ABSTRACT
Objective To observe the effects of uric acid (UA) on mitochondrial oxidative damage and apoptosis in renal tubular epithelial cells (HK-2),and investigate the possible mechanism.Methods HK-2 cells were exposed to UA (480 μmol/L,720 μmol/L) for different time (0 h,24 h,48 h)in vitro.The mitochondrial ROS production was detected by MitoSOX staining.The mitochondrial membrane potential was measured by JC-1 staining.The expressions of prohibitin and AIF were examined by Western blotting and irnmunofluorescence cytochemistry.The cell apoptosis was measured by Annexin V-FITC/PI staining.Results The mitochondrial ROS production in HK-2 cells exposed to 480 μ mol/L UA was increased than that of control group at 24 h (P ＜ 0.05),and increased gradually with UA concentration and incubation time increasing,while the mitochondrial membrane potential was reduced at the same time.There were no significant changes in AIF expression and apoptosis rate of HK -2 cells exposed to 480 μmol/L UA for 24 h compared with that of control group (P ＞ 0.05),while both of them were up-regulated when HK-2 cells were exposed to 480 μmol/L UA for 48 h and 720 μmol/L JA for 24 h and 48 h (P ＜ 0.05).The prohibitin expression in HK-2 cells exposed to 480 μmol/L UA was reduced than that of control group at 24 h (P ＜ 0.05),and down-regulated gradually with UA concentration and incubation time increasing.Conclusion Uric acid can induce the mitochondrial ROS production increased,the mitochondrial membrane potential reduced,the prohibitin expression down-regulated and the mitochondrial apoptosis pathway activated in HK-2 cells.
ABSTRACT
Objective To investigate the effect of benazepril on intergrin-linked kinase (ILK) and α-smooth muscle actin (α-SMA) expression in glomerular mesangial cells induced by high-glucose.Methods The mesangial cells from SD rat (HBZY-1) were cultured conventionally and randomly divided into four groups:normal glucose (D-glucose 5.5 mmol/L,group NG),mannitol-treated group (mannitol 20 mmol/L,group MG),high glucose (D-glucose 30 mmol/L,group HG),Benazepril-treated high glucose group (D-glucose 30 mmol/L + Benazepril 10 μmol/L,group ACEI).Cells from NG,MG,HG,ACEI gronps were harvested after 3,6,12,24,48 and 72 hours of treatment respectively.The mRNA expressions of ILK and α-SMA were detected by RT-PCR.The protein levels of ILK and α-SMA were detected by Western blotting and immunofluorescence.Results The expressions of ILK mRNA and protein in HG group were significantly increased compared with those in NG group (all P ＜ 0.05).The increased expressions of ILK and α-SMA in HG group were time-dependent and the expression reached the peak at 48 h (ILK,P ＜ 0.05) or 72 h (α-SMA,P ＜ 0.01).The expressions of ILK and α-SMA in ACEI group were lower than those in HG group (all P ＜ 0.01),but failed to rescue to the same level as those in NG.There was no significant differences of ILK expressions between MG group and NG group at the same time point (P ＞ 0.05).The expressions of α-SMA mRNA and protein in MG were higher than that in NG (P ＜ 0.05),which suggest that high osmotic pressure could cause the increasing of α-SMA.Conclusions Benazepril can decrease the expressions of ILK and α-SMA to inhibit the process of fibrosis in DN and mediate the phenotypic transformation of glomerular mesangial cells.The phenotypic transformation of glomerular mesangial cells in glucose may also depend on high osmotic pressure in DN.