Maladaptive Role of IL-6 in Ischemic Acute Renal Failure
Mariusz L. Kielar*,
Reji John*,
Michael Bennett,
James A. Richardson,,
John M. Shelton,,
Liying Chen*,
D. Rohan Jeyarajah||,
Xin J. Zhou,
Hui Zhou,
Brett Chiquett*,
Glenn T. Nagami¶ and
Christopher Y. Lu*,#
Departments of * Internal Medicine (Nephrology), Pathology, Molecular Biology, Internal Medicine (Cardiology), || Surgery, # Graduate Program in Immunology, University of Texas Southwestern Medical Center, Dallas, Texas, and ¶ Nephrology Section, Medical Research Services, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California
Address correspondence to: Dr. Christopher Y. Lu, Department of Internal Medicine (Nephrology), University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75390-8856. Phone: 214-648-3959; Fax: 214-648-2071; E-mail: christopher.lu{at}utsouthwestern.edu
Received for publication September 14, 2003.
Accepted for publication August 18, 2005.
The role of IL-6 was investigated in murine ischemic acute renalfailure. The renal pedicles were clamped for 17 min, and themice were studied at various times after reperfusion. We foundthat serum IL-6 increased after murine ischemic renal injury.This increase was associated with increased IL-6 mRNA in theischemic kidney but not in the contralateral kidney or the liver.Maximal IL-6 production occurred at 4 to 8 h and decreased tobaseline by 24 h. Reperfusion of the kidney was required forIL-6 production. In situ hybridization and immunohistochemistryshowed that macrophages infiltrated areas adjacent to the vascularbundles in the outer medulla within hours of reperfusion andshowed that these macrophages produced IL-6 mRNA. For understandinghow macrophages were stimulated to produce IL-6, an in vitromodel in which S3 proximal tubular cells were injured by reactiveoxygen species was set up. These injured cells released moleculesthat activated macrophages to produce IL-6 in vitro. IL-6 thatwas produced in response to renal ischemia was maladaptive becausetransgenic knockout of IL-6 ameliorated renal injury as measuredby serum creatinine and histology. IL-6 transgenic knockoutmice were lethally irradiated, and their bone marrow was reconstitutedwith wild-type IL-6 cells. Such bone marrow transfers abolishedthe protective effects of transgenic IL-6 knockout. It is concludedthat macrophages infiltrate the area of the vascular bundlesof the outer medulla, these macrophages produce IL-6, and thisIL-6 exacerbates ischemic murine acute renal failure.
After ischemia/reperfusion initiates injury to epithelial andvascular cells during ischemic acute renal failure (ARF), maladaptiveresponses "extend" the injury (see reviews [1,2]). Inflammationis one maladaptive response (2–5), but the regulationof the inflammatory response to ischemic renal injury is notwell understood.
This report focuses on IL-6 because, as reviewed in the Discussionsection, this cytokine is a major regulator of inflammation.Furthermore, IL-6 production may be a common feature of ischemicinjury of any organ. IL-6 not only is found after ischemia ofthe brain (6), gut (7), and heart (8), but also the amount ofIL-6 correlates with the amount of ischemic injury (9). In humanrenal allografts with ischemia-reperfusion injury, IL-6 is detectedin urine, and its level correlates with the severity of thatinjury (10).
The overall goal of this article is to understand the role ofIL-6 on ischemic acute renal failure (ARF). We make the followingpoints: First, IL-6 protein increases in the serum after ischemicrenal injury. This increase is associated with increased IL-6mRNA in the ischemic kidney. Second, in situ hybridization andimmunohistochemistry localize IL-6 production to macrophagesnear the vascular bundles of the outer medulla. Molecules thatare released by injured S3 proximal tubular cells activate macrophagesto produce IL-6 in vitro. Third, transgenic knockout of IL-6ameliorates renal injury as measured by serum creatinine andhistology. Finally, transfer of IL-6–sufficient macrophagesby means of bone marrow transplantation into IL-6 knockout micerestores the susceptibility of the knockout mice to ischemicrenal injury.
Animals and Surgical Protocols
Male C57Bl/6J [IL-6 (+/+)] and C57BL/6J-Il6tm1Kopf [IL-6 (–/–)]6-wk-old mice were purchased from Jackson Laboratories (BarHarbor, ME). The genotype was confirmed by genomic PCR of tailsnips (http://www.jax.org). The IL-6 (+/+) mice have a 174-bpDNA fragment from the wild-type allele, whereas homozygous IL-6(–/–) mice have a single 280-bp band as a resultof insertion of a neocassette. Mice were handled in accordancewith institutional and National Institutes of Health guidelines.
Mice were anesthetized using inhaled isoflurane (Fortec System,Fraser Lake, NY) and maintained at 37°C by a TR100 temperaturecontrolling system with a rectal probe (Fine Science Tools,Foster City, CA). In most mice, first the right kidney was removedand the left renal artery and vein then were clamped for 17min. In some mice, the left renal artery and vein were clamped,but the right ("contralateral") kidney was not. Other mice had"sham" surgery: Laparotomy and dissection but not clamping ofthe left renal pedicle. Peripheral serum was assayed for creatinineby the Refletron automated system (Roche Diagnostics, Indianapolis,IN) and IL-6 by ELISA (Endogen, Woburn, MA). This ELISA measuresan active form of IL-6.
In some experiments, the cortex and outer medulla were isolatedby dissection using a x3 to x30 operating microscope. Thesewere analyzed by RNase Protection Assay.
RNase Protection Assay
Total RNA was extracted from frozen kidneys using RNA-Easy MidiKits (cat. no. 75144; Qiagen, Santa Clara, CA). Total RNA fromS3 proximal tubular cells (see below) was harvested using theRNA-Easy kit (Qiagen). The P32-labeled probes were made usingIn Vitro Transcription Kits, mCK-2b and mCK-3b templates, andRiboquant RPA kits from Pharmingen (Pasadena, CA). The RNaseprotection gels were exposed on a phosphor image screen andanalyzed with a Molecular Dynamics Storm 820 Phosphorimager(Piscataway, NJ). Densitometry analysis was performed usingImageQuant software (Molecular Dynamics).
In Situ Hybridization for IL-6 mRNA and Immunohistology for F4/80 Macrophage
Ischemic and "contralateral" kidneys were harvested 4 h afterreperfusion and fixed in 4% buffered paraformaldehyde in PBS,embedded in paraffin, cut into 3-µm sections, and usedfor in situ hybridization or macrophage immunostaining. TheIL-6 fragment for the in situ probe was amplified from an ischemickidney cDNA library (11). The details of probe preparation andin situ hybridization were published previously by our group(12). To stain with F4/80 antibody, the kidney sections weredeparaffinized, blocked with Protein Block Serum (cat. no. 0909;DAKO Labs, Glostrup, Denmark), and incubated overnight at 4°Cwith 1:200 F4/80 antibody (cat. no. RM2900; Caltag Laboratories,San Francisco, CA). Subsequently, sections were incubated withbiotinylated rabbit anti-rat IgG from DAKO (cat. no. DK-2600)at 1:50, then with streptavidin, horseradish peroxidase, andfinally with DAB as per the manufacturer’s instruction(cat. no. K0377; DAKO Labs).
In Vitro Model of Ischemic ARF
We developed a two-stage assay using the S3 cell line. Thiscell line was originally dissected from the S3 segment of theproximal tubule of the kidney of an SV40 large T antigen transgenicmouse (13). In stage 1, S3 tubules were injured by reactiveoxygen species (ROS) for 12 h; the ROS were generated by theaction of reaction of 0.01 U/ml xanthine oxidase on 5 mM hypoxanthine(both from Sigma Chemical Co., St. Louis, MO), as previouslyreported by our laboratory (12). In stage 2, the supernatantwas cultured with a murine macrophage line (J774), and IL-6mRNA was measured 4 h later by RNase protection assay (Pharmingen).
In Vivo Injections of Anti–IL-6
IL-6–sufficient (C57Bl6/J) mice received 1 dose of monoclonalrat anti–IL-6 antibody (MP5-20F3 cat. no. 16-7061; eBioscience,www.ebioscience.com) in the amount of 72 µg per mouse.The control group received an equivalent dose of rat IgG1 (cat16-4301). The antibodies were suspended in 0.2 ml of 0.1% BSAand injected into the penile vein immediately before the nephrectomy/clampprocedure. Peripheral serum was assayed for creatinine at 24h after reperfusion. In some mice, peripheral blood was obtainedat 4 h after reperfusion and used for measurement of IL-6 levelsby the ELISA method (Endogen).
In addition, we used polyclonal goat neutralizing anti-murineIL-6 (AB-406-NA) and control goat IgG (AB-108-C) from R&Dsystems (www.rndsystems.com). These antibody preparations had<0.01 EU per 1 µg of antibody. According to the manufacturer’sspecifications, 3.2 ng of antibody will neutralize 1 ng of IL-6.According to Figure 1, the IL-6 concentration at 4 h after ischemiais 2500 ng/ml. If the mouse weighs 20 g and the volume of distributionof IL-6 is 50%, then the total IL-6 in the mouse is 25,000 ngand the dose of antibody to neutralize is 80 µg. We administereda dose of 125 µg.
Figure 1. Ischemic acute renal failure (ARF) increased serum IL-6 protein. Serum IL-6 was measured by ELISA (see Materials and Methods) in three different groups of mice: (1) Sham C57BL/6J mice () had their left renal arteries and veins dissected free of surrounding fat, but these vessels were not clamped. (2) Clamp-only mice () had their left renal arteries and veins dissected and also clamped for 17 min; this resulted in an ischemic left kidney and a contralateral nonischemic right kidney. The contralateral kidney maintained a normal GFR; both the clamp-only and sham mice had serum creatinines of <0.5 mg/dl. These mice had renal ischemia but no uremia. (3) Clamp + nephrectomy mice () had their left renal artery clamped like the above group but, in addition, had their right kidney removed. These mice had serum creatinines of 1.9 mg/dl. They had renal ischemia and uremia. Peripheral blood was taken from the sham group at 4 h after surgery and from the clamp and the clamp + nephrectomy groups at the indicated times of reperfusion. The means and SE are shown. *P < 0.01 versus control by t test. There was no statistically significant difference between the clamp-only and the clamp + nephrectomy groups at 4 h of reperfusion.
Histologic Examination
Three IL-6 (–/–) and three IL-6 (+/+) mice werekilled for morphologic studies at 24 h after reperfusion. Thekidneys were fixed in 10% buffered formalin, embedded in paraffin,cut into 5-µm sections, and stained with hematoxylin andeosin. The morphologic analysis was carried out in a blindedmanner as detailed previously (14). Briefly, the cortex andouter stripe of the outer medulla were evaluated for epithelialnecrosis, loss of brush border, tubular dilation, and cast formation.The kidney sections were scored on the basis of the percentageof affected tubules as follows: 0, none; 1, <10%; 2, 11 to25%; 3, 26 to 50%; 4, 51 to 75%; 5, >75%. At least 10 high-powerfields (x400) were reviewed for each slide. In addition, leukocyteinfiltration in the outer stripe of the outer medulla was countedon hematoxylin and eosin–stained sections. The numberof leukocytes were averaged on the basis of at least 5 high-powerfields for each slide.
Bone Marrow Transplant
Bone marrow cells were isolated from femurs and tibias, filteredthrough nylon mesh, counted using an electronic particle counter,and washed, and 8 x 106 cells in 0.5 ml of PBS were injectedintravenously into recipients within 6 h of their receivingtwo doses of 5 Gy separated by 3 h. We transplanted IL-6 +/+bone marrow into IL-6 –/–, or vice versa. The micewere kept in a sterile environment for 8 wk to allow full cellularreconstitution. Full chimerism of each mouse was confirmed bygenotyping of DNA from peripheral blood and tails using JacksonLab protocol (see above). Renal ischemia reperfusion was inducedas described above.
Ischemic ARF Increased Serum IL-6 Protein and IL-6 mRNA in the Ischemic Kidney Figure 1 shows that mice with ischemic kidneys ("clamp only"and "clamp + nephrectomy") had increased serum IL-6 proteinat 4 h after reperfusion compared with mice with nonischemicsham-operated kidneys. The ischemic left kidney had similarlyincreased serum IL-6, in the presence or absence of a functioningright kidney. This finding showed that the increased IL-6 wasnot due to decreased renal elimination by the ischemic leftkidney and suggested hepatic, rather than renal, eliminationof IL-6 (15).
Figure 2A shows that the increased serum IL-6 originated inthe ischemic kidney, not the liver. In these experiments, liverand a kidney were taken from the same mouse. Minimal amountsof IL-6 mRNA were present in the sham-operated kidney and theassociated liver. In the ischemic kidney, IL-6 mRNA abundanceincreased until 8 h of reperfusion and decreased at 24 h ofreperfusion; little IL-6 mRNA was present in the livers of thesemice.
Figure 2. Reperfusion of the ischemic kidney increased IL-6 mRNA in the injured kidney. Ischemic, contralateral, and sham kidneys are defined in the legend to Figure 1. (A) IL-6 mRNA is increased in the ischemic kidney but not in the liver. RNase protection assays for IL-6 mRNA were performed on the total renal RNA at the indicated reperfusion times. In addition, IL-6 mRNA was assayed from livers that were isolated from a mouse with a sham kidney (a), or from mice with ischemic kidneys at the following renal reperfusion times: 1 h (b), 2 h (c), 8 h (d), and 24 h (e). L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are housekeeping genes. (B) IL-6 mRNA is increased in the ischemic but not the contralateral kidney. Total RNA was taken at the indicated reperfusion times from sham, ischemic, and contralateral kidneys; the indicated cytokines were assayed by RNase protection assays. (C) Reperfusion is required for increased renal IL-6 mRNA. Total RNA was harvested from a sham (control) kidney, from an ischemic kidney without releasing the clamp on the renal artery (no reperfusion), and either 1 or 4 h after release of the clamp (reperfusion). (D) IL-6 mRNA is found in the ischemic outer medulla. Total RNA was isolated from the cortex (C) or outer medulla (M) and was analyzed by RNase protection assay.
Figure 2B compares IL-6 mRNA abundance in sham-operated kidneysand ischemic left versus contralateral right nonischemic kidneysat various times after reperfusion. IL-6 mRNA increased in theischemic kidney at 1 h, peaked at 4 to 8 h, and decreased by24 h. The IL-6 mRNA did not increase in the contralateral orin the sham kidneys. These data confirm previous observations(16). Figure 3 summarizes RNase protection assays, quantifiedby densitometry, on four ischemic versus four sham kidneys.The IL-6 mRNA was significantly increased at 1 and 4 h and thendecreased to baseline at 72 h.
Figure 3. Increased IL-6 mRNA abundance in the ischemic kidney after reperfusion: Composite of four experiments. L32 is a housekeeping gene. The means and SE of four kidneys per group are shown. By t test: *P < 0.04 sham versus ischemic 1-h reperfusion; **P < 0.01 sham versus ischemic 4-h reperfusion.
Figure 2C compares IL-6 mRNA abundance in ischemic kidneys beforerelease of the renal arterial clamp ("ischemia, no reperfusion")with ischemic kidneys after 1 or 4 h of reperfusion. IL-6 mRNAabundance did not increase unless the kidney was reperfused.
In addition to IL-6, the Pharmingen RNase protection assaysprovided information about other cytokines. These were not thefocus of our studies, but we comment on them briefly. Figure 2Bshows that mRNA for IL-1, IL-1Ra, and IL-18 increased inthe ischemic kidney and to a lesser extent in the contralateralkidney. These molecules are expressed by the ischemic kidney(16,17). However, expression by the contralateral kidney toour knowledge has not been previously reported. Such expressionis consistent with the relatively minor inflammation there andmay result from the systemic release of cytokines from the ischemickidney (18). In addition, Figure 2D shows increased leukemiainhibitory factor expression in the ischemic kidney and confirmsa previous report (16).
Macrophages in the Ischemic Outer Medulla Express IL-6 mRNA
To determine which part of the ischemic kidney expressed IL-6mRNA, we dissected the cortex and the outer medulla. Figure 2Dshows that the greatest increase in IL-6 mRNA at 4 and 8h was in the ischemic outer medulla.
To identify the cell population expressing IL-6 mRNA in theischemic kidney, we performed in situ hybridization. We selectedthe 4-h reperfusion time point on the basis of the time coursefor IL-6 mRNA expression shown in Figure 2. Low-power darkfieldphotomicrographs localize IL-6 expression to the ischemic outermedulla: Figure 4A shows that the S35-labeled antisense IL-6mRNA bound to IL-6 mRNA in the outer medulla of the ischemickidney, Figure 4B shows absent staining of the ischemic kidneyby control S35-labeled sense IL-6 mRNA, and Figure 4C showsabsent staining of the contralateral nonischemic kidney by S35-labeledantisense IL-6 mRNA.
Figure 4. IL-6 expression by interstitial macrophages near the vascular bundles of the ischemic outer medulla at 4 h of reperfusion. (A through C) These are darkfield, low-magnification photomicrographs. (A) Main panel; shows IL-6 mRNA in the outer medulla of an ischemic kidney at 4 h after reperfusion. The coarse white dots denote silver grains of antisense S35-labeled IL-6 mRNA. (B) Control ischemic kidney with in situ hybridization with a S35-labeled sense IL-6 probe. As expected, this sense probe does not hybridize to the ischemic kidney. (C) Another control: In situ hybridization of a nonischemic contralateral kidney with an antisense S35-labeled IL-6 probe. There was minimal IL-6 mRNA in the nonischemic kidney. (D) A x40 objective brightfield of the outer medulla of the ischemic kidney shown in A. The dark dots denote S35-labeled antisense IL-6 mRNA. The arrows (both black and red) point at boxes that outline macrophages that express IL-6 mRNA. VB, vascular bundle. (E) High-magnification (x80 objective) brightfield photomicrograph of the two macrophages designated by red arrows in D.
Higher power views show that the IL-6–expressing cellsare mononuclear cells and that the mononuclear cells are locatedadjacent to the vascular bundles. Figure 4D is a medium-powerbrightfield photomicrograph of the ischemic outer medulla. Fourmononuclear cells that express IL-6 are designated by arrowsand outlined in boxes. The neighboring vascular bundles aredesignated VB. Figure 4E is a high-power view of the two mononuclearcells designated by red arrows in Figure 4D.
We immunostained some of the above ischemic kidney sectionswith F4/80, an mAb that detects murine macrophages (19). Therewere no detectable F4/80 macrophages in nonischemic kidneys(Figure 5, top). After 4 h of reperfusion, F4/80 macrophageswere found adjacent to the vascular bundles of the outer medulla(Figure 5, bottom).
Figure 5. Macrophages are found adjacent to the ischemic vascular bundles of the outer medulla. F4/80-stained macrophages are found near the vascular bundles in the outer medulla of ischemic (bottom) but not contralateral nonischemic (top) kidneys at 4 h of reperfusion. nt, necrotic tubules; c, casts. White arrows indicate a few of many F4/80+ (brown) macrophages.
Macrophages Express IL-6 in Response to Molecules Released by S3 Proximal Tubular Cells In Vitro
Molecules that are released by injured cells activate macrophagesin vitro (see reviews [20]). To determine whether this occursin the ischemic kidney, we injured S3 renal tubular cells withROS in vitro (Figure 6). Such ROS are produced during ischemicARF (21), and ROS injury of renal tubular cells in vitro haspreviously been used to model ischemic ARF in vitro (e.g., 22).The injured renal tubular cells released molecules into thesupernatant that activated macrophages to express IL-6 mRNA.Control experiments established that resting renal tubular cellsdid not release activating molecules (Figure 6, Grp C). GrpD was ROS and medium incubated overnight in the absence of S3tubules. The ROS of Grp D were so unstable that they degradedduring the overnight incubation and were not a factor in stage2.
Figure 6. In an in vitro model of ischemic ARF, macrophages express IL-6 in response to molecules released by S3 proximal tubular cells. (A) In stage 1, SV40-transformed S3 tubules were injured by reactive oxygen species (ROS); they then released putative macrophage-activating molecules into the supernatant for 12 h. In stage 2, the supernatant was cultured with a murine macrophage line (J774), and the IL-6 mRNA was measured 4 h later by RNase protection assay (Pharmingen). The positive control "B" was endotoxin added directly to the macrophages. The negative controls were "C," supernatant conditioned by S3 tubules in the absence of ROS, and "D," ROS and media incubated overnight in the absence of S3 tubules. (Top) The experimental design and the means and SE of four experiments. *P < 0.01 versus media control. The y axis is the densitometry (ratio of IL-6:L32 housekeeping gene expression). (B) An RNase protection assay from one of the four experiments.
Altogether, these in vitro experiments support the in vivo experimentsof Figures 4 and 5 that show that renal macrophages expressIL-6 in response to ischemic ARF. Further support for macrophagesas the source of IL-6 in ischemic kidneys will be provided whenwe discuss our bone marrow chimera experiments at the end ofthe Results section.
Removal of IL-6 Ameliorates Ischemic ARF
To determine whether IL-6 exacerbates ischemic ARF, we injectedtwo different anti–IL-6 antibodies intravenously at thetime of renal ischemia and measured the effect on renal injury.First, we injected monoclonal rat anti–IL-6 (MP5–20F;eBioscience) intravenously at a dose seven times larger thanthat previously used successfully to neutralize IL-6 and amelioratemurine septic shock (23). Control mice received an equivalentamount of rat IgG. The anti–IL-6 decreased the peripheralblood IL-6 at 4 h of reperfusion from 7000 ± 1000 ng/mlin the rat IgG–injected group to 1600 ± 600 ng/mlin the anti–IL-6–injected group (mean ± SE;n = 6; P < 0.01 by t test). Despite this 77% decrease inperipheral blood IL-6, there was no significant effect on ischemicARF; the serum creatinine in the rat IgG–treated groupwas 0.8 ± 0.2 mg/dl and 0.7 ± 0.1 mg/dl in theanti–IL-6–treated group (mean ± SE; n = 6;P = 0.38 by t test). In a second experiment, we injected 125µg of neutralizing polyclonal goat anti-murine IL-6 (AB-108-C;R&D Systems). This antibody preparation contained <0.01EU of endotoxin per 1 µg of antibody. We chose this dosebecause it exceeds that necessary to neutralize the 2500 ng/mlof IL-6 found in blood at 4 h after ischemia (see Figure 1 andMaterials and Methods). We increased the clamp time to inducegreater injury in these experiments. The serum creatinine was1.6 ± 0.3 mg/dl in the goat control IgG-injected miceand 1.5 ± 0.4 mg/dl in the anti–IL-6–injectedmice. Again, the anti–IL-6 did not ameliorate ischemicARF. The inability of anti–IL-6 to ameliorate ischemicARF most likely represents an inability of sufficient quantitiesof the large antibody molecule (molecular weight = 160 k) toaccess the outer medulla, where the IL-6 is located.
To determine further the role of IL-6 in ischemic ARF, we examinedthe effect of transgenic knockout of IL-6 as assessed by bothfunction (Figure 7) and morphology (Figure 8). We used C57BL/6J-Il6tm1Kopf[IL-6 (–/–)] and C57Bl/6J wild-type [IL-6 (+/+)]mice. These mice are the same except for the nonfunctional IL-6gene in the former. Figure 7 compares renal function duringischemic ARF. The serum creatinine (Scr) was determined at 24h after reperfusion. The mean serum creatinine level in theIL-6 (–/–) group was 0.89 ± 0.136 mg/dl versus1.83 ± 0.1 mg/dl in the wild-type group (mean and SEof at least eight mice per group; P < 0.05).
Figure 7. Transgenic knockout of IL-6 ameliorated ischemic ARF: Renal function. We removed the right kidney and clamped the left renal pedicle for 17 min. The serum creatinine was measured after 24 h of reperfusion. , homozygous IL-6 knockout [IL-6 (–/–)] mice; , wild-type mice [IL-6 (+/+)]. The means and SE are shown.
Figure 8. Transgenic knockout of IL-6 ameliorated ischemic ARF: Pathology. Kidneys were removed after 24 h of reperfusion, fixed in formalin, sectioned, and stained with hematoxylin and eosin. (A) IL-6 (–/–). (B)IL-6 (+/+). (C) IL-6 (–/–) view of the outer medulla. (D) IL-6 (+/+) view of the outer medulla. G, glomeruli; *, necrotic tubules. Arrows show neutrophils. Magnification, x200 in A and B; x400 in C and D.
Figure 8, A and B, are low-power photomicrographs that comparethe pathology of the IL-6 (–/–) versus IL-6 (+/+)kidneys at 24 h of reperfusion. The IL-6 (+/+) kidneys had severeinjury (Figure 8B). There were many necrotic tubules in theouter medulla, indicated by *. These tubules were filled withcasts. There was less but still significant injury in the cortex.There were many neutrophils in the interstitial spaces, indicatedby arrows. In comparison, the IL-6 (–/–) kidneys(Figure 8A) had much less injury by all of the above criteria.
Figure 8, C and D, are high-power photomicrographs of the outermedulla of the ischemic IL-6 (–/–) versus IL-6 (+/+)kidneys. Necrotic tubules and inflammation are present in theIL-6 (+/+) kidneys. The morphometric analysis (Figure 9) confirmedthe decreased medullary and cortical damage and the decreasedinflammation in the IL-6 (–/–) kidneys.
Figure 9. Transgenic knockout of IL-6 ameliorated ischemic ARF: Morphometrics. Morphometric comparison of ischemic kidneys at 24 h of reperfusion; , IL-6 (+/+); , IL-6 (–/–). The means and SE of three kidneys in each group are shown. *P < 0.015 between wild-type and IL-6 knockout groups by t test. Morphometry (see Materials and Methods) was performed according to the method of Thurman et al. (14).
Production of IL-6 by Bone Marrow–Derived Cells Increases Ischemic ARF Figure 10 shows that chimeric mice with IL-6 (–/–)renal parenchymal cells and IL-6 (+/+) macrophages have greaterischemic ARF than chimeric mice with IL-6 (+/+) renal parenchymalcells and IL-6 (–/–) bone marrow–derived cells.In these experiments, C57BL/6J-Il6tm1Kopf [IL-6 (–/–)]mice were lethally irradiated and then received bone marrowtransplants from C57BL/6J [IL-6 (+/+)] mice. Similarly, lethallyirradiated IL-6 (+/+) mice received IL-6 (–/–) bonemarrow. Because these mice differed only in the IL-6 gene, therewas no graft-versus-host or host-versus-graft disease. GenomicPCR (see Materials and Methods) of the radioresistant tail andradiosensitive peripheral blood confirmed the chimerism.
Figure 10. Production of IL-6 by bone marrow–derived cells increases ischemic ARF. Lethally irradiated IL-6 (–/–) mice received IL-6 (+/+) bone marrow; lethally irradiated IL-6 (+/+) mice received IL-6 (–/–) bone marrow. The IL-6 (–/–) and IL-6 (+/+) mice differed only at the IL-6 gene (see Materials and Methods). Top panels are representative genomic PCR that demonstrate full replacement of hematopoietic cells (including macrophages) in the chimeric mice. Genomic DNA was obtained from the tails (recipient radioresistant cells) and blood (donor hematopoietic cells); we used primers specific for the IL-6 gene [IL-6 (+/+)] and for the neocassette in the IL-6 knockout [IL-6 (–/–)] mice. All mice underwent right nephrectomy and left renal pedicle clamp for 17 min. The serum creatinine was measured after 24 h of reperfusion. , IL-6 (+/+) bone marrow to IL-6 (–/–) recipient mice; , IL-6 (–/–) bone marrow to IL-6 (+/+) recipient mice. The means and SE are shown.
This article makes several points. IL-6 is produced by the ischemickidney. Immunohistology and in situ hybridization show thatmacrophages adjacent to the ischemic vascular bundles of theouter medulla produce IL-6. An in vitro model of ischemic ARFsuggests that this production is stimulated by molecules thatare produced by ischemic renal tubular cells. Transgenic knockoutof IL-6 ameliorates renal injury. In chimeric mice whose renalparenchymal cells and macrophages were or were not capable ofproducing IL-6, maximal ischemic injury required IL-6–producingmacrophages.
Our transgenic mice carry the caveat of all such experiments,i.e., mice with transgenic knockout of IL-6 may produce moleculesthat compensate for the absence of IL-6 from birth. It is possiblethat the lesser ischemic ARF of the knockout mice is due tothese compensatory molecules and not the deficiency of IL-6.One way to overcome this caveat is by inhibiting IL-6 in wild-typemice. Although others ameliorated ischemic ARF with anti–IL-6injections (24), we found that exogenous anti–IL-6 didnot ameliorate ischemic ARF. Our antibody experiments are inconclusivebecause the antibodies may not access the interstitial spacesof the injured kidney. We have overcome this caveat using bonemarrow chimeras. These experiments demonstrate a specific effectof wild-type IL-6–producing macrophages. Thus, wild-typeIL-6–producing macrophages in the IL-6 knockout kidneysproduce more ischemic ARF than IL-6 knockout macrophages inwild-type kidneys (Figure 10). In other words, bone marrow transferof IL-6–producing wild-type macrophages overcame the protectiveeffect of IL-6 knockout to ischemic renal injury.
In addition to our group, many other laboratories (25–27)found macrophages in the outer medulla early during ischemicARF. Preventing the macrophage infiltration ameliorates renalinjury (26,28,29). However, how macrophages injure the kidneywas not known. We now suggest that one mechanism is the productionof IL-6.
The maladaptive renal effects of IL-6 remain to be elucidatedin detail. We favor the following hypothesis. Macrophages area major component of the initial inflammatory response to renalinjury, as shown in Figure 5 and previously reported by others(26,27). These macrophages release IL-6, which further increasesrenal inflammation by recruiting more neutrophils into the injuredkidney. This hypothesis is consistent with our observations(Figures 8 and 9), which show decreased inflammation in thekidney at 24 h in the IL-6 (–/–) ischemic kidney.This hypothesis is also consistent with previous reports thatthe peak neutrophilic infiltrate after renal ischemia does notoccur until 12 h (30,31), well after we and others (26) foundIL-6–expressing renal macrophages (Figures 4 and 5).
Also consistent with this hypothesis are the known proinflammatoryeffects of IL-6. These include stimulation of neutrophil releasefrom the bone marrow (32), prevention of neutrophil apoptosis(33), activation of neutrophils to produce toxic enzymes (34),and activation of endothelial cells to express intercellularadhesion molecule 1 and chemokines (35,36). These effects ofIL-6 are confirmed by the decreased inflammatory response intransgenic knockout mice after ischemia in the lung (37), gut(7,38), or brain (6); after injections of sterile turpentine(39) or carrageen (35); and after infections. (40,41). Furthersupport of the proinflammatory effects of IL-6 are the increasedisletitis in mice overexpressing IL-6 driven by the insulinpromoter (42) and the anti-inflammatory effects of removingIL-6 in experimental (43) and clinical arthritis (44). Furthermore,IL-6 contributes to neutrophilic influx and increased damagein the models of hemorrhagic shock (45) and spinal cord injury(46).
Another issue raised by our experiments is what activates themacrophages after they have entered the kidney. Figure 6 showsthat injured renal tubules release molecules that activate macrophagesin vitro. Ongoing experiments in our laboratory aim at identifyingthese molecules. One possibility is that macrophages are stimulatedby molecules that ordinarily reside inside renal tubular cellsbut are released into the extracellular space after these cellsare injured. Heat-shock proteins are examples of such molecules.Extracellular heat-shock proteins do activate macrophages viatheir Toll-like receptor 4 (TLR4) receptor (20,47–49).Consistent with the idea that TLR4 participates in ischemicARF are our data that TLR4-deficient mice suffer less injuryafter renal ischemia (R.J. and C.Y.L, unpublished observations,2005).
Although our data and most reports in the literature, reviewedin the beginning of this article, indicate that IL-6 exacerbatesischemic injury, no fair discussion would be complete withoutacknowledging that, in a few models, IL-6 ameliorates ratherthan exacerbates injury (37,50). Why IL-6 exacerbates injuryin most models but ameliorates injury in a few models remainsto be explained.
In summary, this article makes the following major points: First,IL-6 protein increases in the serum after ischemic renal injury.This increase is associated with increased IL-6 mRNA in theischemic kidney. Second, in situ hybridization and immunohistochemistrylocalize IL-6 production to macrophages near the vascular bundlesof the outer medulla. Molecules that are released by injuredS3 proximal tubular cells activate macrophages to produce IL-6in vitro. Third, transgenic knockout of IL-6 ameliorates renalinjury as measured by serum creatinine and histology. Finally,transfer of IL-6–sufficient macrophages by means of bonemarrow transplantation into IL-6 knockout mice increases ischemicrenal injury.
Acknowledgments
M.L.K. is supported by a KO 8 grant from the National Institutesof Health. C.Y.L. is supported by RO-1 and R21 grants from theNational Institutes of Health.
We thank Kathy Trueman for secretarial assistance, as well asNicole Franz, Vipin Bhagat, and Vanessa Woodward for technicalassistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication September 14, 2003.
Accepted for publication August 18, 2005.
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Top
Abstract
Introduction
(cat 16-4301). The antibodies were suspended in 0.2 ml of 0.1% BSA and injected into the penile vein immediately before the nephrectomy/clamp procedure. Peripheral serum was assayed for creatinine at 24 h after reperfusion. In some mice, peripheral blood was obtained at 4 h after reperfusion and used for measurement of IL-6 levels by the ELISA method (Endogen). 
, IL-1Ra, and IL-18 increased in the ischemic kidney and to a lesser extent in the contralateral kidney. These molecules are expressed by the ischemic kidney (16,17). However, expression by the contralateral kidney to our knowledge has not been previously reported. Such expression is consistent with the relatively minor inflammation there and may result from the systemic release of cytokines from the ischemic kidney (18). In addition, Figure 2D shows increased leukemia inhibitory factor expression in the ischemic kidney and confirms a previous report (16). 
) had their left renal arteries and veins dissected free of surrounding fat, but these vessels were not clamped. (2) Clamp-only mice (
) had their left renal arteries and veins dissected and also clamped for 17 min; this resulted in an ischemic left kidney and a contralateral nonischemic right kidney. The contralateral kidney maintained a normal GFR; both the clamp-only and sham mice had serum creatinines of <0.5 mg/dl. These mice had renal ischemia but no uremia. (3) Clamp + nephrectomy mice (
, wild-type mice [IL-6 (+/+)]. The means and SE are shown.
