| Background & ObjectiveDiabetic nephropathy is one of major chronic complications of diabetes,which is characterized by glomerular hypertrophy,hyperfiltration,basement membrane thickening in the early stage and excessive deposition of extracellular matrix, glomerulosclerosis,renal failure in the end stage.It has been the principal cause of end-stage renal failure in developed countries,which is also increasing in our country.The study about pathogenesis of diabetic nephropathy concentrated on metabolic disorders,hemodynamic abnormalities.However,recently the role of inflammation has been paid more and more attention to.The infiltrative inflammatory cells and kidney resident cells can produce proinflammatory cytokines,chemotatic factors and adhesion molecules.These cytokines amplify the inflammation by autocrine and paracrine, which finally lead to renal lesions.As a cytokine found in 1995,IL-18 has many biologic activities.It can promote excretion of other cytokines,like IL-8,TNF-α,IL-1β,ICAM-1.These cytokines can also impact the production of IL-18,thus cascade reaction is formed. Recently the role of IL-18 in pathogenesis of DN has been paid more and more attention to,but there is few study about the change of IL-18 in nephridial tissue and the relationship with other cytokines in progession of DN.As far as I am concerned,no report has been found in our country.TNF-α,a significant factor of cytokine network,is mainly produced by mononuclear macrophage,vascular endothelial cell and many kinds of cells in kidney,including mesangial cell and epithelial cell of proximal convoluted tubule.It participates the onset of DN.As an important adhesion molecule,ICAM-l expresses in the surface of many cells after stimulus,such as vascular endothelial cell, mesangial cell.It can enhance leucocyte to adhere and through vessel wall,to release more inflammatory cytokines.It is the molecular base of cell infiltration and proliferation,extracellular matrix expanding and glomerulosclerosis.NF-κB is a pleiotropia regulator,which plays an important role in cell signal transmission and gene expression.The activation of NF-κB maybe a key player in inflammatory cytokine network and interaction of many cytokines.It plays an important role in progression of DN.As a new and high-performance immunosuppressive agent,MMF has been widely used in prevention and treatment of graft rejection.Recently,following the futher research of its pharmacological action,its application in nonimplantation kidney disease has been paid more attention to.Vitro experiments have discovered that MMF can inhibit macrophage and mesangial cell proliferation and production of many cytokines.On account of above theories,this study observed the expression of IL-18, TNF-α,ICAM-1 in different stages of renal glomerulus and the relationship between themselves in diabetic rats induced by injection of streptozotocin,in order to understand renal inflammatory lesion process and explore the role of cytokine network in DN development.This study also investigate the superior protective effects of combination of PDTC with MMF on expression of IL-18,TNF-α,ICAM-1 and the possible mechanism,which provide a new way for DN treatment.MethodsAfter 70 SD rats were fasted for 12 hours,they were divided into two groups at random.The rats in one group (n=58) were performed peritoneal injection of 60mg.kg-1STZ and the rats in the other group (n=12) were performed injection of sham citrate bufer with the equal volume.72 hours later,the rat whose blood glucose level was above or equal 16.7mmol/L was identified as diabetic rat.The diabetic rats were randomly divided into 4 groups:diabetes with no treatment (D group),diabetes treated with PDTC (P group),diabetes treated with MMF (M group),diabetes treated with PDTC and MMF (P+M group),and normal rats were controlled(C group). 100mg .kg-1PDTC and (or) 10mg.kg-1 MMF was perfused into stomach once everyday in corresponding group(dissolved in degermed distilled water) and equal volume distilled water was perfused into stomach once everyday in C and D group. Half rats in each group were killed 4 weeks later,and the rest were killed 8 weeks later. Before the rats were killed,they were put in metabolism cage to gather 24 hour urine, in order to detect 24 hour urinary protein(24hUPro) .After the rats were anesthetized, blood from caval vein was collected and serum was separated in order to detect blood glucose(BG),blood urea nitrogen(BUN),serum creatinine(Scr).Kidneys were weight in order to measure kidney weight/body weight ratio (KI) after rats were killed.Kidney pathological changes were observed by light microscope and electron microscope. Expression of interleukin-18(IL-18),tumor necrosis factor-alpha(TNF-α) and intracellar adhesionmolecule-1(ICAM-1) in renal glomerulus were detected by immunohistochemical straining.Correlation analysis were used to evaluate the relationship between expression of IL-18/TNF-α,ICAM-1 and BUN,Scr,24hUPro, also the relationship between the three inflammatory cytokines.Results1.Biochemical indicators For 4 weeks,compared with C group,BG,KI,24hUPro, Scr were significantly higher in D,P,M and P+M group (P<0.05), while body weight was lower(P<0.05).There was no statistic difference of BUN. Compared with D group, there was no statistic difference of above biochemical indicators in P,M and P+M group,except 24hUPro was lower in P+M group (P<0.05).Compared with P+M group, there was no statistic difference of biochemical indicators in P,M group (P>0.05).For 8 weeks,BG,BUN,Scr,24hUPro,KI were significantly higher in D,P,M and P+M group than those in C group(P<0.05),while body weight was lower(P<0.05).Compared with D group,body weight was higher in P,M and P+M group (P<0.05),while BG,KI, 24hUPro,Scr were lower(P<0.05),and there was no statistic difference of BG(P>0.05). Body weight of P+M group was higher than that of P,M group,while 24hUPro was lower (P<0.05).KI,24hUPro,BUN,Scr of D group in 8 weeks were significantly higher than those in 4 weeks.2.Immunohistochemical straining For 4 weeks,expression of IL-18,TNF-α, ICAM-1 in D,P,M and P+M group were higher than those in C group(P<0.05).There was no statistic difference between D group and P,M,P+M group(P>0.05),also no statistic difference between P,M group and P+M group(P>0.05).For 8 weeks, expression of IL-18,TNF-α,ICAM-1 in D group were much higher than those in C group(P<0.05).They were reduced in P,M and P+M group compared with D group, but still higher than those in C group(P<0.05). Expression of IL-18,TNF-α,ICAM-1 in P+M group were lower than those in P group(P<0.05),while expression of IL-18, TNF-αwere lower than those in M group(P<0.05).Though there was no statistic difference of ICAM-1 between M and P+M group,it has decreasing tendency. Expression of IL-18,TNF-α,ICAM-1 in D group For 8 weeks were significantly higher than those for 4 weeks.3.Correlation analysis Average integrated optical density of positive area in renal glomerulus of IL-18,TNF-α,ICAM-1 were positively correlated with BUN,Scr and 24UPro(P<0.05),and average integrated optical density of positive area in renal glomerulus of the three inflammatory cytokines were related with each other(P<0.05).4.Kidney pathological changes HE stain of rat renal glomerulus in C group shows the normal glomerulus.For 4 weeks,There were larger volume of renal glomerulus and more mesangial cells and matrixes in D group,with segmented shape.For 8 weeks,pathological changes were more aggravated.Electron microscope shows normal basement membrance and foot process in C group.In D group, following the diabetes course prolonged,glomerular basement membrance became thickened and foot process became mixed. Compared with D group,pathological changes were attenuated in P,M group,especially P+M group.Conclusions1.High blood glucose induces high expression of IL-18,TNF-α,ICAM-1 in renal glomerulus of rats.2. IL-18,TNF-α,ICAM-1 play an important role in the development of diabetic nephropathy. 3. Both PDTC and MMF have renal protective effects partly through suppressing the expression of IL-18,TNF-α,ICAM-1,independent of glycometabolic regulation.4. The combination of PDTC with MMF can arrest proteinuria earlier,attenuate pathological changes and suppress expression of inflammatory cytokines better,which shows superior protective effects on kidneys of diabetic rats and brings us a new thought for prevention and treatment of early diabetic nephropathy.
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