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Effects of PTKs and Genistein on Human Renal Tubular Epithelial-Myofibroblast Transdifferention in Vitro

Author: MeiZuoMing
Tutor: Zhang
School: Third Military Medical University
Course: Internal Medicine
Keywords: epithelial-myofibroblast transdifferentiation (EMT) acid fibroblast growth factor (aFGF) α-smooth muscle actin (α-SMA) human renal tubular epithelial cell(HKC) genistein protien tyrosine kinases(PTKs) transforming growth factor β1(TGFβ1)
CLC: R692.9
Type: Master's thesis
Year: 2002
Downloads: 51
Quote: 1
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Objective:Interstitial fibrosis has shown a strong correlation with the progression of kidney disease to renal insufficiency.This proccess is associated at least in some forms of interstitial fibrosis with a differentiation of tubular epithelial cells into myofibroblasts.However,the mechanism underlying this differentiation of tubular epithelial cells is poorly understood.This study investigated the possible roles of protein tyrosine kinases(PTKs) in the phenotypic transformation of cultured human renal tubular epithelial cell line(HKC).Methods: The HKC cells were divided into three groups:(1)serum-free (negative control);(2)aFGF(positve control);(3)aFGF-inhibtion(treated with aFGF + genistein).Cells were cultured for 72h on collagen typeⅠ-coated plate.HKC cells were analyzed by immunohitochemistry with monoantibodies(mAbs) to α-smooth muscle actin(α-SMA),vimentin and cytokeratin, respectively.Morphlogy was determined using phase contrast microscopy and electron microscope.The number of HKC cells stained with the anti-α-SMA mAb was determined by counting the number of positive-stained cells and data are expressed as the mean percentage.Cell motility was estimated by the ability of cells located at the edge of an artificial "Scarification ".The expression of FN in the supernatant of HKC cells was assessed with ELISA. Flow cytometry was adopted to measure the activity of PTKs. TGFβ1 mRNA expression was examined by revers transcription- polymerase chain reaction(RT-PCR) after cells were allowed to grow for 24h<WP=5>in the presence or absence of various factors and reagents.Results: HKC cells in serum control showed a classic epithelial morphology.In contrast,culture in 80ng/ml aFGF caused profound changes in morphology.These involved hypertrophy,a loss of apical-basal polarity, identifiable junctional complexes and microvilli,with cells becoming elongated and invasive, and formation of a new front-end back-end polarity, and rich in ribosome, rough endoplasmic reticulum and Golgi body.These morphological changes were companied by phenotypic changes.Immunohistochemistry staining showed that the addition of aFGF to subconfluent cell culture caused a loss of the epithelial marker (cytokeratin)and increased expression of mesenchymal marker (vimentin and α-SMA).However,medium wih aFGF +genistein,these expressions were lower than those in positive controls.The percentage of α-SMA +HKC cells in the positive controls was significantly higher than that in negative controls(60.60±2.70% vs 3.35±1.45%,P<0.01); While the percentage of α-SMA +HKC cells in aFGF +genistein group at genistein dose of 48,96μg/ml(3.90±2.09%,3.98±1.75%) was dramtically lower than that in positive controls(P<0.01).The content of FN in the supernatant in aFGF +genistein group at genistein dose of 24,48μg/ml was lower than that in positive controls(0.571±0.016, 0.499±0.002 vs 0.701±0.047,P<0.05) and FN in positive control was markedly higher than that in negative controls(0.701±0.047 vs 0.498±0.004,P<0.01).There was a dose-dependent increase in the level of cells expressing TGFβ1 mRNA with increasing concentrations of aFGF.The level of TGFβ1 mRNA in cells by the addition of aFGF(40,80 ng/ml) was much higher than negative control(0.583±0.015,0.947±0.015 vs 0.383±0.015,P<0.01);However, the level of TGFβ1 mRNA in aFGF +genistein group at genistein dose of 24,48μg/ml was markedly lower than that in positive controls(0.617±0.025, 0.400±0.026 vs 0.947±0.015,P<0.01).In the course of epithelial cell transdifferent to<WP=6>myofibroblast,PTKs was activated in a aFGF dose-denpendent manner..This represented 1.16,1.17,3.46times increase in the activity of PTKs (aFGF20,40,80ng/ml vs negative controls).On the contrary,inhibition of the activity of PTKs was achieved in aFGF +genistein group at genistein dose of 6,12,24,48μg/ml.(a 45.6%,53.5%,66%,81.9% decrease of PTKs of positive group respectively.)Conclusion:1. The transdifferentiaton of HKC cells can be induced by aFGF .The present model may serve as a useful tool for

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Other diseases
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