| Objective: Hyperuricemia(HUA)is associated with a variety of diseases such as gout,diabetes.There is high prevalence of HUA among type 2 diabetic patients.Various studies have indicated that incidence of HUA in patients with T2 DM was respectively 24.1% and 33.8%.Moreover,HUA has proven to be an independent risk factor for kidney disease progression.Besides,hyperuricemia could cause kidney inflammation,inducing oxidative stress and causing renal tubular damage.Thus it is increasingly urgent to develop a new type of agent to improve hyperglycemia and hyperuricemia.Empagliflozin,a novel inhibitor of sodium-glucose co-transporter 2(SGLT2),inhibits glucose reabsorption by blocking SGLT2 receptors on the surface of renal proximal convoluted tubules and promotes glucose excretion in urine.Empagliflozin has been reported to reduce cardiovascular mortality,reduce blood pressure and serum uric acid and slower progression of kidney disease in addition to glycemic control.In this paper,we propose a possible mechanism to explain how empagliflozin attenuates hyperuricemia and provide an insightful approach for targeted therapies for hyperuricemia.Methods: 1(In vivo):6-week-old male KK-Ay mice(25 ± 1g),a kind of T2 DM mouse model,were randomly assigned to 3 groups: diabetic control group(KK-Ay group,n=10),hyperuricemia group(KKAy+HUA group,n=10),empagliflozin group(KK-Ay+HUA+EMP group,n=10).The hyperuricemic mouse mode was induced by combination of peritoneal injection of potassium oxonate(PO)at dose of 250mg/kg and intragastric administration of hypoxanthine(HX)at dose of 300mg/kg daily,and mice in the KK-Ay+HUA+EMP group were orally administered with empagliflozin(10mg/kg)daily for 8 weeks.At the end of treatment,10 mice from each group were placed in individual metabolic cages for 24 hours to collect the urine.The samples of blood were obtained from the retroorbital venous plexus after 1-hour treatment.HE,PAS method was used to observe the morphological changes of kidney and ileum.The relative expression of UA transporters ABCG2,OAT1,OAT3,GLUT9 and URAT1 in kidney and ileum was detected by RT-PCR and WB assay.The WB method further verified the relative expression of p-AMPK,p-Akt and p-CREB pathway proteins in both tissues.2(In vitro):Human kidney-2(HK-2)cell were cultured with DMEM/F12 medium containing 10%(vol/vol)FBS and 1% penicillin/streptomycin.The cells were divided into four groups:(1)control group incubated in DMEM/HG medium containing 30mmol/l D-glucose(HG group)(2)hyperuricemia group incubated in UA(10mg/dl)added to DMEM/HG medium(HG+ HUA group);(3)UA+ EMP group incubated in DMEM/HG medium with UA and treated with 50 ?M EMP(HG+ HUA+EMP Group);(4)UA+EMP group+ AMPK inhibitor(Dorsomorphin)group incubated in DMEM/HG medium with UA,Dorsomorphin(10?M))and EMP(50 ?M)for 48 hours(HG+ HUA+EMP+ Dorsomorphin group).Western blot assay was used to detect the expression of urate transporters ABCG2 and p-AMPK,p-Akt and p-CREB proteins.The relative expression of the above four groups of ABCG2 was detected by immunofluorescence.HEK-293 A cells were cultured in DMEM/HG medium containing 10% fetal bovine serum(FBS)and 1% penicillin/streptomycin.The transfection efficiency of the ABCG2 promoter plasmid and the CREB overexpression plasmid was examined by PCR or WB.Transient transfection was performed using Lipofectamine 2000.HEK-293 A cells were transiently co-transfected with the following seven groups:(1)ABCG2 plasmid and CREB plasmid(ABCG2+CREB),(2)ABCG2 plasmid and empty vector as control for CREB plasmid(ABCG2+CREBNC),(3)empty vector as control for ABCG2 plasmid and CREB plasmid(ABCG2NC+CREB),(4)empty vector as control for ABCG2 plasmid and empty vector as control for CREB plasmid(ABCG2NC+CREBNC),(5)mutation of the promoter at 511 to 518 sites in ABCG2 promoter region and CREB plasmid(ABCG2mut511+CREB),(6)mutation of the promoter at 833 to 840 sites and CREB plasmid(ABCG2mut833+CREB),(7)mutation of the promoter at 511 to 518 sites and 833 to 840 sites and CREB plasmid(ABCG2 double mut511/833+CREB).Ch IP assay and luciferase secretion assay were used to detect the relationship between CREB and ABCG2 promoter.Results:1.Empagliflozin decreased serum uric acid and increased urinary uric acid excretion.HE and PAS staining showed that mice in the KK-Ay+HUA group displayed drastic tubular dilatation,hydropic degeneration and deposited mesangial matrix compared with mice in the KK-Ay group;however,those pathological features were significantly attenuated in the KK-Ay +HUA+EMP group.We also examined the histological changes in ileum exhibiting a remarkable increase in villus lodging,irregular villi and inflammatory cell infiltration for the KK-Ay +HUA group.However,this phenomenon has partially improved in the KK-Ay +HUA+EMP group.2.RT-PCR and WB assay showed that empagliflozin promoted ABCG2 expression in kidney and ileum.3.While,WB results indicate that empagliflozin increased the protein expression of p-AMPK,p-Akt and p-CREB in kidney and ileum.4.The same trend was also observed in human kidney 2(HK-2)cells.Additionally,by introducing an AMPK inhibitor to cultured HK-2 cells,no sign of up-regulation of ABCG2 was observed,indicating EMP exerted its anti-hyperuricemic effects.5.Ch IP assay and luciferase secretion assay indicated that empagliflozin promoted CREB binding to the ABCG2 promoter.Conclusions: 1.Empagliflozin decreased serum uric acid and increased urinary uric acid excretion.2.Empagliflozin ameliorated histopathologic changes of kidney and ileum.3.Empagliflozin promoted ABCG2 expression in kidney and ileum.4.Empagliflozin promoted in AMPK/Akt/CREB pathway in kidney and ileum.5.Empagliflozin promotes HUA-induced AMPK/Akt/CREB pathway in human kidney 2(HK-2)cells.6.Chromatin immunoprecipitation(Ch IP)assays and luciferase secretion assay demonstrate that CREB may bind to ABCG2 promoter.7.Empagliflozin promoted CREB binding to the ABCG2 promoter.8.Empagliflozin attenuates hyperuricemia by upregulation of ABCG2 via AMPK/Akt/CREB signaling pathway. |
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