The Role Of Of The Ubiquitination Of Histone H2A?h2B In Glomerular Mesangial Cells Induced By High Glucose

Objective:Diabetic nephropathy(DN)is a common and characteristic microvascular complication of diabetes.The mechanism of DN has not been completely clarified and the treatment was limited.The main pathological characteristics of DN are fibrosis of glomerulus and renal interstitium in later stage.Nowadays,researches indicate that transforming growth factor-?(TGF-?)was one of the key cytokines in diabetic renal fibrosis,actived TGF-? could induce the development renal fibrosis in DN by increasing the gene expression of fibronectin,collagen and etc.Histone modification including ubiquitination,small ubiquitination,methylation,acetylation and phosphorylation,were concerned as an important content in epigenetics,which played a vital role in regulation of gene expression.Studies have shown that histone acetylation and methylation invovled in diabetic nephropathy.Histone acetylation can activate TGF-P signaling pathway,which plays an important role in renal fibrosis of DN.Researchers have paid close attention to the connection between histone ubiquitination and tumor occurrence,but wether it is involved in DN through activating TGF-? signaling pathways have not been reported in literature.Therefore,in this study we investigated the ubiquitination of histone H2A K119,H2B K120 and the mRNA levels of H2A ubiquitination,H2B ubiquitination(uH2A?uH2B)and TGF-p in rat glomerular mesangial cells(GMC)inducing by high glucose.It aims to explore the potentail mechanism of uH2A,uH2B in the pathogenesis of diabetic nephropathy.Methods:1.cell culture and grouping:rat glomerular mesangial cells were cultured in vitro,the high glucose as a stimulating factor,ubiquitin-proteasome inhibitor MG 132 as an inhibitor of ubiquitination,and were divided into six groups:? normal control group(NC group):medium containing 5.6 mmol/L glucose,? the 10 mmol/L glucose group(HG1 group):medium containing 10mmol/L glucose,? the 20 mmol/L glucose group(HG2 group):medium containing 20mmol/L glucose ? the 30 mmol/L glucose group(HG3 group):medium containing 30mmol/L glucose;? the osmotic pressure control group(mannitol group):medium containing 5.6 mmol/L glucose + 24.4mmol/L mannitol;? MG 132 intervention group(MI group):medium containing 30mmol/L glucose + 1 ?mol/L MG 132.2.uH2A,uH2B expression were detected respectively by protein immunoblot(Western-blot),cell immunofluorescence staining and confocal laser scanning microscopy in each group after 12h,24h,48h,and the mRNA levels of uH2A,uH2B and TGF-? were measured by RT-PCR in each group.3.Statistics:All experimental data were expressed as mean ±S.D.(x±s),which was analyzed by SPSS 11.5 statistical software.Statistical differences were calculated using one-way-analysis of variance(one-way-ANOVA)for comparison of each groups followed by q test for two comparisons,p<0.05 was defined statistically significant.Result:1.the expression of uH2A in rat renal mesangial cells:H2A ubiquitination is weak in NC group,and it increased from normol group to high glucose group compared with NC group(P<0.01),especially,the expression of H2A ubiquitination was the strongest in HG3 group for 24h,whereasc it was decreased obviously followed ubiquitin proteasom inhibiter MG 132,which could block histone H2A ubiquitination induced by high gluclose compared with HG3 group(P<0.05).There is no difference between mannitol group and NC group about the ubiquitination of H2A(P>0.05).2.the expression of uH2B in renal mesangial cells:,the expression of H2B ubiquitination is strong in NC group.It did not change apparently in HG1 group(P =0.327)but decreased in HG2 and HG3 group in glucose concentration and time dependent manner(P<0.01).Histone H2B ubiquitination was weakest in HG3 group for 48h,but it was incresed in MI group compared with HG3 group(P<0.05).There were no difference between mannitol group and NC group about the ubiquitination of H2B(P>0.05).3.the mRNA levels of uH2A,uH2B and TGF-? in renal mesangial cells:comparing with NC group,the mRNA level of transforming growth factor-(3(TGF-?)was increased(P<0.05)whercase the mRNA level of uH2A and H2B did not have statistical difference in each group.Compared with HG3 group,the expression of TGF-P mRNA decreased in MI group(P<0.05).Conclusion:1.The high glucose may induce the ubiquitination of' histone H2A and reduce the ubiquitination of histone H2B in GMCs.2.The changes of histone ubiquitination in GMCs could activate TGF-? signaling pathway,which involved in the pathogenesis of diabetic nephropathy.