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*
Department of Medicine, Section of Nephrology, The University of Chicago,
Chicago, Illinois
Department of Pathology, The Johns Hopkins University School of Medicine,
Baltimore, Maryland
Department of Medicine, Division of Rheumatology, University of Colorado
Health Sciences Center, Denver, Colorado.
Correspondence to Dr. Richard J. Quigg, University of Chicago, Section of Nephrology, AMB, S-508, MC 5100, 5841 S. Maryland, Chicago, IL 60637. Phone: 773-702-0757; Fax: 773-702-4816; E-mail: rquigg{at}medicine.bsd.uchicago.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human complement receptor 1 (CR1) is a potent complement inhibitor that is active toward C3 and C5 convertases of both the alternative and classical pathways of complement. Given this, CR1 has been produced as a soluble recombinant protein (sCR1). The first use of sCR1 came in a rat model of IRI in the heart (6). In this setting, infusion of sCR1 at the time of arterial ligation protected against the resultant myocardial infarction. Inhibition of complement protects against the neutrophilic influx that occurs (6). Subsequently, the use of sCR1 has been applied successfully in IRI models in intestine, skeletal muscle, and liver (7,8,9,10).
An elegant series of experiments performed by Carroll et al. (11,12) defined events in skeletal muscle and intestinal IRI. Mice that were deficient in complement components C3 and C4, as well as in Ig, were protected from IRI. Reconstitution of IgM in Ig-deficient mice restored IRI. Taken together, these data indicate that IgM natural antibody-mediated activation of the classical pathway on endothelium is a proximate event in IRI in these two organs.
In renal IRI, tubular cells also are prominently injured, which leads to the pathologic picture of acute tubular necrosis. The events that occur are complex but involve complement activation on endothelial cells, endothelial cell P- and E-selectin (but not L-selectin) engagement of neutrophils, and an ensuing inflammatory reaction that ultimately results in necrotic and apoptotic tubular cell death through as-yet incompletely characterized mechanisms (1,2,13,14). A role for C5b-9—mediated tubular injury also has been proposed through the use of mice with both targeted and natural deficiencies of various complement proteins (15).
Given the apparent involvement of the complement system in renal IRI and the current availability of sCR1 as a recombinant complement inhibitor in humans (16), therapeutic blockade of the complement system is a viable option in human IRI. To evaluate the potential utility of complement inhibition in renal IRI, we used the potent rodent complement regulator CR1-related gene/protein y (Crry) (17). For these studies, we used Crry-Ig, which is a chimera of Crry with the Fc region of mouse IgG1 (18). Crry-Ig has an extended half-life and is effective in complement-mediated glomerular injury (18). Unexpectedly, here we show that Crry-Ig is ineffective in a mouse model of renal IRI.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice
All work with mice was approved by the University of Chicago Animal Care
and Use Committee and was performed in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals. Male mice were
used exclusively in these studies because of their higher complement activity
(20) and apparent greater
susceptibility to complement-mediated renal injury
(21). Normal C57BL/6 mice were
obtained from Jackson Laboratories (Bar Harbor, ME). Mice with targeted
deletions of C3 (C3 -/-) (22)
and CR1/CR2 (MCR1/2 -/-) (23)
had been generated previously. Wild-type controls for C3 -/- mice (C3 +/+)
were on a mixed 129SvJ and C57BL/6 background (Jackson Laboratories), whereas
C57BL/6 mice were used as controls for CR1/2 -/- mice, because this strain has
been backcrossed over seven generations onto C57BL/6.
Transgenic mice expressing recombinant soluble Crry directed by the metallothionein-I promoter also were used. Male mice, as used in these studies, have circulating levels of soluble Crry that are complement inhibitory and high intrinsic renal production of Crry, including in glomeruli and tubules (21). The Crry transgenic animals used in this study were derived from a single founder line and are on a CD-1 background. In all animals, the presence of the Crry transgene was documented by PCR, and soluble Crry in sera was identified by enzyme-linked immunosorbent assay (21). As controls, littermates that lacked the Crry transgene were used.
Experimental Protocol
Two h before induction of renal IRI, mice received an intraperitoneal
injection of either 7.5 mg of Crry-Ig or the negative control, K9/4
IgG1. Pilot studies indicated that there was no difference in IRI
severity between animals that were given K9/4 IgG1 and animals that
were given normal saline. Normal saline therefore was used in these studies as
a negative control, except when noted otherwise. One h before IRI, animals
were given 5 U of heparin subcutaneously
(24,25).
To exclude an effect of heparin on IRI, one group of animals was not given
heparin.
Mice that were anesthetized with isoflurane had baseline blood drawn for blood urea nitrogen (BUN) determinations and then underwent laparotomy. Core body temperature was maintained between 38 and 39°C. Both renal arteries were isolated by blunt dissection, and a nontraumatic vascular clamp was applied directly to the arteries, leaving the renal veins unoccluded. Cessation of blood flow was documented by visual inspection and by Doppler ultrasound (Koven Technology, Inc., St. Louis, MO). After ischemia times of 20 to 30 min, the clamps were released and flow was verified to occur by visual inspection of the kidneys and by Doppler ultrasound. Fluid resuscitation was via normal saline given intraperitoneally as well as 1 ml given subcutaneously after closure of the abdomen.
Blood was collected from the retro-orbital plexus at 10, 20, and 30 h after IRI. Then mice were killed, and both kidneys were obtained for the histologic studies described below. In instances when an animal died after 20 h (6 of 128 animals), 20-h BUN measurements were used for 30-h values.
Renal Histology
Sagittal sections of kidneys were fixed in buffered formalin. Four-µm
sections were stained with periodic acid-Schiff. The extent of epithelial
necrosis and neutrophil infiltration was graded according to the schema of
Kelly et al. (2). In
that process, the estimated percentage of tubules in the outer medulla and
corticomedullary junction that had epithelial cell necrosis and/or necrotic
debris was estimated and assigned a score as follows: 0, none; 1+, <10%;
2+, 10 to 25%; 3+, 26 to 75%; and 4+, >75%. The extent of neutrophil
infiltration was derived from the estimated mean number of neutrophils per
high power field (400x) in 5 to 10 consecutive fields from the outer
medulla and corticomedullary junction, starting at the most involved area and
proceeding in the direction of greatest involvement. Scores were assigned on
the basis of these counts as follows: 0, 0 to 1; 1+, 2 to 10; 2+, 11 to 20; 3+
21 to 40; and 4+, >40 or too many to count. In some instances, when scoring
was borderline between two scores, an average value was assigned,
e.g., 2.5+. All sections were provided as coded slides, so the
observer (M.H.) was blinded as to treatment group and duration of ischemic
injury.
Immunofluorescence Microscopy
For immunofluorescence microscopy, 4-µm cryostat sections of frozen
tissue were processed for direct immunofluorescence microscopy as described
elsewhere (26), by use of
FITC-conjugated anti-mouse C3 Cappel, Organon Teknika Corp., Durham, NC).
Because this antiserum reacts with the C3c portion of C3b but not C3d
(27,28),
additional staining was performed for C3d. A dual labeling technique was
performed in which sections were incubated with rabbit anti-human C3d (Dako,
Carpinteria, CA), which is cross-reactive with mouse C3d
(28), followed by
rhodamine-conjugated anti-rabbit IgG (Cappel) and FITC-conjugated anti-mouse
C3. The extent that C3 was present on basolateral aspects of tubules also was
assigned a score. In this case, the numbers of tubules and the percentage of
their total circumference stained by anti-C3 in at least 10 high-power fields
were estimated and assigned scores as follows: 0, none; 1+, <3 tubules with
<30% circumference stained in a discontinuous patter; 2+, 
3 tubules
stained, of which at least 1 had 50% circumference stained in a continuous
pattern; 3+, >60% tubules stained, of which the majority had 75%
circumference stained in a continuous pattern; and 4+, >90% tubules
stained, with the majority having >90% circumference stained. A group of
unmanipulated C57BL/6 mice served as a control for C3 staining.
Measurements from Sera
BUN values were determined with a Beckman Autoanalyzer (Fullerton, CA).
Sera levels of Crry were measured by an enzyme-linked immunosorbent assay
technique described elsewhere
(18). Because native Crry is a
transmembrane protein, normal mice do not have circulating Crry. Complement
inhibition was determined by use of an assay described elsewhere in which sera
were incubated with zymosan particles, leading to complement activation and
C3b deposition, which was quantified by flow cytometry
(18). The capacity of sera
from animals that were given Crry-Ig to prevent complement activation on
zymosan was compared with sera from control animals that were given buffer
alone.
Statistical Analyses
All data are expressed as mean ± SEM. When a particular mouse strain
was compared with its litter-mate control, t testing was used.
Otherwise, one-way ANOVA followed by Tukey's pairwise comparisons were used.
Correlations among variables were examined by regression analysis. Minitab
software (State College, PA) was used for these analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given the ineffectiveness of Crry-Ig in these studies, it was possible that 30 min of ischemia led to complement-independent injury and/or resulted in overwhelming complement activation that could not be constrained with Crry-Ig. To explore these possibilities, we used shorter times of ischemia. Renal injury was dependent on the time of ischemia (Figure 1); for example, BUN levels 30 h after 20 min of ischemia were 54.7 ± 4.5 mg/dl. As shown in Figure 1, there was no reduction in renal injury after varying times of ischemia in animals that received Crry-Ig. This was true of BUN values obtained 10 and 20 h after ischemia (not shown). In those animals, Crry-Ig levels were extremely high (1.81 ± 0.21 mg/ml) at the time of ischemia. To verify that such levels of Crry-Ig were complement inhibitory in these studies, complement activity was measured in a separate group of animals. As expected, complement activity was diminished by 95.9 ± 0.6% in animals given Crry-Ig compared with that in controls (n = 3 each). There was no correlation between Crry levels at the time of ischemia and BUN values after reperfusion.
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To examine the effect of intrinsic production of Crry by renal tubules, upon which injury is focused in IRI, transgenic animals that overexpressed Crry in kidney were subjected to 27.5 min of ischemia. These mice are protected from tubular injury induced by antiendothelial cell antibodies (29). As with the Crry-Ig—treated animals, there was no effect of complement inhibition on BUN values obtained at any time after IRI (30 h BUN = 164.7 ± 27.2 and 196.7 ± 10.3 mg/dl in transgene-negative and -positive animals, respectively; n = 3 each).
Our use of a single low dose of heparin was based on a concern that the vascular manipulation could lead to thromboembolic events, which would affect renal function independently from IRI. Heparin has been used in studies that have implicated the complement system in IRI (24). Nonetheless, to exclude an effect of heparin, a group of animals were not given heparin but were exposed to 27.5 min of ischemia. Thirty h later, BUN values were 157.5 ± 11.3 mg/dl (n = 6), which was slightly higher than that in animals that received Crry-Ig (127.7 ± 28.7 mg/dl; n = 6) or buffer alone (124.4 ± 11.0 mg/dl; n = 7), but these were not statistically different. To the extent that heparin can inhibit the complement system (30), maximum complement inhibition in the former group that received Crry-Ig and heparin was ineffective in reducing renal injury in IRI.
Effect of C3 and MCR1/2 Deficiency on Renal IRI
Given the recent report by Zhou et al.
(15) that animals that were
deficient in C3 were partially protected from renal IRI, C3 -/- mice were
subjected to IRI. In addition, MCR1/2 -/- mice also were examined in these
studies, because these mice are protected from intestinal IRI, apparently
because of a deficient natural antibody repertoire
(31). Activated mouse
neutrophils also bear MCR1
(28,32),
which could recruit neutrophils to C3b deposited in IRI. In C3 -/- animals,
there was reduced injury 10, 20, and 30 h after 27.5 min of ischemia, but this
was only statistically significant at the 20-h time point (P =
0.033). In contrast, MCR1/2 -/- mice were not protected from IRI at any time
after ischemia. Figure 2 shows
BUN values 30 h after ischemia in the two groups of deficient animals and
their corresponding control groups. The extent of BUN elevation was lower in
the C3 +/+ mice, compared with all studies performed in C57BL/6 mice,
including the MCR1/2 +/+ mice, which presumably reflects the contribution of
the 129 strain genes in the C3 +/+ and C3 -/- animals. Nonetheless, these data
are comparable to those reported elsewhere, in which C3 deficiency did
favorably affect renal IRI
(15).
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Histology
The histologic indices of injury measured in this study were epithelial
cell necrosis and the extent of neutrophil infiltration. As expected, both of
these measures were significantly elevated in all groups of animals subjected
to IRI, and there were no differences among any of the groups.
Figure 3A shows a
representative field from a control animal that was exposed to 27.5 min of
ischemia, whereas Figure 3B
shows one from an animal that received Crry-Ig. BUN values positively
correlated with both epithelial cell necrosis (r = 0.52; P
< 0.001) and neutrophil infiltration (r = 0.39; P <
0.001). No animal with an epithelial necrosis score <3 or a neutrophil
infiltration score <2 had a 30-h BUN value >85 mg/dl, which illustrates
the relevance of both of these indicators to the resultant renal failure.
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In C3 -/- mice, the extent of neutrophil infiltration was reduced in parallel with the reduction in BUN, although, as with renal function, this was not statistically significant (neutrophil infiltration scores, 1.6 ± 0.4 in C3 -/- mice and 2.2 ± 0.3 in C3 +/+ mice; P = 0.24). Neutrophil infiltration was no different in MCR1/2 -/- mice, compared with wild-type controls (2.5 ± 0.2 and 2.8 ± 0.3, respectively). Thus, neutrophil infiltration in renal IRI appeared to be independent of CR1-C3b interactions (6,8,9).
C3 Staining
In normal C57BL/6 animals, C3 staining is prominent in basal aspects of
tubules (C3 staining score, 3.2 ± 0.2; n = 6;
Figure 4A). This is a specific
finding because it is absent in C3 -/- animals
(33) and may reflect that the
presence of ammonium in the interstitium can result in C3 activation
(34). Of interest, C3 staining
was diminished in animals with IRI compared with that in unmanipulated
controls, irrespective of whether they received Crry-Ig (P <
0.001; Figure 4B). When all
animals were considered together (with the exception of C3 -/- studies), there
was an inverse correlation between BUN levels and C3 staining (r =
-0.46; P < 0.001). This relationship can be attributed solely to
the results from Crry-Ig—treated animals, as shown in
Figure 5 (r = -0.63;
P < 0.001). One conceivable explanation for this is that Crry-Ig
facilitated factor I—mediated cleavage of iC3b to C3d, which would not
be identified by use of the anti-C3 antibody used for immunofluorescence
(although this second cleavage of C3b has not been shown directly). To address
this, a specific anti-C3d antibody was used. In these studies, there was no
difference in the extent and distribution of C3d staining compared with that
for C3, the latter detecting the C3c chain of C3b and iC3b
(Figure 6). Staining was absent
in C3 -/- animals, which confirms the specificity of the antibody. Thus, in
the renal tissue of animals that received Crry-Ig, C3 was present as C3b or
iC3b, and these were present in reduced amounts in animals with severe
IRI.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, we could not demonstrate a reduction in renal IRI using the potent murine complement inhibitor Crry-Ig (18). This was true for ischemic times of 20 to 30 min, which resulted in a spectrum of renal IRI. Crry-Ig given in this way was present in extremely high levels, well above those required in vitro and in vivo for meaningful complement inhibition (18). Given its large size, Crry-Ig is predicted to be restricted primarily to the intravascular space, which constitutes an advantage to limit complement activation within the vascular system, e.g., the glomerulus, (18) and also raises its half-life to the level of IgG. To the extent that IRI in kidney is focused on the renal endothelium, Crry-Ig present in the quantities documented here should have completely eliminated C3 convertase activity on vascular endothelium. The tubulointerstitial compartment of the kidney is unique in that it starts with a sizable amount of C3 deposition around tubules (34). Of interest, in animals that were given Crry-Ig, C3 deposition on tubules was reduced, and this reduction was proportional to the extent of renal failure. This could reflect vascular damage in IRI, allowing access of Crry-Ig to the renal interstitium. As an alternative explanation, which incorporates the reduction in C3 staining in all animals, access of inflammatory cells to tubular cells that bear C3 deposits may have marked them for phagocytosis.
The complement system in renal IRI may be dissimilar from other organ systems, because it seems to be focused on tubules (15). In a study in rats by Stein et al. (35), depletion of complement with cobra venom factor before renal IRI did not affect tubular necrosis and the decrease in GFR. As an independent verification that complement inhibition was of no benefit, we used Crry transgenic mice that overexpress Crry in tubules, limiting complement activation in this site (29). These animals also were susceptible to renal failure induced with IRI, making complement activation leading directly to renal tubular injury an unlikely culprit in our model system.
In renal IRI, there are many candidate mediator systems that originate from endothelial, tubular epithelial, and infiltrating inflammatory cells (3,4). Renal vascular endothelial cells are stimulated in IRI to express adhesion molecules, which results in recruitment and activation of inflammatory cells, particularly neutrophils. In addition to expressing adhesion molecules, endothelial cells produce phospholipid products, such as leukotriene B4 and platelet-activating factor, which can stimulate inflammatory cells (36). Therefore, blockade of platelet-activating factor as well as the adhesion molecules, intercellular adhesion molecule-1 (2,37), P- and E-selectins (1,38) (but not L-selectin (13)), and ß2 integrins (39) all reduce the extent of renal IRI. The resulting situation, in which endothelial cells and leukocytes are activated, results in production of reactive oxygen species, including superoxide (36). Nitric oxide is also clearly involved in IRI, because mice that are deficient in inducible nitric oxide synthase are protected from renal IRI (40). Tubular epithelial cell alterations in IRI include cell adhesion molecule alterations (41) and cellular death. Although commonly termed acute tubular necrosis, the tubular cell death in renal IRI clearly can proceed through the process of apoptosis (14,42).
Complement activation products have the potential to be involved at various steps in the pathogenesis of renal IRI. C5a and C5b-9 have been shown to stimulate endothelial cell expression of selectins and intercellular adhesion molecule-1 (43,44,45). The anaphylatoxins C3a and C5a are well-known stimulants of leukocytes that act through their specific G-protein—linked receptors (46). Leukocytes also contain CR1 (CD35) and CR3 (CD11/CD18b), which bind C3b and iC3b present at sites of complement activation. Finally, complement can directly affect tubular epithelial cells, either through the formation of C5b-9 (47,48) or by interacting with C5a receptors, which have been identified recently on proximal tubular epithelial cells (49).
The data of Zhou et al. (15) clearly implicate the complement system in renal IRI. Their studies, which used mice that were deficient in C3, C4, C5, and C6, allowed dissection of the roles of various complement activation products and showed that C5b-9—mediated tubular cell damage was etiologic in their model. To account for the lack of effect of complement inhibition in our studies of renal IRI, we need to consider the differences between the two groups of studies. We used a model of selective arterial occlusion, whereas they clamped the renal pedicle, including renal vein and ureter. We did have complete cessation of renal blood flow as shown with a Doppler flow probe. We also used anticoagulation with heparin to eliminate the possible effects of thromboemboli in our experimental system. Although heparin can affect the complement system, we provide evidence here that such did not influence our results. The 20 to 30 min of complete arterial occlusion that we used led to remarkable renal IRI, including elevations in BUN, tubular cell necrosis, and neutrophil infiltration. These ischemic times are more in line with the bulk of published data on mouse IRI (2,13,38,40), compared with the 58 min used in the Zhou study. Finally, we studied the effects of complement inhibition with Crry, rather than used mice with complete deficiencies of individual complement components. The limitation of using animals with complete deficiencies of a given protein, including through gene targeting, is that expression of the particular gene product is eliminated throughout the body beginning with conception, with likely unknown effects, including the possibility that related and parallel pathways can be affected (50).
Overall, it is clear that renal IRI has a complex pathogenesis. It does seem that complete deficiencies of C3, C5, and C6 limit renal IRI by reducing C5b-9 generation on proximal tubular cells (15). Of course, a complement-deficient state cannot be applied to humans, whereas complement inhibitors with activity comparable to Crry are available for use as therapeutic agents. Given the discrepancy between our results and those from complement-deficient animals, further study is warranted to determine whether complement inhibition would be beneficial in renal IRI that affects humans.
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