Glomerular Injury and Proteinuria in Rats After IntrarenalInjection of Cobra Venom Factor

Evidence for the Role of Neutrophil-Derived Oxygen Free Radicals

AHMED REHAN, MD, ROGER C. WIGGINS, MD,ROBIN G. KUNKEL, MS, GERD 0. TILL, MD, and

KENT J. JOHNSON, MD

The purpose of these studies was to determine how in-travascular complement activation could lead to glomer-ular injury. Cobra venom factor (CVF) infused into therenal artery ofrats resulted in increased excretion ofpro-tein in urine, which was maximal over the first 24 hours(51.2 ± 6.0 mg/24 hours in CVF versus 14.1 ± 0.9 mg/24hours in saline-treated animals; P < 0.001). Depletionof circulating neutrophils with anti-neutrophil serumsignificantly reduced the CVF-induced proteinuria in thefirst 24 hours (neutrophil depleted rats 22.7 ± 2.8 mg/24hours versus 63.4 ± 9.9 mg/24 hours in neutrophil intactrats; P< 0.005). Morphologic abnormalities (which werequantitated morphometrically) included accumulation ofneutrophils in glomerular capillary loops, blebbing ofen-dothelial cells, and epithelial cell foot process fusion. The

IN SEVERAL TYPES of glomerulonephritis in manthere is evidence for the participation of the comple-ment system with decreased total serum hemolytic com-plement and complement deposition in glomeruli.Studies by Cochrane et al revealed that certain typesof experimental anti-glomerular basement membranedisease required complement for the development ofthe glomerular injury. 1-3 The exact mechanism by whichcomplement is involved in the pathogenesis of glomer-ular injury is not clear. However, one potential mecha-nim requires immune complexes in the glomeruli to ac-tivate the complement system locally, which in turnwould attract leukocytes (neutrophils and macrophages)into glomeruli as a result of generation of chemotacticpeptides, particularly C5a.4 In most cases where thishas been studied experimentally, as in lung and skin,it appears that the leukocyte is then responsible forresulting tissue injury by means of the generation andrelease of oxygen radicals, proteases, and other inflam-matory mediators.4`6

From the Departments of Internal Medicine and Pathology, University ofMichigan Medical School, Ann Arbor, Michigan

increased protein excretion was reduced by 70% by simul-taneous administration ofcatalase (23 ± 4.3 mg/24 hoursin CVF plus catalase versus 52.1 ± 10 mg/24 hours inCVF alone; P < 0.05). Catalase reduced glomerular en-dothelial cell blebbing and epithelial cell foot process fu-sion but not neutrophil accumulation in glomeruli asassessed by morphometry. In similar experiments super-oxide dismutase, dimethyl sulfoxide, and deferoxaminedid not prevent CVF-induced proteinuria. These studies,therefore, suggest that intravascular activation of com-plement in the rat causes glomerular injury and protein-uria which is dependent on neutrophils and upon thegeneration ofhydrogen peroxide and/or its metabolites.(Am J Pathol 1986, 123:57-66)

To further analyze the events in the glomerulus, weexamined a model of glomerular injury caused by acti-vation of the complement system in the kidney inducedby infusion of cobra venom factor (CVF) directly intothe renal artery.

Materials and MethodsAdult pathogen-free Sprague-Dawley male rats

weighing between 250 and 300 g (Charles River, Port-age, Mich) were anesthetized by intraperitoneal injec-

Supported in part by USPHS Grant AM30673 and a grant-in-aid from the Michigan Branch of the American Heart As-sociation. R. C. Wiggins is in receipt of an Established In-vestigatorship from the American Heart Association.

Accepted for publication November 15, 1985.Address reprint requests to Ahmed Rehan, MD, Depart-

ment of Internal Medicine, Nephrology Division, Universityof Michigan, 3238 M.P.B. Box 19, Ann Arbor, MI 48109.

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58 REHAN ET AL

tion of ketamine (1 g/kg) (Parke-Davis Co., MorrisPlains, NJ). A laparotomy incision was made and thetest material was infused directly into the left renal ar-tery as previously described.7 After closure of the ab-dominal wound the animals were put into metaboliccages and given water ad libitum. Urine was collectedat 12- or 24-hour intervals. Protein excretion was quan-titated by the TCA-Lowry method.8CVF was isolated from crude lyophilized cobra (Naja

naja) venom by ion-exchange chromatography andSephadex G-150 gel separation. This preparation wasfree of any phospholipase A2 or endotoxin contamina-tion.9 For the studies described, CVF (5-40 units) wasinfused directly into the renal artery. One unit of CVFactivity is defined as the amount of CVF required tocause 50Wo inhibition of lysis of sensitized sheeperythrocytes under the conditions defined by Balow andCochrane."0 As little as 5 units of CVF injected intothe renal artery caused complete depletion of the mea-surable complement CH50 hemolytic activity (<5 CH50units), measured at 1, 6, 12, 24, and 36 hours in theperipheral blood. l

Neutrophil depletion was accomplished by a singleintraperitoneal injection of 1.5 ml of rabbit anti-rat neu-trophil serum. 12 Within 12 hours, circulating neutrophilswere depleted to less than 400 neutrophils/cu mm. Thisdegree of neutrophil suppression lasted up to 36 hours.This antibody (in non-CVF-treated rats) did not affectthe number of circulating mononuclear cells, erythro-cytes, or platelets and did not change the complementlevels as measured by CH50 method. We were unableto rule out the effect of this antibody on tissue macro-phages. However, Ward et al have previously shown thatphorbol myristate acetate (PMA)-stimulated generationof superoxide anion rates by alveolar macrophages ob-tained from neutrophil-depleted rats were not effectedby the in vivo exposure of animals to antibody directedagainst rat neutrophils.13

Inhibitor Studies

To study the role of oxygen free radicals in this model,we used several inhibitors.

Polyethylene-glycol coupled catalase (PEG:catalase)was obtained from Enzon Inc. (South Plainfield, NJ). 14The specific activity was measured spectrophotometri-cally.Is The half-life of PEG:catalase was found to beabout 36 hours. Various doses of PEG:catalase (250-1000units) were given intravenously just before the intra-arterial infusion of CVF. PEG:catalase was chemicallyinactivated by reduction and alkylation as described byMeans and Feeney.16 The preparation was then exten-sively dialyzed against phosphate-buffered saline (pH7.4). This preparation retained less than 10% of its ac-

tivity as compared with the untreated preparation ofPEG:catalase.

Superoxide dismutase (SOD) was obtained from DataDiagnostic Inc., (Mountain View, Calif). The specificactivity was assayed by the method of McCordand Fridovich.17 Polyethylene-glycol coupled SOD(PEG:SOD) was obtained from Enzon Inc."8 PEG:SODor SOD was given intravenously just before the intraar-terial infusion of CVF. The half-life of PEG:SOD wasfound to be about 24 hours.

Dimethyl Sulfoxide (DMSO) was obtained fromFisher Scientific Co. (Fair Lawn, NJ). The dosage sched-ule of DMSO was as follows; 1 ml DMSO given in-traperitoneally 10 minutes before the infusion of CVF;this was followed by the intraperitoneal infusion of 0.5ml DMSO at 30 and 60 minutes after the injection ofCVF.

Deferoxamine mesylate (CIBA Pharmaceutical Com-pany, Summit, NJ) was used (5 mg) intravenously 5 and30 minutes before the infusion of CVF. The doses ofthese oxygen radicals inhibitors and iron chelator usedwere based on previous experimental studies.19The amount of protein leakage in the urine was mea-

sured 24 hours after the infusion of CVF.

Morphologic Studies

At least 3 animals from each group were sacrificedat 2-, 4-, 6-, 12-, and 24-hour intervals after the infu-sion of CVF. Sections were obtained from both thetreated (left) and untreated (right) kidney. Sectionsstained with hematoxylin and eosin (H&E) were exam-ined under light microscopy. For electron microscopy,the tissue was fixed in buffered glutaraldehyde andstained with uranyl acetate and lead citrate and ana-lyzed in a Philips 400 T transmission electron micro-scope.

Morphometric Studies

Light Microscopy

Plastic sections 1 j thick stained with toluidine bluewere used in study of the kinetics of neutrophil (PMN)influx into the glomeruli, 2-24 hours after CVF infu-sion alone or after co-instillation of catalase and CVF.Approximately 70 glomeruli per sample were selectedat random, and the number of PMNs per glomeruluswas counted with an Olympus microscope with a 40 xobjective.

Electron Microscopy

Morphometric analysis was conducted at the ultra-structural level on electron micrographs (7840x ) from

AJP * April 1986

COMPLEMENT ACTIVATION IN GLOMERULAR INJURY

Table 1 -Morphometric Analysis of Time Course ofCVF-Induced Neutrophil Influx Into Glomeruli

Number of Number of Number ofMaterial Time glomeruli PMNs PMNs/glomerulusinjected (hours) examined observed (mean SEM)

Saline 2 106 15 0.14 + 0.03CVF 0.5 71 10 0.14 0.03*CVF 1 115 74 0.64 + 0.08tCVF 2 114 102 0.89 0.1OOtCVF 6 111 101 0.80 0O.9tCVF 12 129 67 0.50 0O.67tCVF 24 115 35 0.30 + 0.58t

* Not significant.t P < 0.001, compared with saline control.t P < 0.02, compared with saline control.

glomeruli of rats treated with CVF (n= 17), CVF + Cat(n= 11), and saline (n= 8). Three to six micrographs per

glomerulus were placed on an electronic pad linked toa Carl Zeiss Video-Plan. The instrument was pro-grammed to measure 1) length (in microns) of basementmembrane bordering on epithelial cells, 2) length (inmicrons) of epithelial foot process fusion, 3) length (inmicrons) of endothelial cell surfaces on glomerularcapillaries, and 4) length (in microns) of damaged en-

dothelial cells. Damaged endothelium was defined as

blebbing and denuded basement membranes.The proportion of epithelial foot processes that were

fused and endothelium that was damaged was calcu-lated (Tables 2 and 3).

Glomerular filtration rates (GFR) were measured 2-3hours after infusion of the experimental material bythe use of '251-iothalamate, as described by Sigman etal.20 Results were compared among rats given saline,CVF alone, PEG:catalase alone, and CVF with PEG:catalase.

Statistical Analysis

The Student t test (two-tailed analysis) was used forcomparison of the differences in data derived from thevarious experimental groups. Data are expressed in allfigures as the mean ± the standard error of the mean(SEM).

Table 2-Morphometric Analysis of Effect of Catalase onPMN Influx Into Glomeruli

Number of Number of Number ofglomeruli PMNs PMNs/glomerulus

Material injected examined observed (mean SEM)

Saline 106 15 0.14 0.03CVF 114 102 0.89 + 0.1

CVF + catalase 165 136 0.82 0.1

All measurements made 2 hours after injection. Forty units of CVF were

used in each case. The dose of catalase used was 1000 units.

Table 3-Ultrastructural Morphometric Analysis ofGlomerular Injury

Number ofMaterial glomeruli Endothelial cell Epithelial cell footinjected examined damage* process fusiont

Saline 8 37 p/2476 t 120 g/4018 i(1.5%) (3.0%)

CVF 17 454 p/5742 p 883 p/7848 t(7.9%) (11.3%)

CVF+ 11 78 p/6656 A 272 p/7427 pcatalase (1.2%) (3.7%)

* Data are expressed as surface area of damaged endothelial cells pertotal surface area of endothelial cell measured.

t Data are expressed as length of fused epithelial cell foot processesper total length of epithelial cell bordering on basement membranemeasured.

All measurements made 2 hours after injection. Forty units of CVF wereused in each case. The dose of catalase used was 1000 units.

Results

Proteinuria Induced by the Infusion ofCobra Venom Factor

Infusion of CVF directly into the left renal arterycaused significant proteinuria in the subsequent 24hours. As shown in Figure 1, infusion of 40 units ofCVF caused 51.2 ± 6.0 mg/24 hours of protein excre-tion in urine as compared with 14.5 ± 0.9 mg/24 hoursin animals receiving normal saline (P < 0.001).The ability of CVF to induce proteinuria in a dose-

dependent manner is illustrated in Figure 2. CVF (5units) induced a significant increase in protein excre-tion as compared with saline controls (27.7 ± 0.2 mg/24hours versus 12.3 ± 2.5 mg/24 hours). Increasing thedose of CVF resulted in increased protein excretion inurine and was maximum at a dose of 40 units. A doseof 40 units of CVF was, therefore, used for all subse-quent studies.The duration of the CVF-induced proteinuria is il-

lustrated in Figure 3. The amount of protein excretedin urine peaked within the first 12 hours and then rap-idly decreased, so that by 48 hours the amount of pro-tein present in the urine approached that of saline-treated controls.

Prior depletion of complement by intraperitoneal in-jection of 8 units of CVF prevented the CVF-inducedproteinuria (complement-depleted rats receiving salineexcreted 10.5 ± 5.6 mg of protein/24 hours, comparedwith complement-depleted rats receiving CVF, whichexcreted 6.4 ± 1.0 mg of protein/24 hours (n= 3)).These results show that 1) the injection of CVF into

the renal artery induced increased protein excretion ina dose-dependent fashion, 2) this effect was limited tothe first 24 hours, and 3) circulating complement wasnecessary for the effect.

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Figure 1 -Total urinary protein excretion during the first 24 hours in ratstreated with CVF. Results are expressed as mean proteinuria ± 1 SEM.(n = 8 for each group).

Characterization of CVF-Induced Proteinuria

The next set of experiments were done to determinewhether CVF-induced proteinuria was of glomerularor tubular origin. IThe urinary proteins were first evalu-ated by SDS-slab gel electrophoresis.2" As illustratedin Figure 4, a large percentage of the protein in urinemigrated with an apparent molecular weight of 60,000daltons under nonreducing conditions and was there-fore probably albumin. Higher and lower molecularweight proteins were also present. This pattern of pro-tein excretion is compatible with a glomerular proteinleak, but it does not exclude the possibility of a tubu-lar contribution to the proteinuria.

The concept that glomerulus may be the main source

SALINE RANGE IISDF ''I/////X T /7 X4 0L~~~,, 7"i/ 711" 7p 171

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NORMAL SALINE

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Figure 3-Time course of CVF-induced proteinuria. The proteinuria is max-imal within the first 24 hours. The data are corrected for a 24-hour excre-tion for comparison (n = 5 for each group). Mean - SEM.

of the protein leak into urine is further strengthenedby immunofluorescence studies of the renal tubules ofthe CVF-treated animals. Protein droplets have beendescribed in tubules during periods of protein leakagefrom glomeruli.22 These droplets probably representprotein reabsorption by tubular cells. Their presencewould therefore suggest that the origin of protein leak-age occurred upstream from the proximal tubule. Asillustrated in Figure 5, by using a rabbit anti-rat albu-min serum as the primary antibody, bright fluorescentreabsorption droplets were seen in the proximal tubules

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A B C D EFigure 4-Characterization of urine protein excreted by CVF-treated ratsusing SDS slab gel electrophoresis under nonreducing conditions.Represented are urines from normal rat (A), rats treated with normal sa-line (B), rats treated with CVF alone (40 p) (C), rats treated with PEG:cata-lase (1000 units) and CVF (40 i) (D), and normal rat plasma (E). Note thatthe bulk of the urine protein appears to be albumin as compared with plas-ma control. (The apparent molecular weight of albumin under nonreduc-ing conditions is 60 kilodaltons). Catalase suppression of CVF-inducedproteinuria is illustrated by the markedly smaller amount of albuminpresent.

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COMPLEMENT ACTIVATION IN GLOMERULAR INJURY

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Figure 5-Indirect immunofluorescence of rat kidney tissue using rabbit anti-albumin serum in a CVF-treated rat (A) representing a high concentrationof protein reabsorption droplets, compared with saline control rats (B). (x 400)

of CVF-treated rats. These droplets were not seen whennormal rabbit serum was used instead of rabbit anti-rat albumin. Fine background tubular fluorescence waspresent in both the control and CVF-treated rat tubules.Therefore, both the presence of "reabsorption droplets"in the tubules of CVF-treated rats and the urinary pro-tein being mainly albumin are compatible with the con-clusion that the CVF-induced proteinuria is probablymainly of glomerular origin. However, tubular dysfunc-tion could also have contributed.

Requirement of Neutrophils forCVF-Induced Proteinuria

CVF activates the complement cascade and leads tothe production of C5a.4 C5a has been shown to be apotent chemotactic agent for inflammatory cells as wellas being a potent stimulator of oxygen free radicals andprotease release by these cells. Therefore, it was of in-terest to determine whether the in vivo effects of CVFinfusion into the kidney might be attributable to neu-trophils. Six rats were depleted of circulating PMNs byinjection of rabbit anti-rat neutrophil serum as detailedin the Methods section. These animals then received

40 units of CVF into the renal artery, and the amountof protein excreted by these animals was compared withthat of animals receiving saline alone. As illustrated inFigure 6, animals that were PMN-depleted showed amarked decrease in protein excretion during the first

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24 hours after infusion of CVF when compared withPMN-intact animals (22.7 + 2.8 mg/24 hours inneutrophil-depleted versus 63.4 + 9.9 mg/24 hours inneutrophil-intact animals; P< 0.005). If the backgroundproteinuria (14.5 + 0.9 mg/24 hours) in the saline con-trols is subtracted from both groups, the suppressionof the proteinuria in the neutrophil-depleted animalswas 800%o. Thus, the proteinuria induced by systemiccomplement activation was largely dependent on cir-culating neutrophils.

Effect of Oxygen Radical Inhibitors on theCVF-Induced Proteinuria

With the neutrophil having been identified as beingcritical for the development of the proteinuria in thismodel, a series of studies was then undertaken to de-termine whether oxygen radicals generated by neu-trophils were responsible for the proteinuria.The first oxygen radical inhibitor tested was catalase.

Groups of 6 animals each were injected intravenouslywith 250, 500, and 1000 units of PEG:catalase 10minutes before the infusion of CVF. As shown in Fig-ure 7, the addition of the PEG:catalase suppression ofthe CVF-induced proteinuria appeared to be dose de-pendent, although differences were not statisticallydifferent between the groups. Two hundred fifty unitsof PEG:catalase reduced proteinuria to 34.5 + 1 mg/24hours versus 52.1 + 10.0 mg/24 hours in the animalsreceiving CVF alone (P < 0.05). At the highest doseof PEG:catalase (1000 units) proteinuria was reducedto 23.7 mg + 3.8 mg/24 hours. If the background pro-tein excretion (14.5 + 0.9 mg/24 hours) is subtracted,this represented a reduction in protein excretion by 70%.

0-

T

CVF 140ul CVF 140ul+SOD

CVF 140UI+

PEG: SOD

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Figure 8-Effect of PEG:SOD and uncoupled SOD on CVF-induced pro-teinuria (n = 6 per group).

Infusion of chemically inactivated catalase caused nodiminution of CVF-induced proteinuria. Thus catalasewas able to inhibit CVF-induced proteinuria in a dose-dependent manner, which suggests that hydrogen perox-ide and/or its metabolic products are important medi-ators for the development of the CVF-induced pro-teinuria.As shown in Figure 8, the co-instillation of either cou-

pled or uncoupled SOD with CVF had no effect onCVF-induced proteinuria (52.4 + 15.9 mg/24 hours inSOD + CVF, 50.4 - 7.8 mg/24 hours in PEG:SOD+ CVF and 54.7 + 10.6 mg/24 hours in CVF alone).Therefore, the superoxide anion does not appear to beinvolved in CVF-induced proteinuria.

To assess the role of the hydroxyl radical, both DMSOand deferoxamine were tested. As shown in Figure 9,neither DMSO nor deferoxamine had a suppressiveeffect on CVF-induced proteinuria (58.9 ± 5.5 mg/24hours in CVF + DMSO, 64.2 + 13.1 mg/24 hours inCVF + deferoxamine and 58.9 + 5.5 mg/24 hours inCVF alone). Thus, on the basis of these studies, therewas no evidence that the hydroxyl radical was involvedin the pathogenesis of the CVF-induced proteinuria.

Glomerular Filtration Rates (GFR)

GFR was measured by using '251I-sodium iothalamate.Rats that received saline injection into the left renal ar-tery had an average GFR of 1.2 + 0.1 ml/min/100 gbody weight. Infusion of PEG:catalase (1000 units)alone did not alter the GFR (0.97 ± 0.86 ml/min/100g body weight). Infusion of CVF reduced the GFR to0.20 + 0.1 ml/min/100 g body weight. Co-instillation

A T) * April 1 986

COMPLEMENT ACTIVATION IN GLOMERULAR INJURY

of PEG:catalase together with CVF did not alter theGFR from that seen with CVF alone (0.18 ± 0.11ml/min/100 g body weight). Therefore, although pro-tein excretion following CVF injection was reduced bycatalase, the fall in GFR seen following CVF injectionwas not prevented by catalase. This suggests that differ-ent mechanisms mediate the decrease in GFR and theprotein leak following CVF injection.

Morphologic Alterations

Animals were sacrificed at various intervals duringthe first 24 hours after the infusion of the CVF. Theinjected kidney was compared with the contralateralkidney in the same animal as well as kidneys fromanimals given saline and the various oxygen radical in-hibitors. Light-microscopic examination of the CVF-treated kidneys revealed neutrophils present in theglomeruli. Neutrophil influx was maximum 2-6 hoursafter CVF infusion. Neutrophil accumulation in glo-meruli was also seen in the catalase-treated rats. Theuninjected right kidney of the CVF-treated rats also hadincreased numbers of neutrophils per glomerulus.As shown in Table 1, a minimum of 70 glomeruli were

examined at each time point. There were increased num-bers of neutrophils present in glomeruli by 2 hours af-ter CVF injection, with the maximal number of neu-trophils per glomerulus appearing at 2- and six-hourtime intervals. Therefore, a single injection of CVF in-duced a significant neutrophil influx into the glomeruli.The neutrophil influx was compared in the CVF-treatedversus the CVF + PEG:catalase-treated animals. Wedid this to determine whether the decreased protein ex-cretion observed in the catalase-treated animals was dueto a reduced number of neutrophils. As shown in Ta-ble 2, co-instillation of PEG:catalase with CVF did notaffect neutrophil accumulation in glomeruli at 2 hours.Thus the protective effect seen with catalase was notdue to a decreased number of neutrophils in glomeruli.Ultrastructural studies were performed at times of peakneutrophil influx (2-6 hours). As shown in Figure IOAand B, the glomeruli of the CVF-treated animals showedendothelial cell damage, fibrin generation, and patchy fu-sion of epithelial-cell foot processes. In contrast, asshown in Figure IOC, the CVF + PEG:catalase-treatedanimals showed little in the way of endothelial cell in-jury or foot process fusion and no fibrin formation.These changes were quantitated morphometrically (Ta-ble 3).

Thus, both by traditional morphology and quantita-tive morphometry CVF-induced renal injury wasmanifested by structural glomerular cell alterations.Catalase protected against these changes without affect-ing the influx neutrophils.

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Figure 9-Effect of hydroxyl radical inhibitors on CVF-induced proteinur-ia (n = 6 per group).

Discussion

The data presented in this study provide evidence thatintravascular activation of the complement system canlead to proteinuria and that this occurs in a dose-dependent manner. This protein excretion is maximalover the first 24 hours and is neutrophil-dependent.It is accompanied by morphologic changes in glomeruliwhich include endothelial cell swelling and epithelialcell foot process fusion. Both the proteinuria and themorphologic alterations, but not the decrease in GFR,appear to be caused by the production of H202 and/orits metabolic products as shown by the protective effectsof catalase.

Morphologic changes, including leukocyte aggrega-tion, were present in both the CVF-infused and nonin-fused kidneys. Since Blantz et al have shown that com-plement (C3) does not bind to the basement followingadministration of CVF,23 we hypothesize that neu-trophils aggregate in response to generation of C5a af-ter CVF infusion. We further hypothesize that the lo-cal concentration of C5a is greater in the infused kidney,thereby explaining the increased leukocyte accumula-tion and endothelial cell changes seen on that side ascompared with the noninfused side.

Similar studies have previously shown that oxygenfree radicals (more specifically H202 and/or its meta-bolic products derived from neutrophils) are importantin causing protein excretion and glomerular changesin models of both the heterologous phase of nephro-toxic nephritis7 and after infusion ofPMA into the re-nal artery of the rat.22 In quantitative terms, the amountof protein excreted in these models of glomerular in-

63Vol. 123 * No. 1

64 REHAN ET AL

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Figure 10-Glomerular morphologic alterations associated with CVF in-fusion. A-Electron micrograph of a glomerulus from a rat given CVF2 hours previously. Note the presence of neutrophils (N), swelling of theendothelial cell (E), fibrin deposition (F), and patchy fusion of the epithelialfoot processes (Ep). (x3900) (8)-Note the glomerular endothelial cell(E) swelling of detachment from the basement membrane 2 hours after theinfusion of CVF. (x4900) C-Catalase suppression of CVF-inducedglomerular alterations. Note that while neutrophils (N) are still present inthe glomerulus, there is minimal endothelial cell damage and epithelial footprocess are intact. (x 3700)

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jury and the suppression of protein excretion by cata-lase (but not by other inhibitors of oxygen radicals) weresimilar. We hypothesize that neutrophils are attractedinto the glomeruli by various stimuli, such as C5a andPMA, and that they subsequently produce oxygen rad-icals (particularly H202 and/or its metabolic products)which appear to mediate damage to the glomerulus ei-ther directly or indirectly. The major anatomic abnor-malities were seen in the endothelial cell; however, wecannot exclude effects on other structures such as base-ment membrane, mesangial cells, or epithelial cells. Invitro studies have previously shown that the endothelialcell is susceptible to damage by H20224; therefore, ourobservations are compatible with that in vitro data.However, cultured mesangial cells have also been shownto produce oxygen radical in association with phago-cytosis of zymosan.25 Therefore, oxygen radicals produc-tion by intrinsic glomerular cells might cause glomeru-lar injury and proteinuria under some circumstances.The fact that neutrophil depletion prevented the pro-tein leak by 70-80% suggests that in this particularmodel oxidant production by intrinsic glomerular cellswas not an important mediator.The amount of CVF-induced proteinuria which in-

volved mainly albumin suggested that glomerular in-jury was the major source of the protein in the urine.This, together with the presence of reabsorptiondroplets of albumin in the proximal tubules, and mor-phologic data which was confined to the glomerulus,provide further evidence that the glomerulus is the ma-jor site of proteinuria. Both kidneys probably con-tributed to the proteinuria, because morphologicchanges were present bilaterally. We cannot exclude thepossibility that tubular dysfunction also contributedto the proteins measured in the urine.What is not clear from these studies is the exact mech-

anism of action of these radicals in causing tissue in-jury. H202 can directly interact with a halide group,forming hypochlorous products in the presence of themyeloperoxidase enzyme system. These products arehighly toxic, with effects on cells via lipid peroxidationand by other mechanisms.26 There is evidence for syn-ergism between oxygen radicals and lysosomal pro-teases.3'24 In addition, oxygen radicals are capable ofinactivating a1-anti-trypsin, which is a major neutralprotease inhibitor of plasma. Furthermore, oxygen rad-icals may act by rendering substrates such as the base-ment membrane more susceptible to subsequent degra-dation by proteolytic enzymes.27 Modulation of thefunction of glomerular cells (eg, by production ofarachidonate metabolites or other substances which al-ter glomerular cell function) might also have inducedthe protein leak. We cannot distinguish between thesepossibilities from our studies. The fact that the fall in

GFR seen after CVF injection was not prevented bycatalase suggests that H202 production was not respon-sible for this change. Thus, complement activation inthe glomerulus probably modifies glomerular functionby various mechanisms.

In conclusion, we have shown that infusion of CVFinto the rat renal artery results in neutrophil aggrega-tion along with glomerular morphologic alterations andproteinuria. Because of the protective effect of thespecific enzyme catalase, we have inferred that hydro-gen peroxide (and/or its metabolic products) producedin the presence of circulating neutrophils is an impor-tant mediator of injury in this model. This mechanismmight also play a role in some forms of acute glomeru-lar injury in man.

References

1. Cochrane CG: Immune complex mediated tissue injury,Mechanisms of Immunopathology. New York, JohnWiley & Sons, 1979

2. Cochrane CG, Janoff A: The Arthus reaction: A modelof neutrophil and complement mediated injury, TheInflammatory Process, Vol 3, 2nd edition. New York, Ac-ademic Press, 1974

3. Cochrane CG, Koffler D: Immune complex disease in ex-perimental animals and man. Adv Immunol 1973, 16:185

4. Ward PA, Hill JH: Biologic role of complement prod-ucts: Complement-derived leucotactic activity extracta-ble from lesions of immunologic vasculitis. J Immunol1972, 108:1137-1145

5. Fantone JC, Ward PA: Role of oxygen derived free radi-cals and metabolites in leukocyte dependent inflamma-tory reactions. Am J Pathol 1982, 107:395-418

6. JanoffA, Carp H: Proteases, Anti proteases and oxidants:Pathways of tissue injury during inflammation, CurrentTopics in Inflammation and Infection. Baltimore, Wil-liams and Wilkins, 1982, p 62

7. Rehan A, Johnson KJ, Wiggins RC, Kunkel RG, WardPA: Evidence for the role of oxygen radicals in acutenephrotoxic nephritis. Lab Invest 1984, 51:396-403

8. Lowry OH, Rosenbrough NJ, Farr AL, Randal RJ: Pro-tein measurement with the folin phenol reagent. J BiolChem 1951, 193:265-273

9. Till GO, Johnson KJ, Kunkel R, Ward PA: Intravascularactivation of complement and acute lung injury. J ClinInvest 1982, 69:1126-1135

10. Ballow M, Cochrane CG: Two anti-complementary fac-tors in cobra venom: Hemolysis of guinea pig erythro-cytes by one of them. J Immunol 1969, 103:944-952

11. Garvey, Crewer, Sussdorf: Complement fixation assays,Methods in Immunology. 3rd edition. Benjamin, 1977,pp 379-410

12. Johnson KJ, Ward PA: Role of oxygen metabolites in im-mune complex injury of lungs. J Immunol 1981,126:2365-2369

13. Ward PA, Duque RE, Sulavik MC, Johnson KJ: In vitroand in vivo stimulation of rat neutrophils and alveolarmacrophages by immune complexes. Am J Pathol 1983,110:297-309

14. Abuchowski A, McCoy JR, Davis FF: Effect of covalentattachment of polyethylene glycol on immunogenicity andcirculating life of bovine liver catalase. J Biol Chem 1977,252:3582-3586

15. Beers RF Jr, Sizer LW: A spectrophotometric method for

66 REHAN ET AL AJP * April 1986

measuring the breakdown of hydrogen peroxide by cata-lase. J Biol Chem 1952, 195:133-140

16. Means GE, Feeney RE: Reductive alkylation of aminogroups in proteins. Biochemistry 1977, 7:2192-2201

17. McCord JM, Fridovich I: Superoxide dismutase, an en-zymatic function for erythrocuprein (hemocuprein). JBiol Chem 1969, 244:6049-6055

18. Pyatak PS, Abuchowski A, Davis FF: Preparation of apolyethylene glycol: superoxide dismutase adduct and anexamination of its blood circulating life and anti-inflam-matory activity. Res Commun Chem Pathol Pharmacol1980, 29:113-127

19. Ward PA, Till GO, Kunkel R, Beauchamp C: Evidencefor role of hydroxyl radical in complement and neutrophl-dependent tissue injury. J Clin Invest 1983, 72:789-801

20. Sigman EM, Elwood CM, Khan F: The measurement ofglomerular filtration with sodium iothalamate. J NuclMed 1965, 7:60-68

21. Laemmil UK: Damage of structural protein during theassembly of the head of bacteriophage T4. Nature 1970,227:680-685

22. Rehan A, Johnson KJ, Kunkel RG, Wiggins RC: Roleof oxygen radicals in phorbol myristate acetate inducedglomerular injury. Kidney Int 1985, 27:503-511

23. Blantz RC, Tucker BJ, Wilson CB: The acute effects of

antiglomerular basement membrane antibody upon glo-merular filtration in the rat: The influence of dose andcomplement depletion. J Clin Invest 1976, 58:910-921

24. Weiss SJ, Young J, LoBuglio AF, Shivak A: Role of hydro-gen peroxide in neutrophil mediated destruction of cul-tured endothelial cells. J Clin Invest 1981, 68:714-721

25. Baud L, Hagege J, Sraer J, Rondeau E, Perez J, Ardail-lou R: Reactive oxygen production by cultured rat glo-merular mesangial cells during phagocytosis is associatedwith stimulation lipoxygenase activity. J Exp Med 1983,158:1836-1852

26. Weiss SJ, Klein R, Shivra A, Wei M: Chlorination of tau-rine by human neutrophils. Evidence for hypochlorousacid generation. J Clin Invest 1982, 70:598-607

27. Fligiel SE, Lee EC, McCoy JP, Johnson KJ, Varani J:Protein degradation following treatment with hydrogenperoxide. Am J Pathol 1984, 115:418-425

Acknowledgments

We are grateful to Mr. Craig Biddle and Mr. Eddie Burkefor photographic work and to Ms. MaryAnn Byrnes forsecretarial assistance.