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.
