| PART I STUDY OF LOW DENSITY LIPOPROTEIN INDUCEDRAT KIDNEY TUBULAR EPITHELIAL- MENSENCHYMAL TRANSITIONObjective: To observe the effect of the low-density lipoprotein (LDL) in vitro on rat proximal tubular epithelial cells (NRK-52E cells) trans-differentiation, as well as further explore the mechanisms and a new treatment for LDL induced rat kidey tubular epithelial-mensenchymal transition.Methods: NRK-52E cells with 10%FBS DMEM culture medium were cultured in 37℃,5%CO2 incubator for 24 hours. The cells were divided into (1)normal control group:cultured in serum-free DMEM medium;(2)lovastatin (Lovastatin, Lov)(1μmol/L) cultured group:Lov was added to the serum-free culture medium at final concentration of 1μmol/L;(3)LDL cultured group:LDL was added to the serum-free culture medium at different final concentrations of 50 mg/L, 150 mg/L, 250 mg/L;(4)LDL (250 mg/L) and Lov (1μmol/L) cocultured group: 250 mg/L LDL and 1μmol/L Lov were despectively added to the serum-free culture medium as a final concentration.Cell morphological changes were observed by inverted microscope.Expressions ofα-Smooth Muscle Actin(α-SMA),E-cadherin mRNA were assessed by RT-PCR. Expressions ofα-SMA,E-cadherin proteins were detected by Western blotting.Results:1. inverted microscope analysis findings: NRK-52E cells were cubic or polygonal with typical morphological characteristics of cobblestone in the normal control group. With different final concentrations of LDL (50 mg/L,150 mg/L,250 mg/L) cultured for 24 h, epithelial cells were obviously changed in cell morphology as the development of an elongated and spindle morphology, lossing the cobblestone growth pattern, showing the fibroblast-like appearance.With the increase of the doses induced by LDL, more NRK-52E cells changed into spindle-shaped cells. In 250 mg/L LDL and 1μmol/L Lov cocultured group,most still maintained a normal epithelial cell morphology.But there was no significant changes in Lovastatin group.2.RT-PCR results showed that: NRK-52E cells hardly expressedα-SMA mRNA in the normal control group.With an increase of the final concentration range of 50 mg/L,150 mg/L,250 mg/L LDL,the a-SMA mRNA expression was gradually up-graduated in a dose-dependent manner,peaked at 250 mg/L final concentration.Compared with normal control group, the expressions ofα-SMA mRNA were significantly increased (P <0.01). 3.RT-PCR results indicated that:E-cadherin mRNA was highly expressed in normal control group. With an increase of the final concentration range of 50 mg/L,150 mg/L,250 mg/L LDL,the expressions of E-cadherin mRNA were a gradual decline in a dose-dependent manner, as well as a minimum at 250 mg/L LDL.E-cadherin mRNA expressions were significantly reduced compared with normal control group (P <0.05, P <0.01 ).Therefore,the choice of LDL with 250 mg/L as the best final concentration.4.Western blot and RT-PCR assays showed that: NRK-52E cells hardly expressedα-SMA mRNA and protein in normal control group,high expression of E-cadherin mRNA and protein. Compared with normal control group,with the stimulation of 250 mg/L LDL,α-SMA mRNA and protein expression were respectively raised 5.2-fold and 5.0-fold(P <0.01),while E-cadherin mRNA and protein expression were respectively reduced by 48.7% and 24.9% (P <0.01).In LDL and Lov cocultured group,the expressions ofα-SMA mRNA and protein were significantly lower than LDL group(P <0.01),but significantly higher than the normal control group (P <0.01);The levels of E-cadherin mRNA and protein were significantly higher than LDL group(P <0.01),but significantly lower than those in normal control group(P <0.01).There was no significant difference between normal control group and Lov group(P >0.05).Conclusions: LDL induced rat kidey tubular epithelial-mensenchymal transition in a dose-dependent manner,but lovastatin can partly block the LDL-induced tubular epithelial cells transdifferentiation.PART II MECHANISM OF LOW-DENSITY LIOPROTEIN -INDUCED RAT KIDEY TUBULAR EPITHELIAL-MENSENCHYMAL TRANSITIONObjective: To investigate the mechanism of low-density lipoprotein-induced rat kidney tubular epithelial-mensenchymal transitionMethods: Expressions of transforming growth factor beta 1(TGF-β1),Smad2,Smad3 and integrin-linked kinase (ILK) mRNA were assessed by RT-PCR.Level of TGF-β1 in the supernatant of cultured cells was measured by enzyme-linked immunosorbent assay(ELISA).Expression of Phosphorylated Smad2/3 (p-Smad2/3) protein was detected by Western blotting.Results:1.RT-PCR assay showed that: in LDL (250 mg/L) group,TGF-β1 mRNA expression was significantly up-regulated compared with normal control group(P <0.01);In Lov plus LDL group, compared with LDL group,TGF-β1 mRNA expression was markly down-regulated (P <0.01),but significantly higher than normal control group(P <0.01);There was no significant difference between Lov group and normal control group (P >0.05).ELISA method showed that: Level of TGF-β1 protein in the supernatant of cells content was (279.40±28.42)pg/ml in the normal control group,the LDL group increased to (536.89±56.88)pg/ml, compared with normal control group,the difference had statistical significance(P <0.01).In LDL and Lov cocultured group, TGF-β1 protein expression in the cells supernatant was markly down-regraduated(P <0.05), but still higher than normal control group(P <0.05).There was no significant difference between Lov group and normal control group (P >0.05).2.RT-PCR results showed that:in 250 mg/L LDL group, Smad2 and Smad3 mRNA expression were significantly higher than normal control group(P <0.01).In Lov plus LDL group,Smad2 and Smad3 mRNA expression were markly lower than LDL group(P <0.05). There was no significant difference between Lov group and normal control group (P >0.05).Western blot results showed that: LDL up-regulated phosphorylation of Smad2/3 protein expression compared with normal control group(P <0.01).In LDL plus Lov group,p-Smad2/3 protein expression was significantly reduced(P <0.01),but markly higher than normal control group(P <0.01).3.RT-PCR results showed that:LDL increased the expression of ILK mRNA,compared with normal control group,there was statistical significance (P <0.01).Lov partially inhibited the LDL-mediated expression of ILK mRNA compared with LDL group(P <0.05),but markly higher than normal control group (P <0.05).There was no significant difference between Lov group and normal control group (P >0.05).Conclusions: LDL induced renal tubular epithelial-mensenchymal transition and the mechanism was related to the activation of TGF-β1/Smads/ILK signaling pathway in NRK-52E cells. |
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