Xu Yan-fang, Wan Jian-xin, Jiang De-wen
Department of Nephrology, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China.
Zhonghua Yi Xue Za Zhi. 2009 Jun 16;89(23):1639-44.
To investigate the effects of bone morphogenic protein (BMP)-7 upon epithelial-to-mesenchymal transition (EMT) and the expression of connective tissue growth factor (CTGF) in human renal proximal tubular epithelial cells (HK-2) induced by transforming growth factor-beta1, (TGF-beta1) and to explore the possible mechanisms of BMP-7 for the inhibition and reversal of renal interstitial fibrosis.
HK-2 cells were treated with TGF-beta1 or a combination of TGF-beta1 and BMP-7. RT-PCR and Western blot were used to determine the mRNA and protein expression of alpha-SMA, E-cadherin and CTGF. For EMT reversal experiments, when TGF-beta1-induced EMT occurred, then the medium was removed and replaced with medium containing 200 ng/ml BMP-7. After 48 h, the morphological changes and the expression of E-cadherin were assessed by phase contrast microscopy or immunofluorescent microscopy.
The control cells displayed typical cobblestone morphology of epithelial cells. 3 ng/ml TGF-beta1 induced profound morphologic changes after 48 h, with cells becoming elongated in shape, but addition of 200 ng/ml BMP-7 for 48 h restored the epithelial morphology of HK-2 cells. Indirect immunofluorescence showed that treatment of 3 ng/ml TGF-beta1 resulted in a distinct loss of E-cadherin staining in the plasma membrane of HK-2 cells but subsequent treatment with 200 ng/ml BMP-7 largely restored the E-cadherin protein staining. 3 ng/ml TGF-beta1 significantly up-regulated the mRNA and protein expression of alpha-SMA, reduced the expression of E-cadherin and increased the expression of CTGF after 48 h (vs. control, P < 0.01). BMP-7 dramatically suppressed the mRNA and protein expression of alpha-SMA, restored the expression of E-cadherin and prevented the expression of CTGF in a dose-dependent manner after co-incubation with TGF-beta1 for 48 h (400 ng/ml BMP-7 + TGF-beta1 vs. TGF-beta1, alone, P < 0.01).
BMP-7 exerts its antifibrotic effect partially through blocking and reversing TGF-beta1-induced EMT and downregulating the expression of CTGF in human renal tubular epithelial cells.
研究骨形态发生蛋白（BMP）-7对转化生长因子-β1（TGF-β1）诱导的人肾近端小管上皮细胞（HK-2）上皮-间质转化（EMT）及结缔组织生长因子（CTGF）表达的影响，探讨BMP-7抑制和逆转肾间质纤维化的可能机制。
用TGF-β1或TGF-β1与BMP-7联合处理HK-2细胞。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测α-平滑肌肌动蛋白（α-SMA）、E-钙黏蛋白和CTGF的mRNA及蛋白表达。在EMT逆转实验中，当TGF-β1诱导EMT发生后，去除培养基，换为含200 ng/ml BMP-7的培养基。48小时后，通过相差显微镜或免疫荧光显微镜评估形态学变化及E-钙黏蛋白的表达。
对照细胞呈现典型的上皮细胞鹅卵石样形态。3 ng/ml TGF-β1处理48小时后诱导了显著的形态学变化，细胞变长，但加入200 ng/ml BMP-7处理48小时可恢复HK-2细胞的上皮形态。间接免疫荧光显示，3 ng/ml TGF-β1处理导致HK-2细胞质膜上E-钙黏蛋白染色明显缺失，但随后用200 ng/ml BMP-7处理可在很大程度上恢复E-钙黏蛋白蛋白染色。3 ng/ml TGF-β1在48小时后显著上调α-SMA的mRNA和蛋白表达，降低E-钙黏蛋白表达并增加CTGF表达（与对照组相比，P<0.01）。与TGF-β1共同孵育48小时后，BMP-7以剂量依赖方式显著抑制α-SMA的mRNA和蛋白表达，恢复E-钙黏蛋白表达并阻止CTGF表达（400 ng/ml BMP-7+TGF-β1与单独TGF-β1相比，P<0.01）。
BMP-7部分通过阻断和逆转TGF-β1诱导的EMT以及下调人肾小管上皮细胞中CTGF的表达发挥抗纤维化作用。
