Dapagliflozin Reverse The Imbalance Between Th17 And Tregs By Inhibiting Serum/Glucocorticoid Regulated Kinase 1 In Diabetes Kidney Disease

Background:Diabetic Kidney Disease(DKD)is one of the most common chronic complications of diabetes mellitus(DM)and is widely regarded as the main reason of end-stage renal disease(ERSD).By far,the mechanism of DKD is still not completely clear.Studies have shown that T-cell immune plays an important role in the occurrence and development of DKD.Particularly,the imbalance between Helper T cell 17(Th 17)and Regulatory T cell(Tregs)can promote the occurrence and development of DKD.Serum/Glucocorticoid Regulated Kinase 1(SGKI),kind of serine/threonine kinase,is widely expressed in various tissues and organs and can be activated by the high salt condition.It has been reported that the activation of SGK1 can promote the polarization of Th17 by activating the Foxol/IL-23r pathway,leading to the imbalance between Th17 and Tregs.Sodium/Glucose cotransporter 2(SGLT2)is a kind of cotransporters expressed in renal tubular epithelial cells.Under high-sugar condition,the expression of SGLT2 significantly increased,promoting renal tubular epithelial cells to reabsorb Sodium ions and glucose and finally leading to form a local high salt environment.Therefore,this study aims to investigate whether SGLT2 activates SGK1 by augmenting the absorption of sodium ions and then causes the imbalance between Th17 and Tregs in renal tissue and ultimately promote the progression of DKD..Methods:1.Animals8-weeks-old db/db mice(n=18)and 8-weeks-old C576 wild mice(n=6)were used in this study.Db/db mice were evenly divided into three groups which respectively received nonnal saline(1mg/kg),Dapagliflozin(1mg/kg)and Voglibose(0.6mg/kg)by gavage.C57L6 mice were received normal saline(lmg/kg)as vehicle control.Weight and glucose were measured once a week.24-hours urine were collected at the age of 14 and 20 weeks.All the mice were killed at the age of 22 weeks.2.ImmunohistochemistryMice kidneys were collected and fixed in 4%paraformaldehyde for 24 hours.Then the kidney samples were embedded in paraffin and cut into 3 um series slices using a microtome.Slices were then mounted onto glass slides,stained with PAS and Masson chrome stain afterwards.For immunohistochemistry analysis,the slices were stained with anti-SGK1 polyclonal antibody or anti-SGLT2 polyclonal antibody,followed by HRP-conjugated secondary antibody.All images were obtained using an Olympus microscope.3.ELISAMice were housed in the metabolic chambers with free access to drinking water for 24h to collect urine samples.After being centrifuged for 10 minutes at 3000g,the urine samples determined for urinary albumin and creatinine levels with the ELISA assay kit.Blood samples were collected from orbital venous plexus.After being centrifuged for lOminutes at 3000g,samples were placed into an EP tube with anticoagulation at4?.Serum IL-10 and IL-17 concentrations were measured using an ELISA Kit.Absorbance was measured by Bio-Rad iMark microplate reader.4.Flow cytometrySingle cell suspensions were incubated with labeled anti-mouse IL-17A antibody at 37?for 20 min in dark.After washed by 2ml staining buffer,the samples were fixed with lml 1 X ROR y t Fix/Perm buffer for 20min at 37? in dark,and then washed by 2ml staining buffer twice and 1ml 1 ×ROR? t Perm buffer once successively.Then the samples were fixed with 1ml 1 × ROR ?t Perm buffer for 15min at 37? in dark.After centrifugation,the samples were resuspended in 100ul 1 ×ROR? t Perm buffer and stained with anti-mouse ROR ? t antibody for 30 min in the dark.After being washed by 2ml staining buffer,stained samples were added with 500ul stain buffer and then assessed on a Millpore Guava Easy Cyte cytometer.Tregs were evaluated using the same protocol with antibodies against CD4 and FoxP3.5.Magnetic bead cell sortingAnti-mouse CD4 and CD62 Micro Beads were used to select CD4+CD62L+ primary T cells from single cell suspensions.Selected cells were resuspended in 1 ml 1 X FOXP3 Fix/Perm buffer and incubated for 20 min in the dark at indoor temperature.Wash the cells with lml cell staining buffer and 1mlml 1× FOXP3 Perm buffer successively for once.After that the cells were added to 1ml 1 × FOXP3 Perm buffer and incubated for 15 min in the dark at indoor temperature.After centrifugation,cells were resuspended in 100ul 1× FOXP3 Perm buffer and then incubated with antibodies against ROR y t and IL-17 for 30min.Centrifugation again.Finally,the selected cells were resuspended in 200ul PBS for next detection with cytometry flow technology.6.Western BlottingProtein was extracted from Th17 by RIPA and measured by BCA assay.Equal amounts of protein(20ug)was electrophoresed on 10%SDS-PAGE gels,transferred to PVDF membrane,blocked with 5%skim milk,and then incubated at 4? overnight with the primary antibodies against SGK1,p-FOXO1 and IL-23R respectively.PVDF membrane was then incubated with the HRP-conjugated secondary antibody at indoor temperature for 1 h.Afterwards,PVDF membrane was performed by enhanced chemilumin escence reagents and visualized by a Bio-rad Gel Doc EZ imaging system.All western Blots were developed repeatedly for at least 3 times to quantify the density using ImageJ software.7.StatisticsExperimental results were presented as mean ± standard deviation(x ± sd).One-Way-ANOVA with SNK test or Dunnett's T3 test was used to assess statistical significance between four groups.Comparisons between groups for body weight and random blood glucose were performed for repeated measurements(RM)followed by SNK test or Dunnett's T3 test.SPSS22.0 statistical software was used for statistical analysis.P<0.05 was considered statistically significant.Result:1.Effects of dapagliflozin on urinary albumin/creatinine ratio and renal fibrosis in db/db miceCompared with wild type mice,db/db mice showed a distinctly increase in urinary albumin/creatinine ratio.At the same time,the fibrosis of kidney in db/db mice was apparently server than wild type mice.Dapagliflozin could lower urinary albumin/creatinine ratio and finally improve the fibrosis of kidney.2.Changes caused by dapagliflozin on the imbalance between Th17 and Tregs in db/db miceIn db/db mice,the number of Th17 increased in both peripheral blood and kidney,while the number of Tregs decreased.Being interfered by dapagliflozin,the imbalance between Th17 and Tregs was revealed as the fibrosis of kidney was relieved in db/db mice.3.Dapagliflozin was able to influence the differentiation of T cellDifferent from the wild type mice,the expression of SGK1?p-FOXO1 and IL-23R were increased in renal Th17 in db/db mice.Dapagliflozin could inhibit the expression of SGK1?p-FOXO1 and IL-23R.Conclusion:1.Dapagliflozin improves the pathological changes of diabetic kidney disease2.Dapagliflozin reverses the imbalance between Th17 and Tregs both in peripheral blood and renal tissues of db/db mice3.Dapagliflozin inhibits the activation of SGK1/p-Foxol/IL-23R pathway in renal Th17 in ab/ab mice.