Abstract
BackgroundEverolimus (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.MethodsSeveral 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.ResultsBiomolecular 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.ConclusionsOur 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.
Highlights
Everolimus (EVE) belongs to the group of drugs called mammalian target of rapamycin inhibitors, a group of proliferation signal inhibitors used in several de novo and maintenance renal transplant immunosuppressive protocols and to treat some tumors [1].The main mechanism of action of this drug is the inhibition of mTOR, a regulatory protein kinase involved in lymphocyte proliferation, developmental processes such as neurologic and muscle generation, and tumor cell growth [2,3]
Everolimus-induced matrix metalloproteinase 9 (MMP9) gene expression To evaluate whether EVE treatment was able to modulate MMP9 transcription in wild-type (WT) and HPSEsilenced HK-2 cells [Figure 1], we first treated for 6 hours both cell lines with EVE
Role of AKT Since mTORC1 inhibition may lead to AKT activation and since AKT pathway has a central role in epithelial to mesenchymal transition (EMT), we investigated the effect of EVE in AKT-silenced cells
Summary
Everolimus (EVE) belongs to the group of drugs called mammalian target of rapamycin inhibitors (mTOR-I), a group of proliferation signal inhibitors used in several de novo and maintenance renal transplant immunosuppressive protocols and to treat some tumors [1].The main mechanism of action of this drug is the inhibition of mTOR, a regulatory protein kinase involved in lymphocyte proliferation, developmental processes such as neurologic and muscle generation, and tumor cell growth [2,3]. Because of its relative low nephrotoxicity, EVE is a valid option to calcineurin inhibitors for maintenance immune suppression in patients with chronic allograft nephropathy [10]. It is clear the clinical utility of this drug category, as other antineoplastic/immunosuppressive drugs, mTOR-I may induce the development of several renal (e.g., proteinuria) and systemic side effects including hematological disorders (e.g., anemia, leukopenia and thrombocytopenia), dismetabolism (e.g., hyperlipidemia, post-transplant diabetes), lymphedema, stomatitis and fertility/gonadic toxicity [11,12,13]. Heparanase, an enzyme recently associated with the progression of chronic allograft nephropathy, could contribute to activate this machinery in renal cells
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