Role of xanthine

oxidase in ischemia/reperfusion

injury

STUART L. LINAS, DENNIS WHITTENBURG, AND JOHN E. REPINE University of Colorado School of Medicine, Webb- Waring Lung Institute, Denver General Hospital, Denver, Colorado 80204-4507

LINAS, STUART L., DENNIS WHITTENBURG,AND JOHN E. REPINE.Role of xanthine oxidase in ischemialreperfusion injury. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F711F716, 1990.-Oxygen metabolites formed during reperfusion of ischemic kidneys prevent recovery of renal function after short periods of renal ischemia. Xanthine oxidase has been proposed as a source of toxic oxygen metabolites during reperfusion of ischemic kidneys. To determine whether the enzyme is converted from the non-oxygen metabolite-producing dehydrogenase (type D) to the oxygen metabolite-producing oxidase (type 0), we measured type D and type 0 (total, reversible, and irreversible) xanthine oxidase in renal cortical homogenates after 30 min of ischemia in vivo and 60 min of reperfusion by the isolated perfused kidney technique. Total enzyme activity (type D plus type 0) was not altered by ischemia or reperfusion. Compared with nonischemic conditions, ischemia increased total type 0 (53 t 5 vs. 21 t 3%, P < 0.01) and reversible type 0 (15.4 & 1.5 vs. 2.1 t 1.4 U/g) xanthine oxidase activities. Reperfusion further increased total type 0 (82 t 3%) and reversible type 0 (27.7 t 3.3 U /g, both P < 0.01 vs. nonischemic perfusions) xanthine oxidase activities. To determine the physiological role of xanthine oxidase in renal ischemia, we depleted rats of xanthine oxidase by feeding tungsten. After 4 wk of tungsten, renal xanthine oxidase levels were reduced by >90% and renal function was markedly improved during reperfusion. For example, after 30 min of ischemia and 60 min of reperfusion, glomerular filtration rate was 457 t 25 &min-l l g-l and tubular sodium reabsorption was 92 t 2% in tungsten-treated rats comparedwith 144 & 15 &min-l l g-l and 71 t 4% (both P c 0.01) in non-tungsten-treated rats. We conclude that xanthine oxidase is converted from type D to type 0 (predom-

inantly reversible) during both ischemia and reperfusion and that xanthine oxidase is an important source of deleterious oxygen metabolites during reperfusion of ischemickidneys. oxygen metabolites;

enzyme activity

ALTHOUGHCELLULAREVENTS occurringduringischemia have long been recognized to contribute to the pathogenesis of organ injury, the contribution of reperfusion to the pathogenesis of injury has only recently been appreciated. Several studies have suggested that O2 metabolites are formed during reperfusion of ischemic organs and contribute to reperfusion injury (1, 13, 16, 25, 33, 37, 39). The source of O2 metabolites formed during reperfusion of ischemic kidneys has not been determined but there are multiple possibilities. For example, O2 metabolites could be produced by kidney tissue or by circulating cells such as polymorphonuclear leukocytes. However, since reperfusion injury occurs in isolated, blood-free, perfused kidneys, O2 metabolites must be generated in part from intrinsic renal sources such as xanthine oxi0363-6127/90

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dase (27-29), catecholamine metabolism (7), arachidonic acid metabolism (10, 17), and/or various subcellular organelles (5, 11). Granger et al. (13) postulated that xanthine formed from the degradation of ATP during ischemia would be metabolized by xanthine oxidase to superoxide and uric acid following the reintroduction of O2 during reperfusion (13). Since the usual substrate for xanthine oxidase is NAD, a pivotal component of this hypothesis was the prediction that the enzyme would be converted from a dehydrogenase (type D xanthine oxidase) to an oxidase (type 0 xanthine oxidase) during either ischemia or reperfusion. In vitro, type D can be converted to type 0 xanthine oxidase by a number of mechanisms including limited proteolysis and chemical modification resulting in the formation of an irreversible oxidase (4,8,9, 38) or by oxidation of sulfhydryl groups or chemically induced changes in sulfhydryl groups resulting in the formation of a reversible oxidase (6,9, 38). Reversible and irreversible oxidases can be distinguished on the basis of their reversibility by addition of thiol reducing agents, such as dithioerythreitol (DTE). Reversible type 0 is reconverted to type D, whereas irreversible type 0 is not reconverted to type D in the presence of thiol reducing agents (4, 9). The purpose of the current study was to determine the effects of ischemia and reperfusion on type D and type 0 xanthine oxidase levels in the kidney and to determine the role of xanthine oxidase in renal ischemia/reperfusion injury. METHODS Sources of reagents. Albumin (fraction V bovine, Reheis Chemical, Phoenix, AZ); (hydroxy [‘*Cl methyl) inulin (Amersham, Arlington Heights, IL); phenylmethylsulfonyl fluoride (PMSF), DTE, xanthine, NAD, allopurinol (Sigma Chemical, St. Louis, MO); normal protein test diet for rat, custom prepared with 0.7 g/kg sodium tungstate (ICN Biochemicals, Cleveland, OH); and Sephadex G-25 (Pharmacia, Piscataway, NJ) were all used. Preparation of rats for ischemia-reperfusion. Studies were performed using male Sprague-Dawley rats (Sasco Laboratories, Omaha, NE) weighing 300-350 g. Rats were placed on standard chow, a normal protein test diet, or the test diet supplemented with 0.7 g/kg sodium tungstate for a period of 28 days before the experiment. During this period, rats were allowed free access to the tungstate-containing diet. Rats were also allowed free access to glass-distilled deionized water supplemented with 0.6 mM sodium tungstate. After pentobarbital so-

0 1990 the American

Physiological

Society

F711

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F712

XANTHINE

OXIDASE

IN

ISCHEMIA/REPERFUSION

dium anesthesia (60 mg/kg ip), the peritoneal cavity was opened and a nontraumatic vascular clamp was placed across the right renal artery of some animals for 30 or 60 min (ischemic and nonischemic kidneys). During this time, the kidney was wrapped in warmed saline-moistened gauze and rat body temperature was maintained at 38°C. For reperfusion studies, a 3-cm length of PE-10 tubing was placed in the right ureter and the right adrenal gland was removed. Following ischemic or nonischemic exposures, the kidney was perfused for 60 min by the isolated kidney technique (reperfusion). The vascular clamp was not removed until the renal artery had been successfully cannulated. Tissue preparation for xanthine oxidase assays. Samples of superficial renal cortex (100-200 mg) were rapidly excised, snap-frozen in liquid nitrogen and processed immediately. The tissue was ground into a fine powder using a mortar and pestle and homogenized (Polytron model PT IOST, Brinkman Instruments, Westbury, NY) in 4 ml ice-cold homogenizing buffer containing 50 mM K phosphate solution (containing 1 mM PMSF, 1 mM EDTA, pH 7.0 with or without 5 mM DTE). The homogenate was then centrifuged at 15,000 g at 4°C in a Sorval RC-5 B centrifuge (DuPont Instruments, Newton, CT) for 20 min and the pellet discarded. The supernatant was chromatographed on Sephadex G-25-80 column in the same buffer at 4°C to remove endogenous substrates. The resultant eluate was kept at 4°C and used for measurement of total xanthine oxidase activity as well as total and reversible type 0 xanthine oxidase activities. Perfusion of isolated rat kidneys. Perfusion of the isolated rat kidney was conducted according to Nishiitsutsuji-Uwo et al. (32) as modified by Little and Cohen (24) and previously described in our laboratory (20). Briefly, after the right renal artery was cannulated, the kidney was transferred to a perfusion chamber and perfused using a pulsatile pump at a constant mean arterial pressure (distal to the tip of the cannula) of 100 mmHg. Following a 15-min equilibration period, three urine collections and perfusion samples were obtained at 15-min intervals. Perfusion samples were placed in a test tube for subsequent determinations of sodium and [“Cl inulin. The total duration of each perfusion was 60 min. Urine and perfusate samples were stored at -20°C within 60 min of collection. For each perfusion, 150 ml of a KrebsRinger bicarbonate solution was used that contained urea, (hydroxy [“Cl methyl)inulin, and albumin. The final composition of the perfusion medium (in mM unless otherwise noted) was 140 Na, 5.0 K, 1.2 ionized Ca, 1 Mg, 105 Cl, 25 bicarbonate, 1 sulfate, 1 phosphate, 7 urea, 70 mg/ml albumin, 5 glucose, and 1 ml/l00 ml perfusate of Aminosyn 8.5% (Abbott Laboratories, North Chicago, IL). Measurement of renal injury. Measurements of perfusion flow rate (PFR), glomerular filtration rate (GFR), and tubular sodium reabsorption (T& were used to assessrenal injury. Urinary clearances of [14C]inulin and sodium were calculated from their respective urine and plasma concentration ratios. Sodium was measured with an IL 343 flame photometer (Instrumentation Laboratory, Lexington, MA). Radioactivity of [14C]inulin was

INJURY

counted in a Packard Tri-Carb model 460 liquid scintillation counter (Packard Instruments, Laguna Hills, CA). Measurement of xanthine oxidase activities. Enzyme assays were performed in duplicate within 90 min of completion of sample processing. Xanthine oxidase activities were assayed by continuous measurement of the rate of uric acid formation at 295 nm at 37°C as described by Della Corte and Stripe (9). The basic reaction mixture contained 100 PM xanthine, 100 PM EDTA, 50 mM K phosphate, pH 7.8. Allopurinol (50 PM) was used to confirm that the rates were specific for xanthine oxidase. Total enzyme activity consisted of type D plus type 0 activities. Type 0 consisted of DTE-reversible plus DTEirreversible activities. For determination of total enzyme activity 600 PM NAD and 0, were utilized as electron acceptors. For determination of type 0 activity (reversible plus irreversible), 0, was the only electron acceptor. Type D was calculated by subtracting total type 0 from total enzyme activity. Irreversible type 0 was determined from samples prepared with DTE. Reversible type 0 was calculated by subtracting irreversible type 0 from total type 0 activity. One unit of enzyme activity is defined as the amount of enzyme required to convert 1 pmol of xanthine to uric acid per min at 37°C pH 7.8. Specific activity is defined as unit per gram wet weight. Balance studies. After 2 wk of control or tungstenenriched diets, food and water intake was determined for 7 additional days in seven control and seven tungstentreated rats as previously described (21). Statistical analyses. Statistical analyses were performed using one-way analysis of variance in conjunction with a Scheffe test for comparisons of multiple means and the Student’s t test for single comparisons. A P value of < 0.05 was considered significant. Data were expressed as means t SE. RESULTS

Sample preparation. Data in Table 1 demonstrate the effects of addition to the homogenization buffer of a protease inhibitor PMSF and a thiol reductant DTE on xanthine oxidase activity in nonischemic tissues. Total enzyme activity (type D plus type 0) was not altered by the inclusion of PMSF or DTE in the homogenization buffer of nonischemic kidneys. However, following addition of PMSF, type 0 activity decreased from more than 40% to 22% of total enzyme activity. By comparison addition of DTE had no further effect on type 0 activity. Because these results suggest that protease-mediated type 0 activity is formed during tissue preparation, PMSF was always included in the homogenization buffer. Effect of ischemialreperfusion on xanthine oxidase activity. Data in Table 2 demonstrate the effects of ischemia on renal cortical xanthine oxidase activity. There was no change in total enzyme (type D plus type 0) activity after 30 min of renal ischemia (51.1 t 4.1 nonischemic; 47.9 t 3.5 U/g ischemic). However, following ischemia, type 0 activity increased by more than twofold compared with nonischemic conditions. Increases in type 0 activity after ischemia were caused by increases in reversible type 0 activity since no changes occurred in irreversible type 0 activity.

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XANTHINE

1. Influence total type 0 activity,

TABLE

ISCHEMIA/REPERFUSION

Tme 0, % total activity

45.8t3.8 46.7k3.2 48.2k3.4

43.0t7.5” 22.1t4.8 19.9t3.4

k SE for 6 kidneys. 0 IR, irreversible type

PMSF, phenylmethylsulfonyl 0 xanthine oxidase activity.

F713

INJURY

and thiol reductant (DTE) type 0 in rat renal cortex

Total Xanthine Oxidase Activity (Type D and Twe O), U/g

+ DTE

Values are means oxidase activity; type

IN

of protease inhibition (PMSF) and reversible and irreversible

Additions

None PMSF PMSF

OXIDASE

on total xanthine

oxidase activity,

Type OR, u/g

Type OIR, u/g

8.1t2.4 3.8t1.8 2.4k1.2

11.6t2.3 6.5t1.9 7.2k2.4

fluoride; DTE, dithioerythritol; * P < 0.05 compared with other

type groups.

OR, reversible

type

0 xanthine

TABLE 2. Effect of 30 min of ischemia on type D, total type 0, and reversible and irreversible type 0 xanthine oxidase activity in rat renal cortex

Tw Q Nonischemic Ischemic P Values activity.

are means

21.3t3.2 52.7t4.8 co.01

10.9tl.6 25.2t2.1 co.01

CO.01

TABLE 3. Effect of reperfusion and reversible and irreversible

% total activity

u/g

40.2k4.4 22.723.8 ~fi SE for 8 kidneys.

Type 0,

Tme 0,

u/g

Type

0 R, reversible

type

0 xanthine

Nonischemic perfusion Ischemic reperfusion P are means

activity;

type

Type OIR, u/g

2.1t1.4 15.4t1.5

reperfusion injury.

Oxygen metabolites formed during reperfusion of ischemic kidneys prevent recovery of renal function after short periods of renal ischemia. Xanthine ox...
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