Treatment with an Inhibitory Monoclonal Antibody to Mouse Factor B Protects Mice from Induction of Apoptosis and Renal Ischemia/Reperfusion Injury
Joshua M. Thurman*,
Pamela A. Royer*,
Danica Ljubanovic,
Belda Dursun*,
Amanda M. Lenderink*,
Charles L. Edelstein* and
V. Michael Holers*
* Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado; and Department of Pathology, University Hospital Dubrava, Zagreb, Croatia
Address correspondence to: Dr. Joshua M. Thurman, University of Colorado Health Sciences Center, Division of Renal Diseases and Hypertension, 4200 E. 9th Avenue, B-115, Denver, CO 80262. Phone: 303-315-0171; Fax: 303-315-5540; joshua.thurman{at}uchsc.edu
Received for publication July 8, 2005.
Accepted for publication December 10, 2005.
Complement activation in the kidney after ischemia/reperfusion(I/R) seems to occur primarily via the alternative complementpathway. The ability of an inhibitory mAb to mouse factor B,a necessary component of the alternative pathway, to protectmice from ischemic acute renal failure was tested. Treatmentwith the mAb prevented the deposition of C3b on the tubularepithelium and the generation of systemic C3a after renal I/R.Treated mice had significantly lower increases in serum ureanitrogen and developed significantly less morphologic injuryof the kidney after I/R. For gaining insight into potentialmechanisms of protection, the activity of caspases within thekidney also was measured, and it was found that caspases-2,-3, and -9 increased in a complement-dependent manner afterrenal I/R. Apoptotic cells were detected by terminal deoxynucleotidyltransferase catalyzed labeling of DNA fragments, and mice inwhich the alternative pathway was inhibited demonstrated significantlyless apoptosis than control mice. Thus, use of an inhibitorymAb to mouse factor B effectively prevented activation of complementin the kidney after I/R and protected the mice from necroticand apoptotic injury of the tubules.
The alternative pathway of complement is activated after ischemia/reperfusion(I/R) of the kidneys in rodents and humans (1,2), and complementdeficiency protects mice from the full development of ischemicacute renal failure (ARF) (2,3). Complement activation by theclassical and lectin pathways has been demonstrated after I/Rof the heart (4), intestine (5,6), and skeletal muscle (7).Mannose-binding lectin is deposited in the postischemic kidney(8), perhaps indicating activation of this pathway. Complementactivation after I/R of the kidney is critically dependent onan intact alternative pathway (2,3), however, and does not requirethe protein C4 (3). Selective inhibition of the alternativepathway, therefore, should confer protection against the developmentof ischemic ARF while leaving the classical and lectin pathwaysintact.
Renal I/R results in both epithelial cell necrosis and apoptosis(9). Although necrosis is often the more prominent morphologicfinding, inhibition of apoptosis has been shown to protect micefrom ischemic ARF (10). The caspases are a group of cysteineproteases that execute many of the processes that are necessaryfor cellular apoptosis (11). Studies have demonstrated thatcomplement activation directly activates caspases within renalcells (12) and may also induce caspase activity within othertissues after I/R (13). Furthermore, the use of an anti-C5 antibody(which prevents formation of C5a and the membrane attack complex)has been shown to prevent the development of apoptosis afterrenal (14) and cardiac (13) I/R. Thus, there is reason to suspectthat complement activation in the kidney after I/R mediatesthe development of apoptosis through the caspases and that thisis an important mechanism of complement-mediated injury to thekidney.
We recently developed an inhibitory mAb to mouse factor B, designated1379, that selectively blocks the alternative pathway of complement(15). This reagent may be of particular therapeutic benefitin injury states, such as renal I/R, that critically dependon an intact alternative pathway for complement activation toproceed. In the current study, we tested the ability of 1379to prevent activation of complement in the kidney after I/Rand to protect mice from ischemic ARF. Caspase activity andapoptosis within the kidneys were also measured to determinewhether these are downstream effectors of complement-inducedinjury after renal I/R.
Protocol for Induction of Renal I/R
Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) wereused for all experiments. Eight- to 10-wk-old mice that weighed20 to 25 g received intraperitoneal injections of 1 mg of controlmouse polyclonal IgG (Sigma-Aldrich, St. Louis, MO) or 1 mgof 1379 anti-mouse factor B (mouse IgG1), which was generatedas described previously (15). Two hours later, the mice wereanesthetized with 300 µl of 2,2,2-Tribromoethanol (Sigma)injected intraperitoneally. Additional anesthetic (50 µl)was administered as necessary to keep the mice sedated for theentire procedure. Mice were placed on a heating pad to maintaintheir body temperature during surgery. Laparotomies then wereperformed, and the renal pedicles were isolated by blunt dissectionas described previously (2). The pedicles were clamped withsurgical clips (Miltex Instrument Co., Inc., Bethpage, NY) for24 min, and occlusion of blood flow was confirmed by visualinspection of the kidneys. The kidneys were observed for approximately1 min to ensure blood reflow, then fascia and skin were suturedwith 4-0 silk (United States Surgical, Norwalk, CT). Sham surgerywas performed in an identical manner and for the same duration,except that the renal pedicles were not clamped. The mice werevolume resuscitated with 0.5 ml of normal saline and kept inan incubator at 29°C to maintain body temperature. After24 h, the mice were anesthetized, blood was obtained by cardiacpuncture, and the kidneys were harvested. All animal procedureswere in adherence to the National Institutes of Health Guidefor the Care and Use of Laboratory Animals.
Serum and Plasma Measurements
Serum urea nitrogen was determined for each mouse using a BeckmanAutoanalyzer (Beckman, Fullerton, CA). Plasma C3adesArg (a stabledegradation product of C3a) levels were measured by an ELISAaccording to the manufacturer’s instructions (CedarlaneLaboratories Ltd., ON, Canada). Alternative pathway activityin mouse serum was measured using an in vitro analysis of C3deposition on zymosan A particles (Sigma-Aldrich) as describedpreviously (15,16). Briefly, 10 µl of serum from eachmouse to be tested was incubated with 109 zymosan particlesat 37°C for 30 min in a master mix that contained 5 mM MgCl2and 10 mM EGTA (to prevent classical pathway activation). C3deposition on the particles was analyzed by flow cytometry.
Renal Morphology
Sagittal kidney sections were fixed and embedded in paraffin,and 4-µm sections were stained with periodic acid-Schiff(PAS). The sections were evaluated by a renal pathologist (D.L.)in a blinded manner. The cortex and the outer stripe of theouter medulla were assessed for epithelial necrosis, loss ofbrush border, tubular dilation, and cast formation. At least10 fields (x400) were reviewed for each slide, and the percentageof tubules that displayed these findings was determined. Kidneysections were scored as follows on the basis of the percentageof affected tubules: 0, none; 1, <10%, 2, 11 to 25%, 3, 26to 45%, 4, 46 to 75%, and 5, >75% (2).
In Vitro Assay of Complement-Mediated Tubular Cell Injury
BUMPT cells, a mouse proximal tubular epithelial cell (PTEC)line (17), were provided by Dr. John Schwartz (Boston University,Boston, MA). Cells were grown to confluence in DMEM supplementedwith 10% FBS, penicillin-streptomycin, and 0.2 U/ml IFN- (Peprotech,Rocky Hill, NJ). After the cells reached confluence, they werechanged to 1:1 DMEM/Hams F12 supplemented with transferrin 5mg/L (Invitrogen Corp., Carlsbad, CA), hydrocortisone 50 nM(Sigma), and insulin 5 mg/L for 2 d. Lactate dehydrogenase (LDH)was measured by the Cytotox-ONE assay (Promega, Madison, WI),and the results were read on a Wallac 1420 fluorescence platereader with an excitation wavelength of 530 nm and an emissionwavelength of 590 nm. To induce chemical hypoxia, we incubatedcells for 2 h with 1 µM antimycin (Sigma) in DMEM withoutglucose (18). As a source of complement components, serum fromC57BL/6J mice was added to a final concentration of 10%. Cellswere treated for 15 min with serum that had been preincubatedfor 10 min with 40 µg of 1379 or control mouse IgG (Sigma)for each 100 µl of serum or with serum that had been heatedto 55°C for 1 h to inactivate complement, and supernatantthen was tested for LDH. Background LDH was measured in unmanipulatedcells and serum to be added to the cells and also in cells afterfull lysis with 9% Triton X-100. The percentage of lysis ofcells in each sample was calculated according to the equation% lysis = (ODsample – ODserum – ODbackground) x100/(ODfull lysis – ODbackground).
Immunofluorescence and Detection of Apoptotic Cells
Kidneys were snap-frozen in OCT compound (Sakura Finetek, U.S.A.,Inc., Torrance, CA). Four-micrometer sections were cut witha cryostat and stored at –70°C. The slides later werefixed with acetone and stained with FITC-conjugated anti-mouseC3 (Cappel, Durham, NC) or FITC-conjugated F(ab')2 anti-mouseIgG (Cappel) diluted 1:150 and counterstained with hematoxylin(Vector Laboratories, Inc., Burlingame, CA). Detection of apoptoticcells was performed using a Flurescein-FragEL DNA fragmentationdetection kit (Calbiochem, San Diego, CA) in which deoxynucleotidyltransferase binds to the 3'-OH ends of DNA exposed by apoptoticendonucleases, and sections were counterstained with hematoxylin(Vector Laboratories). Six to 10 high-power fields in the outerstripe of the outer medulla were examined. The area of positivelabeling for each field was determined using SlideBook software4.0 (Intelligent Imaging Innovations, Denver, CO), and the resultsfor each sample were averaged.
Western Blot Analysis
Renal tissue was homogenized in RIPA lysis buffer as describedelsewhere (19). Homogenates were centrifuged at 14,000 rpm for15 min at 4°C, and the supernatant was collected. Purifiedfactor B (15), 100 µg of protein lysates, or 2.5 µlof serum from C57BL/6, fB–/– or C3–/–mice (20) were run on 10% Bis-Tris gels, transferred to nitrocellulosemembranes, and probed with horseradish peroxidase–conjugatedanti-mouse C3 (Cappel) or with anti-mouse factor B (clone 1379).For blots that were probed with the 1379 antibody, the proteinthen was detected with an horseradish peroxidase–conjugatedsheep anti-mouse IgG (Amersham Biosciences, Piscataway, NJ).Because this antibody identifies proteins in mouse serum, fB–/–serum was used to confirm specificity of the identified protein.The proteins were detected with Western Lightening chemiluminescencereagent (PerkinElmer Life Sciences, Boston, MA).
Caspase Measurement
The activity of caspases-2, -3, -8, and -9 were determined byusing fluorescence substrates as described previously (21,22).Briefly, whole kidney was mixed with lysis buffer and homogenizedwith 10 strokes in a glass-Teflon homogenizer. The lysate thenwas centrifuged at 4°C at 100,000 x g in a Beckman Ti70rotor for 1 h. The resultant supernatants (cytosolic extracts)immediately were aliqotted, frozen in liquid N2 and stored at–70°C. Lysate protein was measured by the Bradfordmethod with BSA as standards. Next, 200 µg of proteinextract (20 to 50 ml of lysate) was mixed with 10 µl ofthe substrate (final concentration, 50 µM). The assayvolume was made up to 200 µl with assay buffer, and thereaction was initiated by addition of substrate. Peptide cleavagewas measured over 1 h at 30°C using a Cytofluor 4000 seriesfluorescence plate reader (Perseptive Biosystems, Framingham,MA) at an excitation wavelength of 380 nm and an emission wavelengthof 460 nm. A 7-amido-4-methyl coumarin (AMC) standard curvewas determined for each experiment. Caspase activity was expressedin nanomoles of AMC released per minute of incubation time permilligram of lysate protein.
Statistical Analyses
Multiple group comparisons were performed using ANOVA with posttest according to Student-Newman-Keuls. Comparison between thecontrol- and 1379-treated groups was performed with unpairedt testing. P < 0.05 was considered statistically significant.Results are reported as mean ± SEM.
Treatment with 1379 Prevents Activation of Complement in the Kidney after I/R
Mice were treated with 1 mg of control mouse IgG or 1379 andthen subjected to renal I/R. Similar to previous studies (2),sham-treated mice (Figure 1, A and B) demonstrated intermittentdeposition of C3 along the tubular basement membrane (TBM),and mice that were subjected to I/R demonstrated extensive depositionof C3 along the TBM of tubules in the outer medulla (Figure 1C).We did not see C3 deposition in the large vessels or peritubularcapillaries. Pretreatment with 1379, however, prevented thedeposition of C3 along the TBM after I/R (Figure 1D). Westernblot analysis demonstrated that activation of complement inserum using zymosan generated an approximately 40-kD C3 fragmentthat was minimally present in serum that contained 10 mM EDTA(which prevents complement activation) and absent in serum fromC3–/– mice (Figure 1E). In kidney lysates, the approximately40-kD C3 degradation fragment was present at baseline and increasedafter renal I/R. Treatment with 1379 prevented formation ofthis fragment, however, further demonstrating that 1379 preventedcomplement activation within the kidney after I/R.
Figure 1. mAb 1379 inhibits complement activation in the kidney after ischemia/reperfusion (I/R). Deposition of C3 along the tubular basement membrane (TBM) was evaluated by immunofluorescence microscopy. Sham-treated mice that received control IgG (A) or 1379 (B) had intermittent deposition of C3 along TBM of tubules in the outer medulla. In mice that received control IgG before I/R (C), there was extensive C3 deposition along damaged tubules after 24 h. Mice that received 1379 before I/R (D) had only sparse patches of C3. (E) Western blot analysis using an antibody to C3 demonstrated that activation of the complement in mouse serum generated an approximately 40-kD activation fragment that was absent in C3–/– serum. This activation fragment of C3 was also present in lysates of kidneys subjected to I/R but was lower in mice treated with mAb 1379. Treatment with 1379 also prevented systemic rise in complement activation fragments. (F) Levels of C3adesArg were measured in plasma of mice after ischemia and 8 h of reperfusion. I/R resulted in a significant increase in systemic levels of C3adesArg. Treatment with 1379, however, prevented this rise in C3adesArg, and levels in the 1379-treated group were not significantly different from those in the sham-treated group. Representative images are shown (A through E); n = 4 for each experimental group. Magnification, x200 in A through D.
Renal I/R resulted in a significant increase in the level ofC3adesArg in plasma after 8 h of reperfusion (874 ± 343versus 45 ± 13 ng/ml in sham-treated animals; P <0.05; Figure 1F). By 24 h of reperfusion, the plasma C3adesArglevels had returned to baseline (data not shown). Treatmentwith 1379, however, prevented the rise in plasma C3adesArg at8 h of reperfusion, and the levels were not significantly differentfrom those in sham-treated animals (96 ± 37 versus 45± 13 ng/ml in sham-treated animals; NS).
Treatment with 1379 Protects Mice from Ischemic ARF
Treatment with 1379 attenuated the functional and morphologicinjury of the kidney after I/R. The normal serum urea nitrogen(SUN) for unmanipulated C57BL/6J mice is 19 ± 1 (n =7). After ischemia and 24 h of reperfusion, the 1379 treatedanimals had significantly lower SUN than mice that had receivedcontrol IgG (78 ± 16 versus 119 ± 15 mg/dl, P< 0.05; Figure 2), although SUN in this group still weresignificantly higher than in sham-treated animals (22.5 ±2.6 for the sham group that received control IgG; P < 0.05).Treatment with 1379 also ameliorated the morphologic damageseen after renal I/R (Figure 3). We previously found that thisduration of ischemia causes reversible renal injury that peaksafter 24 h of reperfusion and that mice recover within 2 to3 d (19). When graded after 24 h of reperfusion by a pathologistin a blinded manner, the control-treated mice demonstrated extensivetubular injury in the outer stripe of the outer medulla (Figure 3D),and the damage was significantly milder in mice that hadreceived 1379.
Figure 2. Treatment with 1379 blunts the rise in serum urea nitrogen (SUN) after renal I/R. Mice were treated with control IgG or 1379 and subjected to renal I/R. After 24 h of reperfusion (the time point of peak injury in this model), mice treated with 1379 and subjected to I/R had significantly lower SUN than control IgG-treated mice subjected to I/R (78 ± 16 versus 119 ± 15 mg/dl). Mice that received 1379 and were subjected to I/R still had significantly higher SUN than mice that received sham treatment after 1379 (21 ± 1 mg/dl) or sham treatment after control IgG (23 ± 3 mg/dl). The SUN in unmanipulated C57BL/6 mice was 19 ± 1; n = 7.
Figure 3. Treatment with 1379 attenuates proximal tubular injury after I/R. Mice were treated with control IgG or 1379 and subjected to renal I/R. The morphologic injury in the outer stripe of the outer medulla was graded by a pathologist in a blinded manner and scored from 0 to 5 with increasing injury (see Materials and Methods). (A) Sham-treated mice had intact brush border and no visible necrosis. (B) Mice treated with control IgG and subjected to I/R showed extensive epithelial necrosis (arrows) and epithelial simplification (arrowhead). Mice treated with mAb 1379 (C) demonstrated significantly milder histologic injury on average (D). Representative images are shown (A through C); n = 4 for A, and n = 10 for B and C. Magnification, x400 in A through C.
Alternative Pathway Was Fully Inhibited in Serum of Mice that Received 1379
Kidney sections from mice that were treated with control IgGor 1379 were examined for bound IgG by immunofluorescence. Inmice that received 1379 before sham surgery (Figure 4B) andrenal I/R (Figure 4C), IgG was detectable in the glomeruli butcould not be seen in the tubulointerstitium. In unmanipulatedmice that received 1379, the alternative pathway was measuredusing an in vitro assay of alternative pathway activity (16)and found to be inhibited for at least 24 h after injection(data not shown), and activity was fully suppressed in micethat received 1379 before I/R (Figure 4D). Control mice thatwere subjected to I/R had reduced alternative pathway activitycompared with serum from unmanipulated mice (42 ± 6 versus100 ± 4%; P < 0.001). This likely is due to consumptionof alternative pathway components in the kidney (as evidencedby increased C3a in the plasma), and previous studies have showndecreased hemolytic activity in the serum of mice after I/R(14). Factor B levels in the serum of 1379-treated mice didnot detectably decrease by Western blot analysis (Figure 4E),however, indicating that 1379 inhibits factor B but is nondepleting.
Figure 4. 1379 inhibits circulating factor B but is not detectable in the tubulointerstitium. Immunofluorescence for mouse IgG was performed using kidneys of sham-treated mice that received control IgG (A) and 1379 (B) and mice that received 1379 before undergoing I/R (C). Mice that received 1379 demonstrated IgG in the glomeruli (arrowheads), but IgG was not detectable in the tubulointerstitium. (D) Alternative pathway activity was measured in the serum of mice after ischemia (or sham surgery) and 24 h of reperfusion. Alternative pathway activity was significantly lower in mice subjected to I/R than in sham-treated controls. Mice that received 1379 had nearly undetectable alternative pathway activity, significantly lower than in either other group. (E) Western blot analysis for factor B in the serum of mice treated with 1379 showed that the levels of circulating factor B (approximately 90 kD) did not decrease during the period of inhibition. Purified factor B and serum from an fB–/– mouse were used to confirm that the middle band shown for the serum samples was factor B. Representative images are shown (A through C); n = 4 for each experimental group. Serum from two mice was tested at each time point for E, as shown.
Treatment with 1379 Protects PTEC from Complement-Mediated Cytotoxicity after Chemical Hypoxia
Complement activation directly can cause cytotoxicity, or itindirectly can contribute to injury through inflammatory orvasoactive effects. Immortalized murine PTEC were grown in cultureand exposed to whole mouse serum, and LDH release into the supernatantwas measured as a marker of cell injury. The LDH readings werecorrected for background levels that already were present inthe medium and serum that were added to the cells. We have foundthat the addition of serum to the cells does not cause a significantrelease of LDH by the cells (after correcting for the LDH presentin the serum). When the cells were exposed to antimycin A toinduce chemical hypoxia before incubation with serum, however,LDH release rose significantly (Figure 5). This increase wassignificantly attenuated when the serum was treated with 1379before incubation with the cells. We believe that hypoxia mayalter PTEC expression of the complement inhibitor Crry, renderingthe cells susceptible to alternative pathway–mediatedinjury. Thus, chemical hypoxia and the alternative pathway actsynergistically to injure epithelial cells. Furthermore, thecytotoxicity is a direct result of alternative pathway activationand can occur independent of inflammatory cells or altered hemodynamics.
Figure 5. 1379 protects proximal tubular epithelial cells (PTEC) from autologous injury by the alternative pathway after chemical hypoxia. Cells were subjected to chemical hypoxia with 1 µM antimycin A and exposed to mouse serum with or without the mAb 1379 or to heat-inactivated serum (HIS). Lactate dehydrogenase was measured as a marker of cell lysis. The percentage of cells lysed in each group was calculated in relation to full lysis of the cells using Triton X-100 (see Materials and Methods). Exposure to hypoxia and mouse serum resulted in a significantly greater percentage of lysed cells than exposure to serum alone. Treatment with 1379 or HIS, however, resulted in significantly less cell lysis than treatment with control IgG.
Treatment with 1379 Attenuates Activity of Caspases-2, -3, and -9 in the Kidney after I/R
We next assessed the effect of alternative pathway inhibitionon caspase activity within the kidney. I/R induced an increasein the activity of caspases-2, -3, and -9 (Figure 6), and theactivity of these caspases was significantly lower in the groupthat received 1379 compared with the group that received controlIgG. Caspase-8 activity did not increase after I/R (Figure 6),and complement inhibition did not have an effect on caspase-1activity (data not shown). To determine whether treatment with1379 altered the development of apoptosis after I/R, kidneysections from mice that were subjected to I/R was evaluatedby transferase-mediated dUTP nick-end labeling (TUNEL) staining.Apoptosis was not a prominent finding in either group after8 h of reperfusion. After 24 h of reperfusion, however, TUNEL-positivenuclei could be seen in both groups (Figure 7), but apoptosiswas significantly more extensive in the control group than inthe group that had received 1379. Activation of the alternativecomplement pathway after renal I/R, therefore, seems to mediatecaspase activation and the development of epithelial apoptosis.
Figure 6. Alternative pathway inhibition prevents increased caspase activity in the kidney after I/R. Kidneys were subjected to sham surgery or ischemia, and after 24 h of reperfusion caspase activity in the kidneys was measured. Activity of caspases-2 and -3 was significantly greater in postischemic mice than in sham-treated controls. Treatment with 1379 resulted in significantly lower activity of caspases-2, -3, and -9 after 24 h of reperfusion.
Figure 7. Alternative pathway inhibition prevents cells from undergoing apoptosis after I/R. Transferase-mediated dUTP nick-end labeling staining was performed and quantified as described in Materials and Methods. Sections from mice that received control IgG (A) had greater evidence of apoptotic cells in the outer medulla after 24 h of reperfusion than those that received 1379 (B). (Inset) Concurrent light image of the hematoxylin-stained tissue. When the area of positive staining for each field was measured and averaged for all sections (C), there was significantly less positive staining in the mice that had received 1379. Representative images are shown (A and B); n = 9 for each experimental group. Magnification, x200.
We have demonstrated that an inhibitory mAb to mouse factorB prevents activation of the alternative pathway in the kidneyafter I/R. Treatment with 1379 before I/R almost completelyprevented the deposition of C3 along the TBM and generationof systemic C3adesArg (which may mediate systemic effects afterlocal injury). This antibody therefore is capable of preventingthe generation of local and systemic complement activation fragmentsafter renal I/R.
Treatment of mice with 1379 ameliorated the functional and morphologicrenal injury that is caused by I/R. Treatment with 1379 significantlyattenuated the rise in SUN and morphologic injury seen at 24h, although the protection was only partial. Although complementantagonists have been shown previously to ameliorate ischemicARF (14,23,24), those agents nonselectively block the functionof downstream activation fragments that are generated by anyactivation pathway. The mAb used in our study is a specificand potent inhibitor of the alternative complement pathway,and its effects can be presumed to be due to inhibition of thispathway.
Using an in vitro model of chemical hypoxia, we also showedthat an intact alternative pathway acts synergistically to amplifythe hypoxic injury to tubular epithelial cells. Thus, in thesetting of hypoxia, an intact alternative pathway is harmfuldirectly to renal tubular epithelial cells and does not requireinflammatory cells to cause injury. Renal tubular epithelialcells have membrane-bound complement inhibitors on their surface.We recently showed that I/R alters the polarized expressionof these inhibitors and permits alternative pathway activationto occur (19). The in vitro studies presented here further supportthe hypothesis that I/R alters the mechanisms whereby renaltubular epithelial cells protect themselves from autologousalternative pathway activation. These results are also consistentwith previous studies that demonstrated that injury to renaltubular epithelial cells caused them to become complement activating(25,26).
Several studies have shown that the caspases are important mediatorsof ischemic ARF (10,22,27–29), and the complement systemhas been shown previously to induce apoptosis of rat mesangialcells via caspase-3 (12). Previous studies, however, have notdetermined specifically whether proapoptotic caspase activityis a mechanism of complement-mediated injury to the kidney afterI/R. We measured the activity of several caspases in the kidneysof mice, and caspases-2, -3, and -9 increased in a complement-dependentmanner in postischemic kidneys.
Two pathways of caspase-3–mediated apoptosis have beendescribed. The "death receptor" pathway involves signaling throughcell surface receptors. The mitochondrial pathway, in contrast,is initiated by caspase-2–mediated permeabilization ofmitochondria and the release of cytochrome C or by changes inthe Bcl-1 family proteins (30). This pathway is activated inresponse to intracellular stress, including oxidative injury,and catalyzes the conversion of pro–caspase-9 to its activeform (9). Treatment with 1379 also significantly reduced theextent of TUNEL-positive staining seen in the outer medullaafter I/R. This method of detection may not always discriminateapoptotic cells from necrotic or sublethally injured cells (9).Nevertheless, the reduction in caspase-2, -3, and -9 activityand the decreased TUNEL-positive staining that was seen in micethat received 1379 suggest that inhibition of the alternativepathway prevented apoptosis, perhaps via the mitochondrial pathway.
A previous study in which mice were treated with soluble Crry-Ig(a complement inhibitor that works at the level of the C3 andC5 convertases) did not show significant protection with useof the inhibitor (31). Both groups of mice in that study receivedheparin, however perhaps diluting any benefit conferred by theCrry-Ig because heparin is an effective complement inhibitor(32). C3–/– mice were protected compared with C3+/+controls, suggesting that despite the heparin, some of the injurywas complement mediated. It is possible that the Crry-Ig, whichis a fairly large molecule, was restricted to the intravascularspace. Because Crry must be present at the location of the C3and C5 convertases, tissue penetration is critical to its function.The mAb 1379 may bind factor B intravascularly, however, andthereby deprive the tubulointerstitium of this factor even withoutpenetration of the tissue. Nevertheless, treatment with 1379did not seem to confer protection equivalent to that seen previouslyin fB–/– mice in which the increase in SUN and theacute tubular necrosis scores were approximately half thoseseen in wild-type mice (2). This suggests that the antibodydid not prevent all of the complement-mediated injury, perhapsas a result of its inability to penetrate the tubulointerstitiumand inhibit locally produced factor B (Figure 4). Furthermore,given the incomplete protection seen in both of these studies,there likely are complement-independent pathways of injury.Determining which pathways of injury are independent of thealternative pathway may help in devising combinations of agentsthat can be expected to confer additive protection.
Complement inhibitors have proved protective in models of I/Rinjury to the heart (4) and intestine (5). Because complementactivation after renal I/R critically depends on the functionof factor B (2), we tested an inhibitory mAb to factor B ina model of ischemic ARF. Pretreatment with this agent preventedcomplement activation and protected mice from the developmentof functional and morphologic renal injury after I/R. Alternativepathway inhibition reduced the necrotic injury of hypoxic PTECin vivo and in vitro. It is interesting that alternative pathwayinhibition also reduced apoptotic injury of the tubules, andcomplement-induced apoptosis seems to involve the mitochondrialpathway. Alternative pathway activation is an early event afterrenal I/R, and treatment with 1379 may be an effective therapeuticagent for the prevention of ischemic ARF.
Acknowledgments
This work was supported by National Institutes of Health grantsAI31105 (V.M.H.), DK064790-02 (J.M.T.), and DK56851 (C.L.E.),as well as a postdoctoral fellowship from the InternationalSociety of Nephrology (D.L.).
This work was presented in part in abstract form at the annualmeeting of the American Society of Nephrology; St. Louis, MO;October 27 to November 1, 2003.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
), which was generated as described previously (15). Two hours later, the mice were anesthetized with 300 µl of 2,2,2-Tribromoethanol (Sigma) injected intraperitoneally. Additional anesthetic (50 µl) was administered as necessary to keep the mice sedated for the entire procedure. Mice were placed on a heating pad to maintain their body temperature during surgery. Laparotomies then were performed, and the renal pedicles were isolated by blunt dissection as described previously (2). The pedicles were clamped with surgical clips (Miltex Instrument Co., Inc., Bethpage, NY) for 24 min, and occlusion of blood flow was confirmed by visual inspection of the kidneys. The kidneys were observed for approximately 1 min to ensure blood reflow, then fascia and skin were sutured with 4-0 silk (United States Surgical, Norwalk, CT). Sham surgery was performed in an identical manner and for the same duration, except that the renal pedicles were not clamped. The mice were volume resuscitated with 0.5 ml of normal saline and kept in an incubator at 29°C to maintain body temperature. After 24 h, the mice were anesthetized, blood was obtained by cardiac puncture, and the kidneys were harvested. All animal procedures were in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 
(Peprotech, Rocky Hill, NJ). After the cells reached confluence, they were changed to 1:1 DMEM/Hams F12 supplemented with transferrin 5 mg/L (Invitrogen Corp., Carlsbad, CA), hydrocortisone 50 nM (Sigma), and insulin 5 mg/L for 2 d. Lactate dehydrogenase (LDH) was measured by the Cytotox-ONE assay (Promega, Madison, WI), and the results were read on a Wallac 1420 fluorescence plate reader with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. To induce chemical hypoxia, we incubated cells for 2 h with 1 µM antimycin (Sigma) in DMEM without glucose (18). As a source of complement components, serum from C57BL/6J mice was added to a final concentration of 10%. Cells were treated for 15 min with serum that had been preincubated for 10 min with 40 µg of 1379 or control mouse IgG (Sigma) for each 100 µl of serum or with serum that had been heated to 55°C for 1 h to inactivate complement, and supernatant then was tested for LDH. Background LDH was measured in unmanipulated cells and serum to be added to the cells and also in cells after full lysis with 9% Triton X-100. The percentage of lysis of cells in each sample was calculated according to the equation % lysis = (ODsample – ODserum – ODbackground) x 100/(ODfull lysis – ODbackground). 
