Zhiqiang Wei1, Juan Cao2, Xu Zhang2, Di Yin2, Deyu Xu1, Guoyuan Lu1. 1. Department of Nephrology, The First Affiliated Hospital of Soochow University Suzhou, People's Republic of China. 2. Department of Nephrology, Affiliated Taixing People's Hospital of Yangzhou University Taixing, People's Republic of China.
Abstract
BACKGROUND: It was reported that eicosapentaenoic acid (EPA) could prevent tubulointerstitial injury in kidney. EPA could also inhibit the epithelial-mesenchymal transition (EMT) of HK-2 cells stimulated by albumin (Alb) in vitro. However, the regulating molecular mechanism of EPA remains to be elucidated. METHODS: An immortalized human proximal tubular cell line (human kidney-2 (HK-2) cells) was used in all experiments. MTT assay was employed to determine the effect of Alb or EPA on the cell viability of HK-2 cells. The miR-541 expression, the mRNA levels of EMT markers E-cadherin, α-smooth muscle actin (α-SMA), and fibrogenesis markers Collagen I and fibronectin (FN) were examined by RT-qPCR assay. The protein levels of E-cadherin, α-SMA and Collagen I, transforming growth factor β1 (TGF-β1)/Smad3/integrin-linked kinase (ILK) pathway-related protein TGF-β1, pSmad2/3, Smad7 and ILK were measured by western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to detect FN expression. The target relationship between miR-541 and TGF-β1 was confirmed by bioinformatics, luciferase reporter assay and western blot. RESULTS: Low doses of Alb had no effect on the cell viability of HK-2 cells, while EPA repressed the cell viability of HK-2 cells in a concentration-dependent manner. EPA could inhibit EMT and fibrosis and increase the miR-541 expression of HK-2 cells exposed to Alb. Interestingly, introduction of miR-541 effectively abolished the EMT and fibrosis of HK-2 cells stimulated by Alb. Bioinformatics analysis predicted TGF-β1 as a target gene of miR-541, and subsequent luciferase reporter assay and western blot further supported the prediction. miR-541 counter-regulated TGF-β1 expression, and inhibited the TGF-β1/Smad3/ILK pathway. Alb treatment activated the TGF-β1/Smad3/ILK pathway, while EPA inhibited the activation of the pathway. miR-541 inhibitors reversed the effects of EPA on EMT, fibrosis, and TGF-β1/Smad3/ILK pathway-related protein expression induced by Alb. CONCLUSION: EPA attenuates EMT and renal fibrosis through the TGF-β1/Smad3/ILK pathway in renal epithelial cells by targeting miR-541. IJCEP
BACKGROUND: It was reported that eicosapentaenoic acid (EPA) could prevent tubulointerstitial injury in kidney. EPA could also inhibit the epithelial-mesenchymal transition (EMT) of HK-2 cells stimulated by albumin (Alb) in vitro. However, the regulating molecular mechanism of EPA remains to be elucidated. METHODS: An immortalized human proximal tubular cell line (human kidney-2 (HK-2) cells) was used in all experiments. MTT assay was employed to determine the effect of Alb or EPA on the cell viability of HK-2 cells. The miR-541 expression, the mRNA levels of EMT markers E-cadherin, α-smooth muscle actin (α-SMA), and fibrogenesis markers Collagen I and fibronectin (FN) were examined by RT-qPCR assay. The protein levels of E-cadherin, α-SMA and Collagen I, transforming growth factor β1 (TGF-β1)/Smad3/integrin-linked kinase (ILK) pathway-related protein TGF-β1, pSmad2/3, Smad7 and ILK were measured by western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to detect FN expression. The target relationship between miR-541 and TGF-β1 was confirmed by bioinformatics, luciferase reporter assay and western blot. RESULTS: Low doses of Alb had no effect on the cell viability of HK-2 cells, while EPA repressed the cell viability of HK-2 cells in a concentration-dependent manner. EPA could inhibit EMT and fibrosis and increase the miR-541 expression of HK-2 cells exposed to Alb. Interestingly, introduction of miR-541 effectively abolished the EMT and fibrosis of HK-2 cells stimulated by Alb. Bioinformatics analysis predicted TGF-β1 as a target gene of miR-541, and subsequent luciferase reporter assay and western blot further supported the prediction. miR-541 counter-regulated TGF-β1 expression, and inhibited the TGF-β1/Smad3/ILK pathway. Alb treatment activated the TGF-β1/Smad3/ILK pathway, while EPA inhibited the activation of the pathway. miR-541 inhibitors reversed the effects of EPA on EMT, fibrosis, and TGF-β1/Smad3/ILK pathway-related protein expression induced by Alb. CONCLUSION:EPA attenuates EMT and renal fibrosis through the TGF-β1/Smad3/ILK pathway in renal epithelial cells by targeting miR-541. IJCEP
Authors: J P Grande; H J Walker; B J Holub; G M Warner; D M Keller; J D Haugen; J V Donadio; T P Dousa Journal: Kidney Int Date: 2000-03 Impact factor: 10.612