Spin trap (N-t-butyl-alpha-phenylnitrone)-mediated suprainduction of heme oxygenase-1 in kidney ischemia/reperfusion model: Role of the oxygenase in protection against oxidative injury

In mammals the rate-limiting step in heme catabolism is the heme oxygenase (HO) system. Two isozymes, HO-1 and HO-2, oxidatively cleave the substrate to form biliverdin, and the potential cellular messenger, CO; the chelated iron is released as the result of the tetrapyrrole ring opening. Biliverdin is subsequently reduced to bilirubin, an antioxidant, by biliverdin reduct ase. The aim of the present study was to investigate the involvement of HO- 1, a heat shock/stress protein, in protection offered by the spin trap agen t, N-tert-butyl-alpha-phenyl-nitrone (PBN), against kidney ischemia/reperfu sion injury. For this, HO-1 expression and assessment of the parameters ass ociated with tissue-oxidative injury were compared in the presence or absen ce of PBN pretreatment of rats (100 mg/kg i.p., 30 min) before the onset of 30-min ischemia. Twenty-four hours after reperfusion, Northern blot analys is showed an unprecedented;37-fold increase in 1.8-kb HO-1 mRNA in PBN pret reated rat kidney; HO-2 mRNA levels did not increase. At 48 h, the levels o f HO-1 mRNA remained nearly 14-fold higher than the control value. In the a bsence of PBN, the levels measured approximately 5- and 2-fold higher than control values at the 24- and 48-h intervals, respectively. PBN pretreatmen t also resulted in a most impressive increase in the levels of HO-1 protein as judged by Western blot analysis and measurement of enzyme activity at t he 24-h time point. As detected by immunohistochemical analysis, PBN pretre atment caused an increase in HO-1 and biliverdin reductase-immunoreactive p roteins in the cortex and in the outer stripe of the outer medulla. In the absence of PBN pretreatment, there was an intense immunostaining for HO-1 i n the medullary rays, which corresponded with iron and lipid peroxidation s taining of the region; these observations were not made with PBN-pretreated kidneys. Collectively, the findings are consistent with the likelihood tha t suprainduction of HO-1 gene expression protects the kidney from free radi cal-mediated injury by increasing the capacity to produce the potent cellul ar antioxidant bilirubin. We also suggest spin trap-mediated protection aga inst ischemia/reperfusion injury is likely due to a sustained elevation of HO-1 gene expression by formation of stable radicals.