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. 2016 Jul 28;11(7):e0160074.
doi: 10.1371/journal.pone.0160074. eCollection 2016.

Heparanase: A Potential New Factor Involved in the Renal Epithelial Mesenchymal Transition (EMT) Induced by Ischemia/Reperfusion (I/R) Injury

Valentina Masola  1 , Gianluigi Zaza  1 , Giovanni Gambaro  2 , Maurizio Onisto  3 , Gloria Bellin  1 , Gisella Vischini  2 , Iyad Khamaysi  4 , Ahmad Hassan  5 , Shadi Hamoud  6 , Omri Nativ  7 , Samuel Heyman  8 , Antonio Lupo  1 , Israel Vlodavsky  9 , Zaid Abassi  7   10
Affiliations

Heparanase: A Potential New Factor Involved in the Renal Epithelial Mesenchymal Transition (EMT) Induced by Ischemia/Reperfusion (I/R) Injury

Valentina Masola et al. PLoS One. .

Erratum in

Abstract

Background: Ischemia/reperfusion (I/R) is an important cause of acute renal failure and delayed graft function, and it may induce chronic renal damage by activating epithelial to mesenchymal transition (EMT) of renal tubular cells. Heparanase (HPSE), an endoglycosidase that regulates FGF-2 and TGFβ-induced EMT, may have an important role. Therefore, aim of this study was to evaluate its role in the I/R-induced renal pro-fibrotic machinery by employing in vitro and in vivo models.

Methods: Wild type (WT) and HPSE-silenced renal tubular cells were subjected to hypoxia and reoxygenation in the presence or absence of SST0001, an inhibitor of HPSE. In vivo, I/R injury was induced by bilateral clamping of renal arteries for 30 min in transgenic mice over-expressing HPSE (HPA-tg) and in their WT littermates. Mice were sacrificed 48 and 72 h after I/R. Gene and protein EMT markers (α-SMA, VIM and FN) were evaluated by bio-molecular and histological methodologies.

Results: In vitro: hypoxia/reoxygenation (H/R) significantly increased the expression of EMT-markers in WT, but not in HPSE-silenced tubular cells. Notably, EMT was prevented in WT cells by SST0001 treatment. In vivo: I/R induced a remarkable up-regulation of EMT markers in HPA-tg mice after 48-72 h. Noteworthy, these effects were absent in WT animals.

Conclusions: In conclusion, our results add new insights towards understanding the renal biological mechanisms activated by I/R and they demonstrate, for the first time, that HPSE is a pivotal factor involved in the onset and development of I/R-induced EMT. It is plausible that in future the inhibition of this endoglycosidase may represent a new therapeutic approach to minimize/prevent fibrosis and slow down chronic renal disease progression in native and transplanted kidneys.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic representation of in vitro experimental design.
Serum starved HK-2 cells were exposed to 24 h of hypoxia and subsequently to reoxygenation (6 h for gene expression and 24 h for protein expression analysis). Cells were also treated or not with 200 μg/ml SST0001 (red balls) before the hypoxia (point #4; herein after referred to as H/R1) and before reoxygenation (point #5; herein after called H/R2). Control cells were cultured in normoxic conditions with or without SST0001 (points #1 and 2).
Fig 2
Fig 2. Heparanase (HPSE) and epithelial to mesenchymal transition (EMT)-associated gene expression in tubular cells exposed to hypoxia and reoxygenation (H/R).
Wild type (WT) and Heparanase-silenced (HPSE-sil) HK2 cells exposed to hypoxia and reoxygenation (H/R); WT HK-2 cells were also treated or not with 200 μg/ml SST0001. Gene expression analysis of A) Heparanase (HPSE), B) alpha smooth muscle Actin (α-SMA) and C) Fibronectin (FN) evaluated by real-time PCR. Data were normalized to GAPDH expression. NORM = normoxia. Mean ± S.D (error bars) of two separate experiments performed in triplicate. **p<0.001, *p<0.05 vs. WT CTR NORM; ##p<0.001, # p<0.05 vs WT CTR H/R.
Fig 3
Fig 3. Heparanase (HPSE) protein expression in tubular cells exposed to hypoxia and reoxygenation (H/R).
Wild type (WT) and Heparanase-silenced (HPSE-sil) HK2 cells exposed to hypoxia and reoxygenation (H/R); WT HK-2 cells were also treated or not with 200 μg/ml SST0001. A) Quantification of HPSE protein levels assessed by Western blot. GAPDH was included as loading control. NORM = normoxia. B) Representative image of western blot experiments. **p<0.001, *p<0.05 vs. WT CTR NORM; ##p<0.001, # p<0.05 vs WT CTR H/R.
Fig 4
Fig 4. Epithelial to mesenchymal transition (EMT)-associated protein expression in tubular cells exposed to hypoxia and reoxygenation (H/R).
Wild type (WT) and Heparanase-silenced (HPSE-sil) HK2 cells exposed to hypoxia and reoxygenation (H/R); WT HK-2 cells were also treated or not with 200 μg/ml SST0001. A) alpha smooth muscle Actin (α-SMA) and B) Fibronectin (FN) protein levels quantification assessed by western blot analysis. GAPDH was included as loading control. NORM = normoxia. C) Representative image of western blot experiments. **p<0.001, *p<0.05 vs. WT CTR NORM; # #p<0.001 vs. WT CTR H/R. **p<0.001, *p<0.05 vs. WT CTR NORM; ##p<0.001, # p<0.05 vs WT CTR H/R.
Fig 5
Fig 5. Morphological changes in tubular cells exposed to hypoxia and reoxygenation (H/R) and Hypoxia-inducible factor 1-alpha (HIF-1α) expression.
Wild type (WT) and Heparanase-silenced (HPSE-sil) HK2 cells exposed to hypoxia and reoxygenation (H/R); WT HK-2 cells were also treated or not with 200 μg/ml SST0001. A) HIF-1α protein levels assessed by Western blot analysis. GAPDH was included as loading control. NORM: Normoxia. Representative image of western blot experiments. B) Representative images of WT cells treated or untreated with SST0001 and HPSE-silenced HK2 cells grown on a glass surface. Cells were exposed to normoxia or 24 h hypoxia followed by 24 h reoxygenation.
Fig 6
Fig 6. In vivo Heparanase (HPSE) expression induced by Ischemia and Reperfusion (I/R) kidney injury.
A) Box plot representing relative gene expression of HPSE evaluated by real-time PCR in renal tissue extract from Wild type (WT) mice. Results were normalized to GAPDH expression. Mean ± S.E (error bars) of two separate experiments performed in triplicate. B) Representative immunofluorescence staining for HPSE in cortical renal tissues of WT mice 72 h after sham operation or I/R kidney injury. I/R, ischemia/reperfusion. C) Quantification of the immunofluorescence staining. **p<0.001vs. WT SHAM.
Fig 7
Fig 7. Epithelial to mesenchymal transition (EMT) markers in control (SHAM) mice vs. mice after Ischemia and Reperfusion (I/R) kidney injury.
Relative gene expression of A) alpha smooth muscle Actin (α-SMA) and B) Vimentin (VIM) evaluated by real-time PCR in renal tissue extracts from WT and HPA-tg mice subjected to I/R kidney injury. Mean ± S.E (error bars) of two separate experiments performed in triplicate. **p<0.001 vs WT SHAM. Representative immunofluorescence staining for C) α-SMA and D) VIM in renal tissue of WT and HPA-tg mice subjected to I/R. E) Quantification of the immunofluorescence staining. **p<0.001vs. WT HPA-tg SHAM.
Fig 8
Fig 8. Renal function in Wild Type (WT) and HPA-tg mice subjected to Ischemia and Reperfusion (I/R).
Effects of Acute kidney Injury (AKI) on serum creatinine (SCr) after I/R insult in WT and HPA-Tg mice. Results are expressed as Mean±SEM. *p<0.05, **p<0.001 vs. SHAM; ##p<0.001vs. WT I/R 72 h.
Fig 9
Fig 9. Ischemia and Reperfusion (I/R) kidney injury in wild type (wt) and HPA-tg mice.
I/R kidney injury was induced in wt and HPA-tg mice by clamping of both renal arteries for 30 minutes. Mice were sacrificed after 48 or 72 hours. Shown are representative images of PAS staining of paraffin-embedded cortex and medulla sections from various experimental groups. Magnification 40X. I/R, ischemia/reperfusion.
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