The Expression And Relationship Between Midkine And HGF In Tubular-interstitial Of Rat Model Of Type2Diabetic Nephropathy

Objectives:Diabetic nephropathy is one of the chronic complication of the diabetes mellitus,which is now the leading cause of end stage renal diseases.The exact pathogenesis of diabetic nephropathy remain unclear:glomerular pathological caused by abnormalities in glucose metabolism, oxidative stress, disturbance of lipidis metabolism, cytokines and genetic background contribute significantly to the development of diabetic nephropathy. In recent years many studies have shown that the injury of nenal tubular especially serious in diabetic nephropathy and there is a close correlation between the degree of worsening renal function and renal tubular damages. Mikine is a new member of heparin-binding cytokine,right now,much more MK research focus on the acute renal damage,but little on the chronic renal damage,especially on diabetic nephropathy.New research has demonstrated that MK is induced by high glucose and MK can promore migration macrophages and neutrophilis,mediated them though endothelium into kidney interstitium and cause kidney interstitial inflammation and then cause kidney fibrosis,MK play an important role in the development of diabetic nephropathy.hepatocyte growth factor is a kind of induce、regulate、antifibrotic factor,which play a role in protection of renal fibrosis.At present,it is not clear about how midkine paticipate in the diabetic nephropathy and the relationship between midkine and hepatocyte growth factor. Midkine antisense oligodeoxynucleortides is a compound that can prevent diseases at mRNA level, and can decrease the synthesis of MK by intravenous injection.This experiment is going to observe the expression of midkine(MK) and hepatocyte growth factor (HGF)in the rat model of type2diabetic nephropathy(T2DN) and explor their relationships between them.In addition,with the antisense oligodeoxynucleortides intervene the diabetic nephropathy cat, reveal the role of MK AS-ODNs in the treatment of diabetic nephropathy.Methods:Sixty six male Sprague-Dawlry rats were randomly divided into two groups:control group and model group.in model group,T2DN were induced by high-glucose and high-fat diet and intraperitoneal STZ after unilateral nephritic excision,(two rats died after postoperation). while normal control group were treated with general feed and intraparitoneal citric acid buffer after incision surgery. Diabetic rat was confirmed by fasting blood glucose大于等于16.7mmol/L after three days of STZ administration(four cases failed to achieve the standard of diabetes were excluded).Diabetic rats were randomly divided into two groups:diabetic nephropathy group and MK AS-ODNs treatment group. Each group was divided into6th week group and the12th week group. After STZ injected6th week and12th week, MK AS-ODNs was injected to the treatment group at dosage of lmg/kg,normal control group and diabetic nephropathy group received saline at dosage of0.16ml/kg. Four days after6th week and12th week,fasting blood glucose(FBG) and24hours urine protein(24hUMA) were detected and then rats were killed to collect the kidney,the ratio of kidney weigh to body weigh (KW/BW) were calculated. The tubular-interstitial pathomorphology changes were examined with HE staining and the expression of MK and HGF intubular-interstitial were determined by immunohistochemistry and real time PCR.Results:(1) Four days after6th week and12th week,in normal control group the FBG,KW/BW and24hours urine protein were obviously different compared with diabetic nephropathy group and MK AS-ODNs treatment group.(2) HE staining: normal control group had regular renal tubular structure, There were severe structural alterations of tubular-interstitium in diabetic nephropathy group. MK AS-ODNs treatment group is better than diabetic nephropathy group.(3) Immunohistochemistry: MK and HGF expressed in all of the three groups;compared with normal control group, at6th week and12th week there were more MK and HGF espressed in diabetic nephropathy group (P<0.01); compared with MK AS-ODNs treatment group, the expression of MK was significant increase in diabetic nephropathy group at6th week (P<0.01),while the expression of HGF is no significant change (P>0.05). with the course of diabetic nephropathy being longer, in diabetic nephropathy group the express of MK were significant increase compared with MK AS-ODNs treatment group at12th week,but the express of HGF is decreased (P<0.01)(4) RT-PCR:in accord with Immunohistochemistry.(5) Pearson correlation analysis:In diabetic nephropathy group,there were negative correlations between MK and HGF (r=-0.525,P<0.01)Conclusions:(1) There were pathological changes in kidney tissue of diabetic nephropathy group which indicating that there were renal interstitial fibrosis in diabetes.(2) The expression of MK increased in diabetic nephropathy group, while the expression of HGF decreased,which suggest that MK and HGF interaction paticipate in the pathogenesis of diabetic nephropathy.(3) MK AS-ODNs can inhibit the expression of MK and increase the expression of HGF and then slow down the progress of diabetic renal fibrosis.