Effects And Its Mechanisms Of Ace2in The Occurrence And Development Of Rat’s Diabetic Renal Injury

This study focused on the effects of ACE2in the occurrence and development of rat’s diabetic renal injury. The research consists of three parts:1Establishment of Experimental Diabetic Kidney Injury ModelObjective The objective of this study was to establish experimental diabetic kidney injury model and to observe pathological characteristics and tendencies at different points (15days and30days) in disease process, and appraise the pathological degrees of damage to kidney in order to provide reliable experimental animal models for ACE2research later. Methods40healthy adult SD rats were randomly divided into two groups,16rats to the control group and24rats to the model group. The rats in model group were administrated intraperitoneally with streptozotocin (STZ60mg/kg body weight). The rats in control group were only injected with the citrate buffer. After24hours and72hours, the fasting blood glucose was measured respectively. Those rats in the model groups whose fasting blood glucose levels were higher than11.1mmol/L in both measurements were successful rat models with diabetes. The trial period was30days. Body weights, food water intake and mental status of all rats were recorded once daily during the experiment.8successful model rats and8control rats were randomly selected and sacrificed by cervical dislocation, separately on the15th (model group1) and30th (model group2) day of the experiment. Plasma, urine and kidney tissues of these rats were immediately collected for assaying the contents of blood glucose, urine glucose, blood urea nitrogen, urine creatinine and urine protein. Renal injury was evaluated by histopathologic methods. Results (1)48-72hours after injection, the rats performed as polyuria, polydipsia and their activity reduced.(2) Blood glucose levels of all the control rats were between4.9-6.5mmol/L; Blood glucose levels of all the model group1rats (15days) were between14.0-17.5mmol/L; Blood glucose levels of all the model group2rats (30days) were higher than24mmol/L;(3) There was a significant increase in the levels of urine glucose in samples obtained from the rats in model group1(P<0.05), and the levels of urine protein, urine creatinine and urea nitrogen were higher than the control group (P>0.05);The levels of urine glucose, urine protein, urine creatinine and urea nitrogen from the model group2rats were significantly higher than the control group (P<0.01);(4) Histopathologic results showed irregular thickening of glomerular basement membrane, renal tubular structure with normal in model group1rats, the rats in model group2showed mesangial proliferation, diffuse mesangial widening and significant pathological changes;(5) Compared between subgroups(different incubation time point):blood glucose and urine levels had no significant difference between two control groups, the body weight of control rats increased gradually as time expands; The levels of urine protein, blood urea nitrogen and urine creatinine were also not significantly different between two control groups;The body weight of rats in every model subgroups had no significant increase; Compared with the model group1, Urine output and blood glucose levels of the model group2rats were significantly higher (P<0.05). Urine protein of two model groups rats showed a growing tendency along with the proceeding of the disease. Conclusion The intraperitoneal injection of STZ can successfully induced rat models of diabetic renal injury. STZ-induced renal injury gradually aggravated along with the proceeding of disease. The rats of model group2had a greater degree of kidney pathological lesions compared with the rats of model group1.2Relationship Between the Expression Different Of ACE2in the Kidney from Diabetic Rats And Diabetic Renal InjuryObjective This project is designed to the tissue distribution of ACE2and its expression level was observed in diabetes rats and combining the level of Angll in the circulation, to investigate the molecular mechanism of how ACE2involving in the renal injury by diabetic nephropathy. Methods Previous control group1(15days rats), control group2(30days rats), model group1(15days rats) and model group2(30days rats), all the rats were sacrificed, and plasma and kidney tissues of these rats were immediately collected.(1)The mRNA expression levels of ACE2in kidneys were analyzed by RT-PCR;(2)The expression levels of ACE2protein in kidneys were measured by Western blot;(3) The glomerular and cortical expression and location of ACE2were assessed by immunohistochemistry;(4)The contents of Angll in plasma were detected by radioimmunoassay. Results Comparison with the control groups, the expression of ACE2mRNA in the rats of model group1decreased (P<0.05), while ACE2mRNA expression in the rats of model group2was significantly higher than the control group (P<0.05). The expression of ACE2protein in the rats of model group1was significantly higher than the control groups (P<0.05), the expression of ACE2protein in the rats of model group2was lower than the control group (P>0.05). Immunohistochemistry results showed that ACE2mainly located in cell membrane of renal tubular epithelial. Statistics results of ACE2positive staining showed that compared with control group, in the model group1, the expression level of ACE2protein in kidney was significantly higher (P<0.05), in the model group2, the expression level of ACE2protein in kidney was lower than in the control group (P>0.05). This tendency corresponds to the result of Western blot. Compared with the control group, the plasma AngⅡ levels in model group1rats increased slightly, the difference was not significant (P>0.05), while the plasma AngⅡ levels in model group2rats were significantly higher (P<0.05). Conclusion The expression of ACE2in kidney gradually declined with the development of diabetic nephropathy.The expression of ACE2increased in the early stage and decreased in the later stage. AngⅡ levels in circulation also rose in the later stage. The reduction of expression of ACE2and AngⅡ’s accumulation led to aggravating the renal injury. These results suggested that ACE2played an active protection role in the early stage of diabetic nephropathy.3Anti-renal injury Effect of ACE2and its Molecular MechanismsObjective Previous research showed that ACE2participated in the development of renal injury by diabetes. but its mechanism is not well known yet. In this experiment, by observing the differences of the expression levels of ACE-AngⅡ-AT1/ACE2-Ang-(1-7)-Mas and the contents of regulatory factors related to RAS in renal tissues when injury happens, we explored the relationship between negative regulation of ACE/ACE2in the development of renal injury and discussed its possible molecular mechanism. Methods Previous control group1(15days rats), control group2(30days rats), model group1(15days rats) and model group2(30days rats), all the rats were sacrificed, and plasma and kidney tissues of these rats were immediately collected. The mRNA expression levels of ACE, AT1, ACE2and Mas in kidney tissues were analyzed by RT-PCR, the renin activity and the content of AngI,AngⅡ in kidney were detected by radioimmunoassay. Results Comparison with the controlgroup1, the expression of ACE mRNA in model groupl rats decreased (P>0.05) and the same with ACE2, the ratio of ACE/ACE2mRNA decreased; ACE and ACE2mRNA expression in model group2rats were significantly higher than the control group2(P<0.05), the ratio of ACE/ACE2mRNA increased. The expressions of Mas and AT1mRNA had no significant changes in both model group (P>0.05). The renin activity and the content of AngⅠ in kidney tissues of model group1rats were significantly lower than the control group1rats (P<0.05), while the model group2rats were significantly higher (P<0.01); The content of Angllin kidney tissues increased slightly, but the difference was not significant (P>0.05), that of the model group rats2was significantly higher than the control group2(P<0.05). Conclusion The imbalance between the expressions of ACE and ACE2is one of the major causes in development of renal injury by diabetes. Local RAS in kidney will be activated when kidney was injured. During early stage of injury, ACE2-Ang-(1-7)-Mas was the dominant axis and ACE2functions as a protector. When kidney was injured severely, ACE-Angll-AT1was the dominant axis.the decrease of the ACE2expression contributes to the up-regulated level of Angll. All these lead to the development of renal injury.