Abst ract. During renal ischemia, ATP is degraded to hypoxanthine. When xanthine oxidase converts hypoxanthine to xanthine in the presence of molecular oxygen, superoxide radical (O°) is generated. We studied the role ofO°and its reduction product OH * in mediating renal injury after ischemia. Male Sprague-Dawley rats underwent right nephrectomy followed by 60 min of occlusion of the left renal artery. The O2 scavenger superoxide dismutase (SOD) was given 8 min before clamping and before release of the renal artery clamp. Control rats received 5% dextrose instead. Plasma creatinine was lower in SOD treated rats: 1.5, 1.0, and 0.8 mg/dl vs. 2.5, 2.5, and 2.1 mg/dl at 24, 48, and 72 h postischemia. 24 h after ischemia inulin clearance was higher in SOD treated rats than in controls (399 vs. 185 ,gl/min). Renal blood flow, measured after ischemia plus 15 min of reflow, was also greater in SOD treated than in control rats. Furthermore, tubular injury, judged histologically in perfusion fixed spedimens, was less in SOD treated rats. Rats given SOD inactivated by prior incubation with diethyldithiocarbamate had plasma creatinine values no different from those of control rats. The OH * scavenger dimethylthiourea (DMTU) was given before renal artery occlusion. DMTU treated rats had lower plasma creatinine than did controls: 1.7, 1.7, and 1.3 mg/dl vs. 3.2, 2.2, and 2.4 mg/dl at 24, 48, and 72 h postischemia. Neither SOD nor DMTU caused an increase in renal blood flow, urine flow rate, or solute excretion in normal rats. The xanthine oxidase inhibitor allopurinol was given before ischemia to prevent the generation of oxygen free radicals. Plasma creatinine This work was presented in abstract form to the Central Society for Clinical Research, Chicago, 1983 and to the American Society of Nephrology, 16th annual meeting, Washington DC, 1983. Received for publication 13 December 1983 and in revised form 6 June 1984.was lower in allopurinol treated rats: 2.7, 2.2, and 1.4 mg/dl vs. 3.6, 3.5, and 2.3 mg/dl at 24, 48, and 72 h postischemia. Catalase treatment did not protect against renal ischemia, perhaps because its large size limits glomerular filtration and access to the tubular lumen. Superoxide-mediated lipid peroxidation was studied after renal ischemia. 60 min of ischemia did not increase the renal content of the lipid peroxide malondialdehyde, whereas ischemia plus 15 min reflow resulted in a large increase in kidney lipid peroxides. Treatment with SOD before renal ischemia prevented the reflow-induced increase in lipid peroxidation in renal cortical mitochondria but not in crude cortical homogenates. In summary, the oxygen free radical scavengers SOD and DMTU, and allopurinol, which inhibits free radical generation, protected renal function after ischemia. Reperfusion after ischemia resulted in lipid peroxidation; SOD decreased lipid peroxidation in cortical mitochondria after renal ischemia and reflow. We conclude that restoration of oxygen supply to ischemic kidney results in the production of oxygen free rad...

In ischemic acute renal failure oxygen free radicals may mediate injury. In addition, iron appears to play a critical role in hydroxyl radical formation and lipid peroxidation during reperfusion of ischemic kidneys. To determine whether iron may play a similar role in pigment (heme protein)-induced acute renal failure, we studied the effects of the iron chelator deferoxamine in two experimental models of pigment-induced acute renal failure, intramuscular glycerol injection and intravenous hemoglobin infusion without and with concurrent ischemia in the rat. Intramuscular injection of 50% glycerol (5 ml/kg) caused inulin clearance to fall to 0.13 +/- 0.03 (SE) ml/min (normal value, 1.0-1.2 ml/min). Continuous infusion of deferoxamine beginning at the time of glycerol injection significantly attenuated this renal dysfunction. Deferoxamine-treated animals had an inulin clearance of 0.37 +/- 0.06 ml/min (P less than 0.01). Glycerol injection was also associated with significant lipid peroxidation, measured as renal malondialdehyde content. Deferoxamine-treated glycerol-injected rats had renal malondialdehyde content not significantly different from control animals. In another model of heme pigment-induced renal injury, hemoglobin was infused to produce hemoglobinuria. Inulin clearance 1 h after hemoglobin infusion was significantly reduced to 0.84 +/- 0.5 ml/min (P less than 0.025). Infusion of deferoxamine after hemoglobin prevented the hemoglobin-induced decrease in inulin clearance. Thirty minutes of renal ischemia followed by infusion of hemoglobin resulted in more severe renal dysfunction with inulin clearance of 0.54 +/- 0.08 ml/min. Deferoxamine infused at the time of reperfusion attenuated the fall in glomerular filtration rate after ischemia and hemoglobin infusion:inulin clearance 1.04 +/- 0.07 (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of acute and chronic administration of cyclosporine on systemic and renal hemodynamics was studied in conscious rats. Infusion of cyclosporine in a dose of 20 mg/kg (Cy 20) resulted in a significant fall in renal blood flow (RBF) (3.4 vs. 6.5 ml/min/g, P less than 0.05) and a rise in renal vascular resistance (RVR) (36.9 vs. 20.6 mm Hg/ml/min/g, P less than 0.05). Infusion of cyclosporine at a dose of 10 mg/kg (Cy 10) did not result in a significant change in RBF or RVR. Both doses of cyclosporine resulted in stimulation of plasma renin activity (PRA) from control values of 5.6 +/- 0.8 ng/ml/hr to 11.6 +/- 2.0 with 10 mg/kg and 26.7 +/- 5.6 with 20 mg/kg. Urinary 6-keto-PGF1 alpha excretion increased from control values of 14.0 +/- 2.0 ng/6 hr to 22.7 +/- 2.2 with 10 mg/kg and 25.0 +/- 2.0 with 20 mg/kg. Similar effects on RBF, RVR, PRA, and 6-keto-PGF1 alpha excretion were seen after chronic administration of cyclosporine (20 mg/kg i.p. for 7 days). Pretreatment of animals with captopril did not prevent the fall in RBF after cyclosporine, suggesting that the vasoconstriction was not mediated by angiotensin II. Animals treated with meclofenamate demonstrated reduction in RBF with 10 mg/kg cyclosporine (4.3 vs. 7.0 ml/min/g, P less than 0.05), suggesting that prostaglandins protect against the vasoconstrictor effect of cyclosporine. Administration of phenoxybenzamine after cyclosporine improved RBF (5.0 vs. 3.4 ml/min/g) and restored RVR to normal. Similarly, renal denervation dramatically reduced the fall in RBF after cyclosporine (innervated right kidney 3.6 vs. denervated left kidney 6.0 ml/min/g, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether iron participates in free radical-mediated postischemic renal injury and lipid peroxidation, we examined the effects of removal of endogenous iron or provision of exogenous iron following renal ischemia, as well as the effects of renal ischemia and reperfusion on renal venous and urinary "free" iron. Rats underwent 60 minutes of renal ischemia and were studied after either 24 hours (inulin clearance) or 15 minutes (renal malondialdehyde content) of reperfusion. Infusion of the iron chelator deferoxamine (200 mg/kg/hr) during the first 60 minutes of reperfusion resulted in a marked improvement in renal function (inulin clearance: 879 +/- 154 vs. 314 +/- 74 microliter/min; P less than 0.025) and a reduction in lipid peroxidation (renal malondialdehyde: 0.449 +/- 0.06 vs. 0.698 +/- 0.08 mmol/mg prot; P less than 0.05) compared to control animals. Infusion of 50 mg/kg/hr deferoxamine also protected renal function after ischemia (inulin clearance: 624 +/- 116 vs. 285 +/- 90 microliter/min; P less than 0.05) and resulted in less histologic injury. Iron-saturated deferoxamine had no protective effect. Conversely, infusion of the iron complex EDTA-FeCl3 during reperfusion exacerbated postischemic renal dysfunction and lipid peroxidation. Following renal ischemia there was no detectable increase in "free" iron in arterial or renal venous plasma. However, urinary "free" iron increased 10- to 20-fold following reperfusion. Iron chelators which underwent filtration and gained access to this free iron in the urine (free deferoxamine or inulin-conjugated deferoxamine) provided protection, whereas a chelator confined to the vascular space (dextran-conjugated deferoxamine) did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Reperfusion of tissues after interruption of their vascular supply causes free-radical generation that leads to tissue damage, a scenario referred to as “reperfusion injury.” Because sickle disease involves repeated transient ischemic episodes, we sought evidence for excessive free-radical generation in sickle transgenic mice. Compared with normal mice, sickle mice at ambient air had a higher ethane excretion (marker of lipid peroxidation) and greater conversion of salicylic acid to 2,3-dihydroxybenzoic acid (marker of hydroxyl radical generation). During hypoxia (11% O2), only sickle mice converted tissue xanthine dehydrogenase to oxidase. Only the sickle mice exhibited a further increase in ethane excretion during restitution of normal oxygen tension after 2 hours of hypoxia. Only the sickle mice showed abnormal activation of nuclear factor–κB after exposure to hypoxia-reoxygenation. Allopurinol, a potential therapeutic agent, decreased ethane excretion in the sickle mice. Thus, sickle transgenic mice exhibit biochemical footprints consistent with excessive free-radical generation even at ambient air and following a transient induction of enhanced sickling. We suggest that reperfusion injury physiology may contribute to the evolution of the chronic organ damage characteristic of sickle cell disease. If so, novel therapeutic approaches might be of value.

To study the importance of oxygen free radical production by and injury to proximal tubule epithelial cells, an in vitro model was established. Rat renal proximal tubule epithelial cells in primary culture were subjected to normoxic conditions or 60 minutes of hypoxia and 30 minutes of reoxygenation. Under normoxic conditions, these cells produced superoxide radical, hydrogen peroxide, and hydroxyl radical. During hypoxia and reoxygenation, there was an increase in the production of these reactive oxygen species, detected in the extracellular medium, of 252, 226, and 45 percent, respectively. The production rate of superoxide radical was most markedly increased in the first five minutes of reoxygenation. Studies employing 2,7-dichlorofluorescein which fluoresces when oxidized by peroxides revealed a seven-fold increase in cellular fluorescence in cells studied after hypoxia and reoxygenation compared with control cells. That increased production of reactive oxygen species played a role in cellular injury was demonstrated by an increase in lipid peroxidation during hypoxia and reoxygenation, as well as substantial injury during hypoxia and reoxygenation which could be largely prevented by the addition of superoxide dismutase, catalase, dimethylthiourea, or deferoxamine to the cells. These studies demonstrate that proximal tubule epithelial cells produce reactive oxygen species in increased amounts during hypoxia and reoxygenation, and that these reactive oxygen species are injurious to the cells under these conditions.

The mechanism of decreased pressor responsiveness to pressor agents was examined serially throughout pregnancy in conscious rats. Rats, 15 and 20 days pregnant, showed marked blunting of the pressor response to graded doses of angiotensin, whereas after only 5 days of pregnancy there was a normal response and at 10 days an intermediate pressor response. A role for prior occupancy of vascular angiotensin II receptors for the blunted pressor response was made less likely by the observation that treatment with captopril to decrease endogenous angiotensin II did not improve the angiotensin II pressor response in 15-day pregnant rats. Studies of smooth muscle receptor binding of angiotensin II showed that, in pregnancy, receptor affinity and number was not changed. Inhibition of prostaglandin synthesis with meclofenamate increased the pressor response to angiotensin II toward normal in pregnant animals. The blunted vascular response in pregnancy was not specific for angiotensin since pregnant animals showed a similar decrease in the response to both norepinephrine and arginine vasopressin. Furthermore, meclofenamate increased the pressor response to norepinephrine and vasopressin in pregnant rats. We conclude that pressor hyporesponsiveness in pregnancy is not specific for angiotensin II and is not caused by alterations in vascular receptor occupancy or binding. In pregnancy there is a decreased pressor response to all three major pressor agents that is improved by inhibition of prostaglandin production.

Renal reperfusion injury results from oxygen radical generation. During reoxygenation of hypoxic kidney cells, xanthine oxidase produces superoxide radical, which eventuates in hydroxyl radical formation by the Fenton reaction. This reaction, catalyzed by transition metals such as iron, is particularly important because hydroxyl radical Is highly reactive with a wide variety of biomolecules. We tested the hypothesis that this catalytic function is fostered by iron released from the heme moiety of cytochrome P-450. Primary cultures of rat proximal tubule epithellal cells studied in a subconfluent stage were subjected to 60 min of hypoxia and 30 min of reoxygenation. When cells were pretreated with one of three cytochrome P-450 inhibitors (piperonyl butoxide, cimetidine, or ketoconazole), lethal cell injury was attenuated. There was the exped increase in 2O production during hypoxia/reoxygenation that cytochrome P-450 inhibitors did not prevent; on the other hand, inhibitors did prevent reoxygenation-induced hydroxyl radical formation. Analogously, the increase in catalytic iron (bleomycin-detectable iron) that accompanies hypoxia/reoxygenation did not occur in the presence of cytochrome P-450 inhibitors. In vivo studies confirmed a protective effect of cytochrome P-450 inhibition because glomerular filtration rate was better preserved in rats pretreated with cimetidine and then subjected to renal artery occlusion. In summary, several chemically distinct cytochrome P-450 inhibitors reduced iron release, and thereby, hydroxyl radical formation and reoxygenation-induced lethal cell injury, without inhibiting superoxide radical formation. We conclude that highly labile P-450 may act as an Fe-donating catalyst for Fenton reaction production of HO-mediated reperfusion injury.Oxidative injury is an important mechanism in reperfusion injury of a number of organs including the kidney (1-5). The molecular mechanisms for generation of reactive oxygen species have recently received intense scrutiny. In the kidney, during hypoxic conditions, the high-energy molecule ATP is sequentially dephosphorylated and then further degraded to nucleosides to eventually yield the base hypoxanthine. Cytoplasmic xanthine oxidase converts hypoxanthine to xanthine and then to uric acid. A parallel phenomenon that occurs during hypoxia is the conversion of xanthine dehydrogenase to xanthine oxidase through limited proteolysis by a calcium/calmodulin-dependent serine protease (6, 7). Xanthine oxidase uses molecular oxygen as an electron acceptor during these reactions to yield superoxide radical (O-j), which is subsequently dismutated to hydrogen peroxide and further reduced to the highly reactive hydroxyl radical (HO') in the Fenton reaction (8). This final step of the pathway is catalyzed by transition metals such as iron or copper.There are several potential cellular sources of reactive iron. The iron storage protein ferritin is an obvious candidate (9). The kidney, particularly in the proximal tubule, is rich in ferritin (10, 11 (15...