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

Masola V, Zaza G, Granata S, Gambaro G, Onisto M, Lupo A - J Transl Med (2013)

Bottom Line: 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, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Renal Unit, Department of Medicine, University-Hospital of Verona, Piazzale A, Stefani 1, 37126 Verona, Italy. gianluigi.zaza@univr.it.

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.

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Matrix metalloproteinase-9 (MMP9) gene expression and activity after Everolimus (EVE) treatment. (A) Histograms represent MMP9 mRNA expression levels determined by real-time PCR in WT and HPSE silenced HK-2 cells (shHPSE). WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 6 h. FGF-2 (10 ng/ml), a well-known EMT inducer, was used as positive control. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR. (B)Upper, gelatin zymography shows MMP-9 activity bands in the serum-free conditioned media of WT and silenced HK2 cells. WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 24 h, then conditioned media were harvested. Lower, histogram represents the densitometric analysis of relative MMP-9 activity. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR.
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Figure 2: Matrix metalloproteinase-9 (MMP9) gene expression and activity after Everolimus (EVE) treatment. (A) Histograms represent MMP9 mRNA expression levels determined by real-time PCR in WT and HPSE silenced HK-2 cells (shHPSE). WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 6 h. FGF-2 (10 ng/ml), a well-known EMT inducer, was used as positive control. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR. (B)Upper, gelatin zymography shows MMP-9 activity bands in the serum-free conditioned media of WT and silenced HK2 cells. WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 24 h, then conditioned media were harvested. Lower, histogram represents the densitometric analysis of relative MMP-9 activity. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR.

Mentions: As showed in Figure 2A, only high EVE dosages (100, 200 and 500 nM) significantly increased the MMP9 expression level, while 10 nM EVE (corresponding to therapeutic concentration) did not induce any modulation of this EMT marker.


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

Masola V, Zaza G, Granata S, Gambaro G, Onisto M, Lupo A - J Transl Med (2013)

Matrix metalloproteinase-9 (MMP9) gene expression and activity after Everolimus (EVE) treatment. (A) Histograms represent MMP9 mRNA expression levels determined by real-time PCR in WT and HPSE silenced HK-2 cells (shHPSE). WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 6 h. FGF-2 (10 ng/ml), a well-known EMT inducer, was used as positive control. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR. (B)Upper, gelatin zymography shows MMP-9 activity bands in the serum-free conditioned media of WT and silenced HK2 cells. WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 24 h, then conditioned media were harvested. Lower, histogram represents the densitometric analysis of relative MMP-9 activity. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR.
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Figure 2: Matrix metalloproteinase-9 (MMP9) gene expression and activity after Everolimus (EVE) treatment. (A) Histograms represent MMP9 mRNA expression levels determined by real-time PCR in WT and HPSE silenced HK-2 cells (shHPSE). WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 6 h. FGF-2 (10 ng/ml), a well-known EMT inducer, was used as positive control. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR. (B)Upper, gelatin zymography shows MMP-9 activity bands in the serum-free conditioned media of WT and silenced HK2 cells. WT and silenced cell lines were cultured with 10, 100, 200 and 500 nM Everolimus for 24 h, then conditioned media were harvested. Lower, histogram represents the densitometric analysis of relative MMP-9 activity. Mean ± S.D. (error bars) of three separate experiments performed in triplicate. *p < 0.05 vs WT control cells (CTR); **p < 0.001 versus WT CTR.
Mentions: As showed in Figure 2A, only high EVE dosages (100, 200 and 500 nM) significantly increased the MMP9 expression level, while 10 nM EVE (corresponding to therapeutic concentration) did not induce any modulation of this EMT marker.

Bottom Line: 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, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Renal Unit, Department of Medicine, University-Hospital of Verona, Piazzale A, Stefani 1, 37126 Verona, Italy. gianluigi.zaza@univr.it.

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.

Show MeSH
Related in: MedlinePlus