Effect Of Sirt1 Overexpression On TGF-β1-Induced Human Kidney Proximal Tubular Cell Apoptosis And Its Mechanism

Objective: Renal interstitial fibrosis is the common pathological procedure to renal function failure of many kinds of renal diseases. Apoptosis is a process of normal cell death that maintains tissue homeostasis, but excessive apoptosis or its dysregulation can lead to various pathological fibrosis processes. To investigate the effect of Sirt1 overexpression on TGF-β1-induced apoptosis in human kidney proximal tubular cell line(HK-2) and its mechanism.Methods: Cultured HK-2 cells were divided into normal group(NG), TGF-β1(5ng/ml) group, TGF-β1(5ng/ml) +Sirt1 overexpression plasmid group. Apoptosis of HK-2 cells was analyzed by Dead End? Fluorometric TUNEL System. Meanwhile, apoptoic cell percentage was also observed by flow cytometry of Annexin V/PI staining assay and hoechst 33258 staining. The expression levels of Sirt1, CTGF, Bax, Bcl-2 were observed by western blot. The expression levels of Sirt1, CTGF, Bax, Bcl-2 m RNA were observed by real time-PCR. Sirt1 deacetylase activity was colorimetrically measured by the Sirt1 deacetylase activity assay kit. Immunocytochemistry was used to investigate the location and level of expression of Sirt1 protein.Results:1 Western blot analysis indicated that TGF-β1 stimulation could decrease the expression of Sirt1(P<0.01). RT-PCR analysis also showed that the expression of Sirt1 m RNA was lower after TGF-β1 stimulation than control group(P<0.01). Immunocytochemistry staining showed that widely expression for Sirt1 was observed in nuclei of HK-2 cells in control group. stimulation with 5ng/ml TGF-β1, the positive expression mainly in cell nucleus was greatly down-regulated.2 Compared with NG, the activity of Sirt1 decreased dramatically in TGF-β1 group measured by the Sirt1 deacetylase activity assay kit(P<0.01).3 TGF-β1 stimulation of HK-2 cells for 24 h decreased Bcl-2 levels and increased Bax, CTGF levels compared to their expression levels in normal medium(P<0.01). Compared with the cells treated with TGF-β1, delivery of Sirt1 overexpression plasmid significantly ameliorated the decrease in Bcl-2 and increase in Bax, CTGF(P<0.01).4 TGF-β1 enhanced the apoptosis rate of HK-2 cells after 24h(P<0.01); The cell apoptoic rate of Hoechst 33258 staining, TUNEL and FCM was respectively 2.0%±0.08%, 2.2%±0.10% and 3.14±0.21% in normal group. In TGF-β1 stimulation group, the apoptoic rate was 13.59%±1.47%, 17.66%±0.96% and 22.34%±1.03%. This increase was efficiently inhibited by transfection with Sirt1 overexpression plasmid(10.85%±1.09%, 7.35%±0.88% and 14.76%±1.62% respectively by Hoechst 33258 staining, TUNEL and FCM)(P<0.01).Conclusions:From above results we can draw the following conclusions:1 The expression of CTGF protein, m RNA, and the number of apoptotic cells were significantly increased, the expression of Sirt1 was decreased, in the renal tissues with interstitial fibrosis. These suggest that TGF-β/CTGF signaling and Sirt1 may be involved in the progression of renal tubulointerstitial fibrosis.2 Sirt1 overexpression plasmid significantly ameliorated the apoptosis, partly through inhibiting CTGF.