Institute of Nephrology of Chongqing and Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
Toxicology. 2012 Jul 16;297(1-3):68-75. doi: 10.1016/j.tox.2012.04.004. Epub 2012 Apr 15.
Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by progressive tubulointerstitial fibrosis, chronic renal failure and urothelial cancer. Our previous study showed that bone morphogenetic protein-7 (BMP-7) could attenuate AA-induced epithelial-to-mesenchymal transition (EMT) in human proximal tubule epithelial cells (PTEC). However, how gremlin (a BMP-7 antagonist) antagonizes the BMP-7 action in PTEC remained unsolved. The aim of the current study was to investigate the role of gremlin in AA-induced EMT in PTEC (HK-2 cells). HK-2 cells were treated with AA (10 μmol/L) for periods up to 72 h. Cell viability was determined by tetrazolium dye (MTT) assay. Morphological changes were assessed by phase-contrast microscopy. Markers of EMT, including E-cadherin and α-smooth muscle actin (α-SMA) were detected by indirect immunofluorescence stains. The BMP-7 and gremlin mRNA and protein expression in HK-2 cells were analyzed by quantitative real-time PCR (real-time RT-PCR) and western blotting after exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7 activity, was also determined by western blot analysis. Cells were transfected with gremlin siRNA to determine the effects of gremlin knockdown on markers of EMT following treatment with AA. Our results indicated that AA-induced EMT was associated with acquisition of fibroblast-like cell shape, loss of E-cadherin, and increases of alpha-SMA and collagen type I. Interestingly, exposure of HK-2 cells to 10 μmol/L AA increased the mRNA and protein expression of gremlin in HK-2 cells. This increase was in parallel with a decrease in BMP-7 expression and a down-regulation of phosphorylated Smad1/5/8 protein levels. Moreover, transfection with siRNA to gremlin was able to recover BMP-7 signaling activity, and attenuate EMT-associated phenotypic changes induced by AA. Together, these observations strongly suggest that gremlin plays a critical role in the modulation of reno-protective action of BMP, and that inhibition of gremlin will be a promising means of developmenting novel treatments for AAN.
摄入马兜铃酸（AA）与马兜铃酸肾病（AAN）的发生有关，其特征是进行性肾小管间质纤维化、慢性肾衰竭和尿路上皮癌。我们之前的研究表明，骨形态发生蛋白-7（BMP-7）可以减轻人近端肾小管上皮细胞（PTEC）中 AA 诱导的上皮间质转化（EMT）。然而，gremlin（BMP-7 的拮抗剂）如何拮抗 BMP-7 在 PTEC 中的作用仍未解决。本研究旨在探讨 gremlin 在 AA 诱导的 PTEC（HK-2 细胞）EMT 中的作用。HK-2 细胞用 AA（10 μmol/L）处理长达 72 h。通过四唑染料（MTT）测定法测定细胞活力。通过相差显微镜评估形态变化。通过间接免疫荧光染色检测 EMT 标志物，包括 E-钙粘蛋白和α-平滑肌肌动蛋白（α-SMA）。用定量实时 PCR（实时 RT-PCR）和 Western blot 分析 AA 暴露后 HK-2 细胞中 BMP-7 和 gremlin 的 mRNA 和蛋白表达。通过 Western blot 分析测定 BMP-7 活性的标记物磷酸化 Smad1/5/8 的水平。用 gremlin siRNA 转染细胞，以确定在 AA 处理后，gremlin 敲低对 EMT 标志物的影响。结果表明，AA 诱导的 EMT 与获得成纤维细胞样细胞形状、E-钙粘蛋白丢失以及α-SMA 和胶原 I 增加有关。有趣的是，HK-2 细胞暴露于 10 μmol/L AA 会增加 HK-2 细胞中 gremlin 的 mRNA 和蛋白表达。这种增加与 BMP-7 表达减少和磷酸化 Smad1/5/8 蛋白水平下调平行。此外，用 gremlin siRNA 转染能够恢复 BMP-7 信号活性，并减轻 AA 诱导的 EMT 相关表型变化。总之，这些观察结果强烈表明，gremlin 在调节 BMP 的肾保护作用中起关键作用，抑制 gremlin 将是开发 AAN 新型治疗方法的有前途的手段。
