Melatonin Lowers HIF-1α Content in Human Proximal Tubular Cells (HK-2) Due to Preventing Its Deacetylation by Sirtuin 1.

Although melatonin is widely known for its nephroprotective properties, there are no reports clearly pointing at its impact on the activity of hypoxia-inducible factor-1 (HIF-1), the main mediator of metabolic responses to hypoxia, in kidneys. The aim of the present study was to elucidate how melatonin affects the expression of the regulatory subunit HIF-1α in renal proximal tubules. HK-2 cells, immortalized human proximal tubular cells, were cultured under hypoxic conditions (1% O). Melatonin was applied at 100 μM concentration. Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. HIF-1α acetylation level was established by means of immunoprecipitation followed by Western blot. Melatonin receptors MT1 and MT2 localization in HK-2 cells was visualized using immunofluorescence confocal analysis. It was found that melatonin in HK-2 cells (1) lowered HIF-1α protein, but not mRNA, content; (2) attenuated expression of HIF-1 target genes; (3) increased HIF-1α acetylation level; and (4) diminished sirtuin 1 expression (both protein and mRNA). Sirtuin 1 involvement in the regulation of HIF-1α level was confirmed applying cells with silenced gene. Moreover, the presence of membrane MT1 and MT2 receptors was identified in HK-2 cells and their ligand, ramelteon, turned out to mimic melatonin action on both HIF-1α and sirtuin 1 levels. Thus, it is concluded that the mechanism of melatonin-evoked decline in HIF-1α content in renal proximal tubular cells involves increased acetylation of this subunit which results from the attenuated expression of sirtuin 1, an enzyme reported to deacetylate HIF-1α. This observation provides a new insight to the understanding of melatonin action in kidneys.