Abstract
Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.
Highlights
Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy
In order to address this issue, we first studied the response under hyperglycemia of the hypoxia-inducible factor-1α (HIF-1α)-hypoxia-responsive elements (HRE) pathway in HK-2 immortalized PTC cells in which the canonical oxygen, iron-PHD-pVHL-ubiquitin-dependent proteasome pathway of HIF-1α degradation had been inhibited at different levels
This reporter gene exhibited a strong hypoxia-dependent activation response in cells exposed to low glucose (LG) which was significantly reduced when cells were incubated with high glucose (HG) but not with osmotic control mannitol (Fig. 1d, left)
Summary
Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). HIF-1α protein accumulates rapidly -in the absence of a significant increase in HIF-1α mRNA- and dimerizes with the β subunit because HIF-1α is stabilized by inhibition of the canonical PHD-pvHL-proteasome degradation pathway This heterodimer, together with transcriptional coactivators p300/CBP, binds to hypoxia-responsive elements (HRE) in target genes thereby activating the transcription of genes involved in adaptative and survival responses to hypoxia (i.e. angiogenesis, cell proliferation, anaerobic metabolism, wound healing, etc). It is conceivable that, the MPs produced by HK-2 cells in the HGHD microenvironment may convey signals that contribute to impair the regulation of HIF-1 in these cells
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