Effect Of Renal Tubular Epithelial-myofibroblast-transdifferentiation And MMP-9/TIMP-1 On DN And The Influence Of Rhein Intervention

Background and objectives:Diabetic nephropathy is one of the main causes of chronic renal failure.Tubulointerstitial lesion is an essential pathological basis in the development of chronic kidney disease including diabetic nephropathy to end-stage renal disease(ESRD),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),Currently.Researches have shown that epithelial-myofibroblast transdifferentiation(EMT)can occur to the renal tubular epithelial cells in pathological condition.During EMT,renal tubular epithelial cells lose their original phenotypic characteristics and gain MyoF characteristics,which is a principal source of renal interstitium MyoF.According to the researches,above to 36%MyoF come from the renal tubular epithelial.Tubular basement membrane is an important structure of renal tubule,which can effect renal tubular epithelial cells biological behaviour such as maintain the phenotypic characteristics under normal condition.Matrix metalloproteinases -9(MMP-9)can degradateⅣcollagen,tubular basement membrane cracking was one of key steps of EMT.On the other hand,over accumulation of extracellular matrix(ECM)is nucleus of renal interstitial fibrosis.Either its composite increase or degradation decrease can lead to development of renal interstitial fibrosis.MMP-9 is the main catabolic enzymes of ECM,which regulate degradation of ECM with Tissue inhibitor of metalloproteinases-1(TIMP-1),together.Ratio of MMP-9 to TIMP-1 can be used infer the severe of renal interstitial fibrosisAs a traditional Chinese medicine rhubarb was frequently used in clinical practice,Rhein is a prosoma component which is extracted from the rhubarb arthraquinone ramifications and is one of the primary active ingredient of rhubarb which can ameliorate the glomerular mesangial expansion and Inhibit the activation of renal fibroblasts.Both clinical and experimental researches manifest that Rhein can effectively slow down the progression of chronic renal disease with positive protecting eff- ects to kidney,and is a potential cure to DN.Yet little has been reported on the effects on the process of EMT and MMP-9/TIMP-1.From the STZ DM model rats,Tubulointerstitial injury was evaluated by HE,MASSON stain and by referring to indexes like 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 EMT and observe MMP-9/TIMP-1 how to affect the ECM accumulation,and illustrate their relationship with the pathological changes of DN tubulointerstitial lesion, and to observe the influence of Rhein as an intervener in the above-mentioned processes,the effects on the renal protection between Rhein and Valsartan were compared.Through the new method of EMT, we try to investigate the mechanism of the renal protection of Rhein,with the purpose of finding an effective way to control DN.Methods:Male Wistar rats were randomly divided into normal control group (n=12),diabetic group(n=12),Rhein intervention group(n=12)and Valsartan intervention group(n=12),and total number was forty-eight. Diabetic mellitus rat models were induced by intraperitoneal injection of 55mg·kg^-1STZ.Diabetic rats were continuously treated with rhein 100 mg·kg^-1·d^-1or Valsartan 30 mg·kg^-1·d^-1for 16 weeks after the models were successful established.Six rats of each group were killed respectively at the end of 8^th,16^thweeks.Urinary protein,serum creatinine,blood triglyceride and cholesterol were detected,renal histopathology was evaluated under light microscopy by HE and Masson staining.The protein expression of E-Cadherin,α-SMA,MMP-9 and TIMP-1 in renal tubular epithelial cells were assessed by immunohistochemistry.Results:1.Compared with normal control group,the excretion of urinary protein serum creatinine,tubulointerstitial injury index and interstitial collagen area increased significantly in diabetic group,Rhein intervention group and Valsartan intervention group(P＜0.01);Both rhein and Valsartan can reduce these lesions.At 16^thweeks,the renal protection effect became significantly obviously compared with the diabetic group (P＜0.01),there were no significance between Rhein group and Valsartan group.2.The expression of E-Cadherin and MMP-9/TIMP-1 in tubular epithelial cells decreased,while the expression ofα-SMA and MMP-9/TIMP-1 increased significantly in diabetic group.Rhein intervention group and Valsartan intervention group,compared with normal control group(P＜0.05),Rhein and Valsartan intervention can improve the changes.The indicators improvement become significant in Rhein and Valsartan group,compared with diabetic group(P＜0.05,P＜0.01),there were no significance between Rhein group and Valsartan group.Conclusion:1.In the progression of diabetic nephropathy tubulointerstitial lesion, the expression of E-Cadherin decreased,whileα-SMA increased in tubular epithelial cells,which can illustrate the occurence of EMT.2.The increased expression of MMP-9 disrupted the renal Tubular basement membrane and accelerated the occurrence of EMT,while Ratio of MMP-9 to TIMP-1 in renal tubular epithelial cells of diabetic rats decreased,which indicates disequilibrium of MMP-9/TIMP-1 is an important aspect in progress of renal interstitial fibrosis in diabetic nephropathy.3.The similar renal protective effect of rhein compared with Valsartan that the inhibition of process of EMT,and the the melioration of disequilibrium of MMP-9/TIMP-1,which may be the important functioning mechanism of Rhein for the renal protection.