Effect Of Cordyceps Sinensis On The Renal Tubular Epithelial-myofibroblast Transdifferentiation In DN

Background and objectives:The rapid increased incidence of Diabetic nephropathy(DN)has become the important reason of the rising incidence of End stage ranal disesae(ESRD).Tubulointerstitial lesion is an essential pathological basis in Diabetic nephroapthy.The basic pathological character of which is excessive extracellular matrix(ECM)accumulation in the renal interstitium.It is considered that the renal interstitial ECM is mainly secreted by the myofibroblast(MyoF).Researches have shown that tubular epithelial-myofibroblast transdif- ferentiation(TEMT)can occur to the renal tubular epithelial cells in pathological condition.During TEMT,renal tubular epithelial cells lose their original phenotypic characteristics and gain MyoF characteristics.Researchers have found that TEMT play an important role in the occurrence and progress in diabetic nephropathy.It is of great significance to study the mechanism of TEMT and find effective means to block it.TGF-β₁ is an essential factor in the initiation and promotion of TEMT.The biological functions of TGF-β₁ are extensive and complex, which is shown the diversity of positive and negative effects.Inhibiting only TGF-β₁ as an upstream factor could bring negative side effects together with positive effects.Therefore,researchers' attention has been turned to a new hot spot:looking for more specific and effective downstream factors or segments.Integrin-linked kinase(ILK),discovered in 1996,is a kind of Serine/Threonine protein kinase that combines with the integrin intracellular domain.Instead of focusing on glomerulus, recent experiments on the UUO model rats and cells culture in Vitro indicated that the accumulation of renal interstitial fibrosis ECM and the ILK expression are positively correlated,and that the mediation of ILK in TGF-β/Smad signal transduction path participates the EMT of nephric tubule.Yet little is known about the ILK expression and its relationship with EMT in the DN renal tubular epithelial cell.Since TEMT play an important role in the progress of diabetic nephropathy and other chronic kidney disease,then it maybe possible to delay or reverse the occurrence and development of renal interstitial fibrosis by interfering or preventing the process of TEMT.Traditional Chinese medicine is frequently used in clinical prescription,Cordyceps sinensis is one of famous traditional Chinese medicine.Both clinical and experimental researches manifest that Cordyceps sinensis can effectively slow down the progression of chronic renal disease with positive protecting effects to kidney,and is a potential cure to DN.Yet little has been reported on the effects and influence on intertubular pathological changes and the process of TEMT.From the two aspects,DM model rats and Cells Culture in Vitro,and by refering to indexes like FN,which is one of the ingredients of ECM, E-Cadherin andα-SMA,which respectively reflects the phenotypic characteristics of renal tubular epithelial cells and of MyoF,the objective of this research is to investigate the states of TEMT and ILK expression, and illustrate their relationship that exists in the pathological changes of DN tubulointerstitial lesion,and to observe the influence of Cordyceps sinensis as an intervener in the above-mentioned processes.Through the new method of TEMT,we try to investigate a new mechanism of the pathological progress of diabetic nephropathy and how the Cordyceps sinensis protect kidney. Methods:(1)Cell experimentsHuman proximal tubular epithelial cells(HK-2)were divided into six groups:Group N,cultured with sugar(5.5mmol/L)DMEM;Group M, cultured with 24.5mmol·l^-1mannitol and 5.5mmol·l^-1glucose DMEM;Group H,cultured with 30mmol·l^-1glucose DMEM;Group H+5C,cultured with 30mmol·l^-1glucose DMEM + Cordyceps sinensis 5ug/ml;Group H+10C,cultured with 30mmol·l^-1glucose DMEM + Cordyceps sinensis 10ug/ml;Group H+20C,cultured with 30mmol·l^-1 glucose DMEM + Cordyceps sinensis 20ug/ml.Each group cells were collected at 48 hours.The protein of E-Cadherin,α-SMA,FN and ILK were assessed by Western blotting.(2)Animal experimentsMale Wistar rats were randomly divided into normal control group (Group N,n=12),diabetic group(Group D,n=12)and Cordyceps sinensis intervention group(Group C,n=12).Diabetic mellitus rat Models were induced by intraperitoneal injection of 55mg·kg^-1STZ.Cordyceps sinensis intervention rats were received daily intragastric administration of 1000mg·kg^-1of Cordyceps sinensis after the models were successfully established.Six rats of each group were killed respectively at 8,16 weeks. Urinary protein,serum creatinine,blood triglyceride and cholesterol were detected,and HE and Masson stain were employed to observe renal histological changes.The protein expression of E-Cadherin,α-SMA,FN and ILK in renal tubular epithelial cells were assessed by immunohistochemistry while the protein expression of E-Cadherin,α-SMA,FN and ILK was examined by Western blotting.Results:1.Compare with group N,the protein expression of E-cadherin in HK-2 cells decreased(P<0.05);while the protein expression of integrin-linked kinase,α-SMA and FN increased(P<0.05)in high glucose environment.The protein expression of E-cadherin increased (P<0.05)while the protein expression ofα-SMA,FN decreased(P<0.05) in Cordyceps sinensis treated group(group C)with a dose-dependent manner,compared with group H.(P<0.05).2.Excretion of urinary protein,serum creatinine,tubulointerstitial injury index and interstitial collagen relative area increased significantly in group D and group C,compared with group N(P<0.01);Cordyceps sinensis can improve these lesions.At 16 weeks,the renal protection effect of Cordyceps sinensis became significantly obviously compared with the same period in group D(P<0.05).3.The expression of E-cadherin in tubular epithelial cells decreased, while the expression ofα-SMA and FN increased significantly in group D and group C,compared with group N(P<0.05,P<0.01);Meanwhile,the expression of integrin-linked kinase(ILK)significantly increased (P<0.05)and was inversely correlated with the E-cadherin(r_s =-0.81,P <0.05),but which was directly correlated withα-SMA and FN(r_s=0.84,P <0.05;r_s =0.93,P<0.01).Cordyceps sinensis intervention can improve the changes.At 16 week,the indicators improvement become significant in group C,compared with group D(P<0.01).Conclusion:1.In the progression of diabetic nephropathy tubulointerstitial lesion, the existence of EMT in renal tubule cell was observed.2.The expression of ILK was up-regulated in renal tubular epithelial cell of DN rats,which is highly related to EMT.As a mediating agent, ILK has important effects on the positive regulation of process of EMT.3.Cordyceps sinensis can reduce the ILK expression in HK-2 cultured with high sugar.4.Cordyceps sinensis can alleviate the pathological changes of DN tubulointerstitial lesion,reduce the ILK expression of DN renal tubular epithelial cells,inhibit the process of TEMT,and which may be an important functioning mechanism of Cordyceps sinensis as a cure to DN.