Epoxyeicosatrienoic Acids Improve Glucose Homeostasis By Preventing NF-κB-mediated Transcription Of SGLT2 In Renal Tubular Epithelial Cells

Object: To study the effects and underlying mechanisms of epoxyeicosatrienoic acid(EETs)on sodium-glucose cotransporter 2(SGLT2)and glucose homeostasis.Methods: In in vivo research,male diabetic mice(db/db)aged 8 week and control C57BLKS/JNju mice were administered with soluble epoxide hydrolase(s EH)inhibitor1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea(TPPU)in drinking water(1mg/kg/day)every day for 8 weeks.Dynamic blood glucose,glucose tolerance,body weight and blood pressure of the mice in each group were measured to evaluate the systemic metabolism.The urine of the mice were collected in a metabolic cage to measure the urine sodium excretion and glucose excretion of the mice.Finally,blood and kidney samples were harvested from mice anesthetized with chloral hydrate for further analysis.14,15-EET expression levels in kidney and urine were detected by Elisa;Western blot,Real-time PCR and immunohistochemistry were conducted to detect the expression of SGLT2,P65 and p-P65 in kidney.In the in vitro research,human proximal tubular epithelial cells HK-2 were treated with 14,15-EET(1umol/L)and/or NF-κB inhibitor Bay 11-7082 under 50ng/ml insulin stimulation: SGLT2 、 P65 and p-P65 expression were detected by Western blot and Real-time PCR;2-NBDG(100u M)was used to incubate HK-2 for another 15 min to analyse glucose uptake;the nuclear translocation of NF-κB was analyzed by immunofluorescence staining.Western blot and Real-time PCR were also adopted to evaluate SGLT2 protein and m RNA levels in human proximal renal tubular epithelial cells treated with P65 expression vector or Bay 11-7082,respectively.According to the JASPAR database,the binding of NF-κB-P65 to the SGLT2 promoter was analyzed.Different lengths of SGLT2 promoter luciferase reporter gene vectors and corresponding mutant vector were constructed and transfected into 293-T and HK-2 cells to analyze the binding site of NF-κB-P65 at the SGLT2 promoter.The combination of the above sequence and its functionality were further confirmed through EMSA and Ch IP assay.Results: At the body level,there was no significant difference in various indicators between control mice and TPPU treatment mice.The s EH inhibitor TPPU,nevertheless,lowered blood glucose and plasma insulin levels,alongside with improved glucose tolerance in diabetic mice;The systolic blood pressure were decreased and weight gain were restrained in diabetic mice.14,15-EET concentrations as well as 24 h urine volume,urinary glucose excretion(UGE),and urinary sodium excretion were increased.Adversely,the expression of SGLT2 and NF-κB were inhibited in diabetic mice.No significant difference was observed between the control mice and TPPU group.In HK-2 cells,14,15-EET inhibited insulin-induced SGLT2 expression,glucose uptake and nuclear translocation of NF-κB;Bay 11-7082 reduced insulin-mediated SGLT2expression;Insulin-induced NF-κB activation can be blocked by 14.15-EET or Bay11-7082;Overexpression or inhibition of NF-κB-P65 can promote or inhibit SGLT2 expression,respectively.The double luciferase reporter gene combined with EMSA and Chip showed that 14,15-EET inhibited the binding of NF-κB to the SGLT2 promoter,thereby inhibiting the transcriptional activity and expression of SGLT2.Conclusion: Our results showed that TPPU intervention can increase the activity of EETs in vivo,and further inhibit NF-κB-mediated SGLT2 transcriptional activity to attenuate corresponding glucose reabsorption,thereby improving glucose homeostasis in diabetic mice.