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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)
58
REHAN ET AL
AJP
April 1986
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
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
Not significant.
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%)
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-
601
50-
C'd
EE 40-
D 30-
40-
300
cc 200.
20-T-
10n
-,T-
10-
SALINE
NONE
SALINE
NORMAL SALINE
OL
0-12
12-24
MATERIAL INFUSED
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.
Mr x I.-.3
--
70
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SALINE RANGE
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20
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No. I
*5.'wo*P_^ikt.nv,zW*'-w_=
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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 concentration
of protein reabsorption droplets, compared with saline control rats (B). (x 400)
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
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
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).
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%.
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
-we< i
h^s"'=
\X
LSKI /'4
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
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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.