| BackgroundDiabetic kidney disease (DKD) is a major complication of diabetic microangiopathy with early lesions in the glomeruli. Studies in recent years showed that the onset and pathogenesis of tubulointerstitial lesions in DKD were independent. The development of tubulointerstitial lesions and renal interstitial fibrosis are mone closely related to the change of renal function. Renal interstitial fibrosis is an independent risk for DKD progression which is involved in local renal inflammation. The type1and type2diabetes have different pathogenesis but both are associated with cellular immunity. Is there any difference between them in the pathogenesis of DKD? The purpose of this study is to observe the changes of interleukin-17A level in diabetic patients, to investigate the relationship among the expression of interleukin-17A and TGF-β1, NF-κ B-p65, a-SMA in DKD through diabetic rat model and to explore the role of interleukin-17A in diabetic tubulointerstitial injury.Part ⅠObjective To evaluate the interleukin-17(IL-17A) level in the type1and type2diabetes mellitus and analyze the relationship between IL-17A and diabetic nephropathy.Methods39type2diabetes mellitus patients (T1DM group,21males,18females, mean age (31±13) years),40type2diabetes mellitus patients (T2DM group,28males,12females, mean age (36±8) years) were admitted to endocrinological department from2009.1to2010.12.36healthy volunteers (control group,19males,17females, mean age (29±12) years) were from Health Examination Department at the same time. The diagnosis of diabetes is according to the WHO standards (1999version). Levels of interleukin-17A (IL-17A) and B cell activating factor both in plasma and urine were measured by enzyme linked immunosorbent assay (ELISA). Urinary albumin/creatinine ratio was analyzed.Results There was no significant difference in duration of disease, glycosylated hemoglobin levels, triglyceride and low density lipoprotein cholesterol in the TI DM group and T2DM group, but age of patients in T2DM group were older than that in TI DM group. Plasma Interleukin-17A (IL-17A) and B cell activating factor level of TI DM group were significantly higher than Control group and also higher than T2DM group. In comparison with the control group, plasma IL-17A and B cell activating factor level of the TI DM group were slightly higher without statistical difference. Urine IL-17A and B cell activating factor level of both TI DM and T2DM group were higher than Control group. Clinical albuminuria group had highest Urine IL-17A level followed by microalbuminuria group, and the lowest was detected in the normal albuminuria group. Pearson analysis showed that there was positive correlation between urine IL-17A level and urinary albumin/creatinine ratio.Conclusion The aberrant increase of IL-17A may play an important role in the development of both T1DM and T2DM through stimulating secretion of B cell activating factor to affect the survival of B cell.Part ⅡObjective To establish rat model of T1DM and T2DM and to evaluate change of IL-17A level in blood and urine of animals and its expression in kidney.Methods90male Sprague-Dawley rats with1week adaptive feeding were randomly divided into three groups after fasting for12h and weighted:Control group (20rats), type1diabetic rat group (T1DM group,35rats),and type2diabetic rat group (T2DM group,35rats).T1DM group was induced by intraperitoneal injection of50mg/kg streptozoticin (STZ)(0.1mmol/L; pH4.2). T2DM group was fed with a diet enriched with sugar and fat (ordinary feed:sugar:Lard:cholesterol: cholic acid salt=66.5:20:10:2.5:1) for4weeks and induced by intraperitoneal injection of25mg/kg streptozoticin (STZ). The diabetes models were confirmed by checking blood sugar (>16.7mmol/L) and urinary glucose (3+for3days) after1week. Serum and urine IL-17A were measured with enzyme-linked immunosorbent assay (ELISA) at the end of2nd an4th week after models were established, The expression of IL-17A in the kidney were assessed by immunohistochemisty staining and Western blotting at the end of4th week.Results In comparison with control group, serum cholesterol, low density lipoprotein,24hours urinary albumin excretion rate and blood sugar were increased in T1DM and T2DM group and there was no statistical difference between diabetes group. In comparison with control group, the serum and urine IL-17A level were obviously higher in diabetic rats. The urine IL-17A level of T1DM group at the end of2nd week was more significantly increased comparing with T2DM group. Immunohistochemistry showed no IL-17A expression in normal renal tissues; there was positive staining of IL-17A at proximal tubule, renal interstitial region of diabetic rat. Correlation analysis showed that IL-17A of renal tissue in diabetic rats was positively correlated with the expression of IL-17A in the urine.Conclusion The serum and urine IL-17A level were increased in both TIDM and T2DM rats. The aberrant increase of IL-17A expression may play an important role in diabetic tubulointerstitial jnjury.Part ⅢObjective To investigate the relationship between the expression of IL-17A and growth factor-β1(TGF-β1), nuclear factor-kappa B-p65(NF-κBp65), a-smooth muscle actin (a-SMA) in diabetic kidney. To analyze the role of IL-17A in renal tubulointerstitial injury.Methods TGF-β1,NF-κB-p65and a-SMA expression in renal tissue of TIDM and T2DM rats were semi-quantitative analyzed with enzyme-linked immunosorbent assay (ELISA) at the end of4th week after models established. They were measured by Western blotting.Results TGF-β1and a-SMA expression were observed in both glomeruli and renal tubule of diabetic rats, There was no NF-κBp65expression in glomeruli of diabetic rats but positive nuclei staining in renal tubules. In comparison with control group, TGF-β1, NF-κBp65and a-SMA expression were obviously increased in TIDM and T2DM group. In comparison with T2DM group, TGF-β1expression was decreased in TIDM group; There was no statistical difference between diabetic groups for NF-κBp65and a-SMA. Correlation analysis showed that IL-17A expression was positively correlated with NF-κBp65and a-SMA expression in renal tissue of both diabetic groups; There was a significantly positive correlation between TGF-β1and IL-17A expression in renal tissue from T2DM group.Conclusion The aberrant increase of IL-17A expression may play an important role in early diabetic tubulointerstitial inflammation and fibrosis. The mechanism of IL-17A and TGF-β1in the development of type1and type2diabetes were not the same.SummaryOur results demonstrate that tubulointerstitial inflammation and renal interstitial fibrosis were independent risk factor for the progression of DKD. Although the pathogenesis of type1and type2diabetes were different, abnormal changes of inflammatory and fibrogenic factors appeared in both of types. The present study found that urine IL-17A was increased in both type1and type2diabetic patients. The serum level of IL-17A was increased only in type1diabetes and there was no difference between type2diabetes and healthy control group. For animal models, serum and urine IL-17A level was increased in diabetic rats, which were more obvious in T1DM group. There was positive staining of IL-17A in proximal tubules and renal interstitium of diabetic rats, which was mainly expressed in the cytoplasm. There was no IL-17A expression in glomeruli. Correlation analysis of diabetic rats showed that the expression of IL-17A in renal tissue was positively correlated with that in urine and NF-Kb-p65and a-SMA expression in renal tissue. There was no positive correlation between IL-17A and TGF-β1in diabetic renal tissue of TIDM group. The expression of TGF-β1and IL-17A was positively correlated in T2DM group. This study indicates that IL-17A may play an important role in the development of early diabetic renal tubulointerstitial inflammation and fibrosis, and the roles of IL-17A and TGF-β1in the development of type1and type2diabetes may be different. Further study is still needed to uncover the undergoing mechanism of signal transduction which regulates various cytokines. |
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