Zhang Wei, Yi Bin, Zhang Ke, Li Aimei, Yang Shikun, Huang Jing, Liu Jishi, Zhang Hao
Department of Nephrology, Xiangya Third Hospital of Central South University, Changsha, Hunan 410013, P.R. China.
Exp Ther Med. 2017 May;13(5):1969-1976. doi: 10.3892/etm.2017.4211. Epub 2017 Mar 9.
It has previously been reported that 1,25-(OH)D inhibits high glucose-induced epithelial-to-mesenchymal transition (EMT) in HK-2 cells. However, the mechanism of this renoprotective action remains unclear. Elocalcitol (BXL-628), a vitamin D analog, has been suggested to be effective on the RhoA/Rho associated protein kinase (ROCK) pathway, which serves a crucial role in high glucose-induced EMT. The aim of the present study was to investigate the effect of 1,25-(OH)D and its analogue BXL-628 on high glucose-induced activation of the RhoA/ROCK pathway in human renal proximal tubular cells. HK-2 cells were co-treated with high glucose and either 1,25-(OH)2D3 or BXL-628. The RhoA expression levels and ROCK activity of the membrane were assessed via western blot analysis or immunofluorescence. α-smooth muscle actin (α-SMA) and epithelial (E)-cadherin were detected using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), whereas collagen I and fibronectin levels were measured by ELISA and RT-qPCR. The results demonstrated that 1,25-(OH)D and BXL-628 both significantly downregulated the expression of active RhoA and ROCK activity induced by high glucose (P<0.05). Furthermore, the expressions of α-SMA, collagen I, and fibronectin were significantly downregulated at both protein and mRNA (P<0.05) levels, whereas the expression of E-cadherin was significantly increased (P<0.05) by 1,25-(OH)D or BXL-628 treatment. In conclusion, the vitamin D receptor agonist 1,25-(OH)D and its analogue BXL-628 were both able to attenuate high glucose-induced EMT and extracellular matrix accumulation of HK-2 cells by suppressing the RhoA/ROCK signaling pathway .
此前有报道称，1,25-(OH)D可抑制高糖诱导的HK-2细胞上皮-间质转化（EMT）。然而，这种肾脏保护作用的机制仍不清楚。维生素D类似物依洛卡醇（BXL-628）已被证明对RhoA/Rho相关蛋白激酶（ROCK）途径有效，该途径在高糖诱导的EMT中起关键作用。本研究的目的是探讨1,25-(OH)D及其类似物BXL-628对高糖诱导的人肾近端小管细胞RhoA/ROCK途径激活的影响。HK-2细胞与高糖以及1,25-(OH)2D3或BXL-628共同处理。通过蛋白质印迹分析或免疫荧光评估膜上的RhoA表达水平和ROCK活性。使用蛋白质印迹法和逆转录-定量聚合酶链反应（RT-qPCR）检测α-平滑肌肌动蛋白（α-SMA）和上皮（E）-钙黏蛋白，而通过酶联免疫吸附测定（ELISA）和RT-qPCR测量I型胶原蛋白和纤连蛋白水平。结果表明，1,25-(OH)D和BXL-628均显著下调高糖诱导的活性RhoA表达和ROCK活性（P<0.05）。此外，1,25-(OH)D或BXL-628处理后，α-SMA、I型胶原蛋白和纤连蛋白的表达在蛋白质和mRNA水平均显著下调（P<0.05），而E-钙黏蛋白的表达显著增加（P<0.05）。总之，维生素D受体激动剂1,25-(OH)D及其类似物BXL-628均能够通过抑制RhoA/ROCK信号通路减轻高糖诱导的HK-2细胞EMT和细胞外基质积聚。
