| To investigate the correlation between interleukin-2(IL-2)and leptin in DKD patients,clarify the effect of leptin on IL-2 expression in DKD,and confirm that leptin mediate renal tubular injury by upregulating IL-2 expression.1There were 48 patients with DKD from the Department of Nephrology of the People's Hospital affiliated Guizhou University School of Medicine and 24 healthy subjects from the physical examination were enrolled from July 2017 to July 2018.General information,blood,and urine samples were collected.There were 105 Cytokines of serum and 38 renal injury markers of urine were detect by arrays of inflammatory factors and renal injury markers,respectively.The correlation between IL-2 and leptin was analysed by Spearman correlation.2 DKD model induced by Streptozocin(STZ): 63 C57 mice with about 18 g body weight were randomly divided into 3 groups(Control,Con;Diabetic kidney disease,DKD;DKD with IL-2 receptor antibody;DKD+ IL-2Ri).DKD and DKD+ IL-2Ri mice were fed with high-fat and sugar for 8 weeks,then 50mg/kg STZ were intraperitoneally injected.Con mice were fed with ordinary feed and intraperitoneally injected with equal volume of STZ solvent.The mice with random blood glucose>16.7 mmol/L were determined as Type 2 Diabetes mellitus(T2DM).The DKD+ IL-2Ri mice were given injection of 50?g IL-2 receptor blocker for once every 7 days,lasting 4 times.Leptin resistance mice model: There were 63 C57 BLKS mice at the age of 5 week-old were leptin long receptor gene mutant(Leprdb mice);21 wild-type mice from the same litter(WT group),Leprdb mice were divided into 3 groups,21 mice/ group,(Leprdb,Leprdb+ Ins;and Leprdb+ IL-2Ri).The Leprdb+ Ins mice were given intraperitoneal insulin injection 1-5U/ 24 h to control the fasting glucose at < 7.8mmol /L.Blood,urine,and kidney samples were collected after 8,12,and 16 weeks.The blood glucose,creatinine,glycosuria,and urea were detected by biochemical kit.Pathological changes of renal tissue were detected by Hematoxylin-eosin(HE),dyeing Period-Schiff(PAS),Massion,and Sirius Red(SR)stain.CD68,F4/80,E-cadherin,and ?-SMA were detected by Immunohistochemistory and in situ hybridization in renal tissue.The percentages of IL-2 cells were determined by flow cytometry.3 NRK-52 E was cultured with DMEM medium containing 5% fetal bovine serum,1% mixture of streptomycin and penicillin at conditions of 37 ?,95% humidity,5% CO2.The leptin concentration and time of treatment were determined: NRK-52 E cells inoculated with the dose of 25,50,100,150,and 200 ng/ml leptin for 48 and 72 h.NRK-52 E was treated with 150 ng/ml leptin for 24,48,72,and 96 h,respectively.NRK-52 E could be divided into four groups,NRK-52 E were treated with 150 ng/ml of leptin,25 ?mol receptor antagonist,and150 ng/ml of leptin with 25 ?mol receptor antagonist for 48 h and 72 h,NRK-52 E cultured in DMEM as control.ELISA and q PCR were applied detecting the expression of IL-2,Western blotting was used to detected p65,STAT3,and p STAT3 protein expression.4 The leptin and it's potential receptors on the renal tubular epithelial cells were analysed by Molecular docking.1Compared with healthy,the expression of IL-2 and leptin in DKD were significantly increased,and with a positive relation of leptin and IL-2 concentration.2 In both models,blocking the IL-2 receptor did not reduce the random blood glucose,urine glucose,and the ratio of kidney/body of mice.Urea and 24 h urinary protein were significantly increased at each stage of DKD,and decreased with intervention of insulin at 8 and 12 weeks,but not at 16 weeks.The urea and urinary protein levels were reduced by blocking the IL-2 receptor at 12 and 16 weeks.The urinary protein creatinine ratio was increased in STZ-induced DKD at 12 and 16 weeks,decreased by blocking IL-2 receptor at 12 weeks,increased in the leptin-resistant mice at 16 weeks,and were reduced by insulin and blocking of IL-2 receptors.Renal pathology showed that the glomerular dilatation,mesangial matrix amplified,tubule swelling,and exfoliation of tubule epithelium occurred in the two models at all stages.The pathological changes of renal tissue were reversed by insulin and blocking IL-2 receptor.The mesangial index of STZ-induced mice increased significantly at each stage and decreased significantly after blocking IL-2 signal,while the difference of mesangial index between leptin resistant mice groups was not statistically significant.The glomerular area was increased in STZ-induced DKD at 12 and 16 weeks,decreased by blocking IL-2 receptor at 12 weeks,glomerular area increased at all stages in leptin-resistant mice,but it was decreased by insulin at 8 and 12 weeks,and not blockade of IL-2 receptor.The results of Masson stain and Srius Red staining showed that there were significant increases of fibrous collagen in glomerular and tubulointerstitium at all stages of two model,which were significantly improved by insulin intervention and blockade of IL-2 receptor.However,quantitative analysis of collagen fibers showed that the collagen fibers was increased statistically in STZ-induced DKD at only 16 weeks,and could be reduced by blockade of IL-2.Collagen was significantly increased in leptin resistant mice at 12 and 16 week,could not be reduced insulin,but could be reduced by blocking IL-2 receptor.The expression of e-cadherin was significantly decreased in both models at each stage of DKD,and not changed by insulin intervention and blocking IL-2 receptor.The-SMA protein was significantly increased in both DKD models at each stage,was down-regulated insulin intervention and blocking IL-2 receptor at 12 and 16 weeks.The results of HE stain showed that the number of nucleated cells in renal tissue increased significantly in each stage of STZ-induced DKD.The number of nuclear cells increased significantly at each period,and decreased after blocking IL-2 receptor at 16 weeks.There was no significant increase in the number of nucleated cells in the kidney of leptin resistant mice.The number of nucleated cells increased significantly in the kidney of leptin-resistant mice at 8 and 12 weeks,not decreased by blocking IL-2 receptor.The number of CD68+ cells of renal tissue was increased significantly in the two DKD models,reduced by blocking IL-2 receptor in STZ-induced DKD at 12 weeks,leptin resistance DKD at 12 and 16 weeks,and not by insulin.In the two DKD models,the number of F4/80+ cells increased in each period.The number of F4/80+ cells was decreased by blocking IL-2 receptor in STZ-induced DKD,but decreased in leptin resistant mice.3The results showed,compared with the control group,The IL-2 expression up-regulated of NRK-52 E cells after treating with leptin and leptin+receptor antagonist culture secreted.The expression of IL-2 was not significantly different from between NRK-52 E cells treated with leptin and leptin+receptor antagonist.The expression of the downstream protein molecule(STAT3,p STAT3,and p65)of leptin receptor,had not difference between the control and leptin groups.However,the expression of p STAT3 and p65 were significantly lower in leptin receptor antagonist group than that of the leptin group and the control group.4There were 8 antigenic peptides,11 MHC-I antigenic peptides,and 4 MHC-II antigenic peptides were produced.It was found that 4 antigenic peptides were closely bound to TLR2 by molecular docking.The expression of IL-2 in DKD was positively correlated with leptin.It was verified that leptin induced the renal tubular injury of DKD by upregulating IL-2 expression and IL-2 was involved in renal tubular injury. |
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