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Objective To explore the effect of transforming growth factor β1 (TGF-β1) on epigenetic histone lysine acetylation in the plasminogen activator inhibitor 1 (PAI-1) promoter and transcribe regions in glomerular mesangial cells (GMCs).Methods Chromatin immunoprecipitation assay and real-time quantitative PCR were used to detect Histone3K9 acetylation (H3K9Ac) in the PAI-1 promoter and transcribe regions induced by TGF-β1 and high glucose.Immunoprecipitation was also used to see the cooperation of Smad3,CBP and Sp1 proteins.Results In the four target regions of PAI-1 promoter,TGF-β1 treatment enhanced H3K9Ac at P1,P2 and P3 in GMCs (P ＜ 0.05),but no change was seen in the P4 region which was far from the transcription starting site.TGF-β1 obviously induced H3K9Ac in the T1 transcribe region of PAI-1 instead of T2 (P ＜ 0.05).High glucose increased PAI-1 mRNA expression and H3K9Ac around P1 promoter region (P＜ 0.05).TGF-β1 neutralizing antibody abrogated high glucose-induced H3K9Ac at PAI-1 promoter (P ＜ 0.01).TGF-β1 treatment could recruit Smad3 and CBP protein binding to the PAI-1 promoter regions (P1,P2,P3),and induce their cooperation in GMCs,which were responsing to TGF-β1 associated H3K9Ac.Conclusion TGF-β1 can induce H3K9Ac in the promoter and transcribe regions of PAI-1,promote Smad3 recruition and cooperation with Sp1 and CBP,which are associated with PAI-1 gene's regulation in GMCs.
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Objective To investigate the effect of histone acetylation change on the transforming growth factor β1 (TGF-β 1)-associated plasminogen activator inhibitor 1 (PAI-1) regulation in mesangial cells (MCs).Methods MCs were transfected with histone acetyltransferase (HAT)CREB-binding protein(CBP),P300 or histone deacetylase (HDAC) 1,3,5 expression vectors,followed by luciferase assay,real-time PCR and Western blotting to see the change of PAI-1 gene's transcriptional activity,mRNA and protein in response to TGF-β1.HDAC inhibitor trichostatin A(TSA)was used and TGF-β1-Smad signaling activity was detected also in this experiment.Results TGF-β1 enhanced PAI-1 transcriptional activity and mRNA expression (P ＜ 0.05).Over-expression of CBP or P300 significantly increased TGF-β1-associated PAI-1 transcriptional activity,mRNA and protein expression (P ＜ 0.05).On the contrary,over-expression of HDAC1,3,5 or dominant negative CBP or dominant negative P300 obviously reduced PAI-1 gene's expression induced by TGF-β1 (P ＜ 0.05).Change of HAT/HDAC did not affect TGF-β1-associated Smad2/3 phosphorylation.TSA enhanced TGF -β1 induced PAI-1 gene's regulation markedly,but did not change TGF-β1-Smad2/3 signaling activity.Conclusion Change of histone acetylation can affect TGF-β1 associated PAI-1 gene's regulation in MCs.
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Objective:To investigate the effect of 12(S)-HETE on the p27~(kip1) expression in mesangial cells and glomeruli.Methods:Mesangial cells were exposed to 12(S)-HETE.12(S)-HETE was infused to rats by osmotic mini-pump.Total protein content measurement for cell hypertrophy,RT-PCR for mRNA expression and Western blot for protein expression were performed respectively.Results:12(S)-HETE stimulation induced mesangial cell hypertrophy and p27~(kip1) protein expression,but not p27~(kip1) mRNA expression.Furthermore,p27~(kip1) mRNA and protein expression in the glomeruli were significantly increased by 12(S)-HETE stimulation using osmotic mini-pump.Conclusion:12(S)-HETE plays an important role in the pathogenesis of glomerular cell hypertrophy and senescence through upregulation of p27~(kip1) expression.
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Objective To investigate the effect of 12-lipoxygenase(12-LO) on the p27Kip1 expression in diabetic glomeruli. Methods Mesangial cells were exposed to 12-LO product 12 (S)-HETE (10-7 mmol/L) with or without p38 MAPK (p38) inhibitor (SB203580, 1 μmol/L) for 24 hours. Rats fed with high fat diet received low dose streptozotoein (ST-Z, 35 mg/kg, IP injection) to develop type 2 diabetes and were divided into 2 groups: low dose STZ, low dose STZ+12-LO inhibitor cinnamyl-3,4-dihydroxy-α-cynanocinnamate (CDC, 8 mg/kg) treatment. Rats fed with regular chow were divided into two groups: controls, CDC treatment. The rats received injection of CDC or vehicle subcutaneously in the hind leg. CDC or vehicle injection was performed three times weekly on alternate days. All the rats were sacrificed after 4 weeks, Wild type and 12-LO knockout C57BL/6 mice were divided into 4 groups: wild type control, 12-LO knockout, STZ-induced wild type type 1 diabetes and STZ-induced 12-LO knockout type 1 diabetes. All the mice were sacrificed after 16 weeks. Urine, blood, kidney cortical tissue and isolated glomeruli by sieving method were collected at the end of study respectively. Western blot and immunohistochemistry for target protein were performed respectively. Results Inhibition of p38 activation could significantly reduce p27Kip1 expression induced by 12 (S)-HETE in mesangial cells (P<0.01). Increased glomerular volume, microalbuminuria, elevated glomeluli p38 activation, p27Kip1 expresssion in type 2 diabetic glomeruli was decreased after CDC treatment (P<0.01). Compared with wild type diabetic mice, glomerular p38 activation, p27Kip1 exprcsssion and extracellular matrix accumulation in the 12-LO knockout diabetic mice were significantly decreased (P <0.01, respectively). Conclusions 12-LO induces p27kipl expression via p38 pathway in diabetic glomeruli.
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Objective:To investigate the effect of Tacroliums(FK506) on the significance of IL-2 and sIL-2R expression in refractory nephrotic syndrome. Methods: The expression of IL-2 and sIL-2R were evaluated by enzyme-linked immunosorbent assay (ELISA) on both pretreatment and post-treatment groups, and supervise the changes of 24hours urinary protein,serum albumin and lipid. Results:Serum IL-2 and sIL-2R level were higher in pre-treatment group than in normal control group(P