Cellular Biology in Renal Diseases Laboratory, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain.
Nephron Exp Nephrol. 2012;122(1-2):62-74. doi: 10.1159/000346575. Epub 2013 Mar 14.
BACKGROUND/AIMS: Chronic kidney disease is characterized by accumulation of extracellular matrix in the tubulointerstitial area. Fibroblasts are the main matrix-producing cells. One source of activated fibroblasts is the epithelial mesenchymal transition (EMT). In cultured tubular epithelial cells, transforming growth factor-β (TGF-β1) induced Gremlin production associated with EMT phenotypic changes, and therefore Gremlin has been proposed as a downstream TGF-β1 mediator. Gremlin is a developmental gene upregulated in chronic kidney diseases associated with matrix accumulation, but its direct role in the modulation of renal fibrosis and its relation with TGF-β has not been investigated.
Murine renal fibroblasts and human tubular epithelial cells were studied. Renal fibrosis was determined by evaluation of key profibrotic factors, extracellular matrix proteins (ECM) and EMT markers by Western blot/confocal microscopy or real-time PCR. Endogenous Gremlin was targeted with small interfering RNA.
In murine fibroblasts, stimulation with recombinant Gremlin upregulated profibrotic genes, such as TGF-β1, and augmented the production of ECM proteins, including type I collagen. The blockade of endogenous Gremlin with small interfering RNA inhibited TGF-β1-induced ECM upregulation. In tubular epithelial cells Gremlin also increased profibrotic genes and caused EMT changes: phenotypic modulation to myofibroblast-like morphology, loss of epithelial markers and in-duction of mesenchymal markers. Moreover, Gremlin gene silencing inhibited TGF-β1-induced EMT changes.
Gremlin directly activates profibrotic events in cul-tured renal fibroblasts and tubular epithelial cells. Moreover, endogenous Gremlin blockade inhibited TGF-β-mediated matrix production and EMT, suggesting that Gremlin could be a novel therapeutic target for renal fibrosis.
背景/目的：慢性肾脏病的特征是细胞外基质在肾小管间质区域的积累。成纤维细胞是产生基质的主要细胞。激活的成纤维细胞的一个来源是上皮间质转化（EMT）。在培养的肾小管上皮细胞中，转化生长因子-β（TGF-β1）诱导Gremlin 的产生与 EMT 表型变化相关，因此 Gremlin 被提出作为 TGF-β1 的下游介质。Gremlin 是一种在与基质积累相关的慢性肾脏病中上调的发育基因，但它在调节肾脏纤维化及其与 TGF-β的关系方面的直接作用尚未被研究。
研究了鼠肾成纤维细胞和人肾小管上皮细胞。通过 Western blot/共聚焦显微镜或实时 PCR 评估关键的致纤维化因子、细胞外基质蛋白（ECM）和 EMT 标志物来确定肾纤维化。用小干扰 RNA 靶向内源性 Gremlin。
在鼠成纤维细胞中，重组 Gremlin 的刺激上调了致纤维化基因，如 TGF-β1，并增加了 ECM 蛋白的产生，包括 I 型胶原。用小干扰 RNA 阻断内源性 Gremlin 抑制了 TGF-β1 诱导的 ECM 上调。在肾小管上皮细胞中，Gremlin 也增加了致纤维化基因并引起 EMT 变化：向肌成纤维细胞样形态的表型调节、上皮标志物的丧失和间质标志物的诱导。此外，Gremlin 基因沉默抑制了 TGF-β1 诱导的 EMT 变化。
Gremlin 直接激活培养的肾成纤维细胞和肾小管上皮细胞中的致纤维化事件。此外，内源性 Gremlin 阻断抑制了 TGF-β 介导的基质产生和 EMT，这表明 Gremlin 可能是肾脏纤维化的一种新的治疗靶点。
