The Effect Of GSK-3? On The High Glucose Induced Expression Of Inflammatory Cytokines And Injury Via Regulating RNA-binding Protein HuR-TTP In Podocyte Under High Glucose Conditions

BackgroundDiabetic nephropathy has replaced glomerulonephritis as the leading cause of end-stage kidney disease(ESRD).Diabetic nephropathy often accompaned with microvascular disease of other organs or systems,such as diabetic retinopathy and peripheral neuropathy,which significantly affect the survival time and quality of life of patients.As a consequence,exploring the pathogenesis of diabetic nephropathy is of great significance to the prevention and treatment of diabetic nephropathy,which may improve the quality of life of patients with diabetic nephropathy.Podocyte is an important part of glomerular filtration barrier,which structure and function change play a crucial role in the pathogenesis of DN.Inflammation response is the vital cause of injury of podocyte in diabetic nephropathy.Glycogen synthase kinase 3?(GSK-3?)is a ubiquitously expressed,constitutively active,proline-directed serine/threonine kinase involved in diverse cellular processes,affecting the proliferation,differentiation and apoptosis of cells.It has been reported that the expression and activity of GSK-3? were significantly increased in the kidney tissues of patients with DN.Tristetraprolin(TTP)is one of RNA binding proteins,which selectively binds to mRNAs with 3?untranslated regions that contain adenosine-uridine(AU)-rich elements(AREs).Studies have shown that TTP is one of the substrate for GSK-3?,and could be phosphorylated by GSK-3?.interestingly,our studies has confirmed that the the level of TTP is obviously reduced in patients with diabetic nephropathy.Human antigen R(HuR)is also a typical RNA binding protein,which stabilize mRNAs and promote the progression of translation.In recent years,it was reported that the mRNA of TNF-?,cox-2 and other inflammatory factors was regulated by the TTP/HuR axis in tumor,cardiovascular and immunological diseases.It is worth noting that researchers have reported the expression of Claudin-1could be regulated by TTP-HuR in colorectal cancer.As a newly discovered marker of renal injury in recent years,Claudin-1 was widely studied.However,the effects of GSK-3? on the expression of TTP and HuR,and the impact of TTP and HuR on injury and inflammatory response of podocyte have not been documented.The purpose of this study was to investigate the effect TTP-HuR on the expression of claudin-1 and inflammatory cytokines in podocyte under high glucose conditions.ObjectiveThe study is intended to figure out the impact of GSK-3? on the expression of TTP and HuR,and the impact of TTP and HuR on injury and inflammatory response of podocyt.In addition,the research aims to investigate impact of GSK-3? on the expression of Claudin-1 and inflammatory cytokine via regulating HuR-TTP in podocyte under high glucose conditions.MethodsConditionally immortalized mouse podocytes cell line in vitro were adopted.1.The effect of high glucose on the inflammatory response of podocyte and injury.Mature mouse podocytes were randomly divided into three groups: normal glucose group(5.6 mmol/L D-glucose),mannitol control group(30mmol/L D-glucose + 24.4 mmol/L D-mannitol),and high glucose group(30mmol/L D-glucose).The protein expression of Nephrin,IL-17,Claudin-1 and Cleaved caspase-3 were detected using Western blot,and localization and expression Nephrin,IL-17,cytoskeleton protein,F-actin were observed by immunofluorescence.2.The impact of TTP and HuR on the Claudin-1 and IL-17To knock down the expression of TTP and HuR,the small interfering RNA against TTP and HuR(si TTP and siHuR)were transfected into the mouse podocytes under normal glucose conditions.Western blot were used to detect the level of TTP,HuR,Nephrin,claudin-1,IL-17,Cleaved caspase-3.3.The impact of TTP and HuR on the injury and inflammatory response of podocytes treated with high glucose(1)The expression of TTP and HuR under normal glucose conditions and high glucose conditions were detected using Western blot.(2)Lentiviral vetor with over-expression of TTP were used to up-regulate TTP of mouce podocytes treated with normal glucose,and HuR siRNA was transfected to podocytes incubated with high glucose to knock down the expression of HuR.And control groups were set up.Western blot were used to detect the level of TTP,HuR,Nephrin,Claudin-1,IL-17 and Cleaved caspase-3.4.GSK3? promotes the injury and inflammatory response of podocytes treated with high glucose by regulating the expression of TTP-HuR(1)The expression of GSK3? and Phosphorylation of GSK3? at 9 serine site(inhibitory site)under normal glucose conditions and high glucose conditions were detect by western blot.(2)The localization and expression of GSK3? and TTP or HuR were observed by immunofluorescence.To clarify whether GSK3? could combined with TTP and HuR,respectively,co-immunoprecipitation were used.(3)Plasmid with over-expression of GSK3? were used to up-regulate GSK3? of mouce podocytes treated with normal glucose,and GSK-3? siRNA was transfected to podocytes incubated with high glucose to knock down the expression of GSK-3?.And control groups were set up.Western blot was used to detect the expression levels of related proteins.Results1.The results of both western-blot indicate that: compared with normal glucose group,the expression of podocytes marker Nephrin decreased significantly,and the level of IL-17,Cleaved caspase-3 and Claudin-1 increased in podocyte cells incubated with high glucose.(P < 0.05).Down-regulated Nephrin and up-regulated Claudin-1 were observed by immunofluorescence,and cytoskeletal protein f-actin integrity was destroyed as well.2.The results of western-blot indicate that: compared with normal glucose group,the expression of TTP down-regulated,and the expression of claudin-1,IL-17 and Cleaved caspase-3 was increased in podocytes incubated with high glucose.Knock down the expression of HuR,the level of Claudin-1,IL-17,Cleaved caspase-3 decreased.(P < 0.05).3.Compared with normal glucose group,the expression of TTP decreased,accompanied with elevation expression of HuR in podocytes incubated with high glucose.The difference was statistically significant(P<0.05).What is more,compared with high glucose group,the expression of Nephrin up-regulated,and the expression of Claudin-1,IL-17 and Cleaved caspase-3 decreased in podocytes infected with lentiviral vetor with over-expression of TTP.The high glucose induced injury and inflammation of podocytes were relieved,after the expression of HuR was knocked down(P< 0.05).4.(1)The protein expression of GSK3? increased under the high glucose conditions(P< 0.05).(2)The results of immunofluorescence showed that GSK3? was colocalization with TTP and HuR,respectively,and GSK3? could bind to the TTP and HuR is confirmed by immunoprecipitation.The combination of GSK3? and TTP was reduced in podocytes treated with high glucose,accompanied with elevated combination with HuR.(3)Compared with normal glucose group,injury and inflammation occurred in podocytes transfected with plasmid with over-expression of GSK3?,with the elevated expression of Nephrin and the down-regulation of Claudin-1,IL-17 and Cleaved caspase-3.The difference was statistically significant(P < 0.05).However,The high glucose induced injury and inflammation of podocytes were relieved,after the expression of GSK3? was knocked down(P < 0.05).Conclusion1.Claudin-1 and IL-17 play a role in high glucose induced inflammatory response of podocyte and injury,which could be regulated by TTP an HuR.2.GSK-3? is able to combine with TTP and HuR and regulate its expression.3.GSK-3? promotes the expression of inflammatory factor IL-17 and injury via regulating TTP-HuR in podocyte treated with high glucose.