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Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.
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Objective The aim of this study is to examine the effect of myoophenolate mofetil (MMF) on epithelial-myofibroblast transiation(EMT) in adenine-induced chronic renal failure (CRF) rat model and the role of vascular endothelial growth factor(VEGF) and inhibitor of differentiation (Id2 and Id3) in EMT in the rat kidney. Methods Sixty-four male Wistar rats were randomly assigned to the following groups: normal control (n=16), CRF (n=24) and MMF(n=24). CRF was induced by gastric gavage of adenine (125 mg·kg-1·d-1) to rats for eight weeks. CRF rats were treated with MMF (15 mg·kg-1·d-1) as "MMF" group. The rats were sacrificed at week 2, 4, 6 and 8, respectively.Urinary protein and serum ereatinine levels were measured, and the histopathologic degrees of interstitial fibrosis were evaluated in Massen-stained sections. Expressions of a-smooth muscle actin (α-SMA),transforming growth factor β1 (TGFβ1), VEGF and Id (Id2 and Id3) in the kidney tissue were assessed by immunohistochemistry, RT-PCR and/or Western blot methods. Results The urinary protein level in MMF group was evidently lower than that in CRF group (P<0.01), whereas no statistically significant difference was observed in serum creatinine level between the two groups. Renal interstitial fibrosis was reduced significantly with MMF treatment (P<0.01). Expression of α-SMA in MMF group was lower than that in CRF rats at week 6, 8 (P<0.01), while expression of TGFβ1 was decreased markedly at week 2, 4,6 (P<0.01). The expressions of VEGF in MMF rats were increased significantly at week 6,8 (P<0.01),and Id2,Id3 in MMF rats were increased significantly at week 4,6 (P<0.05). Conclusions MMF may ameliorate chronic renal fibrosis and EMT in adenine-induced CRF rats. This effect of MMF on EMT is probably related to upregulation of VEGF, Id2 and Id3 expressions and suppressing overexpression of TGFβ1 in renal tissue. The exact mechanism needs to be studied further.
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Objective Rapamycin (RAPA) is an anti-proliferative immunosuppressant and has been used to suppress rejection of transplanted organs. In present study, we observed the effect of rapamycin on epithelial-myofibroblast transition (EMT)of cultured HKC cells in vitro. Methods Cultured human proximal tubular epithelial cells (HKCs) were divided into three groups: blank control, treated with TGF-?1 (1 ?g/L) and treated with TGF-?1 (1 ?g/L) plus rapamycin (0.1, 1, 10, 100 ?g/L). The protein and mRNA for ?-SMA and E-cadherin in HKC cells were determined by Western Blot and RT-PCR.The mRNA level of Snail in HKC was detected by RT-PCR. Results Rapamycin dramatically abrogated TGF-?1 induced ?-SMA expression and restored E-cadherin expressionin HKC cells in a dose-dependent manner. At a concentration of 100 ?g/L, rapamycin almost completely blocked ?-SMA mRNA and protein expression induced by TGF-?1(1 ?g/L). Rapamycin also suppressed expression of ?-SMA in HKC cells at both mRNA and protein level in a time dependent manner.We also found rapamycin dramatically abrogated TGF-?1 induced Snail mRNA expression in HKC cells in a dose-dependent manner. Conclusion Rapamycin may inhibit EMT of tubular cells in vitro. The downregulation of Snail expression might be one of the mechanisms of rapamycin blocking EMT.
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<p><b>OBJECTIVE</b>To examine the possible role of aristolochic acid (AA) in transdifferentiation and apoptisis of human tubular epithelial cell line (HKC).</p><p><b>METHODS</b>Cultured HKC cells were divided into five groups: serum-free (negative control) and treatment with AA at the concentrations of 5 mg/L, 10 mg/L, 20 mg/L and 40 mg/L for 48 hours, respectively. Transdifferentiation of HKC cells was observed with the following methods: detection of the expression of vimentin and cytokeratin of HKC cells with indirect immunoflourescence, determination of expression of E-cadherin and alpha-smooth muscle actin (alpha-SMA) by indirect immunohistochemical double staining, and determination of the proportion of alpha-SMA (+) HKC cells by flow cytometry. The apoptosis of HKC cells was observed with Giemsa staining, TUNEL reaction and agarose gel electrophoresis, and the ratio of apoptotic HKC cells was quantitatively analyzed by flow cytometry with propidium iodide staining.</p><p><b>RESULTS</b>The expression of cytokeratin and E-cadherin reduced and that of vimentin increased in HKC cells treated with 10 mg/L of AA for 48 hours, and the expression of alpha-SMA (+) in HKC cells treated with 10 mg/L of AA (14.17 +/- 0.61)% was significantly higher than that in serum-free controls (3.57 +/- 0.52)%. Apoptosis of HKC cell treated with 40 mg/L of AA for 48 hours was 53.4%, significantly higher than that in serum-free controls (2%). Treatment with 5 mg/L of AA and 20 mg/L of AA could not induce apoptosis and transdifferentiation of cells.</p><p><b>CONCLUSIONS</b>Treatment with relatively low concentration of AA (10 mg/L) might induce slight transdifferentiation in cultured HKC cells and that with higher concentration of AA (40 mg/L) for 48 hours might induce apparent apoptosis of these cells, which suggested that transdifferentiation and apoptosis of tubular epithelial cells probably played important roles in aristolochic acid-induced nephropathy.</p>
Subject(s)
Humans , Actins , Apoptosis , Genetics , Aristolochic Acids , Pharmacology , Carcinogens , Pharmacology , Cell Differentiation , Cell Line , DNA Fragmentation , Epithelial Cells , Metabolism , Immunohistochemistry , In Situ Nick-End Labeling , Kidney Tubules , Cell Biology , Microscopy, Electron , Muscle, Smooth , ChemistryABSTRACT
Objective To investigate the possible role of valsartan(Val) in transdifferentiation of human renal tubular epithelial cell line(HKC ). Methods HKC cells were divided into four groups: (1) serum-free (negative control); (2) MCP-1 + AAI-treated (positive control); (3) Val-treated alone; (4) Val-inhibition(treated with MCP-1 + AAI + Val). Then the expression of vimentin, ?-smooth muscle actin (?-SMA ) of HKC cells were assessed by indirect enzyme immunohistochemistry (IEI), and the percentage of ?-SMA(+ ) HKC cells was assessed by flow cytometry. Results No difference in expressions of vimentin and ?-SMA by IEI and the percentage of ?-SMA(+ ) HKC cells by cytometry were found between serum-free control HKC cells and Val treated ones. The expressions of vimentin and ?-SMA in positive controls were markedly stronger than negative controls; while these expressions in Val + MCP-1 + AAI-treated HKC cells were less strong than those in positive controls. The percentage of ?-SMA(+ ) HKC cells in the positive controls was significantly higher than that in negative controls(91. 8% vs 3. 1%, P
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In non-dialytic composite treatment of chronic renal failure, wheat (or corn) starch should be used for low protein (20-40 g/day) diet in substituting grains, to increase high biological value protein intake (about 50-70% of total protein intake). This principle has been used in 30 Patients (33 times) for 1-2 monthes. Daily total calorie intake should be maintained within 2000-3000 kcal. According to the status of the patient, EAA-TR2 (13.8 g/day I.V.) and other supporting measurements may be used for most patients. In all the patients, except six (2 cases useless, observation of 4 cases discontinued), uremic manifestations were ameliorated, nutritional status was improved and azotemia markedly alleviated, life span may be prolonged. According to nitrogen balance studied, in 6 cases it is indicated that nitrogen balance can be maintained if protein intake is not less than 0.5 g/kg/day (with sufficient calorie intake) for chronic renal failure patients (Cr clearance 5-10 ml/min).