| Effect of rHuEPO on Epithelial- Mesenchymal Transition of human proximal tubular epithelial cells induced by transforming growth factorβ1Background Epithelial-mesenchymal transition (EMT) is considered as one of the major mechanism of renal interstitium fibrosis. The reduction of VEGF was found to be closely correlated with tubulointerstitial fibrosis. Much clinical and experimental data indicate that erythropoietin(EPO) may slow the rate of progression of chronic renal failure and ameliorate the interstitial fibrosis. However, little information is available regarding its effect on EMT.Objective Our aim is to test the effect of rHuEPO on EMT of cultured human renal proximal tubular epithelail cells and elucidate the relationship between EMT and VEGF expression of the HKC cells in vitro. Methods Part one: Cultured HKC cells were divided into 5 groups: (1)negative control, (2)TGF-β1(8 ng/mL) treated as positive control, (3) concomitant treatment with EPO (0.1, 1.0, 10, 50 and 100 IU/mL) and TGF-β1(8 ng/mL), (4) EPO(0.1, 1.0, 10, 50 and 100 IU/mL) treated alone, (5) pre-treated for 24hr with rHuEPO (0.1, 1.0, 10, 50 and 100 IU/mL) in prior to adding TGF-β1(8 ng/mL). The expression of keratin were assessed by indirect immunofluorescence, and the expression ofα-SMAand E-cadherin were scaled by immunocytochemistry. Western blot was used to evaluate the expressions ofα-SMA,E-cadherin and VEGF of HKC cells. RT-PCR was used to evaluate the expressions ofα-SMA mRNA and VEGFmRNA of HKC cells. Part two: Cultured HKC cells were divided into 4 groups: (1) TGF-β1 treated alone, (2) pre-treated with rHuEPO(100 IU/mL) in prior to adding TGF-β1 (8 ng/mL) into, (3) concomitantly treated with both rHuEPO (100 IU/mL) and TGF-β1 (8 ng/mL) for 72hr, (4) post-treated with rHuEPO(100 IU/mL) after stimulation with TGF-β1 (8 ng/mL) for 12hr, 24hr, 36hr, 48hr. VEGF mRNA andα-SMA mRNA expressions were assessed with semi-quantitative RT-PCR.α-SMA, E-cadherin, VEGF protein expressions were assessed by Western blot.Results Part one: In HKC cells, concomitantly treated or pre-treated with rHuEPO (50 and 100 IU/mL), the expression of keratin was stronger than the positive control. Immunocytochemistry method showed that the expression ofα-SMAincreased and the expression of E-cadherin decreased in the cells mentioned above. The VEGF mRNA, VEGF and E-cadherin protein expressions significantly increased (p＜0.01) while theα-SMA mRNA and a -SMAprotein expressions significantly decreased(p＜0.01) in HKC cells concomitantly treated with rHuEPO (0.1, 1.0, 10, 50, 100 IU/mL) and TCF-β1(8 ng/mL) when comparring with the positive control. Part two: The expressions ofα-SMA mRNA andα-SMA protein were significantly decreased in rHuEPO (100 IU/mL) pre-treated, rHuEPO(lO0 IU/mL) post-treated and rHuEPO(100 IU/mL) concomitantly treated with TGF-β1(8 ng/mL) when compared with the TGF-β1(8 ng/mL) treated alone (p＜0.05). However, the expressions of VEGF mRNA, VEGF and E-cadherin protein were significantly increased (p＜0.01) in the ceils mentioned above. Conclusions rHuEPO may inhibit TGF-β1 induced EMT of cultured HKC cells in dose- and time-dependent manner. This effect is probably related to up-regulated expression of VEGF induced by rHuEPO. Effect of rHuEPO on Epithelial- Mesenchymal Transition and renal interstitial fibrosis in Unilateral Uretheral Obstruction RatBackground It is well-known that overexpressions of MCP-1 and TGFβ1 play a pivot role in the progression of interstitial fibrosis and epithelial-mesenchymal transition (EMT). It is proven that VEGF is beneficial to inhibite EMT of renal tubular epithielia cells induced by TGFβ1. Clinical and experimental studies suggest that rHuEPO may slow progression of chronic renal failure, rHuEPO may attenuate the extent of renal interstitial fibrosis in different rat modal of renal disease. However, the effect of rHuEPO on EMT has not been reported.Objective To examine the effect of rHuEPO on EMTand renal interstitial fibrosis in the rat model of unilateral uretheral obstruction and elucidate its possible mechanisms.Methods Sixty male Wistar rats were divided into three groups: (1).sham-operated group (n=20) , (2) UUO group(n=20), (3) UUO + rHuEPO group (n=20). UUO rats treated with rHuEPO 100IU/Kg, thrice weekly. All the rats sacrificed at day 3, 7, 14, 21. we monitored hemoglobin, hematocrit, serum creatinine and body weight. Masson stain of renal tissue was performed to grade the extent of renal interstitial fibrosis. Immuno histological method and Western blot were used to evaluated the extent of expressions of alpha smooth muscle actin(α-SMA), E-cadherin, transform growth factor-β1(TGF-β1), monocyte chemoattractant protein-1(MCP-1), vascular endothelial growth factor (VEGF) and thrombospondin-1(TSP-1).Results The serum creatinine levels and body weight were not different between the three groups over time. The hemoglobin and hematocrit levels of UUO + rHuEPO group were significantly higher than that in UUO group and sham group at day 7, 14 and 21. The renal fibrosis scores and expressions ofα-SMA,MCP-1,TGF-β1 and TSP-1 were increased in UUO group compared with sham group at day 3, 7, 14 and 21 respectively(p＜0.05). But the expressions of E-cadherin and VEGF were reduced in UUO group compared with sham group at day 3, 7, 14 and 21 respectively(p＜0.05). The extent of interstitial fibrosis (p＜0.05) andα-SMA expression of renal tissue were significantly lower in UUO + rHuEPO group at day 3 and 7 after UUO operation compared with the UUO group (p＜0.05), while the extent of E-cadherin expression of renal tissue were significantly higher in UUO + rHuEPO group at day 3 and 7 (p＜0.05). The MCP-1, TGF-β1 and TSP-1 expressions of renal tissue were significantly lower in UUO + rHuEPO group than that in the UUO rats at day 3, 7 and 14 (p＜0.05), respectively. Meanwhile VEGF expression of renal tissue was significantly higher in UUO + rHuEPO group than that in the UUO rats at day 3, 7 and 14 (p＜0.05). No differences were found in a -SMA, E-cadherin, TGF-β1, MCP-1, VEGF and TSP-1 expressions between UUO+ rHuEPO group and UUO group at day 21.Conclusions rHuEPO may ameliorate EMT and the renal fibrosis in rat UUO model. This effect is probably related to down-regulated expressions of TGF-β1, MCP-1 and TSP-1, and up-regulated expression of VEGF. The exact machenism needs to be studied further. Altered expresstion of integrin-linked kinase in EMT of HKC cells induced by MCP-1Background Epithelial-mesenchymal transition (EMT) is considered as one of the major mechanism of renal interstitium fibrosis. We have found that MCP-1, the potent chemoattractant cytokine for the modulating infiltration of monocyte macrophage in the renal interstitium, may also induce EMT. Integrin-linked kinase (ILK) is an intracellular serine/threonine protein kinase that interacts with the cytoplasmic domains ofβ-integrins and numerous cytoskeleton-associated proteins. ILK has been shown to be involved in the regulation of a number of integrin-mediated processes including EMT induced by TGF-β1. However, the relationship between MCP-1 and ILK during the course of EMT is not clear. Objectives To examine the role of ILK in MCP-1 induced EMT of tubular epithelial cells.Methods Cultured human renal tubular epithelial cells (HKC) were divided into three groups, including negative control, TGF-β1(8 ng/ml) treated (positive control) andMCP-1 (0.1, 1.0, 10, 50, 100ng/ml) treated. HKC cells were incubated with either the same concentration of MCP-1 (1.0 ng/ml) for various periods of time (Oh, 12h, 24h, 36h, 48h, 72h). The effect of various chemical inhibitors, including PD98059 (Mekl 10μM), sb203580 (p38 MAPK 5μM), Y27632 (ROCK 500nM), on ILK expression was also tested respectively, followed by incubating in the absence or presence of MCP-1(1 ng/ml) for 24 hours. MCP-1 inducedα-SMA mRNA expression was assessed by RT-PCR. MCP-1 inducedα-SMAand ILK protein expressions were assessed by Western blot.Results MCP-1 inducedα-SMA mRNA,α-SMAand ILK expression increased in a time and dose-dependent manner, and reached the peak at 48h after MCP-1 treatment. The various chemical inhibitors or vehicle does not affect ILK expression induced by MCP-I(1 ng/ml). Conclusion MCP-1 may induce EMT of cultured renal epithelial cell with the up-regulation of ILK. The mechanism of altered expression of ILK needs to be studied further. |
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