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

BACKGROUND: Prolonged cold ischemia time, the period from the start of perfusion with cold preservation fluid after cessation of circulation due to arterial clamping until transplantation in the recipient, could induce epithelial-to-mesenchymal transition (EMT) in renal tubular cells, a process associated with chronic graft damage. In this context, everolimus (EVE) and sulodexide (SUL) could potentially slow down this process.METHODS: To assess whether SUL (50 μg/ml), EVE (at 5, 10, 100 nM) or their combination were able to inhibit EMT in human renal epithelial proximal tubular cells (HK-2) reoxygenated after 24 h under hypoxic conditions, we used classical biomolecular strategies.RESULTS: Hypoxia induced upregulation of alpha smooth muscle actin (α-SMA), fibronectin (FN) and vimentin at gene-expression and α-SMA and FN at protein levels. However, the addition, after reoxygenation, of SUL plus low-dose EVE (5 nM) to the cell culture reversed this condition. Moreover, SUL and EVE were able to inhibit the hypoxia-induced Akt phosphorylation in HK2 cells and their morphological changes. Similarly, SUL was able to reverse the hyper-expression of EMT markers induced by high EVE dosage (100 nM) in cells cultured under both normoxic and hypoxic conditions.CONCLUSIONS: Our data reveal, for the first time, that sulodexide, alone or combined to low doses of everolimus, may hinder EMT in renal cells following hypoxia or minimize fibrotic complications due to high dosage of mammalian target of rapamycin inhibitors.