Everolimus-induced epithelial to mesenchymal transition in immortalized human renal proximal tubular epithelial cells: key role of heparanase

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.

Sulodexide alone or in combination with low doses of everolimus inhibits the hypoxia-mediated epithelial to mesenchymal transition in human renal proximal tubular cells

“…Notably, SUL alone or combined with 5 nM EVE caused the downregulation of all three EMT markers. On the contrary, a high concentration EVE (100 nM) was able to significantly upregulate all genes encoding for the three mesenchymal markers (confirming our previous published results [24]). However, the co-treatment with SUL reversed the pro-fibrotic effects induced by the high dose of EVE.…”

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“…6,7,11 Epithelial to mesenchymal transition (EMT), a phenotypic conversion of epithelium to a fibroblastic or myofibroblastic phenotype, induced by suppression of the Akt protein kinase signaling pathway, may play a central role in the mTOR inhibitor-associated profibrotic process. 10 Masola and associates 10 recently demonstrated that high concentrations of everolimus induced an up-regulation of all EMT markers in human renal epithelial proximal tubular cells, including alpha-smooth muscle actin, vimentin, fibronectin, and matrix metalloproteinase 9, at both the gene and protein levels. The promotion of EMT may be facilitated 10 or not 12 by a transforming growth factor β-dependent mechanism and appears to be mediated through a heparanase-stimulating process 10 known to be involved in the pathogenesis of several proteinuric nephropathies.…”

“…10 Masola and associates 10 recently demonstrated that high concentrations of everolimus induced an up-regulation of all EMT markers in human renal epithelial proximal tubular cells, including alpha-smooth muscle actin, vimentin, fibronectin, and matrix metalloproteinase 9, at both the gene and protein levels. The promotion of EMT may be facilitated 10 or not 12 by a transforming growth factor β-dependent mechanism and appears to be mediated through a heparanase-stimulating process 10 known to be involved in the pathogenesis of several proteinuric nephropathies. 13,14 In contrast, low dose of everolimus, commonly used during organ transplant, failed to induce EMT in the same experimental settings.…”

“…7,8 High-dose of everolimus in animal models are associated with glomeulosclerosis 9 and tubulointerstitial fibrosis. 10 The exact molecular and/or biologic mechanisms associated with these profibrotic effects remain unknown. Suppression of mTOR Complex 1 activity may lead to a marked inhibition of the endothelial cell through an alteration in vascular endothelial growth factor balance at the podocyte and may cause reduced expression of several slit diaphragm-associated proteins and key podocyte structures, such as nephrin, podocin, and CD2ap, leading to disruption of the filtration barrier, proteinuria, and glomerulosclerosis.…”

“…13,14 In contrast, low dose of everolimus, commonly used during organ transplant, failed to induce EMT in the same experimental settings. 10 Therefore, mTOR inhibitor-associated nephrotoxicity is characterized predominantly by a possible apoptotic renal epithelial cell injury 15 that affects both podocytes [7][8][9]11,[16][17][18][19] in the glomerular compartment and renal tubular epithelial cells 10,[20][21][22][23][24] in the tubulointerstitial compartment. Consequently, the IFTA observed with mTOR inhibitor therapy may be the result of a cumulative toxic renal tubular injury induced by the podocytopathy-associated proteinuria and/or by the direct toxic tubular insult of the drug itself.…”