高糖通过ERK1/2途径调节足细胞产生明胶酶B

目的探讨高糖持续刺激对足细胞培养上清液明胶酶活性、Ⅳ型胶原的影响及可能的信号途径.方法小鼠足细胞系分为正常糖组、高糖组和甘露醇高渗对照组.应用明胶酶谱法观察三组细胞培养液上清明胶酶活性的动态变化,Western印迹分析法检测细胞培养液上清中Ⅳ型胶原α5链蛋白水平及足细胞ERK信号途径活化的变化.RT-PCR方法检测MMP-9 mRNA的表达.结果正常糖组和甘露醇高渗对照组培养液上清MMP-2和MMP-9活性在10天实验时间内无明显变化.高糖组细胞培养液上清MMP-2的活性没有明显变化;MMP-9活性于第2天开始升高,第3天达高峰为正常糖组的144.2±18.7%(P=0.006),第5天下降,第7天回到基础水平为正常糖组的76.6%±16.4%(P=0.218),直至第10天.足细胞培养上清中有较高基础水平的Ⅳ型胶原α5链蛋白;高糖组细胞培养上清Ⅳ型胶原α5链蛋白表达第2天开始下降,第3天达最低水平41.9%±25.5%(P=0.047),第5天回升到基础水平持续至第7天.上清MMP-9的活性与Ⅳ型胶原α5链蛋白的表达量呈负相关(r=-0.577,P=0.006).正常糖组和甘露醇高渗对照组细胞培养上清Ⅳ型胶原α5链蛋白无变化.高糖刺激足细胞2天MMP-9 mRNA水平增加为正常糖组的199.8%±40.2%(P=0.003),刺激5天时为正常糖组的90.9±8.8%(P=0.411).高糖刺激足细胞30 min可以活化ERK1/2,6 h达高峰,持续至24 h,48 h降至基础水平;在足细胞上未能检测到p38和JNK活化.ERK活化特异抑制剂PD98059预处理细胞后,能阻断高糖引起的MMP-9的活性和MMP-9 mRNA增加及Ⅳ型胶原α5链蛋白的下降.结论ERK信号传导途径介导了高糖刺激足细胞MMP-9生成及相应的Ⅳ型胶原α5链蛋白动态反向改变.

High glucose regulates the production of MMP-9 in podocyte through ERK1/2 signal pathway

OBJECTIVE: To assess the effect of high glucose on the production of gelatinase and collagen alpha (IV) protein in podocytes and its possible signal pathway. METHODS: Mouse podocytes of an immortalized cell line were cultured and divided into 3 groups: NG group, treated with normal concentration of D-glucose (100 mg/dl), HG group, treated with high concentration of D-glucose (450 mg/dl), and MN group, treated with mannitol (350 mg/dl) plus D-glucose (100 mg/dl). The culture medium supernatants were collected every day. The activity of MMP-9 and MMP-2 was detected by gelatin zymography, the level of collagen alpha5 (IV) protein and the activation of MAPKs (Erk, p38, and JNK) signaling pathway in podocytes were detected by Western blot analysis, and the level of MMP-9 mRNA was detected by RT-PCR. Another podocytes were pretreated by PD9805, a specific inhibitor of MEK1 activation, and then divided into 3 groups as mentioned above so as to detect the effects of high glucose on the MMP-9 activation, and expression of MMP-9 mRNA and collagen alpha5 (IV) protein. RESULTS: The MMP-2 and MMP-9 activity in the medium supernatants of the NG and MN groups remained constant during the 10 days' incubation. High glucose incubation also did not affect the activity of MMP-2. The MMP-9 activity in the supernatant of the HG group began to increase in the 2nd day, reached the maximum in the 3rd day (144.2 +/- 18.1% that of the NG group, P = 0.006), then began to decline since the 5th day, back to the basal level in the 7th day (76.6 +/- 16.4% that of the NG group, P = 0.218), and remained at the basal level until the 10 th day. The basal level of collagen alpha5 (IV) protein in the supernatant of the NG group was quite high. The collagen alpha5 (IV) protein level in the supernatant of the HG group began to decrease since the 2nd day, reached the minimum in the 3rd day (41.9 +/- 25.5% that of the NG group, P = 0.047), then backed to the basal levels in the 5th day, and retained at that level to the 7th days. The MMP9 activity in the supernatant of the HG group had a strongly negative correlation with the levels of collagen alpha5 (IV) protein (r = -0.577, P < 0.006). The levels of collagen alpha5 (IV) protein in the supernatant of NG and MN groups showed no significant change during the 7 days' incubation. The level of MMP-9 mRNA of the HG group was 199.8 +/- 40.2% that of the NG group (P = 0.003) 2 days after stimulation, and was 90.9 +/- 8.8% that of the NG group 5 days after incubation (P = 0.411). Phosphorylation of ERK1/2 occurred as early as 30 min after simulation by high glucose, reached the peak level 6 hours later, remained at this level for 24 hours, then backed to the basal level 48 hours later, whereas the activation of p38 and JNK remained undetectable. Pretreatment with PD98059, for 30 min abolished the HG-stimulated increase of MMP-9 activity and MMP-9 mRNA, as well as the decrease of collagen alpha5 (IV) protein. CONCLUSION: The production of MMP-9 and the levels of collagen alpha5 (IV) protein can be regulated by high glucose, and the ERK1/2 transduction pathway mediate such regulation.