Ischemia/reperfusion induces oxidative injury to proximal and distal renal tubular cells. We hypothesize that Bcl-2 protein augmentation with adenovirus vector mediated bcl-2 (Adv-bcl-2) gene transfer may improve ischemia/reperfusion induced renal proximal and distal tubular apoptosis through the mitochondrial control of Bax and cytochrome C translocation. Twentyfour hours of Adv-bcl-2 transfection to proximal and distal tubular cells in vitro upregulated Bcl-2/Bax ratio and inhibited hypoxia/reoxygenation induced cytochrome C translocation, O 2 − production and tubular apoptosis. Intra-renal arterial Adv-bcl-2 administration with renal venous clamping augmented Bcl-2 protein of rat kidney in vivo in a time-dependent manner. The maximal Bcl-2 protein expression appeared at 7 days after Adv-bcl-2 administration and the primary location of Bcl-2 augmentation was in proximal and distal tubules, but not in glomeruli. With a realtime monitoring O 2 − production and apoptosis analysis of rat kidneys, ischemia/reperfusion increased renal O 2 − level, potentiated proapoptotic mechanisms, including decrease in Bcl-2/Bax ratio, increases in caspase 3 expression and poly-(ADP-ribose)-polymerase fragments and subsequent proximal and distal tubular apoptosis. However, Adv-bcl-2 administration significantly enhanced Bcl-2/Bax ratio, decreased ischemia/reperfusion induced O 2 − amount, inhibited proximal and distal tubular apoptosis and improved renal function. Our results suggest that Adv-bcl-2 gene transfer significantly reduces ischemia/reperfusion induced oxidative injury in the kidney.

Hypoxia-inducible factor-1 (HIF-1) takes part in the transcriptional activation of hypoxia-responsive genes. HIF-1␣, a subunit of HIF-1, is rapidly degraded under normoxic conditions by the ubiquitin-proteosome system. Hypoxia up-regulates HIF-1␣ by inhibiting its degradation, thereby allowing it to accumulate to high levels with 3-6 h of hypoxia treatment and decreasing thereafter. In vascular tissues, prostacyclin (prostaglandin I 2 (PGI 2 )) is a potent vasodilator and inhibitor of platelet aggregation and is known as a vasoprotective molecule. However, the role of PGI 2 in HIF-1 activation has not been studied. In the present study, we investigated the effect of PGI 2 on HIF-1 regulation in human umbilical vein endothelial cells under prolonged hypoxia (12 h). Augmentation of PGI 2 via adenovirus-mediated gene transfer of both cyclooxygenase-1 and PGI 2 synthase activated HIF-1 by stabilizing HIF-1␣ in cells under prolonged hypoxia or the hypoxia-normoxia transition but not under normoxia. Exogenous H 2 O 2 abolished PGI 2 -and catalase-induced HIF-1␣ up-regulation, which suggests that degradation of HIF-1␣ under prolonged hypoxia is through a reactive oxygen species-dependent pathway. Moreover, PGI 2 attenuated NADPH oxidase activity by suppressing Rac1 and p47 phox expression under hypoxia. These data demonstrate a novel function of PGI 2 in down-regulating reactive oxygen species production by attenuating NADPH oxidase activity, which stabilizes HIF-1␣ in human umbilical vein endothelial cells exposed to prolonged hypoxia.Hypoxia induces a number of cellular responses, such as angiogenesis, erythropoiesis, and glycolysis, through both gene regulation and post-translational modification of proteins. Hypoxia-inducible factor-1 (HIF-1) 2 takes part in the transcriptional activation of hypoxia-responsive genes through binding to the hypoxia-responsive element (HRE) in the promoter or enhancer regions and activating a number of genes (1-4). HIF-1 is a heterodimer composed of HIF-1␣ and HIF-1␤. HIF-1␣ is rapidly degraded under normoxic conditions by the ubiquitin-proteosome system, whereas HIF-1␤ is constitutively expressed (5). Under hypoxia, HIF-1␣ has been shown to be up-regulated to high levels at 3-6 h and decrease thereafter (6, 7). A number of studies have focused on the mechanism of HIF-1␣ stabilization under hypoxia for 4 -6 h. Recently, a natural antisense HIF-1␣ was suggested to down-regulate HIF-1␣ in A549 cells under prolonged hypoxia (6). However, regulation of HIF-1␣ under prolonged hypoxia (12 h) in endothelial cells remains largely unknown.Under normoxic conditions, HIF-1␣ is regulated through hydroxylation of proline residues by prolyl hydroxylase enzymes (8, 9). The von Hippel-Lindau tumor suppressor protein (pVHL) associates with hydroxylated HIF-1␣ and targets it for ubiquitination and rapid degradation (10). Under hypoxia, prolyl hydroxylase is inactivated through oxygen-sensing mechanisms, and the unmodified HIF-1␣ accumulates, permitting dimerization with HIF-1␤ (3, 11). Various redox-de...

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