IL-18 Contributes to Renal Damage after Ischemia-Reperfusion

IL-18 Expression Is Increased in the Kidney

In vivo, WT mice showed significant upregulation of IL-18 expression within kidney at 24 h after ischemia/reperfusion (P < 0.05) compared with sham-operated controls (Figure 1A). In vitro, resident peritoneal macrophages, primary renal tubular epithelial cells (RTEC), and cell lines of both macrophages (RAW264.7) and tubular epithelial cells (MCT) expressed IL-18 mRNA; however, macrophages produced much more (P < 0.001; Figure 1, B and C). Macrophages but not tubular cells submitted to ischemia expressed significantly higher mRNA levels of IL-18 than controls (P < 0.01, Figure 1B, and P < 0.001, Figure 1C). Immunofluorescent double staining demonstrated expression of IL-18 by macrophages (Figure 1D).

IL-18^−/− Mice Are Protected from Renal IRI

As shown in Figure 2, IRI caused kidney dysfunction in WT mice, reflected by significant elevation of serum creatinine at 24 h after IRI. Function was preserved in IL-18^−/− mice, with serum creatinine lower than WT mice at 24 and 72 h after IRI (P < 0.001) and not significantly different from sham-operated controls. Mortality occurred in three of 20 WT mice within 4 d of IRI, whereas no mortalities were observed in the IL-18^−/− and IL-18BP treatment groups.

IL-18 deficiency also afforded protection as assessed by histology. WT mice incurred severe tubular damage, as evidenced by widespread tubular necrosis, loss of the brush border, cast formation, and tubular dilation at the corticomedullary junction at 24 and 72 h after IRI, which was markedly attenuated in IL-18^−/− mice (Figure 3, A and B). Sham-operated mice incurred no tubular injury.

Interstitial Infiltrates Are Reduced in IL-18^−/− Mice

Substantial infiltration of neutrophils was evident in WT mice 24 and 72 h after IRI versus sham-operated controls (P < 0.001; Figure 4B), and this was moderately attenuated in IL-18^−/− mice (Figure 4, A and B). Similarly, significant macrophage accumulation was evident in WT after IRI compared with sham-operated controls (P < 0.001; Figure 4, A and C) but significantly prevented by IL-18 deficiency at 24 and 72 h after IRI (P < 0.001; Figure 4C).

Generation of Proinflammatory Molecules within the Kidney after IRI Is Attenuated by IL-18 Deficiency

To investigate the effects of IL-18 deficiency, we measured mRNA expression of IL-18 downstream effector molecules and chemokines in the kidney by real-time PCR. Sham-operated controls demonstrated detectable levels of TNF-α and iNOS and very low levels of macrophage inflammatory protein 2 (MIP-2) and monocyte chemoattractant protein 1 (MCP-1). There were no significant differences between WT and IL-18^−/− sham-operated controls. WT mice subjected to IRI demonstrated strong upregulation of effector molecules (TNF-α and iNOS) and chemokines (MIP-2 and MCP-1) at 24 h after IRI (TNF-α, P < 0.001; iNOS, P < 0.01; MIP-2 and MCP-1, P < 0.001; Figure 5) compared with sham-operated controls. IFN-γ was not upregulated (data not shown). IL-18^−/− mice showed a significant reduction in TNF-α, iNOS, MIP-2, and MCP-1 expression compared with WT mice at 24 h after IRI (Figure 5).

Macrophage IL-18 Expression Is Induced by the Supernatant of Ischemic Tubular Cells

To determine whether molecules released from ischemic tubular cells can stimulate macrophages to produce IL-18, we measured IL-18 mRNA expression by macrophages stimulated with the supernatant from tubular cells that had been rendered ischemic for 1 h. Macrophage production of IL-18 was modestly enhanced in vitro by injured tubular cell supernatant (P < 0.05; Figure 6).

BM-Derived Cells Play a Critical Role in IL-18–Mediated Kidney IRI

To determine whether BM-derived cells or intrinsic kidney cells play the major role in IL-18 production and subsequent kidney damage caused by IRI, we generated BM chimeric mice: WT host with IL-18^−/− BM (WT/IL-18^−/−BM), IL-18^−/− host with WT BM (IL-18^−/−/WTBM), WT host with WT BM (WT/WTBM), and IL-18^−/− host with IL-18^−/− BM (IL-18^−/−/IL-18^−/−BM). Eight weeks after BM transplantation, chimeric mice were subjected to kidney ischemia. At that time, the genotype of BM-derived cells from the chimeric mice was confirmed by PCR of DNA extracted from peripheral blood cells as shown in Figure 7A.

Both WT/IL-18^−/−BM mice and IL-18^−/−/WTBM mice demonstrated significantly lower serum creatinine and less tubular damage than WT/WTBM mice at 24 h after IRI (P < 0.001; Figure 7, B and C). WT/IL-18^−/−BM mice showed significantly lower serum creatinine and less tubular damages than IL-18^−/−/WTBM mice at 24 h after IRI (P < 0.05; Figure 7, B and C). This indicates that WT/IL-18^−/−BM mice were protected from kidney IRI to a greater extent than IL-18^−/−/WTBM mice, although IL-18^−/−/WTBM were partially protected.

IL-18BP Pretreatment Is Protective in Renal IRI

The protection afforded from renal IRI by IL-18^−/− mice led to the investigation of pretreatment with a neutralizing agent IL-18BP as a possible therapeutic intervention in renal IRI. IL-18BP pretreatment was protective against kidney dysfunction, tubular damage, and cellular infiltration in the kidney at 24 and 72 h after IRI. Serum creatinine levels after IL-18BP pretreatment were significantly less than that of control mice at 24 h and 72 h after IRI (P < 0.01; Figure 8A). IL-18BP pretreatment also significantly reduced tubular damage compared with control mice at 24 h (P < 0.001) and 72 h after IRI (P < 0.05; Figure 8B). Immunohistochemistry demonstrated that IL-18BP pretreatment significantly reduced neutrophil infiltration at 24 h after IRI (P < 0.01; Figure 8C) compared with control mice, but there was no significant difference at 72 h after IRI. IL-18BP pretreatment significantly reduced macrophage accumulation at 24 and 72 h after IRI (Figure 8D) compared with control mice.

IL-18BP Pretreatment Attenuates Expression of Proinflammatory Molecules in the Kidney in Renal IRI

To determine whether neutralizing IL-18 using IL-18BP reduces IL-18 downstream effects, we measured expression of TNF-α, iNOS, MIP-2, and MCP-1. IL-18BP pretreatment significantly reduced iNOS, MIP-2, and MCP-1 expression compared with WT mice at 24 h after IRI (P < 0.05; Figure 9); however, there was no significant difference in TNF-α expression levels.

Animals

IL-18^−/− mice on a C57BL/6 background were generously provided by Animal Services at the Australian National University and were backcrossed more than nine times onto C57BL/6 background. C57BL/6 mice used as WT controls were obtained from the Animal Resource Centre (Perth, Western Australia, Australia). The mice were maintained in a specific pathogen–free facility in the University of Sydney and received food and acidified water ad libitum. Male mice housing at least 1 wk after their arrival and weighing 25 to 30 g were used in all experiments. Experiments were conducted by following established guidelines for animal care and were approved by the animal ethics committee of the University of Sydney.

Induction of Kidney IRI

Induction of kidney IRI was as described previously.⁴⁷ Briefly, using a midline abdominal incision, both renal pedicles were clamped for 21 min with microaneurysm clamps. During the period of ischemia, body temperature was maintained by placing the mice on a 37°C heat pad. After removal of the clamps, the kidneys were inspected for 1 min for restoration of blood flow, returning to their original color. And the abdomen was closed. Sham-operated mice received identical surgical procedures except that microaneurysm clamps were not applied. For maintaining fluid balance, all mice were supplemented with 1 ml of saline administered subcutaneously. Mice were killed 24 and 72 h after reperfusion (n = 6 to 10 per group).

IL-18BP Pretreatment

WT mice received 300 μg of IL-18BP intraperitoneally (provided by Serono Int., Geneva, Switzerland) 40 min before the induction of ischemia and an additional 300 μg of IL-18BP at the time of reperfusion. Control mice received PBS instead of IL-18BP.

Blood and Tissue Samples

Blood and kidney tissues were harvested at time of killing. Tissue slices were divided to be fixed with 10% neutral-buffered formalin for paraffin embedding, fixed with periodate-lysine-paraformaldehyde fixative to be frozen in OCT compound (Sakura Finetek, Torrance, CA), or snap-frozen in liquid nitrogen for subsequent mRNA extraction.

Generation of BM Chimeric Mice

BM chimeric mice were generated by adoptive transfer of donor BM cells into irradiated recipient animals as described previously.⁴⁷ Chimeric mice were produced in the following recipient/donor combinations: WT/WTBM, IL-18^−/−/IL-18^−/−BM, WT/IL-18^−/−BM, and IL-18^−/−/WTBM. The recipient mice were lethally irradiated using a 137Cs irradiator (Gammacell 40 Exactor; Nordion Int., Kanata, Ontario, Canada), with a single dose of 9.5 Gy from a cesium source. Recipient irradiated mice were administered an injection of 1 × 10⁷ BM cells via the tail vein 6 h after irradiation. Mice were housed for 8 to 10 wk to allow full cellular reconstitution before being subjected to 21 min of kidney ischemia. Full chimerism of each mouse was confirmed by genotyping of DNA from whole blood using REDExtract-N-AmpTM Blood PCR kits (Sigma-Aldrich, St. Louis, MO).

Assessment of Renal Function

Serum creatinine was measured using the modified Jaffe rate reaction by the Biochemistry Department of the Royal Prince Alfred Hospital (Sydney, Australia).

Histology and Immunohistochemistry

Kidneys embedded in paraffin were sectioned at 3 μm and stained with hematoxylin and eosin. The percentage of tubules that exhibited cellular necrosis, loss of brush border, and tubule dilation were counted and scored in a blinded manner as follows: 0, none; 1, <10%; 2, 11 to 25%; 3, 26 to 45%; 4, 46 to 75%; and 5, >76%. For each slide, at least 10 fields (×200) of the outer medulla of each kidney were counted.

Periodate-lysine-paraformaldehyde–fixed frozen sections were blocked with a biotin blocker system (Dako Corp., Carpinteria, CA), then blocked with 10% normal horse serum. Primary antibody, either rat anti-mouse neutrophil antibody, clone 7/4, (ABD Serotec, Oxford UK), or macrophage antibody, rat anti-mouse F4/80 antibody (ABD Serotec), was applied to the sections for 60 min. Concentration-matched rat IgG was used as an isotype negative control. Sections were exposed to 1% H₂O₂ in methanol for 5 min, then incubated with the biotinylated secondary antibody anti-rat IgG (BD Biosciences, Pharmingen, San Diego, CA). Vector stain ABC kit (Vector Laboratories, Burlingame, CA) was applied to the tissue followed by 3,3′diaminobenzidine substrate-chromogen solution (Dako). The slides were counterstained with Harris's hematoxylin.

Analysis of the cellular infiltrate was performed in a blinded manner by assessment of 10 consecutive high-power fields (×400 magnification) of the outer medulla and corticomedullary junction on each section. Using an occular grid, the number of cells staining positively for each antibody was counted and expressed as cells per 10 high-power fields.

Primary Culture of Mouse RTEC

Primary mouse RTEC were generated following the method as described previously.⁴⁷ Briefly, kidneys were flushed with saline in vivo to remove blood cells, then removed. The kidney cortices from WT mice were cut into pieces of approximately 1 mm³ and then digested in HBSS containing 3 mg/ml collagenase at 37°C for 25 min and washed in DMEM/F12 (Invitrogen, Grand Island, NY). The kidney digest was washed through a series of sieves (mesh diameters of 250, 150, 75, and 40 μm). The cortical tubular cells were spun down at 300 × g for 5 min and further washed. The cell pellet was resuspended in defined K1 medium.⁴⁸ The cell suspension was then placed on cell culture Petri dishes and incubated at 37°C for 2 to 3 h to facilitate adherence of contaminating glomeruli. The nonadherent tubules were then collected and cultured on collagen-coated Petri dishes (BD Biosciences, Bedford, MA) in K1 medium until epithelial colonies were established. Expression of the epithelial cell marker cytokeratin was verified by immunofluorescent staining with an anti-cytokeratin antibody (Sigma-Aldrich). Cells were 96 to 100% cytokeratin positive as previously reported.⁴⁷ Experiments were commenced after the cells had reached 80 to 90% confluence, which was usually between 5 and 7 d after the isolation procedure.

Isolation of Resident Peritoneal Macrophages

After injection of 10 ml of cold PBS into the peritoneal cavity of WT mice, resident peritoneal macrophages were harvested then plate-adhered in serum-free medium for 4 h, and nonadherent cells were removed by washing. The adherent peritoneal macrophages were cultured in complete DMEM with 10% FBS. Expression of the macrophage marker was verified by staining with PE-conjugated anti-mouse F4/80 antibody (ABD Serotec, Oxford, UK) and analyzed on a calibur flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA). The purity of these cells was defined by positive staining for the macrophage marker (F4/80) and was 97.3%.

Induction of RTEC and Macrophage Ischemia In Vitro

A mouse renal tubular epithelial cell line (MCT) was a gift from Prof. E Neilson.⁴⁹ A macrophage cell line (RAW264.7) was obtained from the American Type Culture Collection (Manassas, VA). MCT cells, primary RTEC, RAW264.7 cells, and peritoneal macrophages were rendered transiently ischemic by immersing the cellular monolayer in mineral oil as described previously.⁴⁷ This immersion induced ischemia by restricting cellular exposure to oxygen and nutrients as well as by limiting metabolite washout. Primary RTEC were placed in serum-free K1 medium and other cells in serum-free DMEM (Invitrogen) for 24 h, washed twice with PBS, and immersed in mineral oil (Sigma-Aldrich) for 40 min at 37°C for cell lines and for 60 min at 37°C for primary RTEC and peritoneal macrophages. After extensive washing with PBS, cells were incubated in K1 medium or complete DMEM with 10% FBS. The cells were collected at 1 h after medium replacement for IL-18 expression. Control group cells were exposed to serum-free K1 or DMEM alone as the nonischemic control. In another set of experiments, the supernatant from ischemic MCT or the nonischemic control was collected at 16 h after medium replacement for macrophage stimulation.

Macrophage Stimulation with the Supernatant from Ischemic MCT

Macrophages (RAW264.7) were placed in serum-free DMEM (Invitrogen) for 24 h and then cultured with the supernatant collected at 16 h from ischemic MCT cells or the nonischemic control. Macrophages were collected at 4 h after co-culture with the supernatant for IL-18 mRNA expression.

Immunofluorescent Double Staining

For macrophage and IL-18 immunofluorescent double staining, adherent peritoneal macrophages on culture chamber slides were blocked with 1% BSA in PBS for 20 min and incubated with rat anti-mouse F4/80 antibody (ABD Serotec) and goat polyclonal antibodies to mouse IL-18 (SC-6177; Santa Cruz Biotechnologies, Santa Cruz, CA) for 60 min followed by anti-rat IgG conjugated with AlexaFluor 595 and anti-goat IgG conjugated with AlexaFluor 488 (Molecular Probes, Eugene, OR). The slides were mounted with DAPI medium.

RNA Extraction and cDNA Synthesis

Total RNA was extracted from kidney tissue and cells using TRIzol (Invitrogen). cDNA was synthesized using oligo d(T)₁₆ (Applied Biosystems, Foster City, CA) and the SuperScript III Reverse Transcriptase kit (Invitrogen).

Real-Time PCR

cDNA was amplified in 1× Universal Master Mix (Applied Biosystems) with gene-specific primers and probe on the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Specific Taqman primers and probes for IL-18 (Mm00434225_m1), TNF-α (Mm00443258_m1), and iNOS (Mm00440485_m1) were obtained from Applied Biosystems. Specific Taqman primers and probes for MCP-1 and MIP-2 were previously described.⁴⁷ The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control to normalize the mRNA content of each sample as described previously.⁴⁷ All results are expressed as ratio to GAPDH.

Statistical Analysis

All data are expressed as means ± SEM. Statistical differences between two groups were analyzed by unpaired, two-tailed t tests, and multiple groups were compared using one-way ANOVA with post hoc Bonferroni correction (Graph Pad Prism 4.0 software; GraphPad, San Diego, CA). P < 0.05 was considered significant.