Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells.

The present study hypothesized that superoxide (O2(-)*) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1alpha expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O2(-)* generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl(2)-induced increase in HIF-1alpha levels and completely blocked the increase in HIF-1alpha levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O2(-)* dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1alpha levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1alpha levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22(phox), into RMICs markedly increased HIF-1alpha levels. In contrast, the OH* scavenger tetramethylthiourea had no effect on the accumulation of HIF-1alpha in these renal cells. By Northern blot analysis, scavenging or dismutation of O2(-)* by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1alpha-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O2(-)* levels induce HIF-1alpha degradation independently of H(2)O(2) and OH* radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1alpha in these cells under physiological conditions.