| Objective:Renal interstitial fibrosis(RIF)is almost the end result of chronic kidney disease,closely associated with chronic renal failure and leading to end-stage renal failure(ESRD).The interstitial fibrosis process is often accompanied by epithelial-mesenchymal transition(EMT)of tubular epithelial cells,by which tubular epithelial cells are converted into the phenotype of myofibroblasts and produce interstitial matrix components.The study found that a variety of causes of renal interstitial fibrosis pathogenic factors,such as metabolic factors,obstruction factors and inflammation,could enhance kidney partial oxidative stress,increase reactive oxygen species(ROS)generation,cause oxidation and antioxidation system imbalance,lead to further injury,and eventually lead to renal tubular atrophy and interstitial fibrosis.Oxidative stress is one of the crucial factors for the formation of renal interstitial fibrosis.It could stimulate various cytokines and activation of multiple signaling pathways.It played an important role in the occurrence of the EMT for the development of renal fibrosis.In recent years,the family of NADPH oxidase(NOXS)in diabetes,high blood pressure,renal damage,5/6 nephrectomy and various renal pathology model has been identified as kidney important promote oxidation system of local oxidative stress reaction.Many studies had shown that transforming growth factor-?1(TGF-?1)and its downstream transcription factor like extracellular signal regulated kinase(ERK)were the key molecules which trigger the process of tubular EMT,but whether the process was regulated by NADPH oxidase still known little.Magnesium sulfate in ischemic stroke and other diseases had antioxidant effect,but whether had effct of protecting renal interstitial fibrosis still known little.The purpose of this study is through the intervention of magnesium sulfate to observe the TGF-?,Nox4,p-ERK1/2 and Col-? expression in rats with unilateral ureteral obstruction model,to research whether the influence of the NADPH oxidase is involved in the regulation of this process,and to explore possible mechanisms of magnesium sulfate on kidney protection.This study will delay the progress of renal interstitial fibrosis and provide a new therapeutic target.Methods: By unilateral ureteral obstruction(UUO)model,the 72 grade clean SD rats(male,180~220 g),were randomly divided into group A,B,C,D: Sham group(separated the left ureter surgery but not ligatured),UUO group(ligatured the left ureter near the renal pelvis section),high dose magnesia group,and low dose magnesia group.Sham group and UUO group were given equal normal saline by intraperitoneal injection.High dose magnesia group and low dose magnesia group were given 300 mg/kg/d magnesium sulfate and 100 mg/kg/d by intraperitoneal injection.During the experiment,the animals drank water freely.We observed general situation of SD rats,such as the diet and activity every day.After surgery,at 3,7 and 14 days,6 SD rats were cut out the left kidney tissue and sacrificed in every group.One part of kidney tissue was fixed by 10% neutral formalin and was detected by immunohistochemisty.Another part of kidney tissue was quickly frozen in liquid nitrogen,preventing protein degradation,and then stored in-80 ?environment,to do western blot experiments.Statistical analysis software SPSS 20.0 was applied for statistical analysis of experimental data,P <0.05 was considered statistically significant.Results: 1 Pathology of kidney 1.1 Visual inspection: The color and size of group A was normal.The other three group especially in the group B had significant figures in the rat kidney increases to some extent,observing the water accumulation in the kidney and the renal cortical thinning.However in C,D group kidney appearance compared with A group were reduce.1.2 Light microscopy: 1.2.1 HemateinEosinThe kidney structure was normal and the glomerulus and renal tubules didn't change in group A.We could observed that the renal tubular epithelial cells fall off and necrosis,inflammatory cell infiltration,renal interstitial broadening and interstitial fibrosis,renal tubular expansion,contraction,collapse,and diffuse thickening of basement membrane.The renal interstitial and tubular structure almost disappear with the development of the lesion,but the degree of glomerular lesions was different.The degree of change of group C and D was lighter than group B.The degree of change of group C was more obviously than group D.1.2.2 Masson stainingGroup A showed that renal tissue structure,renal tubule and renal interstitial normal.Group B showed renal tubular expansion extensively,renal interstitial broadened,interstitial tissue and cell increased,collagen deposition in renal interstitial by Masson staining.With the development of the lesion,collagen fiber gradually extended to the kidney tissue surrouding and interwoven into "mesh".The degree of change of group C and D was lighter than group B.The degree of change of group C was more obviously than group D.2 Immunohistochemistry results 2.1 TGF-?1: In group A,only trace amount of TGF-?1 expression was detected;in group B,the expression level of TGF-?1 went up in a time dependent manner,and there was a significant difference(P <0.05)between group A and group B on the same time point;in group C and D,the expression level of TGF-?1 was not as much as that in group B(P <0.05),but it was still much higher than that of group A(P <0.05),however compared to the group D the expression level of TGF-?1 reduced in group C on the same time point.2.2 Nox4: In group A,a basic expression level of Nox4 existed in tubular cells.In group B,the expression level of Nox4 went up in a time dependent manner,and there was a significant difference(P <0.05)between group A and group B on the same time point;in group C and D,the expression level of Nox4 was not as much as that in group B(P <0.05),but it was still much higher than that of group A(P <0.05),however compared to the group D the expression level of Nox4 reduced in group C on the same time point.2.3 p-ERK1/2:In group A,a basal level of expression was presented in tubular cell nucleus.In group B,the expression level of p-ERK1/2 went up in a time dependent manner,and there was a significant difference(P <0.05)between group A and group B on the same time point.In group C and D,the expression level of p-ERK1/2 was not as much as that in group B(P <0.05),but it was still much higher than that of group A(P <0.05),however compared to the group D the expression level of p-ERK1/2 reduced in group C on the same time point.2.4 Col-?:Under normal circumstances,Col-? was found in the renal tubular epithelial cells,mesenchymal cells and in small amounts of glomerular mesangial cell cytoplasm.In group A,no or only trace amount of TGF-?1 expression was detected.In group B,the expression level of TGF-?1 went up in a time dependent manner,and there was a significant difference(P <0.05)between group A and group B on the same time point.In group C and D,the expression level of TGF-?1 was not as much as that in group B(P <0.05),but it was still much higher than that of group A(P <0.05),however compared to the group D the expression level of TGF-?1 reduced in group C on the same time point.3 results of Western BlotCompared with A group,the production of Nox4 were apparently increased in B group,and there was a significant difference(P <0.05)between group A and group B on the same time point.In group C and D,the expression level of Nox4 was not as much as that in group B(P <0.05),but it was still much higher than that of group A(P <0.05),however compared to the group D the expression level of Nox4 reduced in group C on the same time point.Conclusion:The expression of TGF-?1,Nox4,p-ERK1/2 and Col-? were increased in obstruction kidney after UUO,and they are relaying on each other.While the level of magnesium was decreased by rosuvastatin intervention under the fibrosis factor Nox4 increasing,the expression of TGF-?1,p-ERK1/2 and Col-? was decreased in kidney.It maybe reduce and put off renal interstitial fibrosis.By this experiment,it maybe infer that one of the mechanism of magnesium inhibited renal interstitial fibrosis should be adjust the level of Nox4 protein to reduce RIF,which may protect renal by its isoprene effect to reduce ROS protein expression. |
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