Inhibition of kidney ischemia–reperfusion injury through local infusion of a TLR2 blocker

JIM-11828; No of Pages 5 Journal of Immunological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Immunological Methods

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Hye J. Kim a,b, Sang J. Park a,b,c,d, Soomi Ku a,b, Hee J. Cha a,b,e,f, Jong S. Lee a,b,g,h, Byungsuk Kwon a,b,i,⁎, Hong R. Cho a,b,c,d,⁎ a

Biomedical Research Center, Ulsan University Hospital, University of Ulsan, Republic of Korea Biomedical Research Center, School of Medicine, University of Ulsan, Republic of Korea c Department of Surgery, Ulsan University Hospital, School of Medicine, University of Ulsan, Republic of Korea d Department of Surgery, School of Medicine, University of Ulsan, Republic of Korea e Department of Pathology, Ulsan University Hospital, University of Ulsan, Republic of Korea f Department of Pathology, School of Medicine, University of Ulsan, Republic of Korea g Department of Internal Medicine, Ulsan University Hospital, University of Ulsan, Republic of Korea h Department of Internal Medicine, School of Medicine, University of Ulsan, Republic of Korea i School of Biological Sciences, University of Ulsan, Ulsan, Republic of Korea b

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Article history: Received 14 December 2013 Received in revised form 18 March 2014 Accepted 18 March 2014 Available online xxxx

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Kidney ischemia–reperfusion injury (IRI) occurs as a result of complex interactions of kidney parenchymal cells and immune cells that are initiated by hypoxic damage of parenchymal cells. In particular, tubular epithelial cells (TECs) not only are susceptible to ischemia but also have an auto-loop system to amplify renal inflammation caused by ischemia and reperfusion. Since endogenous TLR2 ligands released from TECs trigger renal inflammation leading to kidney IRI in an autocrine manner, we hypothesized that local infusion of TLR2 blockers would prevent kidney IRI. In this study, we demonstrated that injection of antagonist anti-TLR2 mAb through the renal vein after cross-clamping significantly reduced the recruitment of NK cells to the kidney after IRI, a phenomenon that is governed by TLR2 signaling in TECs. In addition, intrarenal blocking of TLR2 signaling was shown to inhibit NK cell-mediated neutrophil infiltration and subsequent renal damage. Overall, our simple experiment system will be of help in testing the efficacy of candidate blockers targeting kidney parenchymal cells in inhibition of kidney IRI. © 2014 Published by Elsevier B.V.

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Keywords: Kidney ischemia–reperfusion injury Tubular epithelial cell Toll-like receptor 2 blocker

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1. Introduction

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Kidney ischemia–reperfusion injury (IRI) occurs as a result of two sequential events. Firstly, free oxygen radicals produced during ischemia provide an initial damage to parenchymal

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Inhibition of kidney ischemia–reperfusion injury through local infusion of a TLR2 blocker

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Technical note

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journal homepage: www.elsevier.com/locate/jim

⁎ Corresponding authors at: Biomedical Research Center, Ulsan University Hospital and School of Medicine, University of Ulsan, 877 Bangeojin Beltway, Ulsan 682-714, Republic of Korea. Tel.: +82 52 250 7012; fax: +82 52 236 5417. E-mail addresses: [email protected] (B. Kwon), [email protected] (H.R. Cho).

cells such as tubular epithelial cells (TECs) and endothelial cells. Secondly, damaged cells release intracellular damageassociated molecular patterns (DAMPs) and innate receptors that recognize these molecules induce inflammation inflicting a broad range of tissue damage (Rosin and Okusa, 2011). In particular, various endogenous TLR2 and TLR4 ligands are released from parenchymal cells and induce the production of inflammatory mediators in an autocrine manner and by adjacent tissue-resident immune cells in a paracrine manner. We previously demonstrated that signaling via TLR2 in TECs results in the recruitment of NK cells (Kim et al., 2013). CD137 on NK cells in turn stimulates CD137L on TECs to

http://dx.doi.org/10.1016/j.jim.2014.03.014 0022-1759/© 2014 Published by Elsevier B.V.

Please cite this article as: Kim, H.J., et al., Inhibition of kidney ischemia–reperfusion injury through local infusion of a TLR2 blocker, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.014

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2.1. Mice and antibodies

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Female C57BL/6 mice, 7 and 8 weeks of age, were purchased from Orient (Seoul, Korea). Animal studies were approved by the University of Ulsan Animal Care and Use Committee. Fluorescent- or biotin-conjugated mAbs that specifically recognize CD3, CD11b, CD45, Ly6G, NK1.1, or isotype control IgG (eBioscience). Anti-TLR2 mAb and isotype control mouse IgG1 were purchased from Invivogen (Nottingham, UK).

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2.2. Induction of kidney IRI and infusion of antibodies Kidney IRI was induced as previously described (Kim et al., 2012). In brief, following anesthesia, bilateral flank incisions were performed, both kidney vessels were exposed and cross-clamped for 35 min. Immediately after clamping, anti-TLR2 mAb (5 μg in 100 μl of PBS) or the same amount of control mouse IgG1 was injected via the right or left renal

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2. Materials and methods

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produce CXCR2 chemokines that are required for neutrophil recruitment (Kim et al., 2012). Therefore, TECs play a central role in kidney IRI by mediating two key sequential signaling events leading to the recruitment of NK cells and neutrophils. Since kidney IRI is initiated by parenchymal cells and kidney-resident stromal cells, including tissue-resident immune cells, local infusion of candidate blockers into the kidney may provide a valuable tool to screen their effectiveness to inhibit kidney IRI. In this experimental system, 1) a lower dose of candidate drugs can be used for the purpose of the screening; 2) the systemic side effects of testing drugs are minimal due to their confinement to the local organ; and lastly, it is possible to test whether candidate drugs are used as effective components of perfusion solution to prevent potential organ failure caused by IRI. In this study, we showed that injection of neutralizing anti-TLR2 mAb through the renal vein potently inhibited kidney IRI, thus demonstrating that this local infusion experimental system is suitable to test the effectiveness of candidate blockers for innate immune receptors in inhibiting kidney IRI.

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Fig. 1. A local infusion procedure. (A) Exposure of the right kidney after lateral flank incision on the right side. (B) Cross-clamping the right renal vessel. (C and D) The needle of an insulin syringe containing perfusion solution was pierced into the right renal vein in the space between the clamping site and the kidney (C) and bleeding from the injection was allowed until the kidney became pale (D). (E) Injection of the perfusion solution. (F) The appearance of the right kidney after injection.

Please cite this article as: Kim, H.J., et al., Inhibition of kidney ischemia–reperfusion injury through local infusion of a TLR2 blocker, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.014

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2.3. Preparation of kidney cells

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Kidneys were perfused, minced, and placed in DMEM containing 1 mg/ml collagenase IA (Sigma Aldrich) at 37 °C for 30 min. Digested kidney tissues were passed through a 40-μm cell strainer (BD Biosciences, San Jose, CA) using the rubber end of a 1-ml syringe plunger, and a cell suspension was obtained via centrifugation at 1200 rpm for 10 min. Cells were washed in PBS containing 2% BSA and suspended in 36% Percoll (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The suspension was gently overlaid onto 72% Percoll and centrifuged at 2000 rpm at room temperature for 30 min. Cells were retrieved from the Percoll interface and washed twice in DMEM medium and once with staining buffer (PBS containing 0.2% BSA and 0.1% sodium azide). This protocol

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Kidneys were fixed in 10% formalin, embedded in paraffin, 133 and sectioned (5 μm). Paraffin sections were stained with H&E 134 and analyzed. Kidney injury was scored by a single pathologist 135

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2.5. Pathological scoring

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Percoll-purified kidney cells were preincubated in blocking buffer (PBS containing anti-CD16/CD32 [2.4G2] mAbs, 0.2% BSA, 0.01% sodium azide) for 20 min at 4 °C. After washing twice with staining buffer, cells were incubated with the relevant mAbs for 30 min at 4 °C. They were then washed twice with staining buffer and analyzed using FACS Canto II (BD Biosciences). The percent of NK cells was presented by counting CD3−NK1.1+ cells in total kidney cells after gating from CD45+ leukocytes. Similarly, the percent of neutrophils was calculated by counting CD45+CD11b+Ly6G+ cells in total kidney cells.

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2.4. Flow cytometry

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excluded most of the dead kidney cells. We confirmed that 118 harvested cells were 7-AAA-negative live cells. 119

vein, respectively. In some experiments, both sides of kidneys received anti-TLR2 mAb or control IgG1 for the comparison between the anti-TLR2-treated and control IgG1-treated groups of mice. Reperfusion was allowed for 4 h or 24 h through clamp release. During the surgery, mice were placed on a 37 °C heat pad.

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Fig. 2. Local infusion of anti-TLR2 mAb inhibits kidney IRI. Anti-TLR2 mAb or control mouse IgG1 was infused through the right renal or left vein, respectively, immediately after clamping, as described in Materials and methods. The right and left kidneys were compared in a paired way. (A) Percentages of NK cells in total kidney cells isolated 4 h after kidney IR. (B) Percentages of neutrophils in total kidney cells 24 h after kidney IR. (C) Photomicrographs of representative H&E-stained sections of 4-h or 24-h post-IR kidneys. Magnification: 200×. (D) Tubular injury scores 24 h after kidney IR. Data are the mean ± SEM (n = 4–5 per group) and represent two independent experiments. *, P b 0.05; **, P b 0.01.

Please cite this article as: Kim, H.J., et al., Inhibition of kidney ischemia–reperfusion injury through local infusion of a TLR2 blocker, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.014

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2.6. Analysis of renal function

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Kidney function was determined by measuring creatinine and blood urea nitrogen (BUN) concentrations in sera obtained at 24 h after kidney IRI. Creatinine concentrations were measured enzymatically using either the Creatinine Colorimetric/Fluorometric Assay kit (BioVision) or the IDTox™ Creatinine Assay kit (ID Labs). BUN concentrations were measured colorimetrically using the Quantichrom™ Urea Assay kit (Bioassay Systems), according to the manufacturer's instructions.

Systemic injection of anti-TLR2 antibody has been shown to effectively inhibit kidney IRI in a murine model of kidney transplantation (Farrar et al., 2012). We further investigated whether local infusion of anti-TLR2 antibody would be sufficient to inhibit kidney IRI. After clamping the right renal vessel, it was cut using an insulin syringe to allow bleeding. Perfusion solution (100 μl of PBS containing 5 μg of anti-TLR2 mAb) was infused via the right renal vein, as described in Fig. 1. As a paired negative control, the left kidney was infused with 100 μl of PBS containing isotype control mouse IgG1.

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3. Results and discussion

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GraphPad Prism 5 (GraphPad Inc., La Jolla, CA) was used to analyze and present data. Differences between groups were analyzed using a paired parametric t test or a two-way ANOVA test. Values are expressed as the mean ± SEM. A P-value of b0.05 was considered statistically significant.

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2.7. Statistics

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(H.J.C.) as the percentage of damaged tubules in the corticomedullary junction. Criteria for kidney injury included tubular necrosis, cast formation, loss of brush border, tubular dilatation and immune cell infiltration. Scoring for each category was as follows: 0, no change; 1, b 10%; 2, 11– 25%; 3, 25–45%; 4, 46–75%; and 5, N76% area changes. Scores for all the categories were added for the final injury scoring.

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Fig. 3. Locally infused anti-TLR2 mAb is protective from kidney IRI. Anti-TLR2 mAb was infused through both renal veins, immediately after clamping, as described in Materials and methods. Mouse IgG1 was infused as a negative control. Sham-operated mice were also used as negative control. (A) Percentages of NK cells in total kidney cells isolated 4 h after kidney IR. (B) Percentages of neutrophils in total kidney cells 24 h after kidney IR. (C and D) Serum creatinine and BUN levels 24 h after kidney IR. (E) Photomicrographs of representative H&E-sections of 24-h post-IR kidney. Magnification: 200×. (F) Tubular injury scores 24 h after kidney IR. Data are the mean ± SEM (n = 4–5 per group) and represent two independent experiments. *, P b 0.05; **, P b 0.01; ***, P b 0.001.

Please cite this article as: Kim, H.J., et al., Inhibition of kidney ischemia–reperfusion injury through local infusion of a TLR2 blocker, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.014

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Acknowledgments

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This study was supported by a grant of University of Ulsan 264 (2011-0083). 265

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The protocol shown in this manuscript is simple and easy 260 to perform to examine the efficacy of blockers targeting renal 261 stromal and parenchymal cells in inhibiting kidney IRI. 262

References

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4. Conclusion

Farrar, C.A., Keogh, B., McCormack, W., O'Shaughnessy, A., Parker, A., Reilly, M., Sacks, S.H., 2012. Inhibition of TLR2 promotes graft function in a murine model of renal transplant ischemia–reperfusion injury. FASEB J. 26 (2), 799. http://dx.doi.org/10.1096/fj.11-195396. Kim, H.J., Lee, J.S., Kim, J.D., Cha, H.J., Kim, A., Lee, S.K., Lee, S.C., Kwon, B.S., Mittler, R.S., Cho, H.R., Kwon, B., 2012. Reverse signaling through the costimulatory ligand CD137L in epithelial cells is essential for natural killer cell-mediated acute tissue inflammation. Proc. Natl. Acad. Sci. U. S. A. 109 (1), E13. http://dx.doi.org/10.1073/pnas.1112256109 (Jan 3). Kim, H.J., Lee, J.S., Kim, A., Koo, S., Cha, H.J., Han, J.A., Do, Y., Kim, K.M., Kwon, B.S., Mittler, R.S., Cho, H.R., Kwon, B., 2013. TLR2 signaling in tubular epithelial cells regulates NK cell recruitment in kidney ischemia– reperfusion injury. J. Immunol. 191 (5), 2657. http://dx.doi.org/10. 4049/jimmunol.1300358 (Sep 1). Leemans, J.C., Stokman, G., Claessen, N., Rouschop, K.M., Teske, G.J., Kirschning, C.J., Akira, S., van der Poll, T., Weening, J.J., Florquin, S., 2005. Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney. J. Clin. Invest. 115 (10), 2894. Rosin, D.L., Okusa, M.D., 2011. Dangers within: DAMP responses to damage and cell death in kidney disease. J. Am. Soc. Nephrol. 22 (3), 416. http:// dx.doi.org/10.1681/ASN.2010040430. Wolfs, T.G., Buurman, W.A., van Schadewijk, A., de Vries, B., Daemen, M.A., Hiemstra, P.S., van 't Veer, C., 2002. In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation. J. Immunol. 168 (3), 1286. Zhang, Z.X., Wang, S., Huang, X., Min, W.P., Sun, H., Liu, W., Garcia, B., Jevnikar, A.M., 2008. NK cells induce apoptosis in tubular epithelial cells and contribute to renal ischemia–reperfusion injury. J. Immunol. 181 (11), 7489.

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in TECs to recruit neutrophils, suggesting that TECs have an auto-amplification loop for renal inflammation through the two important signaling events (Kim et al., 2013). These observations strongly suggest TECs as a good therapeutic target for kidney IRI. In this study, we convincingly showed that delivery of a TEC TLR2 blocker through the renal vein is an effective way to prevent kidney IRI. This in vivo experimental system will be particularly of value in testing the efficacy of kidney IRI prophylaxis that can be included in perfusion solution.

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In a set of experiments, we harvested kidneys at 4-h post-IR, a peak time for NK cells to infiltrate into the injured kidney (Zhang et al., 2008; Kim et al., 2012), and counted NK cell infiltrate. There was a significant reduction in the percentage of NK cells in the kidney that received anti-TLR2 mAb, compared to the contralateral kidney that received PBS containing control IgG1 (Fig. 2A). This result suggested that blocking of TLR2 signaling in TECs might be sufficient to inhibit the recruitment of NK cells after kidney IRI, as shown by our previous studies (Kim et al., 2013). Since NK cell infiltration occurs prior to and is required for neutrophil recruitment (Kim et al., 2013), it was expected that blocking of TLR2 signaling in TECs should also prevent neutrophils from infiltrating into the kidney after IR. Indeed, we found that the percentage of CD45+CD11b+Ly6G+ neutrophils in total kidney cells was markedly decreased in the 24-h post-IR kidney infused with anti-TLR2 mAb (Fig. 2B). Histopathological analysis showed that proximal tubular morphology was almost intact in the 24-h post-IR kidney injected with anti-TLR2 mAb, whereas proximal tubular necrosis in the outer medulla was evident in the kidney that was infused with PBS only at this time point (Fig. 2C). At 24 h after IRI, the overall tubular injury score of the kidney infused with anti-TLR2 mAb was significantly lower than that of the kidney infused with control IgG1 (Fig. 2D). Taken together, our results indicated that local infusion of anti-TLR2 mAb was effective in preventing renal injury caused by IR. We performed another set of experiments where both kidneys were infused with either anti-TLR2 mAb or control IgG1. Sham-operated mice were also used as a negative control. In this experimental system, kidney function of an individual mouse could be more easily evaluated by measuring serum creatinine or BUN levels, as their difference between experimental groups was more distinguishable when both kidneys were damaged. Local infusion of anti-TLR2 mAb significantly reduced the recruitment of NK cells and neutrophils at 4 h and 24 h after IRI induction, respectively (Fig. 3A and B). Consistently, mice infused with anti-TLR2 mAb had significant decreases in kidney dysfunction and damage after 24 h of IR, as reflected by lower plasma creatinine and BUN levels, less tubular necrosis, and less overall acute tubular injury compared to mice infused with control IgG1 or sham mice (Fig. 3C–F). TLR2 is constitutively expressed in TECs and upregulated by kidney IRI (Wolfs et al., 2002; Leemans et al., 2005). Endogenous TLR2 ligands such as HMGB1 and histones are released from TECs during kidney IRI (Allam et al., 2012; Kim et al., 2013) and initiated renal inflammation by recruiting NK cells in an autocrine manner. NK cells in turn trigger CD137L signaling

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Please cite this article as: Kim, H.J., et al., Inhibition of kidney ischemia–reperfusion injury through local infusion of a TLR2 blocker, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.014

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