| Chronic kidney disease(CKD)is a worldwide clinical medicine problem.In the early stage of CKD,the renal function is still in a compensatory period,but with the progression of the disease;eventually it will progress to end stage kidney disease(ESRD)and the kidney will complete loss of function ensues.The main pathological changes in this time,include glomerulosclerosis,tubular atrophy,renal interstitial fibrosis,etc.;and renal interstitial fibrosis is considered to be the final outcome of all CKD regardless of its etiology.Matrix metalloproteinase-2(MMP-2)is crucial for the development of renal interstitial fibrosis.In the early stage of CKD,MMP-2 activity increases and promotes the mesenchymal transformation of renal tubular epithelial cells,resulting in increased extracellular matrix(ECM)production.With the development of CKD,the expression of MMP-2 increased but the activity of MMP-2decreased.Ultimately,the imbalance between the synthesis and degradation of the ECM leads to its accumulation in the renal interstitium,promoting the renal interstitial fibrosis.However,the precise mechanism underlying the decrease of MMP-2 activity in the advanced stage of CKD remains unclear.Hypoxia plays an important role in the pathogenesis of renal fibrosis.And MMP-2 activation mainly occurs on the surface of the cell membrane.Once activated,MMP-2 can exert its effects,therefore,membrane structure remodeling may affect MMP-2 activity.Endocytosis is enhanced during hypoxia and cell membrane remodeling is also enhanced during hypoxia.So,we hypothesized that the decrease of MMP-2 activity in the advanced stage of CKD is related to the enhanced endocytosis induced by hypoxia.We previously demonstratedthat the endocytosis of human proximal renal tubular epithelial cells(HK-2 cells)is enhanced by hypoxia,while MMP-2 activity in the supernatant decreased.However,inhibition of the key gene involved in endocytosis,Caveolin-1,in HK-2 cells increased MMP-2 activity.Accordingly,we suggest that during hypoxia,endocytosis of HK-2 cells is enhanced,which affects the activation of MMP-2,resulting in decreased MMP-2 activity and aggravation of renal interstitial fibrosis.However,the underlying mechanism is not clear.Src is an important member of the tyrosine kinase family,and hypoxia can induce Src phosphorylate to phospho-Src(Tyr 416).p-Src(Tyr 416)can regulate many kinds of membrane receptor signaling pathways related to fibrosis,such as TGF-/Smad,STAT3,EGFR and others.At present,studies have shown that inhibition of its activation at Tyr 416 can alleviate renal interstitial fibrosis.Therefore,we wondered whether a relationship exists between Src,cell endocytosis and MMP-2 activity during renal interstitial fibrosis? Does the Src which activated by hypoxia in renal interstitial fibrosis may affect the activation of MMP-2 through affect cell endocytosis?This prompts us to do further research.First we establish a cyclosporin A(Cs A)induced hypoxic renal interstitial fibrosis model in mice with a modified approach,then evaluate the method;on the basis of preliminary success,a rat model of renal interstitial fibrosis induced by Cs A was established.Then the MMP-2 activity,the expression of cell endocytosis regulating molecule Caveolin-1,and the expression of Src in the fibrotic renal tissue were observed from the whole level.Finally,the vitro experiments were used to further study the mechanism of how Src regulation of MMP-2 activity.We simulated hypoxia in renal fibrosis by hypoxia culture HK-2 cells,and interfered with Src activation by PP1——a highly potent and selective inhibitor of Src;then detected the changes of Src,Caveolin-1,MMP-2activity;and to explore whether Src affected MMP-2 activity through endocytosis.Part one A modified approach to establish a murine and rat model of hypoxic renal interstitial fibrosisⅠ.A modified approach to establish a murine model of hypoxic renal interstitial fibrosis Objectives A murine model of Cs A induced hypoxic renal interstitial fibrosis is commonly used.A previously introduced approach involves the subcutaneous injection of Cs A combined with a 0.01% low-sodium diet.However,the clinical application of Cs A is mainly oral.Therefore,modeling through gastric administration can simulate the clinical more accurately.Meanwhile,the low-sodium diet is essential in the modeling process;but the production of a low-sodium diet is time-consuming and expensive,and the standardisation of sodium dosages is difficult.However,furosemide is a commonly used drug and easy to get;it can mimic a low-sodium environment because its role in promotes the excretion of sodium.Thus the aim of this study was to provide a modified approach to this model by replacing the subcutaneous injection with gavage and the low-sodium diet with furosemide.Methods The mice were divided into the following 6 groups: Cs A + furosemide group,single Cs A group,single furosemide group,single sunflower oil group,distilled water group,and untreated group.The general condition of the mice was observed daily;the body weight of each mouse was recorded every 4 days and the dosage was adjusted accordingly.After treatment for 14 and 28 days,metabolic cages were used to collect urine,and 6 mice from each group were sacrificed.Their blood and kidneys were obtained.The gross morphology was observed and detection the serum and urine sodium,serum creatinine(Scr),blood urea nitrogen(BUN),urinary protein,and creatinine clearance(Ccr)rate.And Hematoxylin-Eosin(HE),Masson-trichrome,Periodic Acid-Schiff(PAS)stainings were used for renal athological examination;Western blotting method was used to detect he protein levels of Vimetin to know the degree of kidney fibrosis;the immunohistochemical staining was used to detect renin secretion;Western blotting method was also used to detect the protein levels of hypoxia inducible factor-1 alpha(HIF-1α)in different groups to konw the hypoxia situation of kidney tissue.Results After 14 th and 28 th day,compared with other groups,only the kidneys of Cs A+ furosemide group appeared obviously fibrosis appearence.Meanwhile,the blood sodium in this group was lower than that in other groups,and the urine sodium in this group was higher than that in other groups.The renal function indexes in this group was also obviously abnormal than that of other groups.Immunohistochemical staining also indicated that the renin secretion only obvious in the kidneys of Cs A +furosemide group.Western blotting suggested that the obvious protein expression of Vimentin and HIF-1α was also only in this group.Conclusions Altogether,the data suggest that our modified approach is also an effective,alternative way to establish this model.Ⅱ.A modified approach to establish a rat model of hypoxic renal interstitial fibrosis Objectives In experimental I,we used Cs A combined with furosemide to establish a modified murine model of hypoxic renal interstitial fibrosis successfully.However,on one hand,due to the small volume of kidney in mice,the amount of fresh tissue that is advisable is limited at a time,and the more fresh kidney tissue is needed in subsequent part of the experiment;on another hand,the reliability of this modified approach should be verified again.Because of these two,we will use this method to establish the hypoxia renal interstitial fibrosis model in rats,and evaluate the modeling results.Methods The group of rats was optimized based on the experiment I;the rats were divided into the following 4 groups: Cs A + furosemide group,Cs A + distilled water roup,furosemide + sunflower oil group,and untreated group.The general condition of the rats was observed daily;the body weight of each rat was recorded every 4 days and the dosage was adjusted accordingly.After treatment for 14 and 28 days,metabolic cages were used to collect urine,and 6 rats from each group were sacrificed.Their blood and kidneys were obtained.The gross morphology was observed and the Scr,BUN,urinary protein,Ccr were detected.HE,Masson and PAS stainings were used for renal pathological examination.The immunohistochemical staining was used to reveal fibroblast proliferation by detecting Vimentin;Western blotting method was used to detect the protein levels of HIF-1α in different groups to konw the hypoxia situation of kidney tissue.Results After 14 th and 28 th day,compared with other groups,only the kidneys of Cs A+ furosemide group appeared obviously fibrosis appearence.Meanwhile,the renal function indexes in this group was obviously abnormal than that of other groups.Immunohistochemical staining also indicated that the Vimentin positive cells were only increased in the kidneys of Cs A + furosemide group.Western blotting suggested that the obvious protein expression of HIF-1α was also only in this group.Conclusions The model can be sucessfully established by using this method.Part two Observation of correlation between p-Src(Tyr 416)expression and MMP-2 activity in rat kidney tissue Objectives Observing the the change of MMP-2 activity,cell endocytosis regulate molecule Caveolin-1,and Src in the renal tissue on the whole level from different groups of rats.Methods Fresh rat kidney tissues from different groups were lysed to extract protein.Then the Western blotting method was used to detect the protein levels of MMP-2,membrane type 1-matrix metalloproteinase(MT1-MMP),reversion-inducing cysteine-rich protein with kazal motifs(RECK),tissue inhibitor of metalloproteinase-2(TIMP-2),Caveolin-1,and p-Src(Tyr 416)in different groups.The activity change of MMP-2 in each group was detected by zymography.Finally,the correlation between p-Src(Tyr 416)and MMP-2 activity was observed.Results The protein levels of MMP-2,RECK,TIMP-2,p-Src(Tyr 416),and Caveolin-1 in the renal tissue of rats with fibrosis(Cs A+ furosemide group)were significantly higher than other groups;but the protein level of MT1-MMP and the MMP-2 activity in this group were significantly lower than other groups.Conclusions At the whole level,there is a negative correlation between p-Src(Tyr416)and the MMP-2 activity,and p-Src(Tyr 416)may affect the MMP-2 activity by affecting cell endocytosis.Part three Studies on the mechanism of p-Src(Tyr 416)regulating MMP-2 activity Objectives By hypoxia culture HK-2 cells to simulate renal interstitial fibrosis in hypoxic conditions,and interfered with Src activation by PP1——a highly potent and selective inhibitor of Src.Then detect the changes of p-Src(Tyr416),endocytosis level,Caveolin-1 and MMP-2 activity;to explore whether Src affected MMP-2activity through cell endocytosis.Methods The HK-2 cells were divided into four groups: normoxia group;hypoxia group;hypoxia + DMSO group;and hypoxia + PP1 group.The PP1 dose and treatment time were determined by a Cell Counting Kit-8(CCK-8)cytotoxicity experiment.Then Western blotting method was used to detect the protein levels of HIF-1α,MMP-2,RECK,TIMP-2,MT1-MMP,p-Src(Tyr 416),and Caveolin-1.And the dye FM4-64 FX was used to observe the endocytosis of each group.And the protein levels of RECK,TIMP-2 and MT1-MMP in each group was verified by immunofluorescence again.Finally,the MMP-2 activity in supernatant of each group was detected by zymography.Results Compared with HK-2 cells grown in normoxia,in cells treated with hypoxia,the protein levels of p-Src(Tyr 416)and Caveolin-1 increased,as did the protein levels of the MMP-2 activity regulated molecules RECK and TIMP-2,while the protein level of MT1-MMP and MMP-2 activity were decreased.Compared with HK-2 cells grown in hypoxia,in cells treated with PP1,the protein levels of p-Src(Tyr 416)and Caveolin-1 decreased,as did the protein levels of the MMP-2 activity regulated molecules RECK and TIMP-2,while the protein level of MT1-MMP increased and MMP-2 activity was enhanced.Conclusions Hypoxia promotes the phosphorylation of Src;the activation of Src during hypoxia can stimulate the endocytosis of HK-2 cells and influence MMP-2activation,leading to decreased MMP-2 activity and aggravation of renal interstitial ibrosis. |
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