title: Everolimus-induced epithelial to mesenchymal transition in immortalized human renal proximal tubular epithelial cells: key role of heparanase.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Valentina
authors.initials: V
authors.lastname: Masola
authors.affiliation: Renal Unit, Department of Medicine, University-Hospital of Verona, Piazzale A, Stefani 1, 37126 Verona, Italy. gianluigi.zaza@univr.it.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Zaza
authors.forename: Simona
authors.initials: S
authors.lastname: Granata
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gambaro
authors.forename: Maurizio
authors.initials: M
authors.lastname: Onisto
authors.forename: Antonio
authors.initials: A
authors.lastname: Lupo
mesh_terms: D000199:Actins / Q000235:genetics; D002461:Cell Line, Transformed; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D000068338:Everolimus; D005353:Fibronectins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000494:pharmacology; D014746:Vimentin / Q000235:genetics; D000099175:Heparanase
chemical_list: D000199:Actins; D000068338:Everolimus; D005353:Fibronectins; D005966:Glucuronidase; D007166:Immunosuppressive Agents; D020780:Matrix Metalloproteinase 9; D020123:Sirolimus; D014746:Vimentin; D000099175:Heparanase; C541116:ACTA2 protein, human