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Effect of Renal Tubular Epithelial-myofibroblast Transdifferentiation on DN and the Influence of Rhein Intervention

Author: NingJianPing
Tutor: ZhangGuiYing
School: Central South University
Course: Internal Medicine
Keywords: Diabetic nephropathy renal tubular epithelial cell epithelial-myofibroblast transdifferentiation Rhein Integrin-linked kinase
CLC: R587.2
Type: PhD thesis
Year: 2007
Downloads: 259
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Abstract


Background and objectives:Tubulointerstitial lesion is an essential pathological basis in thedevelopment of diabetic nephropathy to end-stage renal disease (ESRD),the basic pathological character of which is excessive extracellular matrix(ECM) accumulation in the renal interstitium. Currently, it is consideredthat the renal interstitial ECM is mainly secreted by the myofibroblast(MyoF). Researches have shown that epithelial-myofibroblast transdif-ferentiation (EMT) can occur to the renal tubular epithelial cells inpathological condition. During EMT, renal tubular epithelial cells losetheir original phenotypic characteristics and gain MyoF characteristics,which is a principal source of renal interstitium MyoF. In researches onmodel rats with unilateral urethral obstruction (UUO), EMT wasobserved and participated in the occurrence and development of renalinterstitial fibrosis. However, it was still unknown whether the same EMTprocess would take place in the DN tubulointerstitial lesion development.TGF-β1 is an essential factor in the initiation and promotion of EMT.The biological functions of TGF-β1 are extensive and complex, which isshown through the diversity of positive and negative effects. Inhibitingonly TGF-β1 as an upstream factor could bring negative side effectstogether with positive effects. Therefore, researchers’ attention has been turned to a new hot spot: looking for more specific and effectivedownstream factors or segments. Integrin-linked kinase (ILK), discoveredin 1996, is a kind of Serine/Threonine protein kinase that combines withthe integrin intracellular domain. Researches showed evidence of ILK’srole as the junction and important effectors of the signal transduction pathof integrin, growth factor, Wnt, etc. Instead of focusing on glomerulus,.recent experiments on the UUO model rats and cells culture in Vitroindicated that the accumulation of renal interstitial fibrosis ECM and theILK expression are positively correlated, and that the mediation of ILK inTGF-β/Smad signal transduction path participates the EMT of nephrictubule. Yet little is known about the ILK expression and its relationshipwith EMT in the DN renal tubular epithelial cell.A traditional Chinese medicine frequently used in clinicalprescription, Rhein is a prosoma component which is extracted from therhubarb arthraquinone ramifications and is the primary active ingredientof rhubarb. Both clinical and experimental researches manifest that Rheincan effectively slow down the progression of chronic renal disease withpositive protecting effects to kidney, and is a potential cure to DN. Yetlittle has been reported on the effects and influence on intertubularpathological changes and the process of EMT.From the two aspects, DM model rats and Cells Culture in Vitro, andby refering to indexes like FN, which is one of the ingredients of ECM,E-Cadherin andα-SMA, which respectively reflects the phenotypiccharacteristics of renal tubular epithelial cells and of MyoF, the objectiveof this research is to investigate the states of EMT and ILK expression,and illustrate their relationship that exists in the pathological changes ofDN tubulointerstitial lesion, and to observe the influence of Rhein as anintervener in the above-mentioned processes. Through the new method ofEMT, we try to investigate a new mechanism of the pathological progress of DN, with the purpose of finding an effective way to control DN.Methods:(1) Animal experimentsMale Wistar rats were randomly divided into normal control group(Group N, n=12), diabetic group (Group D, n=12) and Rhein interventiongroup (Group R, n=12). Diabetic mellitus rat Models were induced byintraperitoneal injection of 55mg·kg-1 STZ. Rhein intervention rats werereceived daily intragastric administration of 100mg·kg-1 of Rhein afterthe models were successfully established. Six rats of each group werekilled respectively at 8, 16 weeks. Urinary protein, serum creatinine,blood triglyceride and cholesterol were detected, and HE and Massonstain were employed to observe renal histological changes. The proteinexpression of E-Cadherin,α-SMA, FN and ILK in renal tubular epithelialcells were assessed by immunohistochemistry and, while the proteinexpression of E-Cadherin,α-SMA, FN and ILK was examined byWestern blotting.(2) Cell experimentsHuman proximal tubular epithelial cells (HK-2) were divided intofive groups: Group N, cultured with sugar (5.5mmol/L) DMEM; GroupD, cultured with 30mmol·l-1 glucose DMEM; Group R-25, cultured with30mmol·l-1 glucose DMEM+Rhein 25ug/ml; Group R-50, cultured with30mmol·l-1 glucose DMEM+Rhein 50ug/ml; Group R-100, culturedwith 30mmol·l-1 glucose DMEM+Rhein 100ug/ml. Each group cellswere collected at 24,48 and 72 hours. The protein and mRNA expressionof E-Cadherin,α-SMA, FN and ILK were assessed by Westem blottingand RT-PCR method. Results:1. Excretion of urinary protein, serum creatinine, tubulointerstitialinjury index and interstitial collagen relative area increased significantlyin group D and group R, compared with group N (P<0.01); Rhein canimprove these lesions. At 16 weeks, the renal protection effect of Rheinbecame significantly obviously compared with the same period in groupD (P<0.05).2. The expression of E-cadherin in tubular epithelial cells decreased,while the expression ofα-SMA and FN increased significantly in group Dand group R, compared with group N (P<0.05, P<0.01); Meanwhile, theexpression of integrin-linked kinase (ILK) significantly increased(P<0.05) and was inversely correlated with the E-cadherin (rs=-0.82, P<0.05),but which was directly correlated withα-SMA and FN (rs=0.83, P<0.05; rs=0.94, P<0.01). Rhein intervention can improve the changes. At16 week, the indicators improvement become significant in groupR, compared with group D(P<0.01).3. Compare with group N, the protein expression of E-cadherin inHK-2 cells decreased(P<0.05); while the protein expression ofintegrin-linked kinase,α-SMA and FN increased (P<0.05) in highglucose environment. The protein expression of E-cadherin increased(P<0.05) while the protein expression ofα-SMA, FN decreased (P<0.05)in Rhein treated group (group R) with a time and dose-dependentmanner, compared with group D. (P<0.05).Conclusion:1. In the progression of diabetic nephropathy tubulointerstitial lesion,the existence of EMT in renal tubule cell was observed, which was animportant source of MyoF.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. Rhein can reduce the ILK expression of DN renal tubularepithelial cells, inhibit the process of EMT, and alleviate or meliorate thepathological changes of DN tubulointerstitial lesion, which may be animportant functioning mechanism of Rhein as a cure to DN.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetic coma and other complications
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