Mark S. Paller

Oxygen free radicals in ischemic acute renal failure in the rat.

During renal ischemia, ATP is degraded to hypoxanthine. When xanthine oxidase converts hypoxanthine to xanthine in the presence of molecular oxygen, superoxide radical (O-2) is generated. We studied the role of O-2 and its reduction product OH X 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 O-2 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 microliter/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 specimens, 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 X 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 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 concluded that restoration of oxygen supply to ischemic kidney results in the production of oxygen free radicals, which causes renal injury by lipid peroxidation.

Decreased pressor reactivity to angiotensin II in cirrhotic rats. Evidence for a post-receptor defect in angiotensin action.

We used a model of cirrhosis in the rat, produced by inhalation of carbon tetrachloride for 6 weeks, to investigate the mechanism of resistance to the pressor effects of angiotensin II. The pressor response to angiotensin II was significantly lower in conscious cirrhotic animals than in controls. On the other hand, cirrhotic animals had normal pressor responses to norepinephrine, indicating that a generalized defect in vascular reactivity does not cause the decreased pressor response to angiotensin II. Enhanced baroreceptor activity was not the cause of the decreased pressor response to angiotensin II, since baroreflex control of heart rate after angiotensin II was similar in cirrhotics and controls. Pretreatment with either the converting enzyme inhibitor captopril to reduce circulating angiotensin II or the prostaglandin synthesis inhibitor meclofenamate failed to normalize the response to angiotensin II. Thus, neither prior occupancy of receptors with endogenous angiotensin II nor the production of vasodilatory prostaglandins was responsible for the decreased angiotensin II response. Studies of angiotensin II binding by mesenteric artery smooth muscle particles showed that, in cirrhotic animals, receptor affinity for angiotensin II, was significantly lower than in controls (kd: cirrhosis 1.11 +/- 0.09 nM, control 0.94 +/- 0.13 nM; P less than 0.02), whereas receptor number was significantly increased (cirrhosis 315 +/- 42 fmol/mg protein, control 277 +/- 43 fmol/mg protein, P less than 0.01). However, total binding of AII by vascular receptors from cirrhotics was no different than in controls, since the decrease in affinity negated the increase in receptor number.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-induced nephropathies.

Drug-induced renal disease is a common problem. Drugs cause several renal syndromes, such as prerenal azotemia, fluid and electrolyte abnormalities, acute tubular necrosis, acute interstitial nephritis, and chronic interstitial nephritis. Acute renal failure due to acute tubular necrosis is the most common syndrome and is most frequently caused by aminoglycoside antibiotics, radiographic contrast agents, and amphotericin B. Avoidance of these drugs in volume-depleted or hypotensive patients with preexisting renal disease or in those receiving multiple nephrotoxic drugs is the most effective way to reduce nephrotoxicity. Acute interstitial nephritis is an immune process that is most commonly caused by penicillins, diuretics, allopurinol, nonsteroidal anti-inflammatory drugs, cimetidine, and sulfonamides. Prompt recognition of the disease and cessation of the responsible drug are usually the only necessary therapy. Chronic interstitial nephritis is most often seen after prolonged use of several different types of analgesic agents, including aspirin, acetaminophen, and nonsteroidal anti-inflammatory drugs. These patients develop recurrent papillary necrosis and eventually chronic renal failure. They are also at risk of developing transitional cell carcinomas of the urinary collecting system. Some patients who are receiving cyclosporine also develop chronic renal failure due to interstitial fibrosis.

A randomized double-blind trial of the use of human recombinant superoxide dismutase in renal transplantation

Oxygen free radical generation has been implicated as a possible mediator of the reperfusion injury postulated to occur following revascularization of the cold preserved and transplanted kidney. The superoxide radical (O2—) scavenger, superoxide dismutase, from bovine or recombinant (rh-SOD) sources, may ameliorate oxygen-free-radical mediated reperfusion injury of transplanted kidneys. To test this hypothesis, we performed a prospective, randomized, double-blind trial of the use of human rh-SOD in renal transplantation at three participating centers. Half of a 20 mg/kg solution of rh-SOD or placebo was administered as a bolus intravenous injection immediately prior to renal allograft reperfusion and the remainder as a peripheral intravenous infusion for 1 hr thereafter. Posttransplant renal function was determined using 99Tc-DTPA clearance to measure glomerular filtration rate at 48±24 hr and day 6 post-transplant. A two-tailed t test was used for pooled data, and analysis of variance was used to evaluate between center differences in outcome. One hundred and sixteen patients (58 rh-SOD and 58 placebo) were entered into the study. No adverse reactions to rh-SOD or placebo were noted. No differences were noted between rh-SOD and placebo groups with regard to GFR at 48 hr, serum creatinine or creatinine clearance at day 6, or percentage of patients with GFR ×10 ml/min or ×5 ml/min at 48 hr. The data did not vary when analyzed by center or in aggregate form, and no correlation was noted between storage time and GFR in either group. We conclude that data from this trial provide little basis for the use of rh-SOD as described to ameliorate reperfusion injury in transplanted kidneys.

Free Radical-Mediated Postischemic Injury in Renal Transplantation

Oxygen free radicals are generated during reperfusion of ischemic organs. Studies employing several species of laboratory animal (rat, dog, pig, rabbit, mouse) have documented protective effects of a variety of free-radical scavengers and antioxidants when administered before or immediately preceding reperfusion of ischemic kidneys. These protective agents include superoxide dismutase, dimethylthiorea, dimethyl sulfoxide, alpha-tocopherol, glutathione, the iron chelator deferoxamine, probucol, allopurinol and oxypurinol, and the spin-trapping agent PBN. Furthermore, deficiency of antioxidants (selenium, alpha-tocopherol, or catalase) exacerbates postischemic renal injury. These findings have been applied to renal transplantation in an attempt to decrease the incidence of posttransplantation acute renal failure. This is important because acute renal failure results in morbidity, increases hospital stay and the cost of transplantation, and complicates the use of cyclosporine. In porcine and in canine kidney transplantation, superoxide dismutase and allopurinol have provided renal protection. Transplantation is complicated because there may be prolonged hypoperfusion before harvesting plus a brief period of total ischemia during harvesting, followed by a prolonged period of cold ischemia and/or reperfusion, then followed by another brief period of ischemia and reperfusion during transplantation. Injury may occur at each of these phases by different mechanisms.

Nitric oxide—mediated renal epithelial cell injury during hypoxia and reoxygenation

The potent endothelial-derived vasodilator nitric oxide (NO) has been identified as a protective agent in acute renal failure. However, some recent studies have suggested a detrimental effect of NO on rat proximal tubules exposed to hypoxia and reoxygenation. We determined whether NO metabolites cause intracellular oxidation during hypoxia and reoxygenation and whether this oxidative stress is linked to irreversible cell injury. Primary cultures of rat proximal tubular epithelial cells were studied in a subconfluent stage and subjected to 60 min hypoxia and 30 min reoxygenation. Intracellular oxidation was assessed by monitoring the conversion of nonfluorescent dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123 as a probe for the long-lived oxidant peroxynitrite. Hypoxia and reoxygenation produced a marked increase in cellular generation of oxidant species. Intracellular oxidation of DHR was reduced by approximately 40% when cells were also exposed to the NO synthase inhibitor L-NAME. Oxidation of DHR following hypoxia and reoxygenation was not affected by SOD or DMTU. A combination of SOD and L-NAME was no more effective than L-NAME alone. Hypoxia and reoxygenation produced substantial injury (as LDH release). There was a 40% reduction in LDH release when cells were pretreated with a NO synthase inhibitor. In summary, increased generation of NO capable of inducing intracellular oxidizing reactions and cell death occurred during renal hypoxia and reoxygenation.

Effects of the prostaglandin E1 analog misoprostol on cyclosporine nephrotoxicity

Acute infusion of cyclosporine in rats causes intense renal vasoconstriction and decreased glomerular filtration rate, effects that persist during short-term daily administration. We tested whether the orally active prostaglandin E1 analog misoprostol could reverse cyclosporine-induced renal vasoconstriction and restore the glomerular filtration rate. Male Sprague-Dawley rats were infused with cyclosporine (10 mg/kg) and then given an oral dose of misoprostol (100, 500, or 1000 μg/ kg). Cyclosporine caused large decreases in glomerular filtration rate and renal blood flow and a large increase in renal vascular resistance. Misoprostol decreased renal vascular resistance and increased renal blood flow and glomerular filtration rate to near normal levels. The two highest doses of misoprostol caused severe hypotension only in cyclosporine-treated animals. However, when it was given in a dose of 100 μg/kg hypotension was not a serious problem. This dose of misoprostol resulted in an increase in glomerular filtration rate from 341±57 to 669±104 μl/min (P<0.005) and renal blood flow from 2.23±0.36 to 4.25±0.65 ml/min (P<0.01), as well as a decrease in renal vascular resistance from 73.7±23.8 to 29.4±5.8 mmHg/ml/min (P<0.05) in cyclosporine-treated animals. When given to control animals, misoprostol had no effects on renal hemodynamics or renal function. In summary, acute cyclosporine-in-

EXTRACELLULAR IRON CHELATORS PROTECT KIDNEY CELLS FROM HYPOXIA/REOXYGENATION

Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury.

Lateral mobility of Na,K-ATPase and membrane lipids in renal cells. Importance of cytoskeletal integrity

Because membrane fluidity is an important determinant of membrane function, the lateral diffusion rate (DL) of the membrane protein Na,K-ATPase was determined in intact renal proximal tubule epithelial cells by the technique of fluorescence redistribution after photobleaching (FRAP). In normal cells the DLof Na,K-ATPase in the basal membrane was 3.31×10−10 cm2/ sec. Treatment with cytochalasin D to promote actin filament depolymerization caused a sevenfold increase in DL. Exposure of cells to a Ca2+-free medium or to hypoxia and reoxygenation, which have similar disruptive effects on the cytoskeleton, also caused increases in DL. Disruption of actin microfilament structure also increased the mobile fraction of Na,K-ATPase. Using a confocal laser microscopic technique only 14.9% of total Na,K-ATPase was observed to reside in the apical membrane domain of normal cells. Microfilament depolymerization caused this fraction to increase to 47.7%. Thus, the translocation of Na,K-ATPase from the basolateral to the apical domain induced by cytoskeletal protein dysfunction was enabled by an increased rate of lateral diffusion of Na,K-ATPase. The behavior of a variety of membrane lipids following actin depolymerization was more heterogeneous. Some lipids showed a similar increase in DLwhereas others showed very little dependence upon the cytoskeleton for lateral restraint.

Purinergic receptors mediate cell proliferation and enhanced recovery from renal ischemia by adenosine triphosphate

Kidney dysfunction after ischemia can be improved by either limiting the initial injury or by enhancing the subsequent proliferative repair process. Adenosine triphosphate (ATP) favorably affects kidney function when it is given shortly after ischemia. We tested whether ATP promotes the proliferative repair response. Rats were subjected to occlusion of the left renal artery for 40 minutes and received an infusion of ATP, 12.5 micromol intravenously over 30 minutes, beginning at reperfusion. Control animals received saline solution or the hydroxyl radical scavenger dimethylthiourea (DMTU). Despite comparable functional protection by DMTU and ATP, only ATP specifically increased DNA synthesis (renal incorporation of tritiated thymidine) to an extent greater than that produced by ischemia alone. In other animals, ribonucleic acid was extracted from kidneys for Northern analysis. Expression of the proto-oncogenes c-fos and c-jun was enhanced in ATP-treated animals as compared with controls. Expression of a histone protein gene (H2b) and thymidine kinase was increased by ischemia but was not additionally affected by ATP. In vitro studies of primary cultures of renal proximal tubule epithelial cells confirmed the ability of ATP to stimulate cellular proliferation as a consequence of stimulation of purinergic P2 receptors, possibly of the P2x subclass. In summary, ATP given after ischemia increased new DNA synthesis and augmented expression of genes critical to cellular proliferation. These beneficial effects were not merely a consequence of limiting initial cellular damage, and they suggest a novel mechanism of action for ATP and other purinergic receptor agonists in renal ischemia.