Vitamin D Receptor Agonist Attenuate High Glucose-induced Renal Interstitial Fibrosis By Suppressing The Activation Of RhoA

Objectives:To investigate the role of Vitamin D Receptor Agonist1,25-(OH)2D3and its analogue BXL-628in high glucose-induced activation of RhoA and epithelial-mesenchymal transition in human renal proximal tubular cell.Methods:HK-2cells were cultured with low glucose DMEM/F12and passed to the second generation cells; then HK-2cells were stimulated by high glucose (30mmol/mL) for0min,30min,1h,2h,4h,8h, and12h to determin the activation of RhoA protein by Western Blot. Meanwhile, HK-2cells were stimulated by high glucose (30mmol/mL) for0h,12h,24h,48h, and72h to determin the expression of a-SMA, E-cadherin at both mRNA and protein level by Wetstern Blot and Real Time PCR. According to the results above, the cells were divided into four groups:Normal Control Group (glucose5.5mM/L); High glucose Group (glucose30mM/L); VitD3group(High glucose+1,25-(OH)2D3); BXL-628Group (High glucose+BXL-628); The48h-treated cells were harvested to determine the expression of a-SMA, E-cadherin at mRNA and protein level by Western Blot analysis and Real-Time PCR.,the expression of COL-I, and Fn were determined by ELISA and Real-Time PCR.The2h-treated cells were harvested to determine the expression of active RhoA protein by Western Blot and immunofluorescence.Results:Active RhoA protein was increased by high glucose treatment, compared with0min the expression showed an significantly increase at lh (P<0.01), and came to a heat at2h (P<0.01). And the expression of a-SMA was significantly up regulated at24h by high glucose treatment and48h treatment showed the utmost increase (P<0.01), while the expression of E-cadherin was decreased by high glucose treatment at both protein and mRNA level. Western Blot analysis and Real-Time PCR results showed1,25-(OH)2D3and BXL-628had significantly decreased the expression of a-SMA, COL I and Fibronectin at both protein and mRNA level(P<0.01), meanwhile increased E-cadherin expression compared with high glucose group(P<0.01).Both Western Blot and immunofluorescence results showed Both1,25-(OH)2D3and BXL-628treatment decreased the expression of active RhoA protein at2h compared with high glucose group.Conclusion:High glucose could induce the activation of RhoA/ROCK pathway and mediate epithelial-mesenchymal transition in human renal proximal tubular cell. Vitamin D receptor agonist1,25-(OH)2D3and its analogue BXL-628could attenuate high glucose-induced epithelial-mesenchymal transition in human renal proximal tubular cell by suppressing RhoA/ROCK signaling pathway.