Preliminary Study On The Relationship Of IL-2 With Renal Fibrosis In Diabetic Kidney Disease

Objective: To investigate the expression of interleukin-2(IL-2)in different stages of Diabetic Kidney Disease(DKD),and to explore the relationship of IL-2 with renal fibrosis in DKD.Methods: 1.In vivo: ?1 Type 1 diabetes: single high-dose streptozotocin(STZ)was used to induce type 1 diabetes in C57BL/6J mice [four groups: control group,type 1 diabetic nephropathy(T1DN)group,Insulin group and benazepril(ACEI)group].?2 Type 2 diabetes: C57BL/6J mice were reared with high-fat and high-glucose diet for 8 weeks,then were injected 30 mg/kg STZ intraperitoneally to induce type 2 diabetes [three groups: normal control(NC)group,type 2 diabetic nephropathy(T2DN)group and high-fat and high-glucose(HFG)group].After the establishment of the model of type 2 diabetes,each group was further divided into five periods: 1 week,4 weeks,8 weeks,12 weeks and 16 weeks.Anothor experimental animals model were C57BLKS/J-db/db mice which were spontaneous type 2 diabetes animal model.The mice were divided into three groups: db/m(control)group,db/db(T2DN)group and insulin(db/db+insulin)group,each of which was divided into four periods: 6 weeks old,8 weeks old,12 weeks old and 16 weeks old.Renal pathological changes in mice were examined by Hematoxylin-Eosin(H&E)staining and Masson staining.The localization and expression of IL-2 m RNA and protein in kidney tissues were respectively detected by in situ hybridization and immunohistochemistry.The expression of Snail1,HSP47,?-SMA,E-cad and Col-1 in kidney tissues were detected by immunohistochemistry.The correlation of IL-2 with fibrotic proteins was analyzed by Pearson analysis.2.In vitro: NRK-52 E cells were divided into normal control(NG)group and high glucose(HG)group.NG group was given low-sugar medium(5 m M glucose)and high-sugar group was given high-sugar medium(25 m M glucose).Cells and cell culture media were collected at 12 h,24 h,48 h,72 h and 96 h.The expression of IL-2 in NRK-52 E cell culture medium at different time points were detected by ELISA.Western-Blot was used to detect the levels of Snail1,HSP47,CTGF,?-SMA,E-cad,Col-1,MMP9 and TIMP2 in NRK-52 E cells.Results: 1.Renal pathological changes in T1 DN mice were as follows: glomerular mesangial expansion,glomerular pyknosis and sclerosis,KW nodules formation,interstitial inflammatory cell infiltration and renal interstitial fibrosis.Compared with the control group,the expression of IL-2 was significantly decreased in the T1 DN group(P<0.05),the expression of E-cad was significantly decreased,and the expression of ?-SMA and Col-1 was significantly increased(P<0.05).The interventional effects of insulin and ACEI were not significant.Pearson analysis showed that there was a negative relationship of IL-2 with renal fibrosis in T1 DN mice,suggested that the decreased IL-2 might be associated with renal fibrosis in T1 DN.2.In T2 DN mice,glomerular hypertrophy,basement membrane thickening,mesangial dilatation,interstitial inflammatory cells infiltration occurred in the early stage;glomerular pyknosis and sclerosis,KW nodules formed,tubular injury,vacuolization and particle degeneration,renal interstitial fibrosis occurred in the middle and late stage.IL-2 m RNA and protein in renal tissue were mainly expressed in renal tubular epithelial cells.The expression of IL-2 in T2 DN group was first increased and then decreased with time.Of note,compared with control group at the same time,the expression of IL-2 in T2 DN group was still increased(P<0.05).The expression of E-cad was decreased,and Col-1,?-SMA,HSP47 and Snail1 were increased.In addition,Pearson analysis showed that there was a positive relationship of IL-2 protein with fibrosis in T2 DN mice,indicated that increased IL-2 might be associated with the development of renal fibrosis in T2 DN.3.Compared with NG group at the same time,the level of secreted IL-2 induced by HG-treated NRK-52 E cell cultures was increased with time,as well as Snail1,HSP47,CTGF,?-SMA,Col-1,MMP9 and TIMP2(P<0.05),while the expression of E-cad in HG group was decreased.It was indicated that hyperglycemia may be related to increased IL-2 levels in DKD.Conclusion: 1.The expression of IL-2 in renal tissue of T1 DN mice was decreased,while increased in T2 DN mice and high glucose-treated NRK-52 E cells,which might be associated with renal fibrosis in DKD.2.IL-2 might have played different roles between T1 DN and T2 DN on the development of renal fibrosis.3.During DKD progression,high glucose could be an important factor in the changing level of IL-2,thus,regulating the expression of IL-2 could possibly affect the progression process.