| Background:The incidence of DN has increased year after year,along with complicated pathogenesis resulting in renal fibrosis,which is hard to be treated.But as to the cause of renal fibrosis of signaling pathways and molecular mechanisms are not fully clear.Studies suggest possible PPAR gamma from transcription to participate in the regulation of PTEN in development process in the DN.Phosphatase and tensin homology deleted on chromosome ten(PTEN),which is an important negative regulator of PI3K/AKTand FAK signal pathway,encodes active lipid phosphatase and protein phosphatase.According to reports,the activation of PI3K/AKT signal pathway results in Multiple fibrosis effects,while PTEN may antagonize the effect of fibrosis through negatively regulating PI3K/AKT pathway.However,the mechanisms between the development of DN and the decrease in the expression of PTEN,especially the effects of the decrease in the activation of protein phosphatase on renal fibrosis of DN and the regulatory factors of the expression of PTEN,are still unknown.Therefore,this research intends to through to the diabetic kidney PPAR gamma expression level of intervention,and to study the regulation of PTEN and influence on renal fibrosis,and to identify the effects of up-regulation and down-regulation of the expression of PTEN on tubular epithelial cell phenotype and fibrosis lesions under high concentration glucose.This study of the pathogenesis of renal fibrosis of DN will help us to find a new and effective target for treating DN.Part one Changes of PPAR?and PTEN expressions in diabetes andhigh glucose as well as their association with renal fibrosisObjective: This study investigated the changes of PPAR? and PTEN expressions in renal tubular epithelial cells and diabetic renal tissue cultured by high glucose,as well as their association with EMT and renal fibrosis.Methods:(1)NRK-52 E cells were used as the study subjects to culture three groups,including the normal glucose group,the high glucose group and the high osmotic pressure group.Immunoblotting and real-time quantitative PCR were used to detect the effects of high glucose and osmotic pressure on PPAR? and PTEN proteins along with mRNA expression.The observation groups at 0h,24 h,48h and 72 h were established,and immunoblotting was used to observe the time-dependence of high glucose on the expressions of PPAR? and PTEN proteins.(2)NRK-52 E cells were used as the study subjects,which were cultured in normal glucose and high glucose for 48 h.Immunofluorescence,immunoblotting and real-time quantitative PCR were used to detect PPAR? and PTEN proteins as well as changes of mRNA expression.Moreover,they were also used to detect the changes of the expressions of E-cadherin,Vimentin and ?-SMA proteins.(3)C57BL mice were used as the study subjects to set up the control group and diabetes model group.Automatic biochemical analyzer was used to detect blood glucose and the level of renal function for the two groups of mice.Moreover,pathological changes of renal tissue of the mice were observed.(4)In the control group and the diabetes model group of the above mice,immunohistochemistry,immunoblotting and real-time quantitative PCR were used to detect changes of PPAR? and PTEN proteins as well as mRNA expression in the renal tissue of the two groups.Furthermore,changes of the expressions of Ecadherin,?-SMA and Collagen-? proteins were detected.Results:(1)High glucose can decrease PPAR? and PTEN proteins as well as m RNA expression with statistical significance(P<0.05),but it had no effect on osmotic pressure.Moreover,PPAR? and PTEN expressions in high glucose gradually decreased with the prolongation of time,which showed time-dependence.(2)There were more expressions of E-cadherin protein in normal glucose group.Level of E-cadherin protein after culturing with high glucose for 48 h decreased significantly compared to that in normal glucose group(P<0.05),and expressions of Vimentin together with ?-SMA proteins in high glucose group increased significantly compared to normal glucose group(P<0.05).(3)In diabetes model group,blood glucose level of mice was significantly higher than that in the control group with statistical significance(P<0.01).Moreover,in diabetes model group,the indicators of serum creatinine and blood urea nitrogen reflecting renal function increased significantly compared to the control group with statistical significance(P<0.05).The morphologies of glomeruli and renal tubulars of mice in the control group were normal,while there were obvious lesions in the glomeruli,renal tubules and renal interstitial of mice in the diabetes model group.(4)In normal glucose,there were more expressions of PPAR? and PTEN in renal tissue,while PPAR? and PTEN proteins as well as mRNA expression in the diabetes model group decreased significantly compared to the control group.E-cadherin expression of mice in the diabetes model group decreased significantly compared to the control group,while there were few expressions of ?-SMA and Collagen-IIIin mice in the control group,which increased significantly in the diabetes model group(P<0.05).Conclusion: PPAR? and PTEN expressions in renal tubular epithelial cells and diabetic nephropathy cultured by high glucose decreased significantly compared to the control group,while EMT and renal fibrosis in renal tubular epithelial cells significantly increased.Part two The regulatory mechanism of PPAR? on PTEN expression in diabetes and high glucoseObjective:To investigate the regulatory effect of PPAR? on PTEN in diabetes and high glucose as well as its effect on EMT and fibrosis.Methods:(1)High glucoseinduced NRK-52 E cells were used as the study subject to establish normal glucose group(NG),normal glucose+rosiglitazone group(NR),normal glucose+ GW9662group(NT),high glucose group(HG),high glucose+rosiglitazone group(HR)and high glucose+GW9662 group(HT).Immunoblotting and real-time quantitative PCR were used to detect the changes of PPAR? and PTEN as well as EMT-related indicators.(2)The empty vector group,PPAR? overexpression group and knockdown PPAR? group were set up.Immunofluorescence,immunoblotting and real-time quantitative PCR were used to detect the changes of PPAR? and PTEN expressions as well as EMT-related indicators.(3)C57BL wild-type mice and conditional PPAR? gene knockout mice were used as the study subjects,which were divided into the normal wild-type group,normal gene knockout group,diabetic wild-type group and diabetic gene knockout group.Automatic biochemical analyzer was used to detect blood glucose and renal function level of the two groups of mice.Moreover,light microscope and electron microscope were used to observe the pathological changes of renal tissue.Changes of PPAR? and PTEN proteins as well as mRNA expression in the renal tissues of the two groups of mice were detected using immunohistochemistry,immunoblotting and real-time quantitative PCR,respectively.In addition,E-cadherin,?-SMA and Collagen III were detected as well.Results:(1)After the cells were treated with high glucose and rosiglitazone for 48 h,expressions of PPAR? and PTEN mRNA significantly decreased in HG group compared to NG group(P<0.05).Comparison between HR group and HG group demonstrated that PTEN mRNA expression increased significantly(P<0.05),while there was no significant changes in PPAR? mRNA.Comparison between NR group and NG group showed that there was no significant difference in the expression of PPAR? mRNA,whereas,PTEN expression showed an increasing trend without statistical significance.Comparison between HG group and NG group indicated that expression of PTEN protein decreased significantly,and expression of E-cadherinprotein also decreased,while expressions of Vimentin and ?-SMA proteins elevated significantly(P<0.05).Comparison between HR group and HG group revealed that expressions of PTEN and E-cadherinproteins elevated significantly,while expressions of Vimentin and ?-SMA proteins reduced(P<0.05).There was no significant difference between NR group and NG group.Compared to HG group,expression of PTEN mRNA in HT group further decreased(P<0.05),while there was no significant changes in PPAR? mRNA.Compared to NG group,expressions of PPAR? and PTEN mRNA in NT group were not significantly different.Compared to HG group,expressions of PTEN and E-cadherin proteins in HT group further decreased(P<0.05),and expressions of Vimentinas well as ?-SMA proteins further increased(P<0.05).(2)After overexpressing PPAR?,expressions of mRNA and protein of PPAR? increased.Moreover,expressions of mRNA and protein of PTEN also increased.The expression of E-cadherin in PPAR? overexpression group up-regulated significantly,whereas expressions of Vimentin and ?-SMA proteins down-regulated significantly with statistical significance(P<0.05).Expressions of PPAR? and PTEN proteins as well as m RNA in the PPAR? knockdown group were significantly lower than those in the empty vector group,and the differences were statistically significant(P<0.05).Moreover,expression of E-cadherin also downregulated,while expressions of Vimentin and ?-SMAproteins increased with statistical significance(P<0.05).(3)The blood glucose of mice in the diabetic wild-type group and gene knockout group was significantly higher than that in the normal wild-type group and gene knockout group with statistical significance(P<0.01).In the two groups of diabetic mice,serum creatinine and urea nitrogen were significantly higher than those in the normal groupwith statistical significance(P<0.05).Comparison between the two groups of diabetic mice showed that the levels of creatinine and urea nitrogen in the gene knockout group were higher without statistical significance.Light microscopy showed that there was no obvious lesion in renal tissue in the normal group.There was no obvious lesion in glomeruli and tubule-interstitial in mice in the gene knockout group without diabetes.In diabetic wild-type mice,glomerular mesangial stromal hyperplasia and capillary basement membrane diffuse thickening were visible,and some formed nodular sclerosis.Moreover,there were lumen expansion in renal tubular,swelling in epithelial cells and vacuolar degeneration,as well as irregular thickening of the basement membrane.Furthermore,there were more deposition of extracellular stromal in renal interstitial,and more inflammatory cell infiltration.The above lesions were more serious in the diabetic gene knockout group compared to the diabetic wild-type group.Transmission electron microscopy observed that there was segmental thickening of capillary basement membrane in glomerular of diabetic wild-type and gene knockout mice,but there was no cellularity stromal hyperplasia in the mesangial area.Moreover,there were mass-like electronic compact deposition in the adjacent membrane area,changes of podocyte structure,extensive fusion of foot process,along with enlarged crack between foot processes.(4)There were more PPAR? and PTEN expressions in renal tissueof normal wild-type mice,while the expressions of PPAR? and PTEN proteins as well as mRNA of mice in the diabetes group and gene knockout group without diabetes decreased compared to the normal group.Moreover,protein expression was the lowest in diabetes combined with gene knockout group(P<0.05).E-cadherin expression of mice in the two groups of diabetes decreased significantly compared to the non-diabetes group,and the decrease was the most significant in diabetic gene knockout group.On the contrary,?-SMA and Collagen-IIIwere rarely expressed in mice in the normal group,and there was few expression in normal glucose gene knockout group,while the expression in the diabetes group increased significantly,and the increase was most significant in diabetic gene knockout group(P<0.05).Conclusion:(1)Administration of PPAR? agonist rosiglitazone or PPAR? overexpression can increase the expression level of PTEN in cells,as well as improve EMT of renal tubular epithelial cells and tissue fibrosis.(2)Administration of PPAR? inhibitor GW9662 or knockdown of PPAR? will decrease the expression level of PTEN in cells,and also aggravate EMT of cells and fibrosis.(3)Knockout of PPAR? gene in mice can decrease the expression level of PTEN as well as aggravate fibrosis of renal tissue.Part three The mechanism of PTEN in affecting EMT through regulating AKT and FAK pathways in diabetes and high glucoseObjective: To investigate the mechanism of PTEN in regulating AKT and FAK pathways through the activities of lipopolysaccharide and protein phosphatase as well as its effect on EMT together with tissue fibrosis in vivo and in vitro.Methods:(1)C57BL wild-type mice and conditional PPAR? knockout mice were selected as study subjects to set up normal wild-type group,normal gene knockout group,diabetic wildtype group and diabetic gene knockout group.Immunohistochemistry and immunoblotting were used to detect expression level of PTEN as well as the changes of AKT,p-AKT,FAK and p-FAK expressions in renal tissue of each group.(2)High glucose-induced NRK52 E cells were used to set up the control group,empty vector group,GFP-PTEN group,GFP-PTEN-G129 E group and PTEN-shRNA group..Immunoblotting and quantitative PCR were used to detect changes of proteins and mRNA of AKT and FAK pathways in each group of cells,as well as detect changes of EMT indicators.Scratch test was used to detect cell migration ability.(3)High glucoseinduced NRK52 E cells were used to set up the control group,GW9662 group,GW9662+overexpression PTEN group,rosiglitazone group and the rosiglitazone+PTENshRNA group.Immunoblotting was used to detect the changes of proteins in AKT and FAK pathways in each group of cells.Results:(1)PTEN expression of mice in the two groups of diabetes decreased significantly compared to the non-diabetes group,and the decrease was the most significant in the diabetic gene knockout group.On the contrary,the expressions of p-AKT,FAK and p-FAK were few in the normal group,which were also few in normal glucose gene knockout group,while they increased significantly in diabetes group,and the increase was the most significant in diabetic gene knockout group(P<0.05).However,there was no statistical significance in the expression level of AKT among each group.(2)The expressions of PTEN protein and mRNA increased significantly in GFP-PTEN group and GFP-PTENG129 E group,which were significantly more than those in the control group as well as the empty vector group(P<0.05).In GFP-PTEN group,p-AKT,FAK and p-FAK proteins decreased significantly,which were significantly lower than those in the control group and empty vector group,while FAK mRNA also decreased significantly(P<0.05).However,in GFP-PTEN-G129 E group,the expression of p-FAK was equivalent to that in the GFP-PTEN group,which were both lower than that in the control group and empty vector group,whereas expressions of p-AKT and FAK increased compared to that in the GFP-PTEN group,and there was no statistical significance with the control group and empty vector group.Moreover,there was no significant change in AKT and FAK mRNA.Changes of AKT protein were not statistically significant in the four groups.E-cadherin increased significantly in GFPPTEN group and GFP-PTEN-G129 E group,increased was the most significant in GFPPTEN group(P<0.05).Expression levels of Vimentin and ?-SMA proteins decreased significantly in GFP-PTEN group and GFP-PTEN-G129 E group,decreased was the most significant in GFP-PTEN group(P<0.05),which were opposite to E-cadherin expression.The migration ability decreased significantly in GFP-PTEN group and GFPPTEN-G129 E group.After transfection of PTEN-shRNA,protein and mRNA expression of PTEN decreased significantly compared to the control group and empty vector group,expressions of p-AKT,FAK anpd p-FAK increased significantly,and expression of FAK m RNA increased(P<0.05),while there was no significant change in protein and mRNA of AKT.E-cadherin decreased significantly in PTENshRNAgroup,while expressions of Vimentin and ?-SMA proteins increased significantly in PTEN-shRNA group(P<0.05).The cell migration ability enhanced significantly in PTEN knockdown group.(3)Compared with the control group,PTEN expression in GW9662 group reduced,while expressions of p-AKT,FAK and p-FAK increased significantly with statistical significance(P<0.05).PTEN expression in the GW9662+overexpression PTEN group increased significantly compared to GW9662 group,and expressions of p-AKT,FAK together with p-FAK decreased correspondingly with statistical significance(P<0.05).However,there was no significant difference in AKT among various groups.Compared with the control group,PTEN expression in rosiglitazone group increased,whereas expressions of pAKT,FAK and p-FAK decreased with statistical significance(P<0.05).PTEN expression reduced in rosiglitazone+PTENshRNA group compared to rosiglitazone group,and expressions of p-AKT,FAK and p-FAK increased correspondingly with statistical significance(P<0.05).There was no significant difference in AKTamong various groups.Conclusion:(1)PTEN can affect EMT and tissue fibrosis by regulating AKT and FAK pathways.(2)PTEN had a dual activity of lipid phosphatase and protein phosphatase.The lipid phosphatase activity can regulate the phosphorylation of AKT and FAK,as well as regulate the expression level of total FAK.However,the phosphatase activity can only regulate the phosphorylation of FAK,but it had no effect on total FAK.(3)The regulation of PPAR? on AKT and FAK pathways was mainly achieved through PTEN.Part four The correlation of the expression levels of PPAR? and PTEN together with AKT and FAK signaling pathways in renal tissue of diabetic patients with RTIF and renal functionObjective:To investigate the correlation of the expression levels of PPAR? and PTEN together with AKT and FAK signaling pathways in renal tissue of diabetic nephropathy patients with renal fibrosis and renal function.Methods: Renal tissues of patients with diabetic nephropathy and normal renal tissues were used as the study subjects to set up diabetic nephropathy group and normal control group,so as to observe pathological changes of renal tissue in each group.Immunohistochemical method was used to detect changes of the expressions of PPAR?,PTEN,p-AKT,p-FAK,E-cadherin,?-SMA and Collagen-III in the renal tissues of the two groups.Biochemical analyzer was used to detect the changes of renal function and related biochemical indicators in the two groups.In addition,correlation analysis was performed for the status of renal function and the above indicators.Results:(1)Morphology of renal glomerular in normal tissue was complete with clear structure,and there was no expansion and degeneration as well as necrosis in renal tubular.Moreover,basement membrane was integrity.Basement membrane of the glomerular capillary was thickened in patients with diabetic nephropathy,mesangial stromal hyperplasia occurred,and some formed nodular sclerosis.Lumen expansion in renal tubular,swelling as well as vacuolar degeneration in epithelial cells,irregular thickening of basement membrane,along with more extracellular stromal deposition in the renal interstitial were visible,and there were more inflammatory cell infiltration.(2)In normal renal tissue,there were more PPAR? and PTEN expressions,while expression levels of PPAR? and PTEN proteins decreased significantly compared to the normal control group(P<0.05).There was few p-AKT and p-FAK expression in the normal control group,while their expressions increased indiabetic nephropathy group compared to the normal control group(P<0.05).E-cadherin expression in the renal tissue in diabetic nephropathy group decreased significantly compared to non-diabetes group.On the contrary,there was few expression of ?-SMA and Collagen-III in renal tissue in the normal control group,which increased significantly in renal tissue in diabetic nephropathy group(P<0.05).(3)The clinical and biochemical parameters of the two groups showed there was no statistical significance in age,gender composition,BMI and proportion of smoking.The levels of blood glucose,serum creatinine,blood urea and 24 hours urine protein were significantly higher in patients with diabetic nephropathy than those in normal control group.The estimated glomerular filtration rate was significantly lower than that of normal control group(P <0.05).The relationship between eGFR and the above indicators were analyzed with the estimated glomerular filtration rate as the standard reflecting the degree of renal injury.The results showed that eGFR and PPAR?(r=0.588,P< 0.05)of the patients were positively correlated with PTEN(r=0.791,P< 0.05),but they were negative correlated with p-AKT(r=-0.572,P< 0.05)and p-FAK(r=-0.478,P< 0.05).Conclusion:(1)The expressions of PPAR? and PTEN decreased in renal tissue of patients with diabetic nephropathy,while p-AKT and p-FAK increased,and the change was associated with the aggravation of TIF and the deterioration of renal function.(2)PPAR? and PTEN can be used as a marker of kidney damage and a therapeutic target for diabetic nephropathy to some extent. |
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