Glomerular Injury and Proteinuria

Injection of Cobra Venom Factor

in Rats After Intra renal

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

From the Departments of Internal Medicine and Pathology, University of

Michigan Medical School, Ann Arbor, Michigan

The purpose of these studies was to determine how intravascular complement activation could lead to glomerular injury. Cobra venom factor (CVF) infused into the
renal artery of rats resulted in increased excretion of protein 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/24
hours in saline-treated animals; P < 0.001). Depletion
of circulating neutrophils with anti-neutrophil serum
significantly reduced the CVF-induced proteinuria in the
first 24 hours (neutrophil depleted rats 22.7 2.8 mg/24
hours versus 63.4 9.9 mg/24 hours in neutrophil intact
rats; P < 0.005). Morphologic abnormalities (which were
quantitated morphometrically) included accumulation of
neutrophils in glomerular capillary loops, blebbing of endothelial cells, and epithelial cell foot process fusion. The

increased protein excretion was reduced by 70% by simultaneous administration of catalase (23 4.3 mg/24 hours
in CVF plus catalase versus 52.1 10 mg/24 hours in
CVF alone; P < 0.05). Catalase reduced glomerular endothelial cell blebbing and epithelial cell foot process fusion but not neutrophil accumulation in glomeruli as
assessed by morphometry. In similar experiments superoxide dismutase, dimethyl sulfoxide, and deferoxamine
did not prevent CVF-induced proteinuria. These studies,
therefore, suggest that intravascular activation of complement in the rat causes glomerular injury and proteinuria which is dependent on neutrophils and upon the
generation of hydrogen peroxide and/or its metabolites.
(Am J Pathol 1986, 123:57-66)

IN SEVERAL TYPES of glomerulonephritis in man

there is evidence for the participation of the complement system with decreased total serum hemolytic complement and complement deposition in glomeruli.
Studies by Cochrane et al revealed that certain types
of experimental anti-glomerular basement membrane
disease required complement for the development of
the glomerular injury. 1-3 The exact mechanism by which
complement is involved in the pathogenesis of glomerular injury is not clear. However, one potential mechanim requires immune complexes in the glomeruli to activate the complement system locally, which in turn
would attract leukocytes (neutrophils and macrophages)
into glomeruli as a result of generation of chemotactic
peptides, particularly C5a.4 In most cases where this
has been studied experimentally, as in lung and skin,
it appears that the leukocyte is then responsible for
resulting tissue injury by means of the generation and
release of oxygen radicals, proteases, and other inflammatory mediators.4`6

To further analyze the events in the glomerulus, we

examined a model of glomerular injury caused by activation of the complement system in the kidney induced
by infusion of cobra venom factor (CVF) directly into
the renal artery.

Materials and Methods

Adult pathogen-free Sprague-Dawley male rats
weighing between 250 and 300 g (Charles River, Portage, Mich) were anesthetized by intraperitoneal injecSupported in part by USPHS Grant AM30673 and a grantin-aid from the Michigan Branch of the American Heart Association. R. C. Wiggins is in receipt of an Established Investigatorship from the American Heart Association.
Accepted for publication November 15, 1985.
Address reprint requests to Ahmed Rehan, MD, Department of Internal Medicine, Nephrology Division, University
of Michigan, 3238 M.P.B. Box 19, Ann Arbor, MI 48109.
57

58

REHAN ET AL

AJP

April 1986

tion of ketamine (1 g/kg) (Parke-Davis Co., Morris

Plains, NJ). A laparotomy incision was made and the
test material was infused directly into the left renal artery as previously described.7 After closure of the abdominal wound the animals were put into metabolic
cages and given water ad libitum. Urine was collected
at 12- or 24-hour intervals. Protein excretion was quantitated by the TCA-Lowry method.8
CVF was isolated from crude lyophilized cobra (Naja
naja) venom by ion-exchange chromatography and
Sephadex G-150 gel separation. This preparation was
free of any phospholipase A2 or endotoxin contamination.9 For the studies described, CVF (5-40 units) was
infused directly into the renal artery. One unit of CVF
activity is defined as the amount of CVF required to
cause 50Wo inhibition of lysis of sensitized sheep
erythrocytes under the conditions defined by Balow and
Cochrane."0 As little as 5 units of CVF injected into
the renal artery caused complete depletion of the measurable complement CH50 hemolytic activity (<5 CH50
units), measured at 1, 6, 12, 24, and 36 hours in the
peripheral blood. l
Neutrophil depletion was accomplished by a single
intraperitoneal injection of 1.5 ml of rabbit anti-rat neutrophil serum. 12 Within 12 hours, circulating neutrophils
were depleted to less than 400 neutrophils/cu mm. This
degree of neutrophil suppression lasted up to 36 hours.
This antibody (in non-CVF-treated rats) did not affect
the number of circulating mononuclear cells, erythrocytes, or platelets and did not change the complement
levels as measured by CH50 method. We were unable
to rule out the effect of this antibody on tissue macrophages. However, Ward et al have previously shown that
phorbol myristate acetate (PMA)-stimulated generation
of superoxide anion rates by alveolar macrophages obtained from neutrophil-depleted rats were not effected
by the in vivo exposure of animals to antibody directed
against rat neutrophils.13

tivity as compared with the untreated preparation of

PEG:catalase.
Superoxide dismutase (SOD) was obtained from Data
Diagnostic Inc., (Mountain View, Calif). The specific
activity was assayed by the method of McCord
and Fridovich.17 Polyethylene-glycol coupled SOD
(PEG:SOD) was obtained from Enzon Inc."8 PEG:SOD
or SOD was given intravenously just before the intraarterial infusion of CVF. The half-life of PEG:SOD was
found to be about 24 hours.
Dimethyl Sulfoxide (DMSO) was obtained from
Fisher Scientific Co. (Fair Lawn, NJ). The dosage schedule of DMSO was as follows; 1 ml DMSO given intraperitoneally 10 minutes before the infusion of CVF;
this was followed by the intraperitoneal infusion of 0.5
ml DMSO at 30 and 60 minutes after the injection of
CVF.
Deferoxamine mesylate (CIBA Pharmaceutical Company, Summit, NJ) was used (5 mg) intravenously 5 and
30 minutes before the infusion of CVF. The doses of
these oxygen radicals inhibitors and iron chelator used
were based on previous experimental studies.19
The amount of protein leakage in the urine was measured 24 hours after the infusion of CVF.

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). 14
The specific activity was measured spectrophotometrically.Is The half-life of PEG:catalase was found to be
about 36 hours. Various doses of PEG:catalase (250-1000
units) were given intravenously just before the intraarterial infusion of CVF. PEG:catalase was chemically
inactivated by reduction and alkylation as described by
Means and Feeney.16 The preparation was then extensively dialyzed against phosphate-buffered saline (pH
7.4). This preparation retained less than 10% of its ac-

Morphometric Studies
Light Microscopy
Plastic sections 1 j thick stained with toluidine blue
were used in study of the kinetics of neutrophil (PMN)
influx into the glomeruli, 2-24 hours after CVF infusion alone or after co-instillation of catalase and CVF.
Approximately 70 glomeruli per sample were selected
at random, and the number of PMNs per glomerulus
was counted with an Olympus microscope with a 40 x
objective.

Morphologic Studies
At least 3 animals from each group were sacrificed
at 2-, 4-, 6-, 12-, and 24-hour intervals after the infusion of CVF. Sections were obtained from both the
treated (left) and untreated (right) kidney. Sections
stained with hematoxylin and eosin (H&E) were examined under light microscopy. For electron microscopy,
the tissue was fixed in buffered glutaraldehyde and
stained with uranyl acetate and lead citrate and analyzed in a Philips 400 T transmission electron microscope.

Electron Microscopy
Morphometric analysis was conducted at the ultrastructural level on electron micrographs (7840 x ) from

COMPLEMENT ACTIVATION IN GLOMERULAR INJURY

Vol. 123 * No. I

Table 1 -Morphometric Analysis of Time Course of

CVF-Induced Neutrophil Influx Into Glomeruli
Number of
Number of Number of
PMNs
PMNs/glomerulus
Material Time glomeruli
(mean
SEM)
injected (hours) examined observed
2
0.5
1
2
6
12
24

Saline
CVF
CVF
CVF
CVF
CVF
CVF

106
71
115
114
111

129
115

15
10
74
102
101
67
35

0.14 + 0.03
0.14 0.03*

0.64 + 0.08t
0.89 0.1OOt
0.80 0O.9t
0.50 0O.67t
0.30 + 0.58t

Table 3-Ultrastructural Morphometric Analysis of

Glomerular Injury
Number of
glomeruli Endothelial cell Epithelial cell foot
Material
process fusiont
examined
damage*
injected
37 p/2476 t
120 g/4018 i
8
Saline

Not significant.

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 to
a Carl Zeiss Video-Plan. The instrument was programmed to measure 1) length (in microns) of basement
membrane bordering on epithelial cells, 2) length (in
microns) of epithelial foot process fusion, 3) length (in
microns) of endothelial cell surfaces on glomerular
capillaries, and 4) length (in microns) of damaged endothelial 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 calculated (Tables 2 and 3).
Glomerular filtration rates (GFR) were measured 2-3
hours after infusion of the experimental material by
the use of '251-iothalamate, as described by Sigman et
al.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 for

comparison of the differences in data derived from the
various experimental groups. Data are expressed in all
figures as the mean the standard error of the mean
(SEM).

Table 2-Morphometric Analysis of Effect of Catalase on

PMN Influx Into Glomeruli
Number of
Number of Number of
PMNs
PMNs/glomerulus
glomeruli
(mean
SEM)
Material injected examined observed
Saline

106
114

CVF

catalase

165

15
102
136

17

454

p/5742 p

883

p/7848 t

(7.9%)

(11.3%)

78 p/6656 A
(1.2%)

272 p/7427 p

CVF
+

11

(3.7%)

Data are expressed as surface area of damaged endothelial cells per

total surface area of endothelial cell measured.
t Data are expressed as length of fused epithelial cell foot processes
per total length of epithelial cell bordering on basement membrane
measured.
All measurements made 2 hours after injection. Forty units of CVF were
used in each case. The dose of catalase used was 1000 units.
*

t P < 0.001, compared with saline control.

t P < 0.02, compared with saline control.

CVF

(3.0%)

(1.5%)
CVF

catalase
*

59

0.14
0.03
0.89 + 0.1
0.1
0.82

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

Results
Proteinuria Induced by the Infusion of
Cobra Venom Factor
Infusion of CVF directly into the left renal artery
caused significant proteinuria in the subsequent 24
hours. As shown in Figure 1, infusion of 40 units of
CVF caused 51.2 6.0 mg/24 hours of protein excretion in urine as compared with 14.5 0.9 mg/24 hours
in animals receiving normal saline (P < 0.001).
The ability of CVF to induce proteinuria in a dosedependent manner is illustrated in Figure 2. CVF (5
units) induced a significant increase in protein excretion as compared with saline controls (27.7 0.2 mg/24
hours versus 12.3 2.5 mg/24 hours). Increasing the
dose of CVF resulted in increased protein excretion in
urine and was maximum at a dose of 40 units. A dose
of 40 units of CVF was, therefore, used for all subsequent studies.
The duration of the CVF-induced proteinuria is illustrated in Figure 3. The amount of protein excreted
in urine peaked within the first 12 hours and then rapidly decreased, so that by 48 hours the amount of protein present in the urine approached that of salinetreated controls.
Prior depletion of complement by intraperitoneal injection of 8 units of CVF prevented the CVF-induced
proteinuria (complement-depleted rats receiving saline
excreted 10.5 5.6 mg of protein/24 hours, compared
with complement-depleted rats receiving CVF, which
excreted 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 in
a dose-dependent fashion, 2) this effect was limited to
the first 24 hours, and 3) circulating complement was
necessary for the effect.

60

REHAN ET AL

A,1 1'

*O-

Aprril

19Xfi

p<.O01

70-

50VENOM FACTOR (40units)

601

50-

C'd

EE 40-

D 30-

40-

300

cc 200.

20-T-

10n

-,T-

10-

SALINE

NONE

CYF (40 unIt6)

CVF (40 units)

SALINE

NORMAL SALINE

OL

0-12

12-24

MATERIAL INFUSED

24-48 48-72 72-96

TIME (hours)

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

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

Characterization of CVF-Induced Proteinuria

The next set of experiments were done to determine
whether CVF-induced proteinuria was of glomerular
or tubular origin. IThe urinary proteins were first evaluated by SDS-slab gel electrophoresis.2" As illustrated
in Figure 4, a large percentage of the protein in urine
migrated with an apparent molecular weight of 60,000
daltons under nonreducing conditions and was therefore probably albumin. Higher and lower molecular
weight proteins were also present. This pattern of protein excretion is compatible with a glomerular protein
leak, but it does not exclude the possibility of a tubular contribution to the proteinuria.
The concept that glomerulus may be the main source

of the protein leak into urine is further strengthened

by immunofluorescence studies of the renal tubules of
the CVF-treated animals. Protein droplets have been
described in tubules during periods of protein leakage
from glomeruli.22 These droplets probably represent
protein reabsorption by tubular cells. Their presence
would therefore suggest that the origin of protein leakage occurred upstream from the proximal tubule. As
illustrated in Figure 5, by using a rabbit anti-rat albumin serum as the primary antibody, bright fluorescent
reabsorption droplets were seen in the proximal tubules

Mr x I.-.3

--

70

'.
60

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.Ic:..I1'

* ..
.. *t..,

'*D

w-

^ ,,

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~~~~~~~~~~~- 51-;
~31,
45

cs

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z
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00

SALINE RANGE

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IISD

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7"i/ 711"
7p /7171

''I/////X
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~,

10

20

30

X4
40

0
50

COBRA VENOM FACTOR (units)

Figure 2-Dose response of CVF-induced proteinuria (n

group). Mean + SEM.

8 for each

Figure 4-Characterization of urine protein excreted by CVF-treated rats

using SDS slab gel electrophoresis under nonreducing conditions.
Represented are urines from normal rat (A), rats treated with normal saline (B), rats treated with CVF alone (40 p) (C), rats treated with PEG:catalase (1000 units) and CVF (40 i) (D), and normal rat plasma (E). Note that
the bulk of the urine protein appears to be albumin as compared with plasma control. (The apparent molecular weight of albumin under nonreducing conditions is 60 kilodaltons). Catalase suppression of CVF-induced
proteinuria is illustrated by the markedly smaller amount of albumin
present.

Vol1. 123

No. I

*5.'wo*P_^ikt.nv,zW*'-w_=

COMPLEMENT ACTIVATION IN GLOMERULAR INJURY

v-

*,-

61

w *'

W _L +

.,

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s

; + s;

wK

"*W

' s

1s s s m;

19>*s
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ro
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iF F w -_s

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

of CVF-treated rats. These droplets were not seen when

normal rabbit serum was used instead of rabbit antirat albumin. Fine background tubular fluorescence was
present 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 protein being mainly albumin are compatible with the conclusion that the CVF-induced proteinuria is probably
mainly of glomerular origin. However, tubular dysfunction could also have contributed.
Requirement of Neutrophils for
CVF-Induced Proteinuria
CVF activates the complement cascade and leads to
the production of C5a.4 C5a has been shown to be a
potent chemotactic agent for inflammatory cells as well
as being a potent stimulator of oxygen free radicals and
protease release by these cells. Therefore, it was of interest to determine whether the in vivo effects of CVF
infusion into the kidney might be attributable to neutrophils. Six rats were depleted of circulating PMNs by
injection of rabbit anti-rat neutrophil serum as detailed
in the Methods section. These animals then received

40 units of CVF into the renal artery, and the amount

of protein excreted by these animals was compared with
that of animals receiving saline alone. As illustrated in
Figure 6, animals that were PMN-depleted showed a
marked decrease in protein excretion during the first

70-

60-

04
E

5040

Iz

30

m
0

20-

p<.005

0r
10-

0-

CVF (40u)
PMN INTACT

CVF (40u)
PMN DEPLETED

SALINE
PMN DEPLETED

MATERIAL INFUSED

Figure 6 -The neutrophil requirement for the development of CVF-induced

proteinuria (n = 6 per group). Mean + SEM.

62

REHAN ET AL

60 -

A T) *

April

1 986

70
0-

60-

5 0-

p <.05
0,

E
4
z

4030

50-

p<.05
T

40-

p <.05
z

20 -

0
cc

10

30 20-

0.

CVF

CVF
CVF
+
+
+100Ou
250u
500u
PEG-C ATALA SE
CVF

CVF

10-

INACTIVE
PEG-CATALASE

MATERIAL INFUSED

Figure 7-Effect of PEG:catalase and inactivated catalase on CVF-induced

proteinuria (n = 6 per group). P compared with CVF alone. Mean + 1

0-

CVF 140ul

CVF 140ul
+

SOD

CVF 140UI
+

PEG: SOD

MATERIAL INFUSED

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

24 hours after infusion of CVF when compared with

PMN-intact animals (22.7 + 2.8 mg/24 hours in
neutrophil-depleted versus 63.4 + 9.9 mg/24 hours in
neutrophil-intact animals; P< 0.005). If the background
proteinuria (14.5 + 0.9 mg/24 hours) in the saline controls is subtracted from both groups, the suppression
of the proteinuria in the neutrophil-depleted animals
was 800%o. Thus, the proteinuria induced by systemic
complement activation was largely dependent on cir-

culating neutrophils.
Effect of Oxygen Radical Inhibitors on the
CVF-Induced Proteinuria
With the neutrophil having been identified as being
critical for the development of the proteinuria in this
model, a series of studies was then undertaken to determine whether oxygen radicals generated by neutrophils were responsible for the proteinuria.
The first oxygen radical inhibitor tested was catalase.
Groups of 6 animals each were injected intravenously
with 250, 500, and 1000 units of PEG:catalase 10
minutes before the infusion of CVF. As shown in Figure 7, the addition of the PEG:catalase suppression of
the CVF-induced proteinuria appeared to be dose dependent, although differences were not statistically
different between the groups. Two hundred fifty units
of PEG:catalase reduced proteinuria to 34.5 + 1 mg/24
hours versus 52.1 + 10.0 mg/24 hours in the animals
receiving CVF alone (P < 0.05). At the highest dose
of PEG:catalase (1000 units) proteinuria was reduced
to 23.7 mg + 3.8 mg/24 hours. If the background protein excretion (14.5 + 0.9 mg/24 hours) is subtracted,
this represented a reduction in protein excretion by 70%.

Infusion of chemically inactivated catalase caused no

diminution of CVF-induced proteinuria. Thus catalase
was able to inhibit CVF-induced proteinuria in a dosedependent manner, which suggests that hydrogen peroxide and/or its metabolic products are important mediators for the development of the CVF-induced proteinuria.
As shown in Figure 8, the co-instillation of either coupled or uncoupled SOD with CVF had no effect on
CVF-induced proteinuria (52.4 + 15.9 mg/24 hours in
SOD + 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 be
involved in CVF-induced proteinuria.
To assess the role of the hydroxyl radical, both DMSO
and deferoxamine were tested. As shown in Figure 9,
neither DMSO nor deferoxamine had a suppressive
effect on CVF-induced proteinuria (58.9 5.5 mg/24
hours in CVF + DMSO, 64.2 + 13.1 mg/24 hours in
CVF + deferoxamine and 58.9 + 5.5 mg/24 hours in
CVF alone). Thus, on the basis of these studies, there
was no evidence that the hydroxyl radical was involved
in 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 artery had an average GFR of 1.2 + 0.1 ml/min/100 g
body weight. Infusion of PEG:catalase (1000 units)
alone did not alter the GFR (0.97 0.86 ml/min/100
g body weight). Infusion of CVF reduced the GFR to
0.20 + 0.1 ml/min/100 g body weight. Co-instillation

Vol. 123 * No. 1

COMPLEMENT ACTIVATION IN GLOMERULAR INJURY

of PEG:catalase together with CVF did not alter the

GFR from that seen with CVF alone (0.18 0.11
ml/min/100 g body weight). Therefore, although protein excretion following CVF injection was reduced by
catalase, the fall in GFR seen following CVF injection
was not prevented by catalase. This suggests that different mechanisms mediate the decrease in GFR and the
protein leak following CVF injection.
Morphologic Alterations

Animals were sacrificed at various intervals during

the first 24 hours after the infusion of the CVF. The
injected kidney was compared with the contralateral
kidney in the same animal as well as kidneys from
animals given saline and the various oxygen radical inhibitors. Light-microscopic examination of the CVFtreated kidneys revealed neutrophils present in the
glomeruli. Neutrophil influx was maximum 2-6 hours
after CVF infusion. Neutrophil accumulation in glomeruli was also seen in the catalase-treated rats. The
uninjected right kidney of the CVF-treated rats also had
increased numbers of neutrophils per glomerulus.
As shown in Table 1, a minimum of 70 glomeruli were
examined at each time point. There were increased numbers of neutrophils present in glomeruli by 2 hours after CVF injection, with the maximal number of neutrophils per glomerulus appearing at 2- and six-hour
time intervals. Therefore, a single injection of CVF induced a significant neutrophil influx into the glomeruli.
The neutrophil influx was compared in the CVF-treated
versus the CVF + PEG:catalase-treated animals. We
did this to determine whether the decreased protein excretion observed in the catalase-treated animals was due
to a reduced number of neutrophils. As shown in Table 2, co-instillation of PEG:catalase with CVF did not
affect neutrophil accumulation in glomeruli at 2 hours.
Thus the protective effect seen with catalase was not
due to a decreased number of neutrophils in glomeruli.
Ultrastructural studies were performed at times of peak
neutrophil influx (2-6 hours). As shown in Figure IOA
and B, the glomeruli of the CVF-treated animals showed
endothelial cell damage, fibrin generation, and patchy fusion of epithelial-cell foot processes. In contrast, as
shown in Figure IOC, the CVF + PEG:catalase-treated
animals showed little in the way of endothelial cell injury or foot process fusion and no fibrin formation.
These changes were quantitated morphometrically (Table 3).
Thus, both by traditional morphology and quantitative morphometry CVF-induced renal injury was
manifested by structural glomerular cell alterations.
Catalase protected against these changes without affecting the influx neutrophils.

63

801
70cs

60-

C,'
0,

50-

40-

30

w
0

20-

0.

10-

0-

CVF (40u)

CVF (40u)

CVF (40u)

+
DMS0

DEFEROXAMINE

MATERAL INFUSED
Figure 9-Effect of hydroxyl radical inhibitors on CVF-induced proteinuria (n = 6 per group).

Discussion
The data presented in this study provide evidence that
intravascular activation of the complement system can
lead to proteinuria and that this occurs in a dosedependent manner. This protein excretion is maximal
over the first 24 hours and is neutrophil-dependent.
It is accompanied by morphologic changes in glomeruli
which include endothelial cell swelling and epithelial
cell foot process fusion. Both the proteinuria and the
morphologic alterations, but not the decrease in GFR,
appear to be caused by the production of H202 and/or
its metabolic products as shown by the protective effects
of catalase.
Morphologic changes, including leukocyte aggregation, were present in both the CVF-infused and noninfused kidneys. Since Blantz et al have shown that complement (C3) does not bind to the basement following
administration of CVF,23 we hypothesize that neutrophils aggregate in response to generation of C5a after CVF infusion. We further hypothesize that the local concentration of C5a is greater in the infused kidney,
thereby explaining the increased leukocyte accumulation and endothelial cell changes seen on that side as
compared with the noninfused side.
Similar studies have previously shown that oxygen
free radicals (more specifically H202 and/or its metabolic products derived from neutrophils) are important
in causing protein excretion and glomerular changes
in models of both the heterologous phase of nephrotoxic nephritis7 and after infusion of PMA into the renal artery of the rat.22 In quantitative terms, the amount
of protein excreted in these models of glomerular in-

64

REHAN ET AL

AJP * April 1986

-we< i

h^s"'=

\X

LSKI /'4

Figure 10-Glomerular morphologic alterations associated with CVF infusion.

A-Electron micrograph of a glomerulus from a rat given CVF
2 hours previously. Note the presence of neutrophils (N), swelling of the
endothelial cell (E), fibrin deposition (F), and patchy fusion of the epithelial
foot processes (Ep). (x3900)
(8)-Note the glomerular endothelial cell
(E) swelling of detachment from the basement membrane 2 hours after the
infusion of CVF. (x4900)
C-Catalase suppression of CVF-induced
glomerular alterations. Note that while neutrophils (N) are still present in
the glomerulus, there is minimal endothelial cell damage and epithelial foot
process are intact. (x 3700)

Vol. 123 * No. 1

COMPLEMENT ACTIVATION IN GLOMERULAR INJURY

jury and the suppression of protein excretion by catalase (but not by other inhibitors of oxygen radicals) were
similar. We hypothesize that neutrophils are attracted
into the glomeruli by various stimuli, such as C5a and
PMA, and that they subsequently produce oxygen radicals (particularly H202 and/or its metabolic products)
which appear to mediate damage to the glomerulus either directly or indirectly. The major anatomic abnormalities were seen in the endothelial cell; however, we
cannot exclude effects on other structures such as basement membrane, mesangial cells, or epithelial cells. In
vitro studies have previously shown that the endothelial
cell is susceptible to damage by H20224; therefore, our
observations are compatible with that in vitro data.
However, cultured mesangial cells have also been shown
to produce oxygen radical in association with phagocytosis of zymosan.25 Therefore, oxygen radicals production by intrinsic glomerular cells might cause glomerular injury and proteinuria under some circumstances.
The fact that neutrophil depletion prevented the protein leak by 70-80% suggests that in this particular
model oxidant production by intrinsic glomerular cells
was not an important mediator.
The amount of CVF-induced proteinuria which involved mainly albumin suggested that glomerular injury was the major source of the protein in the urine.
This, together with the presence of reabsorption
droplets of albumin in the proximal tubules, and morphologic data which was confined to the glomerulus,
provide further evidence that the glomerulus is the major site of proteinuria. Both kidneys probably contributed to the proteinuria, because morphologic
changes were present bilaterally. We cannot exclude the
possibility that tubular dysfunction also contributed
to the proteins measured in the urine.
What is not clear from these studies is the exact mechanism of action of these radicals in causing tissue injury. H202 can directly interact with a halide group,
forming hypochlorous products in the presence of the
myeloperoxidase enzyme system. These products are
highly toxic, with effects on cells via lipid peroxidation
and by other mechanisms.26 There is evidence for synergism between oxygen radicals and lysosomal proteases.3'24 In addition, oxygen radicals are capable of
inactivating a1-anti-trypsin, which is a major neutral
protease inhibitor of plasma. Furthermore, oxygen radicals may act by rendering substrates such as the basement membrane more susceptible to subsequent degradation by proteolytic enzymes.27 Modulation of the
function of glomerular cells (eg, by production of
arachidonate metabolites or other substances which alter glomerular cell function) might also have induced
the protein leak. We cannot distinguish between these
possibilities from our studies. The fact that the fall in

65

GFR seen after CVF injection was not prevented by

catalase suggests that H202 production was not responsible for this change. Thus, complement activation in
the glomerulus probably modifies glomerular function
by various mechanisms.
In conclusion, we have shown that infusion of CVF
into the rat renal artery results in neutrophil aggregation along with glomerular morphologic alterations and
proteinuria. Because of the protective effect of the
specific enzyme catalase, we have inferred that hydrogen peroxide (and/or its metabolic products) produced
in the presence of circulating neutrophils is an important mediator of injury in this model. This mechanism
might also play a role in some forms of acute glomerular injury in man.

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Acknowledgments
We are grateful to Mr. Craig Biddle and Mr. Eddie Burke
for photographic work and to Ms. MaryAnn Byrnes for
secretarial assistance.