Masola Valentina, Zaza Gianluigi, Granata Simona, Gambaro Giovanni, Onisto Maurizio, Lupo Antonio
Renal Unit, Department of Medicine, University-Hospital of Verona, Piazzale A, Stefani 1, 37126 Verona, Italy.
J Transl Med. 2013 Nov 20;11:292. doi: 10.1186/1479-5876-11-292.
Everolimus (EVE) is a drug widely used in several renal transplant protocols. Although characterized by a relatively low nephrotoxicity, it may induce several adverse effects including severe fibro-interstitial pneumonitis. The exact molecular/biological mechanism associated to these pro-fibrotic effects is unknown, but epithelial to mesenchymal transition (EMT) may have a central role. Additionally, heparanase, an enzyme recently associated with the progression of chronic allograft nephropathy, could contribute to activate this machinery in renal cells.
Several biomolecular strategies (RT-PCR, immunofluorescence, zymography and migration assay) have been used to assess the capability of EVE (10, 100, 200 and 500 nM) to induce an in vitro heparanase-mediated EMT in wild-type (WT) and Heparanase (HPSE)-silenced immortalized human renal epithelial proximal tubular cells (HK-2). Additionally, microarray technology was used to find additional biological elements involved in EVE-induced EMT.
Biomolecular experiments demonstrated a significant up-regulation (more than 1.5 fold increase) of several genes encoding for well known EMT markers [(alpha-smooth muscle actin (α-SMA), Vimentin (VIM), Fibronectin (FN) and matrix metalloproteinase-9 (MMP9)], enhancement of MMP9 protein level and increment of cells motility in WT HK2 cells treated with high concentrations of EVE (higher than 100 nM). Similarly, immunofluorescence analysis showed that 100 nM of EVE increased α-SMA, VIM and FN protein expression in WT HK2 cells. All these effects were absent in both HPSE- and AKT-silenced cell lines. AKT is a protein having a central role in EMT. Additionally, microarray analysis identified other 2 genes significantly up-regulated in 100 nM EVE-treated cells (p < 0.005 and FDR < 5%): transforming growth factor beta-2 (TGFβ2) and epidermal growth factor receptor (EGFR). Real-time PCR analysis validated microarray.
Our in vitro study reveals new biological/cellular aspects of the pro-fibrotic activity of EVE and it demonstrates, for the first time, that an heparanase-mediated EMT of renal tubular cells may be activated by high doses of this drug. Additionally, our results suggest that clinicians should administer the adequate dosage of EVE in order to increase efficacy and reduce adverse effects. Finally heparanase could be a new potential therapeutic target useful to prevent/minimize drug-related systemic fibrotic adverse effects.
依维莫司（EVE）是一种广泛应用于多种肾移植方案的药物。尽管其肾毒性相对较低，但它可能会引发多种不良反应，包括严重的纤维间质性肺炎。与这些促纤维化作用相关的确切分子/生物学机制尚不清楚，但上皮-间质转化（EMT）可能起核心作用。此外，乙酰肝素酶是一种最近与慢性移植肾肾病进展相关的酶，可能有助于激活肾细胞中的这一机制。
已采用多种生物分子策略（逆转录聚合酶链反应、免疫荧光、酶谱分析和迁移试验）来评估依维莫司（10、100、200和500 nM）在野生型（WT）和乙酰肝素酶（HPSE）沉默的永生化人肾上皮近端小管细胞（HK-2）中诱导体外乙酰肝素酶介导的EMT的能力。此外，利用微阵列技术寻找参与依维莫司诱导的EMT的其他生物元件。
生物分子实验表明，在高浓度依维莫司（高于100 nM）处理的WT HK2细胞中，几种编码著名EMT标志物[α-平滑肌肌动蛋白（α-SMA）、波形蛋白（VIM）、纤连蛋白（FN）和基质金属蛋白酶-9（MMP9）]的基因显著上调（增加超过1.5倍），MMP9蛋白水平增强，细胞运动性增加。同样，免疫荧光分析显示，100 nM依维莫司可增加WT HK2细胞中α-SMA、VIM和FN蛋白的表达。在HPSE和AKT沉默的细胞系中均未出现所有这些效应。AKT是一种在EMT中起核心作用的蛋白质。此外，微阵列分析确定了在100 nM依维莫司处理的细胞中另外2个显著上调的基因（p < 0.005且错误发现率< 5%）：转化生长因子β-2（TGFβ2）和表皮生长因子受体（EGFR）。实时聚合酶链反应分析验证了微阵列结果。
我们的体外研究揭示了依维莫司促纤维化活性的新的生物学/细胞方面，并且首次证明高剂量该药物可能激活肾小管细胞的乙酰肝素酶介导的EMT。此外，我们的结果表明临床医生应给予适当剂量的依维莫司，以提高疗效并减少不良反应。最后，乙酰肝素酶可能是一个新的潜在治疗靶点，有助于预防/最小化药物相关的全身性纤维化不良反应。
