The Effect And Mechanism Of Intermedin On Sodium Glucose Cotransporter 2 Expression And Function Induced By High Sodium

Objective:Excessive dietary sodium is associated with elevation of BP and renal function deterioration,Sodium glucose cotransporter 2(SGLT2),located at renal proximal tubular cell apical membrane,has a pivotal role in sodium and glucose reabsorption.intermedin(IMD)can through enhancing natriuresis,promoting angiogenesis,inhibiting of oxidative stress and inflammation to alleviate kidney injury as well as lower BP level.There is scarcely research on the effect of IMD on SGLT2.In this study,we investigated the mechanism of IMD regulation high sodium induced SGLT2 expression in vivo and vitro.Materials and Methods:This study included animal experiments and cell molecular experiments.In the animal experiments,thirty 8-weeks-old male C57BL/6J mice were randomly divided into five groups.Mice were fed with a high salt diet(8% Na Cl)or a normal salt diet(0.5% Na Cl)by group for 16 weeks,Then Canagliflozin(30mg/kg/day)was administered by gavage for 4 weeks and IMD was administered by an implanted mini-osmotic pump(300ng/kg/h)for 4 weeks,respectively.Mice carotid artery was cannulated for systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean arterial pressure(MAP)monitoring.Mice blood and urine were collected for24 hours urinary protein,creatinine,serum sodium,urinary sodium quantitation.The histology of renal tissue was assessed by Hematoxylin-Eosin(HE),periodic acid schiff base(PAS)and Masson staining.Immunofluorescence was used to determine the expression levels of sodium glucose cotransporter 2(SGLT2)and Hepatocyte nuclear factor-1α(HNF1-α)in kidney tissues of mice in different treatment groups.Western blot and RT-PCR was performed to identify the expression of SGLT2,cyclic adenosine monophosphate(cAMP),protein kinase A(PKA),HNF-1α,Monocyte chemoattractant protein-1(MCP-1),interleukin-6(IL-6),transforming growth factor-β(TGF-β),galectin-3(Gal-3),and galectin-3(Gal-3).In the cell molecular experiments,using IMD small interfering RNA lentivirus to silence HK-2cell IMD gene,The effects of sodium chloride,intermedin and canagliflozin on cytotoxicity were detected by cell counting kit 8,α-smooth muscle actin(α-SMA),tight junction protein(ZO-1)and Vimentin were detected by immunofluorescence.Cell migration was assessed by using cell wound scratch assay.Flow cytometry detected 2-NBDG uptake to evaluate the function of SGLT2.MCP-1,IL-6,TGF-β,Gal-3 were detected by RT-PCR and Western Blot in different treatment groups.At last,AM22-52(IMD receptor inhibitor),DDA(adenylate cyclase inhibitor),H 89(PKA antagonist)and 8-Bromo-cAMP(PKA agonist)were added to cell by group for 30-60 min.The m RNA and protein levels of cAMP,PKA and HNF1-α related to the cAMP-PKA signaling pathway were detected to explore the mechanism of intermedin regulating the expression of SGLT2.Results:First,Intermedin promoted natriuresis and decreased blood pressure,urine microalbumin and urine creatinine.Second,HE,PAS and Masson staining indicated that high salt diet led to glomerular cell hypertrophy,glycogen deposition in renal tubules,collagen fiber deposition in mesangial cells,while intermedin could improve the histological abnormalities.Third,Intermedin inhibited the increased expression of SGLT2 and HNF1-α and the up-regulated expression of cAMP and PKA in mice fed with high salt diet.Forth,both intermedin and canagliflozin could down-regulate the expression of inflammatory factors MCP-1,IL-6 and fibrosis factors TGF-β and Gal-3 induced by high salt diet.Fifth,High sodium chloride treatment induced HK-2 cell SGLT2 protein and m RNA expression as well as expressed more IL-6,TGF-β,MCP-1,Gal-3 along with enhancement 2-NBDG uptake and augment migration.Intermedin was able to inhibit all the abovementioned biological processes.Last,IMD inhibited high sodium chloride induced SGLT2 expression depending on cAMP-PKA signaling pathway.Conclusion:Intermedin could inhibit the upregulated expression of SGLT2 to achieve the effect of decrease blood pressure,plasm glucose,microalbuminuria and urine creatinine by activating cAMP-PKA signaling pathway.Intermedin could also ameliorate renal fibrosis and inflammation,cell migration,epithelial-mesenchymal transformation by inhibiting MCP-1、IL-6、TGF-βand Gal-3 expression.Mice blood pressure,plasm glucose,microalbuminuria and urine creatinine increased in high salt diet group.High salt diet also led to glomerular cell hypertrophy,glycogen deposition in renal tubules,collagen fiber deposition in mesangial cells.HK-2treated with high sodium promoted cell migration and epithelial-mesenchymal transformation,the expression of inflammatory factors MCP-1、IL-6 and fibrosis factors TGF-βand Gal-3 were up-regulated by high sodium.