64±0.12比1.08±0.16，24周1.24±0.11比1.11±0.17)。(2) 糖尿病组Tie-2 mRNA在4~16周显著高于对照组(A，4周87.31±11.69比63.62±5.61，16周81.75±8.58比60.15±2.66)。免疫组化显示Tie-2突出表达于肾小球，糖尿病组各时点均显著高于对照组，高峰在4~8周。(3)糖尿病组仅在第16和20周时检测到明显的Ang-2 mRNA表达。免疫组化显示12周后仅在皮质区肾小管周围有Ang-2染色的微血管，而在第16周时最明显。(4) 糖尿病组2~20周时肾小球TM-1染色显著高于对照组(A对数值，2周0.99±0.15比0.68±0.17，20周1.03±0.17比0.74±0.13)， 第24周时低于对照组，但差异无统计学意义。(5)糖尿病组VEGF mRNA和蛋白表达均较对照组明显升高。(6) 糖尿病组Ang-1、Tie-2、VEGF和TM-1相互间均呈正相关，它们与尿蛋白、肾重/体重、肾小球体积、肾小球面积也均呈正相关。结论 (1)糖尿病肾脏存在VEGF、Ang及Tie-2表达的改变，早期Ang-1、Tie-2表达上调， 后期下调并伴Ang-2表达上调。(2)Ang、Tie及VEGF的改变与糖尿病肾脏新生血管生成有关，其Ang-1下调和VEGF、Ang-2上调起重要作用。(3)糖尿病肾脏中后期皮质区肾小管周围已有Ang-2染色的新生微血管生成。

The correlation of angiopoietins/Tie-2 axis and VEGF with renal microvascular disease in diabetic rats

Objective To observe the expressive changes of angiopoietins(Ang)/Tie-2 axis and vascular endothelial growth factor(VEGF) in kidney tissue and examine the correlation of Ang/Tie-2 axis and VEGF with renal microvascular disease in diabetic rats. Methods SD male rats were randomly divided into streptozotocin-induced diabetes group and control group. The mRNA and protein expression of Ang-1， Ang-2， Tie-2， VEGF and thrombomodulin-1(TM-1)in kidney tissue of two groups was examined by RT-PCR and immunohistochemistry at week 2， 4， 8， 12， 16， 20， and 24 respectively. Results (1) Ang-1 mRNA in diabetic group was up-regulated significantly at week 4 and 8(A，83.58±10.23，88.59±6.97)， but down-regulated significantly at week 24(47.13±8.02 vs 64.53±8.77)， as compared with other time points of diabetic group and the same time points of control group. Ang-1 was expressed outstandingly in glomeruli. From week 4 to week 24， the protein expression of Ang-1 in diabetic glomeruli increased significantly(lgA，1.64±0.12 vs 1.08±0.16，at week 4，1.24±0.11 vs 1.11±0.17 at week 24). (2) From week 4 to week 16， Tie-2 mRNA in diabetic group was up-regulated obviously with the peak between week 8 and week 12(A，87.31±11.69 vs 63.62±5.61 at week 4，81.75±8.58 vs 60.15±2.66 at week 16). The protein expression of Tie-2 was found mainly in glomeruli. Throughout experimental period， the expression of Tie-2 staining in diabetic glomeruli increased apparently with the peak between week 4 and week 8. (3) Ang-2 mRNA in diabetic group was found only at week 16 and week 20. The expression of Ang-2 in peritubular microvessel of diabetic renal cortex was found from week 12 to week 24 with the peak at week 16.(4)compared with control group，TM-1 staining in diabetic glomeruli is much more from week 2 to week 20(lgA，0.99±0.15 vs 0.68±0.17 at week 2，1.03±0.17 vs 0.74±0.13 at week 20)，but a little lower at week 24. (5) The expression of VEGF increased significantly in diabetic group. (6) In diabetic group， correlations were significantly positive among Ang-1， Tie-2， VEGF and TM-1. Meanwhile，they were positively correlated with kidney/body weight， glomerular volume， glomerular area， and urine protein excretion respectively. Conclusions (1) Change of Ang/Tie-2 axis exists in diabetic kidney， which Ang-1 and Tie-2 expression is up-regulated in early stage and down-regulated along with Ang-2 up-regulation in later stage.(2) In diabetic kidney， the change of Ang/Tie-2 axis is partly associated with the renal angiogenesis which is mainly responsible for Ang-1 down-regulation and VEGF， Ang-2 up-regulation. (3) There is a formation of new peritubular microvessels at diabetic renal cortex in middle and later stages.