Transcription Factor KLF15 Protects Podocytes By Suppressing NFATc1 Signaling Pathway

BackgroundIn recent years,the incidence of chronic kidney disease has been increasing year by year in all the world,and has become a public health problem troubling the whole world.Proteinuria is an independent risk factor for the development of chronic kidney disease.How to relieve the level of proteinuria is very important for the prognosis of patients with chronic kidney disease.Podocyte injury is an important cause of proteinuria and a common pathogenesis of many chronic kidney diseases.Previous research groups and domestic and foreign studies have found that Nuclear factor of activated T-cells cytoplasmic 1?NFATc1?is a key factor involved in podocyte injury,and its mechanism may be to promote podocyte apoptosis and glomerulosclerosis by mediating the transcription of downstream target genes^[1].Transcription factor Kr?ppel-like factor 15?KLF15?is a member of the family of transcription factors KLFs,KLF15 can promote cell differentiation,enhance the treatment effect of glucocorticoid^[2],but its specific mechanism to the protection of sertoli cell remains to be elucidated.ObjectiveThis study investigated the protective effect of KLF15 on podocytes by cultured podocytes in vitro.And the regulatory effect of KLF15 on NFATc1,whether KLF15 protects podocytes through NFATc1 and whether glucocorticoid affects NFATc1 protects podocytes through KLF15.Methods1. Immunofluorescence staining was used to observe the expression of KLF15 in normal renal tissues and renal tissues of patients with glomerular disease;2.In vitro podocytes were cultured with adriamycin?ADR?for 24 hours,lipopolysaccharide?LPS?and high glucose?30nmol/L?for 48 hours.m RNA and protein expressions of KLF15 in podocytes were observed by RT-PCR and western blot;3.In vitro cultured podocytes overexpressed KLF15 by adenovirus transfection,the m RNA and protein overexpression efficiency of KLF15,pro-apoptotic protein Bax and anti-apoptotic protein bcl-2 were observed by RT-PCR and western blot.4.After silencing KLF15 in podocytes cultured in vitro,the m RNA and protein silencing efficiency of KLF15 were detected by RT-PCR and western blot,through the Annexin-?/PI double staining and flow cytometry to detect podocytes apoptosis.5.After over-expression and silencing KLF15 in vitro cultured podocytes,m RNA and protein expression of NFATc1 were detected by RT-PCR and western blot;the binding of KLF15 to NFATc1 promoter was detected by chromatin immunoprecipitation and luciferase reporting assay;m RNA expression of downstream target genes of NFATc1 was detected by RT-PCR;6.After podocytes cultured in vitro were cultured for 0 h,6 h,12 h,24 h and 48 h in medium containing 10nmol/L dexamethasone,the m RNA expression of KLF15 was detected by RT-PCR and the m RNA and protein levels of NFATc1 were detected by RT-PCR and western blot,and the m RNA and protein levels of NFATc1 were detected by silencing KLF15 combined with dexamethasone treatment.Results1. KLF15 is decreased in podocytes of patients with glomerular disease.2.In vitro cultured podocytes,the expression of KLF15 decreased after ADR,LPS and HG injury treatment.3.After KLF15 was overexpressed in cultured podocytes,the expression of pro-apoptotic protein Bax was decreased,the expression of anti-apoptotic protein bcl-2 was increased,and the apoptosis of podocytes was decreased,and the apoptosis of podocytes was decreased after the over-expression of KLF15 combined with ADR,LPS and HG injury.4.In vitro cultured podocytes,apoptosis of podocytes increased after KLF15 silencing,and decreased after the addition of NFATc1 inhibitor 11R-VIVIT and NFATc1 silencing.5.In vitro cultured podocytes,after over-expression of KLF15,the expression of NFATc1 decreased,and after silencing KLF15,the expression of NFATc1 increased,and KLF15 was combined with the promoter of NFATc1.After over-expression of KLF15,the expression of downstream target genes of NFATc1 decreased,while after silencing KLF15,the expression of downstream target genes of NFATc1 increased.6.In vitro cultured podocytes,after dexamethasone treatment,the expression of KLF15 increased,the expression of NFATc1 decreased,and the expression of NFATc1 did not change significantly after the silencing of KLF15.ConclusionKLF15 is a protective factor for podocytes.KLF15 inhibits the expression of NFATc1 by binding to the promoter of NFATc1 and plays a protective role in podocytes.Dethasone regulates podocyte injury by KLF15-NFATc1 pathway.