Signaling through the interleukin-18 receptor α attenuates inflammation in cisplatin-induced acute kidney injury

http://www.kidney-international.org

original article

& 2012 International Society of Nephrology

Signaling through the interleukin-18 receptor a attenuates inflammation in cisplatin-induced acute kidney injury Yuji Nozaki1, Koji Kinoshita1, Tomohiro Yano1, Kayo Asato1, Toshihiko Shiga1, Shoichi Hino1, Kaoru Niki1, Yasuaki Nagare1, Kazuya Kishimoto1, Hideki Shimazu1, Masanori Funauchi1 and Itaru Matsumura1 1

Department of Hematology and Rheumatology, Kinki University School of Medicine, Osaka-Sayama, Osaka, Japan

Interleukin (IL)-18 is produced by leukocytes and renal parenchymal cells (tubular epithelial cells, podocytes, and mesangial cells). The IL-18 receptor (IL-18R) is expressed on these cells in cisplatin-induced acute kidney injury, but the role of IL-18R is unknown. To help define this, we compared IL-18Ra knockout with wild-type mice in cisplatin-induced acute kidney injury and found deteriorated kidney function, tubular damage, increased accumulation of leukocytes (CD4 þ and CD8 þ T-cells, macrophages, and neutrophils), upregulation of early kidney injury biomarkers (serum TNF, urinary IL-18, and KIM-1 levels), and increased expression of pro-inflammatory molecules downstream of IL-18. In vitro, leukocytes from the spleen and kidneys of the knockout mice produced greater amounts of pro-inflammatory cytokines upon stimulation with concanavalin A compared to that in wild-type mice. Levels of the suppressor of cytokine signaling 1 and 3 (negative regulators of cytokine signaling) were reduced in the spleen and kidneys of IL-18Ra-deficient compared to wild-type mice. Adoptive transfer of wild-type splenocytes by IL-18Ra-deficient mice led to decreased cisplatin nephrotoxicity compared to control IL-18Radeficient mice. In contrast, anti-IL-18Ra and anti-IL-18Rb antibody treatment tended to increase cisplatin nephrotoxicity in wild-type mice. Thus, signaling through IL-18Ra activates both inflammation-suppressing and pro-injury pathways in cisplatin-induced acute kidney injury. Kidney International advance online publication, 6 June 2012; doi:10.1038/ ki.2012.226 KEYWORDS: acute kidney injury; cisplatin nephrotoxicity; cytokines

Correspondence: Yuji Nozaki, Department of Hematology and Rheumatology, Kinki University School of Medicine, 377-2 Ohno-Higashi, OsakaSayama, Osaka, 589-8511, Japan. E-mail: [email protected] Received 10 February 2011; revised 22 March 2012; accepted 13 April 2012 Kidney International

Cisplatin is widely used to treat carcinoma patients. However, the drug’s use is limited because of its side effects in normal tissues, particularly injury to the kidneys.1 The main site of cisplatin action is in proximal tubule epithelial cells. Although tubular epithelial cell death is recognized as a major determinant of cisplatin nephrotoxicity, it remains unclear how these cells are terminally injured under the inflammatory mechanism. In animals, the in vivo use of cisplatin induces both necrosis and apoptosis in renal tubules.2 In the kidney, interleukin (IL)-18 is a sensitive and early marker of renal tubular damage,3 and in animal models of renal ischemia–reperfusion injury, IL-18 exacerbates acute tubular necrosis.4 IL-18 is known as an interferon (IFN)-g-inducing factor because of its ability to induce IFN-g production in T cells and NK cells. It is synthesized as a biologically inactive precursor (pro-IL-18), similar to IL-1b, which requires cleavage into an active molecule by an intracellular cysteine protease or caspase-1. IL-1b-converting enzyme (caspase-1) cleaves the pro-IL-18 to make a bioactive mature form of IL-18 that recognizes a heterodimeric receptor, which consists of unique a (IL-1 receptor (R)-accessory protein-like) signaling chains and nonbinding b (IL-1R-accessory proteinlike) signaling chains.5 In dextran sodium sulfate-induced colitis, neutralizing antibodies to IL-18 or IL-18-binding protein ameliorate the disease,6,7 whereas in other studies, mice deficient in IL-18Ra exhibit worsening of the disease.8 In addition to rejecting insulin-producing islet allografts, splenocytes and peritoneal macrophages from IL-18Radeficient mice produced significantly greater amounts of several pro-inflammatory cytokines on stimulation with concanavalin A (ConA).9 In this study, we investigated the paradoxical findings of the effect of IL-18R on cytokine production to determine whether knocking out and blocking IL-18R would aggravate or ameliorate cisplatin-induced nephrotoxicity. We found that IL-18Ra-deficient mice had markedly deteriorated renal function and increased histologic injury when they were exposed to cisplatin. The adoptive transfer of splenocytes from wild-type (WT) mice into IL-18Ra-deficient mice decreased cisplatin nephrotoxicity. In contrast, with the 1

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Y Nozaki et al.: IL-18R and acute kidney injury

anti-IL-18Ra and anti-IL-18Rb administration, the nephrotoxicity tended to deteriorate. Renal pro-inflammatory cytokines were increased in IL-18Ra-deficient mice when compared with those in WT mice. The results indicated that IL-18Ra has an inflammation-suppressing signal by a suppressors of cytokine signaling (SOCS) pathway in cisplatininduced acute kidney injury (AKI). RESULTS Functional, structural aggravation from cisplatin nephrotoxicity

Kidneys in IL-18Ra-deficient and WT mice showed normal morphology on day 1 (Figure 1a and c). On day 3, IL-18Radeficient mice developed severe tubular injury with cast formation, loss of brush border membranes, sloughing of tubular epithelial cells, and dilation of tubules (Figure 1d).

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Figure 1 | Representative light microscopy of periodic acidSchiff (PAS)–stained renal sections after cisplatin injection. Kidneys in wild-type (WT) (a, day 1 and b, day 3 after cisplatin injection) and interleukin-18 receptor (IL-18R)a-deficient (IL-18Ra knockout (KO); c, day 1 and d, day 3) mice were showed. Tubular necrosis (e) was scored in the outer medulla of the kidney (see the ‘Materials and Methods’ section for the scoring method), the panels are shown as mean score±s.e. (wPo0.05, the necrosis score on day 1 vs. 3 and *Po0.05, the necrosis score in WT and IL-18Ra KO mice on day 3). Original magnifications  400. Scale bar, 50 mm. ATN, acute tubular necrosis. 2

Serum and urinary biomarker in renal injury

Serum tumor necrosis factor (TNF) and IL-18 production (Figure 2b) and urinary IL-18 and Kim-1 (Figure 2c) were measured as biomarkers in AKI. Serum TNF was not detected in IL-18Ra-deficient mice or WT mice on day 1 (limit of detection of enzyme-linked immunosorbent assay 20 pg/ml). TNF in IL-18Ra-deficient mice was increased significantly on day 3. Serum IL-18 was not detected in mice of either group on day 1 (limit of detection of enzyme-linked immunosorbent assay 15.6 pg/ml). Serum IL-18 in mice of either group was increased significantly on day 3 compared with that on day 1. However, serum IL-18 was the same level in the two groups. We also investigated urinary IL-18 and Kim-1 levels as predictors in cisplatin-induced AKI. Urinary IL-18 levels on day 1 were not detected in any mice, whereas urinary IL-18 levels in IL-18Ra-deficient mice on day 3 were increased significantly compared with those in WT mice. Urinary Kim1 levels on day 1 were the same in the two groups, however, Kim-1 levels in IL-18Ra-deficient mice were increased significantly compared with those in WT mice by day 3. The infiltration of CD4 þ and CD8 þ T cells, macrophages, and neutrophils in cisplatin-induced renal injury

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In WT mice, the damage on day 3 was limited to mild swelling of the tubular epithelial cells, and less histological damage was visible (Figure 1b). Semiquantitative injury scoring in the two groups showed significantly increased necrosis scores between days 1 and 3, respectively, reflecting significant kidney injury. On day 1, the necrosis score was the same in the two groups. On day 3, the necrosis score in IL-18Ra-deficient mice was increased significantly compared with that in WT mice (Figure 1e). The cisplatin-induced rise in blood urea nitrogen (BUN) and serum creatinine levels at day 3 was also significantly increased in IL-18Ra-deficient mice compared with that in WT mice (Figure 2a).

We investigated the infiltration of inflammation cells, that is, CD4 þ and CD8 þ T cells, macrophages, and neutrophils, in the renal interstitium on days 1 and 3 (Figure 3). Interstitial CD4 þ T-cell numbers in IL-18Ra-deficient mice on day 1 showed increased significantly compared those in WT mice. In contrast, interstitial CD8 þ T cells infiltrating the renal interstitium were increased significantly in IL-18Ra-deficient mice on day 3 compared with WT mice. Interstitial macrophage and neutrophil infiltrate shows the greatest increase in cisplatin injected by day 3. In IL-18Ra-deficient mice, macrophage numbers were not significantly increased on day 1. On day 3, the time of peak infiltration in WT mice, renal macrophage, and neutrophil numbers were increased significantly in IL-18Ra-deficient mice. Cisplatin-induced phospho nuclear factor (NF)-jB p65 activation and apoptosis

Figure 4 shows the effect of IL-18Ra on phospho NF-kB p65 as a marker of NF-kB activation and cleaved caspase-3 as a Kidney International

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Y Nozaki et al.: IL-18R and acute kidney injury

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Figure 3 | Interstitial accumulation of leukocytes after cisplatin injection in wild-type (WT) and interleukin-18 receptor (IL-18R)a-deficient (IL-18Ra knockout (KO)) on days 1 and 3. The panels are shown as mean numbers±s.e (c/10hpf, cell numbers per 10 high-power field), (*Po0.05, the cell numbers in WT vs. IL-18Ra KO mice on days 1 and 3). Kidney International

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Figure 2 | Effect of interleukin-18 receptor (IL-18R)a on blood urea nitrogen, serum creatinine, serum cytokines, and urinary biomarkers. Blood samples were collected on day 1 and day 3 after cisplatin injection, then blood urea nitrogen (BUN) and serum creatinine were measured (a). Dotted lines represent mean values from saline-treated mice without cisplatin. Serum tumor necrosis factor (TNF) and interleukin (IL)-18 (b), urinary IL-18 and Kim-1 (c) were measured as early biomarker in acute kidney injury on days 1 and 3. Serum TNF and IL-18, and urinary IL-18 were not detected on day 1, the panels are shown as mean values±s.e. (*Po0.05, wild-type (WT) vs. IL-18 receptor (IL-18R)a knockout (KO) mice on day 3, wPo0.05, WT and IL-18Ra KO mice on day 1 vs. WT and IL-18Ra KO mice on day 3). ND, not determined; sCr, serum creatinine.

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Figure 4 | Effect of interleukin-18 receptor (IL-18R)a on phospho nuclear factor (NF)-jB p65 and cleaved caspase-3 in cisplatin-induced acute kidney injury (AKI) on days 1 and 3. Representative photograph and the scale graph of positive tubule cells (10 fields at  400 magnification) expressing phospho NF-kB p65 (a, b, black arrow) and cleaved caspase-3 (c, d, white arrow) in IL-18Ra-deficient and wild-type (WT) mice on day 3 were shown. Semiquantitative score of positive cells in renal tissue, the panels are shown as mean numbers±s.e. Original magnifications  400 (*Po0.05, **Po0.01, and ***Po0.005, positive tubule cells in WT vs. IL-18Ra-deficient mice on days 1 and 3). Scale bar, 50 mm. c/10hpf, cell numbers per 10 high-power field; ND, not determined.

marker of apoptosis. These markers showed increased expression in tubules after cisplatin injection. In IL-18Radeficient mice, nuclear staining of p65-positive cells was increased significantly in tubules on days 1 and 3 (Figure 4a and b, respectively). Cleaved caspase-3 on day 1 was not detected in either group. In IL-18Ra-deficient mice, cleaved caspase-3 in tubules was significantly increased on day 3 (Figure 4c and d). Renal mRNA expression in cisplatin-induced renal injury

Renal mRNA was measured by real-time quantitative PCR, as described in the Materials and Methods section. It is well documented that inflammatory cytokine and chemokine release contribute to cisplatin nephrotoxicity.10 Figure 5 shows that cisplatin upregulated the pro-inflammatory cytokine expression of IL-1b, IL-12p40, and IL-18 3

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Figure 5 | Effects of interleukin-18 receptor (IL-18R) on gene expression in the kidney. Cytokine and chemokine gene expression was measured 1 and 3 days after cisplatin injection in wild-type (WT) mice and IL-18Ra-deficient (IL-18Ra knockout (KO)) mice by real-time reverse transcriptase (RT)-PCR. In each experiment, the expression levels were normalized to the expression of 18SrRNA and are expressed relative to saline-treated control mice, the panels are shown as mean fold-increase±s.e. (*Po0.05, **Po0.01, and ***Po0.005, WT vs. IL-18Ra KO mice on days 1 and 3). CCL2/MCP-1, monocyte chemoattractant protein-1; IFN, interferon; ND, not determined; TNF, tumor necrosis factor.

significantly in IL-18Ra-deficient mice. IL-10, TNF, and IFNg mRNA cytokine and mRNA chemokine expression of monocyte chemoattractant protein-1 increased after cisplatin injection compared with the expression in WT mice. Renal and urinary Kim-1 expression

Figure 6 shows tubular Kim-1 expression after cisplatin injection. In WT mice, Kim-1 were not detected in the kidney 4

on day 1 (Figure 6a). However, there were more of these cells in tubules on day 3 (Figure 6b). In IL-18Ra-deficient mice, a trace amount of Kim-1 was present on day 1 (Figure 6c, black arrow). The number of Kim-1 was dramatically increased on day 3 compared with that on day 1 (Figure 6d). The numbers of Kim-1 in IL-18Ra-deficient mice were increased significantly on day 3 compared with that on day 1 (Figure 6e). The difference in the number of Kim-1 between the groups Kidney International

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Y Nozaki et al.: IL-18R and acute kidney injury

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Furthermore, activation of CD69 as an early activation marker on CD4 þ and CD8 þ T cell was significantly increased in IL-18Ra-deficient mice on day 3 (Figure 7c).

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Figure 8 shows renal mRNA and protein levels of SOCS1 and SOCS3 in WT and IL-18Ra-deficient mice on day 3 by realtime PCR (Figure 8a) and western blotting (Figure 8b and c). Renal SOCS1 and SOCS3 mRNA in IL-18Ra-deficient mice was decreased significantly compared with that in WT mice. Protein levels of splenic and renal SOCS3 in IL-18Radeficient mice were also significantly reduced compared with those in WT mice. Protein SOCS1 was not detected. Proliferative response and cytokine production in vitro

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Figure 6 | Expression of Kim-1 in the kidney. Representative photograph of Kim-1 expression in the kidney after cisplatin injection (a–d). In wild-type (WT) mice, Kim-1-positive cells were not detected in the kidney on day 1 (a). On day 3, Kim-1-positive cells were present at the apical membrane (b). In interleukin-18 receptor (IL-18R)a-deficient (IL-18Ra knockout (KO)) mice, a trace amount of Kim-1-positive cells is present on day 1 (c, arrow). On day 3, Kim-1-positive cells were progressively increased (d). The number of Kim-1-positive cells in 10  400 fields (e). Expression of renal Kim-1 mRNA were showed on days 1 and 3 (f). The panels are shown as mean numbers and fold increase±s.e. (*Po0.05, WT vs. IL-18Ra-deficient mice on days 1 and 3, wPo0.05, WT and IL-18Ra knockout (KO) mice on day 1 vs. WT and IL-18Ra KO mice on day 3). Scale bar, 50 mm. c/10hpf, cell numbers per 10 high-power field.

was significantly greater on day 3 than on day 1. We also investigated Kim-1 mRNA expression on days 1 and 3 and found that the expression in IL-18Ra-deficient mice was increased significantly from days 1 to 3 (Figure 6f). Apoptosis and activation in cisplatin-induced renal injury

We analyzed apoptosis and activation of splenic and renal CD4 þ and CD8 þ T cells by flow cytometric analysis (FACS), as shown in Figure 7. Apoptosis of CD4 þ and CD8 þ T cells from the spleen and kidney was significantly increased in IL-18Ra-deficient mice on day 1 (Figure 7a). Activation of CD44 as the cell surface expression of an activation marker on CD4 þ T cells was the same in IL-18Ra-deficient and WT mice. However, CD44 on CD8 þ T cells was significantly increased in IL-18Ra-deficient mice on day 1 (Figure 7b). Kidney International

Figure 9a and b show the proliferation and cytokine production by leukocytes in the spleen and kidney with ConA for 4 and 48 h. Splenic and renal leukocytes proliferation in IL-18Ra-deficient mice was increased for 4 h. IFN-g, TNF, and IL-12p40 levels of splenocytes and/or renal leukocytes in IL-18Ra-deficient mice were increased significantly compared with those in WT mice. IL-10 level was the same in the two groups. IL-1b and IL-18 could not be detected. Figure 9c shows IFN-g intracellular staining of CD4 and CD8 þ T cells in the spleen and kidney by FACS. IFN-g-producing splenic CD4 þ cells were the same in the two groups. However, the number of IFN-g-producing splenic CD8 þ cells in IL-18Radeficient mice was increased significantly compared with the number in WT mice. Splenocytes adoptive transfer reconstituted kidney susceptibility to cisplatin nephrotoxicity

To determine whether IL-18Ra deficiency was indeed an exacerbating factor in cisplatin-induced AKI, we transferred 5  106 splenocytes from normal WT mice into each of five IL-18Ra-deficient mice. The successful transfer of splenocytes was confirmed by FACS analysis with IL-18R staining. The mean population of T cells in the WT mouse spleen was 5.1% of total splenocytes. Meanwhile, IL-18Ra-deficient mice had minimal 1.7% splenocytes. Three weeks after the transfer, the splenocytes in IL-18Ra-deficient mice were reconstituted to 3.4%, which was 50% of the level in WT mice. Splenocyte transfers led to a significant improvement of renal dysfunction in IL-18Ra-deficient mice. There was a significant improvement in BUN and acute tubular necrosis score in IL-18Radeficient mice that received a transfer of splenocytes when compared with IL-18Ra-deficient mice without transfer on day 3 after cisplatin injection (Figure 10). In Supplementary Figure S1 online, renal pro-inflammatory cytokines and chemokines expression were trend to increase and increased significantly in IL-18Ra-deficient mice compared with those in the mice received a transfer of splenocytes and WT mice. In the contract, renal SOCS1 and SOCS3 expression were decreased significantly in IL-18Ra-deficient mice and the mice received a transfer of splenocytes compared with WT mice. 5

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Figure 7 | Effect of interleukin-18 receptor (IL-18R) on CD4 þ and CD8 þ T-cell responses. Splenic and renal CD4 þ and CD8 þ T-cell apoptosis and activation were assessed by flow cytometric analysis (FACS) in vivo, annexin-V staining (a), CD44 (b), and CD69 expression (c) and on days 1 and 3, the panels are shown as mean percentages±s.e. (*Po0.05, wild-type (WT) vs. IL-18Ra knockout (KO) mice on days 1 and 3). **Po0.01. PI, propidium iodide.

Blocking the signaling pathway by anti-IL-18Ra and anti-IL-18Rb antibody administration

To determine whether blocking the IL-18Ra and IL-18Rb signaling pathway could contribute to deteriorated renal function, we administrated IL-18Ra and IL-18Rb antibody to WT mice in cisplatin-induced renal injury. These mice showed deteriorated BUN compared with the mice given rat immunoglobulin G (Figure 11). In Supplementary Figure S2 online, renal pro-inflammatory cytokines and chemokines expression were increased in the mice given IL-18Ra and IL18Rb antibody compared with those in control mice. Renal SOCS1 and SOCS3 expression were decreased significantly in the mice given IL-18Ra and IL-18Rb antibody compared with those in control mice. 6

DISCUSSION

We previously reported that MRL/lpr mice cross-bred with mice deficient in IL-18Ra exhibited a reduction in autoantibodies, nephritis, and death.11 However, in some studies, paradoxical findings were reported in IL-18.8,9 In this study, we demonstrated that inflammatory cells were increased in the kidney and deteriorated the renal function in IL-18Radeficient mice. One of our goals was to determine the mechanism of action of IL-18Ra activation and signaling in AKI. The development of cisplatin-induced AKI in WT mice is dependent on the infiltration of activated CD4 þ T cells in the kidney, an event that is accompanied by the influx of other immune cells, including CD8 þ T cells, macrophages and neutrophils. Kidney International

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Figure 8 | Expression of suppressors of cytokine signaling (SOCS)1 and SOCS3 in the kidney on day 3. mRNA (a) and protein levels (b, c) of SOCS1 and SOCS3 in wild-type (WT), interleukin-18 receptor (IL-18R)a-deficient (IL-18Ra knockout (KO)) mice on day 3 were assayed by real-time PCR or western blotting. (a, c) The panels are shown as mean changes±s.e. (*Po0.05 and ***Po0.005, WT vs. IL-18Ra KO mice). (b) The pictures showed representative band in SOCS3. SOCS1 was not detected. ND, not determined.

IL-18R deficiency affected the increasing of renal mRNA pro-inflammatory cytokine and chemokine expressions. Similarly, in vitro, we demonstrated IL-18R deficiency affected ConA-stimulated proliferation and pro-inflammatory cytokine production by leukocytes. We also investigated the intracellular IFN-g secretion on leukocytes from the spleen and kidney by FACS. Intracellular IFN-g staining on splenic CD8 þ T cells in IL-18Ra-deficient mice was increased compared with that in WT mice. Thus, IL-18R is associated with the cytokine pathway in cisplatin-induced AKI. We also demonstrated the expression of p65 NF-kB and cleaved caspase-3 in the kidney after cisplatin injection. Recently, it was reported that phospho NF-kB p65 staining was markedly increased in the renal tubules of cisplatintreated mice.12 Moreover, the expression of multiple proKidney International

inflammatory cytokines leading to neutrophil-mediated inflammation has been linked to NF-kB activation.13 IL-18 also modulates the activity of macrophages14 by the activation of transcription factors including NF-kB.15 We demonstrated that tubular p65 NF-kB and cleaved caspase-3 expression in IL-18Ra-deficient mice on days 1 and 3 was increased significantly compared with that in WT mice (Figure 4). Caspase-3 is known as a major mediator of apoptosis. Cleaved caspase-3 is activated during cisplatininduced renal cell apoptosis as a key step after cisplatin injection.16 Oxidant stress is also contributed to cisplatininduced apoptosis of tubules.17 We investigated the cell activation (CD44 and CD69 as early activate markers) and apoptosis (annexin-V) of splenic and renal CD4 þ and CD8 þ T cells by FACS and found both activation and apoptosis to be increased in IL-18Ra-deficient mice. Taken together, we speculated that CD44 and CD69 þ activated cells on CD4 þ and CD8 þ T cells through pro-inflammatory cytokines and IL-1/Toll-like receptor-mitogen activated protein kinase/NF-kB pathway signaling accumulated to remove the aberrant apoptotic leukocytes (annexin-V þ cells) and epithelial cells (caspase-3 þ cells) for remodeling. These results indicated that IL-18Ra affected intracellular signaling that leads to proliferation, cytokine production, and apoptosis/nuclear translocation of the NF-kB composed of p65 and caspase-3 activation. To confirm our understanding of the functioning of IL-18R is functioning, we performed the splenocytes adoptive transfer and anti-IL-18Ra/b antibody administration. There was a significant improvement in BUN and acute tubular necrosis score in IL-18Ra-deficient mice that received a transfer of splenocytes and a trend toward the deterioration of renal function in the administration of anti-IL-18Ra/b antibody. Inhibition of cytokine signaling by the SOCS family constitutes a major negative feedback mechanism to prevent runaway inflammation. The transcription of SOCS proteins is rapidly upregulated in cells stimulated with pro-inflammatory cytokines; the SOCS then reduce the impact of cytokines by interacting with Jaks and by other mechanisms.18 Furthermore, SOCS1 (ref. 19) and SOCS3 (ref. 20) interfere with IL-1/Toll-like receptor-mitogen activated protein kinase/NF-kB pathway signaling, the increase in activity of these kinases in IL-18Ra-deficient cells may be due to less inhibition resulting from reduced levels of both SOCS. Recently, it was reported that SOCS1 and SOCS3 mRNA expression and protein production were greatly reduced in cells with IL-18Ra-deficient mice compared with cells from WT mice.21 We observed downregulation of splenic/renal SOCS1 and SOCS3 in IL-18Ra-deficient mice compared with WT mice. By downregulation of SOCS, proinflammatory cytokines were increased through IL-1/Toll-like receptor-mitogen activated protein kinase/NF-kB kinases signaling. Our data support that IL-18Ra may induce, through SOCS1 and/or SOCS3, anti-inflammatory responses in addition to pro-injury responses. 7

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Figure 9 | Interleukin-18 receptor (IL-18R)a are potent inducers of proliferation response and cytokine production. Effect of interleukin-18 receptor (IL-18R)a on proliferative response (a) and cytokine production (b). Lymphocytes of spleen and kidney in IL-18Ra-deficient (IL-18Ra knockout (KO)) and wild-type (WT) mice stimulated with concanavalin A (ConA) (1 mg/ml) for 4 and 48 h on day 1 after cisplatin injection. (c) Splenic and renal CD4 þ and CD8 þ T cells intracellular cytokine production were assessed by flow cytometric analysis of IFN-g. The panels are shown as mean values±s.e. (*Po0.05 and ***Po0.005, WT vs. IL-18Ra KO mice). IFN, interferon; TNF, tumor necrosis factor.

MATERIALS AND METHODS Animals IL-18Ra-deficient (IL-1Rrp/) mice (C57BL/6) were kindly provided by Dr S Akira (Osaka University, Osaka, Japan). C57BL/6 mice, used as a WT control in this study, were purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan). All mice were maintained in our specific pathogen-free animal facility. DNA samples were extracted from the tail for PCR-based genotyping. Male mice of 8–10 weeks were used in this study. Cisplatin treatment of animals In the majority of experiments, cisplatin was freshly prepared in saline at 1 mg/ml and injected intraperitoneally in IL-18Ra-deficient 8

(n ¼ 10) and WT mice (n ¼ 11) at a dose of 20 mg/kg as previously described;22 control animals were injected with a comparable volume of saline. This dose of cisplatin produces severe renal failure in mice. Animals were culled at days 1 and 3. Blood was collected from the dorsal aorta in heparinized tubes for measurement of BUN, serum creatinine, and TNF. Assessment of renal injury Tubular injury was assessed in periodic acid-Schiff–stained sections using a semiquantitative scale23 in which the percentage of cortical tubules showing epithelial necrosis was assigned a score: 0 ¼ normal; 1 ¼ o10%; 2 ¼ 10–25%; 3 ¼ 26–75%; 4 ¼ 475%. The individual scoring the slides was blinded to the treatment and strain of the Kidney International

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Y Nozaki et al.: IL-18R and acute kidney injury

IL-18R

a

* 6

*** WT

(%)

*

IL-18Rα KO

3

WT→IL-18Rα KO

0 BUN

b

ATN score

c

*

* ***

4

200

Score

mg/ml

300

100

***

2

0

0

IL-18R KO

WT

e

d

WT→IL-18R KO

f

Figure 10 | Effect of splenocyte transfer on renal function and histological damage in cisplatin-induced renal injury on day 3. Splenocytes were isolated from each mouse on killing and stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse interleukin18 receptor (IL-18R)a antibody and analyzed by flow cytometric analysis (FACS) (a). Blood urea nitrogen (BUN) was measured from blood samples (b) and tubular necrosis was scored in the outer medulla of the kidney (c) in wild-type (WT), IL-18Ra-deficient (IL-18Ra knockout (KO)) mice, and IL-18Ra KO mice with splenocyte transfer. Dotted lines represent the mean values from saline-treated mice without cisplatin. The panels are shown as the mean values±s.e. (*Po0.05, WT vs. IL-18Ra KO mice and IL-18Ra KO mice with splenocyte transfer, and IL-18Ra KO vs. IL-18Ra KO with splenocyte transfer, ***Po0.005, WT vs. IL-18Ra KO mice). Representative light microscopy of periodic acid-Schiff (PAS)–stained renal sections after cisplatin injection. Kidneys in WT (d), IL-18Ra KO (e), and IL-18Ra KO mice with splenocyte transfer mice (f) are shown. Original magnifications  400. Scale bar, 50 mm. ATN, acute tubular necrosis.

ATN score

BUN 4 3

100

Score

mg/dl

150

50

2 1

0

0 Anti-IL 18R

Anti-IL 18R

RatlgG

Figure 11 | Effect of anti-interleukin-18 receptor (IL-18R)a and anti-IL-18Rb antibody administration on renal function and histological damage in cisplatin-induced renal injury on day 3. Blood urea nitrogen (BUN) was measured from blood samples. ATN, acute tubular necrosis; RatIgG, rat immunoglobulin G.

animal. CD4 þ and CD8 þ T cells, macrophages, neutrophils, and Kim-1 were demonstrated by immunoperoxidase staining of periodate lysine paraformaldehyde-fixed frozen 6-mm-thick kidney sections, as described previously.24 The numbers of CD4 þ and CD8 þ T cells, macrophages, and neutrophils were assessed in 10 Kidney International

fields per slide at a magnification of  400, and results are expressed as cells per high-power field. Tubular Kim-1 immunostaining was quantified by counting the number of positively stained tubules in the cortex in 10 fields per slide at a magnification of  200. A positive tubule cross-section was defined as having two or more stained cells. The primary monoclonal antibodies used were rat monoclonal antibody GK1.5 for CD4 þ and 53-6.7 for CD8 þ T cells (Pharmingen, San Diego, CA), for macrophages with F4/80 hybridoma culture supernatant (HB198; American Type Culture Collection, Manassas, VA), RB6-8C5 for neutrophils (anti-Gr-1; Pharmingen), and rat monoclonal antibody TIM-1 (R&D Systems, Minneapolis, MN). Immunohistochemical analysis of cleaved caspase-3 and phospho NF-jB p65 Phospho NF-kB p65 and cleaved caspase-3 were identified in 4-mm-thick formalin fixed kidney sections by using rabbit antibody recognizing phospho NF-kB p65 and the cleaved form of caspase-3 (Cell Signaling Technology, Beverly, MA), as described previously.12,16 The numbers of NF-kB p65-activated and cleaved 9

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Y Nozaki et al.: IL-18R and acute kidney injury

caspase-3-positive cells, in each section were calculated by counting the number of positively stained tubules in 10 fields per slide at a magnification of  400. All tubules in the outer medulla were assessed for each animal. Large blood vessels and glomeruli were excluded from the analysis. The individual scoring the slides was blinded to the treatment. Real-time PCR analysis For measurement of intrarenal mRNA expression of TNF, IL-1b, monocyte chemoattractant protein-1, SOCS1, SOCS3, and 18SrRNA by FastStart DNA master, Sybr Green I (Applied Biosystems, Foster City, CA) and IFN-g, IL-4, IL-10, IL-12p40, IL-18, Kim-1, and 18SrRNA by TaqMan gene (Applied Biosystems), as described previously.24 The sequences of primers and gene database number are listed in Tables 1 and 2. The relative amount of mRNA was calculated using comparative Ct (DDCt) method. All specific amplicons were normalized against 18SrRNA, which was amplified in the same reaction as an internal control using commercial reagents (Applied Biosystems) and was expressed as fold differences relative to saline-treated control animals. Urine IL-18 and Kim-1 level excretion IL-18 (pg/24 h) and Kim-1 (ng/24 h) was measured in the urine collected from mice after cisplatin injection on days 1 and 3 by enzyme-linked immunosorbent assay, as described previously.25 Antibodies were rat anti-mouse IL-18 and TIM-1 monoclonal antibody (R&D Systems), biotinylated goat anti-mouse IL-18 and TIM-1 (R&D Systems), and streptavidin-horseradish peroxidase (Chemicon International, Billerica, MA). Plates were developed using tetramethylbenzidine substrate and optical density was read at 450 nm. Isolation of leukocytes from kidney Previously described methods for leukocyte isolation from murine kidneys were used.26 Kidneys were digested with 1 mg/ml collagenase D (Roche Diagnostics, Penzberg, Germany) and 0.1 mg/ml DNAse I (Roche Diagnostics) in RPMI 1640 medium supplemented

Table 1 | Primer sequences for analysis of mRNA expression Forward primer 18SrRNA IL-1b TNF-a CCL2/MCP-1 SOCS1 SOCS3

0

50 -GCCTCACTAAACCATCCAATCG-30 50 -TCTTCTTTGGGTATTGCTTGG-30 50 -GGTGCCTATGTCTCAGCCTCTT-30 50 -CGGGTCAACTTCACATTCAAAG-30 50 -GGGATGAGGTCTCCAGCCA-30 50 -CGCTCAACGTGAAGAAGTGG-30

Abbreviations: CCL2/MCP-1, monocyte chemoattractant protein-1; IL, interleukin; SOCS, suppressors of cytokine signaling; TNF, tumor necrosis factor.

Table 2 | Gene database number for analysis of mRNA expression Gene database number

Abbreviations: IFN, interferon; IL, interleukin.

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Cell death and activation assay For assessment of apoptosis and activation by FACS (Becton Dickinson, Franklin Lakes, NJ), spleens were obtained from mice on day 1 after cisplatin injection. Apoptosis of CD4 þ and CD8 þ cells was assessed by analysis of annexin-V staining (Roche Diagnostics) and T-cell activation was assessed by analysis of CD44 and CD69 expression on splenic CD4 þ and CD8 þ cells with allophycocyanin-conjugated anti-CD4 (Pharmingen), phycoerythrin-conjugated anti-CD8 (Pharmingen), and fluorescein isothiocyanate-conjugated anti-CD44 and CD69 (Pharmingen). After the cells were washed and resuspended in 0.2 ml of phosphate-buffered saline and propidium iodide (1 mg/ml; Calbiochem, San Diego, CA) using allophycocyanin/Cy-7-conjugated anti-CD4 and allophycocyanin-conjugated anti-CD8 (Pharmingen). Stained cells were analyzed on a FACSCalibur (Becton Dickinson) flow cytometer. Data were analyzed using CELL-QUEST software (Becton Dickinson). Western blotting Proteins were extracted by homogenization of the kidney on day 3 after cisplatin injection in tissue protein extraction reagent (Pierce, Rockford, IL) to determine SOCS1 and SOCS3, as described previously.27 Monoclonal anti-b-actin antibody was obtained from Santa Cruz (St Louis, MO). Anti-mouse SOCS1 and SOCS3 antibody was from Cell Signaling Technology. Peroxidase-conjugated goat immunoglobulin G was from Santa Cruz. Proliferation assay and measurement of cytokines On day 1, the spleen was removed after cisplatin injection, and single-cell suspensions were made in RPMI 1640 medium supplemented with 10% fetal calf serum and adjusted to 2.5  106 cells per well. Spleen cells were plated in triplicate in a flat-bottomed 96-well plate and stimulated with ConA (1 mg/ml) or medium alone for 6 and 48 h, according to the previous report.28 Proliferation was assessed by CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI) and cytokine concentrations (IL-1b, TNF, IFN-g, IL-12p40, and IL-10) in the supernatant were determined by enzyme-linked immunosorbent assay, as described previously.28

Reverse primer 0

5 -GTAACCCGTTGAACCCCATTC-3 50 -TGTAATGAAAGACGGCACACC-30 50 -CGATCACCCCGAAGTTCAGTA-30 50 -AAAAACCTGGATCGGAACCAA-30 50 -GTGGTTGTGGAGGGTGAGATG-30 50 -CCTTTCTTATCCGCGACAGC-30

18SrRNA IFN-g IL-4 IL-10 IL-12p40 IL-18 Kim-1

with 10% fetal calf serum. Viability of the cells was assessed by Trypan blue staining before single-cell suspensions.

NM_026744.3 NM_008337.3 NM_021283.2 NM_010548.1 NM_008352.2 NM_008360.1 NM_134248.1

Splenocyte adoptive transfer IL-18Ra-deficient mice received an adoptive transfer of splenocytes from WT mice. Splenocytes that were collected from C57BL/6 WT mice were minced on a nylon mesh, as described previously.29 Approximately 5  106 spleen cells were injected intraperitoneally into each IL-18Ra-deficient mouse 3 weeks before cisplatin (20 mg/kg) injection. IL-18Ra-deficient (n ¼ 5), WT mice (n ¼ 8), and the mice received a transfer of splenocytes (n ¼ 10) were culled on day 3. To confirm T-cell reconstitution in IL-18Radeficient mice, splenocytes were analyzed for a population of IL-18R-positive cells by FACS. Fluorescein isothiocyanateconjugated IL-18Ra and immunoglobulin G1 were purchased from R&D Systems. Administration of IL-18Ra and b monoclonal antibody Monoclonal antibody to IL-18Ra (R&D Systems), IL-18Rb (Pharmingen), or rat immunoglobulin G as a control was administered (400 mg) 1 day before cisplatin (20 mg/kg) administration. Mice (each group, n ¼ 5) were culled on day 3. Kidney International

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Y Nozaki et al.: IL-18R and acute kidney injury

Statistical analysis Results are expressed as mean±s.e.m. Unpaired t-test was used for statistical analysis (GrandPad Prism; GraphPad Software, San Diego, CA). Differences were considered to be statistically if Po0.05. DISCLOSURE

All the authors declared no competing interests.

11.

12.

13. 14.

ACKNOWLEDGMENTS

We thank K Furukawa, E Honda, S Kurashimo, K Okumoto, and N Mizuguchi for their expert technical assistance.

15. 16.

SUPPLEMENTARY MATERIAL

Figure S1. Effect of the inflammatory pathway by adoptive transfer. Figure S2. Blocking the inflammatory pathway by anti-IL-18Ra, IL-18Rb antibody administration. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki

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18. 19.

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