| Objective: To explore the effect of IGF-1R inhibitor on renal tubular epithelial-to-mesenchymal transition(EMT)of diabetic kidney disease(DKD)by the pathway of IGF-1/ Snail1(Snail zinc finger 1 protein,Snail1).Methods: In vivo: 1.30 mice(C57/BL6)were randomly divided into 5 groups:(1)Control group: Mice fed with general diet;(2)DKD group: Mice fed with high fat and glucose diet for 8weeks and were injected into enterocoelia with streptozotocin(STZ,100mg/kg body weight),then continue to fed with high fat and glucose diet for 8 weeks;(3)IGF-1R group: Mice were given IGF-1R inhibitor(30mg/kg body weight every 48 h for 8weeks by gavage);(4)Angiotensin-converting enzyme inhibitors(ACEI)group:Mice were administrated benazepril(10mg/kg body weight every 48 h by gavage for 8weeks);(5)Insulin group: Mice were administrated 1-2U insulin subcutaneous injection every 48 h for 8 weeks.2.Routine biochemical tests were used to detect blood glucose,urine creatinine(Ucr)and 24 h urinary protein.3.Mice were euthanized at 16 weeks and samples of renal tissues were collected and embedded in paraffin.Histopathology examination:(1)Periodic acid schiff(PAS)staining and Sirius Red(SR)staining were applied to detect the pathological changes of renal tissues.(2)In situ hybridization and immunohistochemistry were carried out to detect the expressions of Snail,IGF-1,ED-1 and Fibronectin(FN)mRNA and protein.In vitro: 1.NRK-52 E cells grouped as follows:(1)Control group(5mmol/L glucose);(2)High glucose group(25mmol / L glucose);(3)NT group(25mmol / L glucose +non-targeting siRNA);(4)IGF-1 siRNA group(25mmol / L glucose +IGF-1 siRNA).2.In situ hybridization and immunofluorescence were applied for examination of the expressions of Snail1,IGF-1,ED-1 and FN mRNAs and proteins after cultured 48 h and 72 h,respectively.One way ANOVA was used to data analysis.Results: In vivo:1.Compared with the control group,the blood glucose(23.14 ± 2.04 mmol/L vs7.33 ± 0.92 mmol/L),the urinary protein/creatinine ratio(697.67 ± 168.78 μg / mg vs97.69 ± 10.68 μg / mg),the urinary protein excretion rate(257 ± 47.56 μg / 24 h vs 25± 2.44 μg/24h)were significantly increased,the difference was statistically significant(P<0.05).Compared with the DKD group,the levels of the urinary protein/creatinine ratio(130.89 ± 27.86 μg/mg)and the urinary protein excretion rate(93 ± 7.98 μg/24h)in IGF-1R group all dramatic declined(P<0.001);the blood glucose(9.65±0.74mmol/L),the urinary protein/creatinine ratio(269.40 ± 13.98 μg/mg)and the urinary protein excretion rate(204 ± 35.98 μg/24h)in Insulin group were significantly lower than those in the DKD group(P<0.05).2 In the control group,the renal tubular epithelial cells of the control group were closely connected with each other,arranged neatly,the basement membrane was intact,the basal membrane was complete,there was no inflammatory cell infiltration and collagen fibril deposition in the tubulointerstitium.DKD group showed renal tubular hypertrophy,renal tubular epithelial injury,renal tubular destruction,a lot of collagen fiber deposition in the interstitium(P<0.01).Compared with DKD group,the renal tubular structure of IGF-1R group was basically complete,the degree of hypertrophy was significantly reduced,and the deposition of interstitial collagen was significantly decreased(P<0.01).There was no significant difference between ACEI group and insulin group(P>0.05).3.The expression of IGF-1,Snail1 and FN mRNA and protein was significantly increased in DKD mice compared with the control group(P<0.05),and the expression of ED-1 mRNA and protein was significantly decreased(P<0.05).Compared with DKD group,the expression of Snail1 and FN mRNA and protein in IGF-1R group were significantly decreased(P<0.05),and the expression of ED-1mRNA and protein was significantly increased.In vitro experiments: 1.The silencing efficiency of IGF-1 mRNA was 61.1%;2.The expression trends of IGF-1,Snail1,FN and ED-1 mRNA and protein in high glucose group renal tubular epithelial cells consistented with DKD group of mice.The expression of Snail1 mRNA and protein in IGF-1 siRNA group were significantly decreased(P<0.01),the expression of FN mRNA and protein was significantly decreased(P<0.05),and the expression of ED-1mRNA and protein was obviously increased.Conclusion: 1.Renal tubular EMT attenuated by inhibition of IGF-1R in DKD.2.Intervention of IGF-1/IGF-1R reduced renal tubular EMT through down-regulation the expression of Snail1.3.Regulation of IGF-1/Snail1 pathway could be an effective therapeutic target for the prevention of DKD. |
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