GC/MS-based Metabonomic Analysis Explores Mechanism Of SGLT2i In Protecting Renal Tubules In Diabetic Kidney Disease

BackgroundDiabetic Kidney Disease(DKD)is an important microvascular complication in patients with diabetes and has become the leading cause of end stage renal disease(ESRD).It has been thought that the main early lesion site of DKD is in the glomerulus,however,recent studies have found that interstitial tubular damage may occur earlier than glomerular damage,becoming an important reference for early renal injury.Sodium-glucose co-transporter2(SGLT2)is a class of sodium-glucose transporter protein mainly found in the renal proximal tubules that mediate the process of renal glucose reabsorption.Adapted hyperexpression of SGLT2 occurs in the early stages of diabetes mellitus and may mediate early renal damage.SGLT2 inhibitor(SGLT2i)is a novel oral glucose-lowering drug that exerts its glucose-lowering effects primarily by selectively inhibiting SGLT2 reabsorption of glucose and increasing urinary sugar excretion.In recent clinical studies,significant protective results have been achieved against DKD renal nodules.The pathogenesis of DKD is not fully understood,but hyperglycemia is an important factor throughout the course of the disease.Prolonged exposure of renal tubule cells to a high-glucose environment leads to activation of the glycogen metabolic bypass and increase in oxidative stress and cytokines,which eventually mediates renal injury.However,the renoprotective effect of the use of SGLT2i is currently unknown.Metabolomics,as it could visualize changes in organismal metabolism,may offer the possibility of targeted therapy for diabetic nephropathy.In recent years,the increasing number of metabolomics studies related to DKD has provided us with an important research strategy for exploring early proximal renal tubule biomarkers in DKD.There is still space for study on the metabolomics mechanism of SGLT2i to improve tubular function in DKD condition,for which,we used gas chromatography-mass spectrometry(GC/MS)method as an investigation strategy.Taken together,in this experiment we observed the protective effect of SGLT2 inhibition on diabetic kidney damage in a db/db mouse animal model,and further explored the metabolomic mechanism of it in tubule cells using a metabolomics-based approach.Method1.Animal experiment8-week-old db/db mice(n=16)and 8-week-old db/m mice(n=8)were used in the previous period.The db/db mice were randomly divided into a diabetic control group(DM group)and a diabetic+dapagliflozin group(DM+DAPA group),respectively,with normal saline(1mg/kg/d)and dapagliflozin(1mg/kg/d)Gavage.The db/m mice were used as a blank control group(NC group),and were administered with saline(1mg/kg/d).All mice were sacrificed after 12 weeks of drug intervention.2.Cell cultureHuman renal tubular epithelial cell line(HK2)was purchased from the cell bank of the Chinese Academy of Sciences in Shanghai,cultured in DMEM medium containing 1.0g/L glucose and 10%FBS,and incubated in a 37℃,5%CO2 incubator.Passage every 2-3 days.3.RNA extraction,reverse transcription and real-time PCR(RT-PCR)Total RNA of mouse kidney tissue and HK-2 cells was extracted with Trizol.M-MLV reverse transcriptase reagent reverse transcribes RNA into cDNA.The SYBR method was used for real-time quantitative PCR reactions.Gene expression was calculated using the 2-??ct method.GAPDH was used as the internal reference.4.Western BlotTotal protein was extracted from tissues and cells by RIPA method.Prepare 10%SDS-PAGE gel with a sample volume of 3 0ug per well.Sample proteins were separated by electrophoresis and wet transferred to a PVDF membrane.PVDF was blocked with 5%skimmed milk powder or 5%BSA.The PVDF membrane was incubated with a primary antibody and shaken at 4 ℃ overnight.The secondary antibodies were incubated for 1 h and washed with TBST.Chemiluminescence was used for luminescence development,and the results were analyzed by a Gel-Pro analyzer.5.Gas chromatography-mass spectrometry(GC/MS)analysisHK2 cells were divided into Normal glucose(NG)group,High glucose(HG)group,High glucose and Canagliflozin(HG+Canagliflozin,HG+CANA)group,and cultured for 72h.After metabolic quenching,GC/MS analysis was performed on the machine.Differential metabolites were finally obtained through quality control and screening of metabolites.6.Statistical AnalysisSPSS 25.0 statistical software was used for data analysis.Each index data was expressed as mean± standard error(x±SEM).Multiple groups of data were compared using single factor analysis of variance.Two samples with normal distribution were compared using two independent sample t tests.Correlation analysis was performed using the Pearson method,and differences were considered statistically significant at P<0.05.Result1.SGLT2i can reduce the expression of SGLT2 protein in renal cortex of db/db mice and high glucose-treated HK2 cellsThe expression of SGLT2 protein was up-regulated in the renal cortex and high glucose-treated HK2 cells in db/db mice.However,SGLT2 protein expression was significantly down-regulated after SGLT2i intervention in db/db mice and HK2 cells cultured in high glucose.2.SGLT2i delays renal fibrosis in db/db miceCompared with the db/m group,the renal cortex fibrosis indicators FN and aSMA in the db/db group were significantly increased.Renal fibrosis was significantly improved in db/db mice after using dapagliflozin.This indicates that SGLT2i can delay DKD progress.3.Metabolomics analysis of SGLT2i intervention in HK2 cells cultured in high glucose(1)Analysis of the metabolites of the glycolytic pathway revealed that glucose and glucose 6-phosphate were up-regulated in the HG group compared with the NG group;compared with the HG group,the glucose,glucose 6-phosphate content,and pyruvate in the HG+CANA group/Lactic acid ratios are all reduced.(2)Analysis of the metabolites of the tricarboxylic acid cycle pathway found that the metabolites of the tricarboxylic acid cycle pathway were down-regulated in the HG group compared with the NG group;there was no significant difference in the HG+CANA group compared with the HG group.(3)Analysis of the glucose metabolism bypass including gluconeogenesis and polyol pathways found that the fructose and 1-phosphate-glucose levels of the HG group were increased compared with the NG group;compared with the HG group,the fructose,1-Phosphate-glucose levels were down-regulated.Conclusion1.SGLT2i protects the kidney by ameliorating DKD renal fibrosis.The specific possible mechanism is related to the changes in metabolites caused by reduced renal tubular glucose consumption.2.Metabolomics analysis results show that SGLT2i improved energy-consuming sugar responses such as glycolysis,gluconeogenesis,and fructose metabolism of renal tubular epithelial cells in the DKD state by restricting glucose from entering cells.