Effects of sense and antisense tissue inhibitor metalloproteinase-1 on apoptosis of rat glomerular mesangial cells and its related-gene expressions]

To investigate the role of tissue inhibitor of metalloproteinase-1 (TIMP-1) in apoptosis of rat glomerular mesangial cells (RMC) in vitro.TIMP-1 full length cDNA cloned in previous work was used to construct two recombinant plasmids, eukaryotic expression rector, pcDNA3-TIMP-1 (PTs) encoding sense TIMP-1 and pcDNA3-ATIMP-1 (PTas) encoding antisense TIMP-1. These two vectors were then transfected into RMC with lipofectin DOTAP. Northern blotting was used to detect the expression of sense and antisense TIMP-1. Apoptosis was induced in normal RMC, RMC transfected with pCDNA3, PTs and PTas by serum deprivation. Those cells were monitored for apoptosis over extended periods of time (12 h approximately 4 d) by TUNEL and DNA electrophoressis. Furthermore, the expression of apoptosis related-genes was also investigated by RT-PCR.Northern blot analysis and RT-PCR demonstrated that cells transfected with exogenous genes effectively expressed sense or antisense TIMP-1 mRNA. RMC transfected with PTas underwent apoptosis after 12 h of serum deprivation, normal RMC and RMC transfected with pcDNA3 underwent apoptosis after 48 hours in serum-free medium, while RMC transfected with PTs did not undergo apoptosis until 4 days after serum deprivation. Furthermore, TIMP-1 upregulation inhibited expression of bax but had no effect on the expression of bcl-2.TIMP-1 suppressed apoptosis of RMC, which suggests a novel activity of TIMP-1 in kidney. Antisense TIMP-1 promotes apoptosis in RMC. The effects of TIMP-1 on RMC apoptosis perhaps correlate with inhibition of bax expression.