Differential regulation of Snail by hypoxia and hyperglycemia in human proximal tubule cells

The centrality of the transcriptional regulator Snail in epithelial-to-mesenchymal transformation (EMT), known to occur in models of diabetic nephropathy, has not been established. Transforming growth factor beta-1 (TGFβ1) is induced in diabetic nephropathy and induces both Snail and EMT. Hypoxia inducible factors (HIFs) are known to induce Snail, independent of TGFβ1. Notch induction is integral to Snail induction and EMT in tumour cells, but its role in the kidney is unknown. The present study was undertaken to determine the upstream regulators of Snail in the kidney in high glucose and hypoxic conditions. HK-2 cells were cultured in normoxic, hypoxic, high glucose and combined hypoxic/high glucose conditions. The expression of HIF1α, NotchIC, Snail, Lysyl oxidase-like 2 (Loxl2), and Hairy and Enhancer Split-1 (Hes1) were measured. We found that hypoxia increased HIF1α expression; however, concurrent exposure to high glucose blunted this effect. A similar pattern was observed in Lox12 expression, suggesting that Loxl2 was downstream of HIF1α, which was confirmed using siRNA techniques. Snail was upregulated by hypoxia and high glucose and in combination the effect was additive, suggesting independent upstream activation pathways by the two stimuli. Hes1 was upregulated by high glucose and to a lesser extent by hypoxia, but the effect of the combined stimuli was no greater than that observed with high glucose alone. NotchIC was downregulated by both hypoxia and high glucose, and in combination the effect was additive. Therefore, this study suggests that hypoxia and high glucose induce Snail expression through distinct pathways, independent of Notch signalling.