Protective Effects And Mechanism Of Action Studies Of Tetramethylpyrazine Nitrone TBN In Diabetic Kidney Disease Models

Objectives:The therapeutic effects of TBN were systematically studied in STZ-induced DKD rats and spontaneous DKD model in db/db mice.In addition,the protective mechanism of action TBN was further investigated,providing experimental basis supporting TBN clinical trials.Studying the effectiveness of TBN in macaca rhesus with spontaneous DKD(DKD-EPI grade:G3,eGFR:30-59 ml/min/1.73m²)provides further evidence that TBN has beneficial effects for DKD.Methods:1.Single intraperitoneal injection of STZ was used to induce islet beta cell injury in SD rats to provide a DKD model to evaluate the efficacy of TBN.In STZ-induced DKD studies,the rats were fasted for 12 h before the injection.Diabetes was induced by single intraperitoneal administration of 55 mg/kg STZ,freshly dissolved in 0.1 M sterile sodium citrate(pH 4.5).The normal control rat group was intraperitoneal injected with equal volume of citrate buffer(pH 4.5).Rats with blood glucose level?16.7 mmol/L were included in this study at 3 weeks of STZ injection.Next,the diabetic rats were randomly divided into 5 groups and were given different drugs.The experiment was completed after 6 weeks of TBN administration.Physiological indicators such as body weight,water intake,food intake,blood glucose,24 h urine protein and microalbuminuria detection,renal function index detection,renal histopathological staining,and pancreatic histopathological analysis were recorded.2.Spontaneous DKD db/db mice was used to evaluate the efficacy of TBN.Animals were divided into 6 groups:wt/wt group,db/db group,TBN low dose group(TBN 10 mg/kg),medium dose group(TBN 30 mg/kg),high dose group(TBN 60 mg/kg)and positive control group(losartan 10 mg/kg).The 7-week-old db/db mouse were randomly assigned and then given TBN by gavage.In the positive control group,losartan was given once a day.The TBN and saline groups were given twice a day,with continuous administration of 6 weeks.Physiological indicators such as body weight,water intake,food intake,blood glucose,24 h urine protein and microalbuminuria detection,renal function index detection,renal histopathological staining,and pancreatic histopathological analysis were recorded.3.Male macaca rhesus(n=7)with spontaneously DKD were included in the study.The selected rhesus monkeys with autonomic stage IIIa DKD met the following criteria:eGFR:30-59 ml/min/1.73m²,CysC?1.3 mg/d L,serum creatinine(Scr)?1.26 mg/d L,weight stable,and have not yet received insulin and any other therapy.Seven rhesus monkeys were assigned to vehicle(n=2)and TBN treatment group(n=5),respectively.TBN were administered to Rhesus monkeys as a subcutaneous injection of 30 mg/kg twice daily for 12 weeks.Bodyweight was measured weekly throughout the course of the study.In order to study the therapeutic effects,major efficacy indicators including plasma creatinine,cystatin C,and glomerular rate filtration were measured every 4 weeks before and after drug administration.Secondary efficacy index include glucolipid metabolism(FPG,FRA,PFI,Hb A1c,LDL,HDL,TG,TC),the urine trace albumin(Malb),urine creatinine(Cr-U),UACR,and blood pressure and hematology indexes.4.To provide a theoretical basis for the therapeutic effects of TBN,H&E staining,PAS staining,Masson staining,immunohistochemistry,ELISA,and Western blot were used to study the possible protective mechanism of action TBN for the treatment of DKD.Results:1.TBN at concentrations of 60 mg/kg significantly attenuated the increased of water consumption and food intake in STZ-induced DKD rats.After STZ injection,rats that developed diabetes exhibited increases in blood glucose level and 24 h urinary protein in a time-dependent manner.At the end of the experiments,TBN treatment significantly attenuated the blood glucose level in a dose-dependent manner and 24 h urinary protein compared with rats in the DKD group.However,treatment with losartan had no effect on blood glucose level,but significantly reduced the increases in 24 h urinary protein.TBN and losartan-treated groups gained slightly less Cr and TC than vehicle-treated group.Importantly,compared with vehicle-treated group,TBN treatment markedly reduced the elevated concentration of BUN and TG by the end of the experiment.Moreover,vehicle-treated DKD rats displayed a significantly lower EPO and serum iron,which also was reversed by TBN.However,the concentration of EPO was lower in the serum of DKD rats given losartan.The results of H&E staining revealed obvious glomerular hypertrophy and mesangial cell proliferation in the vehicle-treated DKD group compared with the ctrl group.PAS and Masson staining further revealed substantial mesangial matrix expansion and glomerulosclerosis in the vehicle-treated DKD group.TBN treatment significantly ameliorated glomerular hypertrophy,mesangial expansion,and glomerulosclerosis compared with the vehicle-treated DKD group.IHC imaging of renal tissue from STZ-induced rats showed the expression levels of the profibrotic molecules TGF-?1 and Col-IV were significantly elevated in the kidneys compared with ctrl rats.TBN-treated DKD rats prevented the increases of TGF-?1and type Col-IV.TBN obviously ameliorated pancreas damage and effectively improved islet mass,which suggests that TBN had a protective effect of pancreas.2.Within 6 weeks of treatment,bodyweight was significantly higher in db/db mice than in wt/wt mice throughout the experiment.There was no difference in bodyweight between db/db and TBN-treated db/db mice.Similar to finding from losartan,bodyweight was no difference between db/db mice and losartan-treated db/db mice.Drinking water and food intake were significantly decreased in the TBN(30 mg/kg)and losartan-treated db/db difference.In addition,TBN treatment markedly reduced the elevated blood glucose level,urinary microalbuminuria,microalbuminuria/creatinine ratio,and 8-OHdG in db/db mice.Images of renal section from wt/wt mice show the normal structure of the glomerular and tubules.Compared to this,renal section section of db/db mice showed a series of indicators of DKD,such as glomerular hypertrophy and mesangial matrix expansion.Treatment with TBN,renal histological changes was remarkably alleviated.IHC imaging of renal tissue from db/db mice showed the expression levels of TGF-?1 and Col-IV were significantly elevated in the kidneys compared with wt/wt mice.TBN treatment prevented the increases of TGF-?1 and Col-IV.We also examined the ultra-microstructure with transmission electron microscope.The GBM thickness and foot process width was increased in vehicle-treated db/db mice,whereas TBN and losartan treatment significantly decreased the GBM thickness and foot process fusion.3.During the whole experiment,there was no significant change in the levels of various indicators in vehicle-treated DKD rhesus macaque,which proved that the model was relatively stable.Mean(±SD)eGFR increased significantly from 48(±5)ml/min/1.73m² at baseline to 53(±6)ml/min/1.73m²(10.4%)in the TBN group,but remained no significant changes in the vehicle group,with values of 50(±6)ml/min/1.73m²and 51(±2)ml/min/1.73m²(2.0%)at baseline and week 12,respectively.Within 12 weeks of treatment with TBN,the concentration of MDA,3-NT,and IL-1?were significantly decreased in comparison to vehicle-treated DKD rhesus macaque.Compared with vehicle-treated DKD group,there was no significant difference in the levels of blood glucose(FPG,FPI,FRA,Hb A1c),serum lipids(LDL,TC,TG and HDL),blood pressure(SBP,DBP)and hematological in DKD rhesus macaque.4.The results of oxidation resistance experiment by chemical method showed that TBN could effectively remove·OH,O₂·^-and ONOO^-,and had a strong scavenging ability for ONOO^-at 5?M.Compared to the vehicle-treated DKD group,the treatment of TBN significantly attenuated the level of serum MDA and urinary 8-OHdG in a dose-dependent manner in STZ-induced DKD rats.At the end of the 6-week treatment,there was significantly decreased8-OHdG in TBN and losartan-treated db/db mice compared with db/db mice.In this study,we used Western blot analyses to assess the effect of TBN on p-AMPK,PGC-1?,Nrf2,and HO-1protein expression levels in DKD rats and db/db mice.We observed significantly reduced p-AMPK/AMPK ratio in the renal cortex of vehicle-treated DKD rats,which was increased after TBN administration with a dose-dependent manner.Similarly,downstream proteins including PGC-1?,Nrf2 and HO-1 were enhanced in response to TBN administration in DKD rats.The expression of p-AMPK,PGC-1?,Nrf2,and HO-1 showed the same results in db/db mice.Conclusions:1.Different doses of TBN can effectively reduce the level of blood glucose,blood lipid,urinary microalbumin,and proteinuria.TBN also significantly improved kidney injury,protected pancreatic beta cells,and increased insulin secretion in STZ-induced SD rats.In addition,TBN provides beneficial effects to diabetic complications such as cataract and anemia.2.TBN at all doses improved blood glucose levels and 24 h microalbumin in db/db mice.Pathological staining showed that TBN effectively improved the kidney damage caused by hyperglycemia,and has beneficial effects on renal anemia.3.In the spontaneous DKD macaca rhesus model,at the end of the 12 weeks,Mean(±SD)eGFR significantly increased in the TBN-treated group,but no significant changes in the vehicle-treated group.Compared to the vehicle-treated group,TBN-treated group had significantly lower the level of serum CysC,TG,MDA,3-NT,and IL-1?.These results show that TBN can improve renal function in chronic DKD macaca rhesus.In this study,the effect of TBN was preliminary due to the small sample size.4.Due to its effects reducing levels of oxidative stress and improving mitochondrial function,TBN may be a novel therapeutic agent for the treatment of DKD.