| Objective:Chronic Kidney Disease（CKD）is a general term for clinically different types of kidney disease.The development of the disease is not easily detectable,and is progressive and irreversible.Therefore,the disease seriously consumes national medical and health resources,increases social burden,and threatens human physical and mental health has become an important public health issue in China,the United States and even the world.Diabetic nephropathy（DN）is the main cause of CKD.Studies have found that plasma S-adenosine homocysteine（SAH）levels may be higher than homocysteine（Hcy）levels.Therefore,the level of SAH is a sensitive indicator of renal insufficiency.SAH is a metabolite after the methyl group is transferred by S-adenosylmethionine（SAM）,and the key enzyme for further SAH metabolism in the body is S-adenosine homocysteine hydrolase（SAH Hydrolase,SAHH）.So the activity of SAHH can affect the level of SAH.Studies have shown that SAH is a risk indicator for the diagnosis of cardiovascular disease,but the effect of SAH levels on type 1 diabetic nephropathy is unknown.The purpose of this study was to investigate the effect of SAH level change on renal injury and its primary mechanism through SAHH inhibitor ADA at animal and cell levels.Methods:In vivo experiment:80 normal C57BL/6 male mice aged 4-6 weeks were fed in SPF animal house with ordinary feed,without restriction of water and food intake.Animals were randomly divided into four groups according to weight:normal group,streptozotocin（STZ）group,adenosine dialdehyde（ADA）group,and ADA+STZ group.The STZ group was intraperitoneally injected with streptozotocin（STZ）(50mg·kg-1),which was treated for 5 days and administered once a day.After 5 days,the STZ group stopped the drug administration and continued to inject the solvent intraperitoneally.ADA group was intraperitoneally injected with adenosine dialdehyde(1mg·kg-1)every other day.ADA+STZ group was treated with streptozotocin combined with intraperitoneal injection of adenosine dialdehyde the next day.The experiment lasted 8weeks,Plasma SAH levels were detected by HPLC-MS/MS.Blood glucose test paper to measure blood sugar.The 24-hour urine protein was detected by Coomassie blue staining.PAS staining was used to observe the pathological changes of renal cortex glomeruli.The ultrastructure of renal tissue was observed by transmission electron microscope.Combined with the above experiments,we explored the effect of inhibiting SAHH on diabetic nephropathy.In vitro experiments:human renal podiatocytes were subcultured,cell slides were prepared in time.The cells were divided into control group,high glucose group,ADA group,and high glucose+ADA group according to the experimental needs.The high glucose group was treated with 20 mmol/L glucose solution（GLU）to induce high glucose injury model of podocytes.The high glucose+ADA group was treated with20mmol/L glucose solution and 20μmol/L adenosine dialdehyde solution（ADA）at the same time.All groups were treated for 24 hours.Each group was provided with two parallel holes.Phalloidin staining was used to observe the expression of actin in different treatment groups.ROS reactive oxygen assay kit was used to detect the production of intracellular reactive oxygen.Apoptotic cells were detected using the apoptosis detection kit.Results:（1）Changes in plasma concentration of SAH:compared with the control group(12.26±3.80 nmol·L-1),the plasma SAH levels in the ADA group(33.77±5.46 nmol·L-1)and the STZ+ADA group(35.92±7.10 nmol·L-1)were significantly increased（p<0.01）,and the difference between the control group and the STZ group was not statistically significant.Compared with STZ group(14.34±3.90nmol·L-1),plasma SAH concentration in the ADA group and STZ+ADA group was significantly increased（P<0.01）.（2）Weight change of mice:after 8 weeks of administration,the coat gloss of mice in STZ and STZ+ADA groups decreased.Compared with the control group（25.769±2.272g）,the body weight of mice in STZ group（18.911±2.374g）and ADA+STZ group（17.922±1.829g）decreased significantly（P<0.05）.（3）Blood glucose,urine protein content and kidney coefficient of mice:compared with the blood glucose of the control group(5.75±1.20 mmol·L-1),the blood glucose of the STZ group(26.60±3.11 mmol·L-1),the blood glucose of the STZ+ADA group(26.12±2.45 mmol·L-1)was significantly increased（P<0.05）,and there was no statistically significant difference in blood glucose in the ADA group.Compared with the control group,the 24-hour urine protein of the STZ group and the ADA group were significantly increased（P<0.05）;compared with the STZ group,the 24-hour urine protein of the STZ+ADA group increased to some extent（P<0.05）.Compared with the control group,the kidney coefficient of the STZ group and the STZ+ADA group increased significantly（P<0.05）;compared with the STZ group,the difference in the kidney coefficient of the STZ+ADA group was not statistically significant..（4）Changes in the histopathological characteristics of the mouse kidney:the PAS staining results showed that the glomerular structure of the control group was clear,the glomerular basement membrane was not thickened,and the mesangial matrix was not increased.Compared with the control group,the glomerular basement membrane of STZ group was thickened and mesangial matrix was increased.Compared with STZ group,PAS staining positive products increased in STZ+ADA group（P<0.05）.By means of projection electron microscopy,the results showed that compared with the control group,the glomerular basement membrane of STZ group,ADA group and STZ+ADA group presented different degrees of fold and thickening,and the glomerular mesangial matrix increased,the podiocyte swelling,and the foot processes fused.Compared with STZ group,STZ+ADA group showed an aggravation of renal histopathological changes（P<0.05）.（5）Effects on skeletal protein F-actin in renal podocytes:compared with the control group,f-actin skeleton in the high-glucose group was disintegrated and collapsed to a certain extent（P<0.05）.Compared with the high-glucose group（0.058±0.005）,the average optical density of the high-glucose+ADA group（0.056±0.004）was slightly reduced,but there was no statistical difference.（6）Effects of ADA on ROS production in renal podocytes:compared with the control group,intracellular reactive oxygen production increased in the high-glucose group（P<0.01）.Compared with the high-glucose group,the ADA group and ADA+STZ group had significantly increased intracellular reactive oxygen species and aggravated oxidative stress damage（P<0.01）.（7）The effect of ADA on apoptosis of renal podocytes:compared with the control group,the apoptosis of the high-glucose group increased（P<0.01）.Compared with the high-glucose group,the apoptosis of cells in the ADA+high-glucose group was significantly increased（P<0.05）.Conclusion:（1）In vivo animal experiments showed that ADA（1mg/kg）induces a significant increase in SAH levels in vivo by inhibiting SAHH,promotes obvious thickening of glomerular basement membrane,seriously damages podocyte structure,increased urinary protein excretion,and aggravated kidney injury.（2）In vitro cell experiments showed that ADA（20μmol/L）inhibites SAHH,enhanced cellular oxidative stress,induced skeletal protein structure disorder,and promoted podocytes apoptosis. |
PDF Full Text Request