Effect Of Sirt1 On The Expression Of TTP And Inflammatory Factors By P38-MAPK In Mouse Podocytes Under High Glucose

Background and ObjectiveAs the most common and serious complications of diabetes mellitus(DM),diabetic nephropathy(DN)has become the leading cause of end stage renal disease(ESRD)worldwide.Inflammation is a key pathophysiological mechanism of diabetic nephropathy,which can lead the podocyte damage and proteinuria and finally accelerate the progression of disease.Inflammatory molecules and mediators are out of control in the early stage of diabetic nephropathy,and then damage renal structure and function through various kinds of mechanisms.Podocyte is the most important component of the renal filtration barrier,and gradually become a medical research hotspot.The inflammatory mechanisms of diabetic nephropathy are complicated,and novel treatment strategies are urgently needed to prevent the progression of diabetic nephropathy.Sirt1(Sirtuins 1)is a nutrient and metabolic protein that regulates epigenetic gene silencing and inhibits rRNA recombination.Studies have shown that Sirt1 is closely related to podocyte injury,and can participate in regulating the inflammation of diabetic nephropathy.However the relevant mechanisms have not been fully elucidated.TTP binds to and destabilizes mRNAs with 3’UTR(untranslated region)that contain AU-rich elements(AREs),including those of TNF-α and IL-6,and many other inflammatory factors.Thus we regard TTP as an anti-inflammatory protein.The expression of TTP protein and its binding capacity to the target mRNA are mainly affected by the level of phosphorylated TTP.TTP has multiple phosphorylation sites that can be phosphorylated by various proteins,such as P38-MAPK,which plays a key role in many cell activities and signaling.A number of studies have shown that Sirt1 can participate in regulating inflammation by P38-MAPK signaling pathway.Whether Sirt1 can regulate the expression of TTP through P38-MAPK signaling pathway,causing the inflammation of diabetic nephropathy and podocyte injury,there are no studies reported.We performed this study to investigate the role and mechanisms of Sirt1 in podocyte injury and inflammatory response under high glucose.MethodsThe "conditionally immortalized mouse podocytes(MPC)" were cultured in vitro.Proliferated MPC were cultured in RPMI 1640 medium containing 10% fetal bovine serum,5.6 mmol/L D-glucose and 4 ng/ml mouse gamma-interferon,and placed in the 33℃、5% CO2 cell incubator.Differentiated MPC were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 5.6 mmol/L D-glucose,and placed in the 37℃、5% CO2 cell incubator.Use 0.25% trypsin to digest MPC according to experiment needs.MPC were randomly assigned to the following groups to receive different treatments:(1)Normal glucose group(containing 5.6mmol/L D-glucose);(2)Mannitol group(containing 5.6mmol/L D-glucose and 19.4 mmol/L D-mannitol);(3)High glucose group(containing 25mmol/L D-glucose);(4)Normal glucose + scramble siRNA group;(5)Normal glucose + Sirt1 siRNA group;(6)Normal glucose + control lentivirus group;(7)Normal glucose + Sirt1 lentivirus group;(8)High glucose + control lentivirus group;(9)High glucose + Sirt1 lentivirus group.The corresponding indexes were measured at 36 th and 48 th hours.The qRT-PCR and Western blot were respectively used to examine the expression of Sirt1,P38-MAPK,TTP,podocyte marker protein nephrin,podocin,injury protein Desmin and inflammatory factors IL-6,IL-18 in mRNA and protein.Results1.Compared with the normal glucose group,the mRNA and protein expression of Sirt1,TTP,Nephrin and Podocin in the high glucose group were decreased(P<0.05).The change of P38-MAPK was of no significance(P>0.05).Conversely,the protein expression of p-P38-MAPK was increased,and the mRNA and protein expression of Desmin,IL-6 and IL-18 were increased(P<0.05).2.The double immunofluorescence staining: There was a co-localization of P38-MAPK and TTP in podocytes under normal/ high glucose.At the same time,Immunoprecipitation also showed that P38-MAPK could be co-immunoprecipited with TTP.3.After using siRNA to down-regulate the expression of Sirt1,the protein and mRNA expression of Sirt1,TTP,Nephrin and podocin in Sirt1 siRNA group were decreased(P<0.05),compared with the normal glucose group.Conversely,the protein expression of p-P38-MAPK was increased,and the mRNA and protein expression of Desmin,IL-6 and IL-18 were increased(P<0.05).4.After using lentivirus to up-regulate the expression of Sirt1,the mRNA and protein expression of Sirt1,TTP,Nephrin and Podocin in Sirt1 lentivirus group were increased(P < 0.05).Conversely,the protein expression of p-P38-MAPK was decreased,and the mRNA and protein expression of Desmin,IL-6 and IL-18 were decreased(P<0.05).Conclusions1.The lack of Sirt1 will induce the podocyte inflammation and injury.2.The over-expression of Sirt1 will alleviate the podocyte inflammation and injury under high glucose.3.Sirt1 may play a role on regulation of TTP by the P38-MAPK signaling pathway,and ultimately regulate the podocyte inflammation and injury.