Changes And Significance Of SIRT3 Expression In Cellular Transdifferentiation Induced By Uric Acid

INTRODUCTIONSilent information regulator 3 (SIRT3) is a kind of NAD+ dependent deacetylase, have deacetylase activity, and it is highly expressed in the kidney, muscle, liver, heart and so on. In recent years, more and more researchers on the study of SIRT3. They view it as a mitochondrial sirtuin which plays an important role in cell metabolism, oxidative stress, apoptosis, aging, etc.Hyperuricemia is one of the leading causes of chronic kidney disease (CKD). Existing research shows that under the high concentration of uric acid stimulation, kidney cells can appear oxidation load increasing, apoptosis, transdifferentiation and so on, cause kidney pathological changes of different level. The ROS tend to be detected in the patient’s cells and tissue. It follows that oxidative stress may participate in the development of uric acid nephropathy. SIRT3 may play an important role in this process. At present, there is few research for uric acid in glomerular mesangial cell’s pathological changes, and the research for the role of SIRT3 in uric acid nephropathy have not been described.Chapter I Effect of uric acid on cell transdifferentiation and changes of SIRT3 expressionObjective:To explore the effect of UA on mouse’s glomerular mesangial cell(MC) oxidative stress,transdifferentiation and the change of SIRT3 expressing in the process.Methods:The MC53 cells were cultured in media containing different concentrations of uric acid (0.05,0.1,0.3 mmol/L)for 48h.The intracellular reactive oxygen species(ROS) level was determined with fluorescence microscope and flow cytometry. The mRNA and protein expression levels of SIRT3,a-smooth muscle actin (a-SMA), transforming growth factor-β1 (TGF-β1),fibronectin (FN) were evaluated through qRT-PCR and western blotting.Results:Compared with the control group,UA increased ROS production in a concentration-dependent manner with a maximal stimulation at 0.3mmol/L(P<0.05).UA stimulated the down regulation of SIRT3 and upregulation of the a-SMA, TGF-β1 and FN mRNA and proteins in a concentration manner(P<0.05).Conclusion:High concentration of uric acid can induce MC53 cells transdifferentiation, and UA can stimulated the down regulation of SIRT3.Chapter II Effect of SIRT3 on ROS level and cell trans-differentiationObjective:Understanding the influence of SIRT3 on MC oxidative stress and transdifferentiation under stimulation of UA.Methods:The siSIRT3 was transfected into MC53 cells by using the siRNA-Mate;the MC53 cells were divided into three groups:0.1mmol/L UA+siSIRT3 group(UA+siSIRT3), 0.1mmol/L UA group(UA) and 0.1mmol/L UA+negative control group(UA+NC).The three groups of cells were incubated with 0.1mmol/L UA for 48h. The ROS level was determined with flow cytometry. The mRNA and protein expression levels of α-SMA, TGF-β1,FN were evaluated through Real-TimePCR and western blotting.Results:SIRT3 siRNA is successful transfected into MC.It was identified through qRT-PCR. Compared with the UA and UA+NC group,The ROS level and the expression of a-SMA, TGF-β1, FN proteins were elevated in cells of UA+siSIRT3 group (P<0.05). While the UA group was no statistically significant difference between UA+NC group(P>0.05).Conclusion:Interference of SIRT3 expression can promote MC ROS generation and differentiation under stimulation of UA. SIRT3 may play an important role in the happening and development of UAN.