To investigate the effects of albumin on the production of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9)in podocytes. Podocytes were treated with bovine serum albumin (BSA) at the concentration of 0.1, 0.5, 1, 2 g/L, respectively. Conditioned media were harvested 12, 24, 48 and 72 h after the treatment. The expression of MMP-2 and MMP-9 was assayed by gelatin zymography, RT-PCR and Western blotting analysis. Our results showed that in comparison with the control group, BSA increased the expression of MMP-2 and MMP-9 mRNA and protein in a doseand time-dependent manner (P<0.05). Meanwhile, the enzymatic activities of MMP-2 and MMP-9 in the culture supernatants of podocytes were also increased (P<0.05). It is concluded that albumin up-regulated the expression of MMP-2 and MMP-9 at gene and protein levels in a time-and dose-dependent manner.
Eukaryotic expression vectors carrying the small hairpin RNA(shRNA)for TRPC6 mRNA were constructed,and the effects of knocking-down TRPC6 on puromycin aminonucleoside (PAN)-induced apoptosis of mouse podocytes were observed.Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized.The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome.The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h.Cultured podocytes were divided into four groups: control group,PAN treatment group,PAN treatment+shRNA transfection group,and PAN treatment+negative control group.The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively.The apoptotic rate of podocytes was measured by flow cytometry.The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A,and pGCsi-TRPC6B.The expression of Bax was increased,and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h.These changes was attenuated by knocking-down TRPC6.Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes.It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes.Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.
