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

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