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

Abstract

To assess the effect of high glucose on the production of gelatinase and collagen alpha (IV) protein in podocytes and its possible signal pathway. 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. 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. 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.