BASIC RESEARCH www.jasn.org The E-Selectin Ligand Basigin/CD147 Is Responsible for Neutrophil Recruitment in Renal Ischemia/ Reperfusion Noritoshi Kato,*† Yukio Yuzawa,† Tomoki Kosugi,*† Akinori Hobo,*† Waichi Sato,† Yuko Miwa,* Kazuma Sakamoto,* Seiichi Matsuo,† and Kenji Kadomatsu* Departments of *Biochemistry and †Nephrology, Nagoya University Graduate School of Medicine, Nagoya, Japan ABSTRACT E-selectin and its ligands are essential for extravasation of leukocytes in inflammation. Here, we report that basigin (Bsg)/CD147 is a ligand for E-selectin that promotes renal inflammation in ischemia/ Ϫ Ϫ reperfusion. Compared with wild-type mice, Bsg-deficient (Bsg / ) mice demonstrated striking suppres- sion of neutrophil infiltration in the kidney after renal ischemia/reperfusion. Although E-selectin expres- Ϫ Ϫ sion increased similarly between the two genotypes, Bsg / mice exhibited less renal damage, suggesting that Bsg on neutrophils contribute to renal injury in this model. Neutrophils expressed Bsg Ϫ Ϫ with N-linked polylactosamine chains and Bsg / neutrophils showed reduced binding to E-selectin. Bsg isolated from HL-60 cells bound to E-selectin, and tunicamycin treatment to abolish N-linked glycans Ϫ Ϫ from Bsg abrogated this binding. Furthermore, Bsg / neutrophils exhibited reduced E-selectin-depen- dent adherence to human umbilical vein endothelial cells in vitro. Injection of labeled neutrophils into Ϫ Ϫ mice showed that Bsg / neutrophils were less readily recruited to the kidney after renal ischemia/ ϩ ϩ reperfusion than Bsg / neutrophils, regardless of the recipient’s genotype. Taken together, these results indicate that Bsg is a physiologic ligand for E-selectin that plays a critical role in the renal damage induced by ischemia/reperfusion. J Am Soc Nephrol 20: 1565–1576, 2009. doi: 10.1681/ASN.2008090957 The selectins and their ligands are essential for leu- and all three play distinct roles during tethering and kocyte tethering/rolling on endothelial cells and the slow rolling of neutrophils on the endothelium.7 A initiation of inflammatory response. The selectins minimal recognition motif for all selectins is sialy- are C-type lectins and consist of three members, i.e., lated and fucosylated glycan determinants, such as P-, L-, and E-selectin.1,2 P-selectin is expressed sialyl Lewis X, that decorate the terminal extensions upon inflammatory stimulation in platelets and en- of carbohydrates of these molecules.8,9 However, dothelial cells. L-selectin is constitutively expressed because of the poor immunogenicity of highly gly- on the tip of leukocyte microvilli and implicated in lymphocyte homing to lymph nodes.3 E-selectin is specifically induced in the endothelium upon in- flammatory stimulation. Thus, E- and P-selectin Received September 12, 2008. Accepted February 27, 2009. closely collaborate with one another and play a ma- Published online ahead of print. Publication date available at jor role in leukocyte recruitment to inflammatory www.jasn.org. sites.4–6 Among the several glycoproteins reported Correspondence: Kenji Kadomatsu, Department of Biochemis- try, Nagoya University Graduate School of Medicine, 65 Tsuruma- to bind to E-selectin, three have been identified as cho, Showa-ku, Nagoya 466-8550, Japan. Phone: ϩ81-52-744- representative physiologic E-selectin ligands on 2060; Fax: ϩ81-52-744-2065; E-mail: [email protected]. neutrophils. There are P-selectin glycoprotein li- ac.jp gand-1 (PSGL-1), E-selectin ligand-1, and CD44, Copyright ᮊ 2009 by the American Society of Nephrology J Am Soc Nephrol 20: 1565–1576, 2009 ISSN : 1046-6673/2007-1565 1565 BASIC RESEARCH www.jasn.org cosylated epitopes, it has proven difficult to identify selectin inflammation in the present study. BsgϪ/Ϫ mice exhibited less ligands. renal damage after ischemia/reperfusion. To our surprise, this Basigin (Bsg)/CD147 (Bsg is the name of the mouse gene) is phenotype was attributable to Bsg on neutrophils, rather than a membrane glycoprotein that belongs to the Ig superfamily. Bsg on other cells in the inflammation area. We found that Bsg was discovered in embryonal carcinoma cells as a receptor highly glycosylated Bsg on neutrophils bound to E-selectin on for Lotus tetragonolobus agglutinin10 and was determined to endothelial cells and led to neutrophil infiltration to the in- have the structure Gal␤134(Fuc␣133)GlcNAc, which is flammatory lesion. Our results may shed light on the mecha- known as the Lewis X structure. But it has been unclear nisms underlying the pathogenesis of AKI. whether Bsg has sialyl Lewis X structure and whether Bsg serves as a selectin ligand. Bsg is expressed in many cell types, e.g., blood cells, epithelial cells, endothelial cells, and germ cells. We RESULTS previously generated Bsg-deficient (BsgϪ/Ϫ) mice and found several abnormalities that included male and female sterility, Bsg Deficiency Preserves Renal Function and progressive retinal degeneration, increased cell proliferation Decreases Renal Injury after Ischemia/Reperfusion upon mixed lymphocyte culture, decreased memory function, We subjected Bsgϩ/ϩ and BsgϪ/Ϫ mice to renal ischemia/reper- and abnormal sensory function.11–13 In addition to these func- fusion injury. As shown in Figure 1, the renal damage was less tions deduced through the study of BsgϪ/Ϫ mice, two addi- pronounced in BsgϪ/Ϫ mice than in Bsgϩ/ϩ mice. Thus, 2 d tional Bsg functions have recently been highlighted. First, Bsg after ischemia/reperfusion, BsgϪ/Ϫ mice showed less tubuloin- activates matrix metaloproteases (MMPs), thereby promoting terstitial injury by all three criteria examined, i.e., tubular cast cancer invasion.14 Second, Bsg functions like a chaperone for formation, dilation, and degeneration (Figure 1, A and B). The monocarboxylate transporters (MCTs).15,16 In the present increase of serum urea nitrogen levels was also significantly study, we found an additional and unexpected role of Bsg; suppressed in BsgϪ/Ϫ mice (Figure 1C). The serum urea nitro- namely, its glycosylation was crucial for inflammation. gen reached the maximum level on day 1 after ischemia in Acute kidney injury (AKI) is a common complication that BsgϪ/Ϫ mice versus day 2 in Bsgϩ/ϩ mice. Thus, postischemic occurs in approximately 5% of hospitalized patients and in renal injury was quickly terminated in BsgϪ/Ϫ mice. approximately 30% of patients in intensive care units. As the mortality of AKI is still unacceptably high, between 40% and Bsg Deficiency Reduces Neutrophil Infiltration into the 60%,17 this disease is being intensively studied. Renal ischemia/ Tubulointerstitium reperfusion injury is characteristic of acute renal inflammation Renal ischemia/reperfusion injury is characterized by a mas- involving marked infiltration of inflammatory cells, such as sive influx of neutrophils early after reperfusion, which plays a neutrophils, and is the most widely used model for human crucial role in the pathogenesis of postischemic renal failure AKI.18 We used this model to investigate the role of Bsg in through the release of cytotoxic proteases and oxygen-derived Ϫ Ϫ Figure 1. Renal injury is less severe in Bsg / mice after renal ischemia/reperfusion. (A) A representative image of tubular lesions 2 d Ϫ Ϫ ϩ ϩ after ischemia/reperfusion injury. Less renal damage was observed in Bsg / mice than in Bsg / mice. Arrow, tubular cast; arrowhead, degeneration of the tubule; asterisk, dilation of the tubule. Scale bar, 50 ␮m. Periodic acid-Schiff staining. (B) Semiquantitative analysis of tubulointerstitial damage 2 d after ischemia/reperfusion injury. The degree of tubular cast formation, tubular dilation, and tubular degeneration were comparatively rated as described in the Concise Methods section. High values indicate more severe damage. White ϩ ϩ Ϫ Ϫ columns, Bsg / mice; black columns, Bsg / mice. cast, cast formation. dil., dilation. degen., degeneration. Data are means (columns) ϩ ϩ Ϫ Ϫ and SEM; bars). *P Ͻ 0.05; **P Ͻ 0.01; n ϭ 6. (C) Blood urea nitrogen (BUN) levels in Bsg / and Bsg / mice after ischemia/ Ϫ Ϫ ϩ ϩ ϩ ϩ Ϫ Ϫ reperfusion. Renal function was better preserved in Bsg / than Bsg / mice. White columns, Bsg / mice; black columns, Bsg / mice. BUN is shown as mg/dl. Data are means (columns) and SEM (bars). *P Ͻ 0.05; n ϭ 6. 1566 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1565–1576, 2009 www.jasn.org BASIC RESEARCH radicals.19 We next compared the amount of neutrophil influx after ischemia/reperfusion, but there was no difference in the and renal function. Bsg deficiency greatly reduced the influx of degree of elevation between the two genotypes (Figure 2C). neutrophils to the kidney 1 d after ischemia/reperfusion (Fig- Neutrophil recruitment in the postischemia/reperfusion ure 2A). The number of infiltrated neutrophils reached the kidney requires adhesion molecules. E-selectin and intercellu- maximum level 1 d after ischemia/reperfusion (Figure 2A). lar adhesion molecule-1 (ICAM-1) on peritubular capillary This was in contrast to the macrophage influx, which peaked cells play particularly crucial roles in this model.23 In the 4 d after ischemia/reperfusion (Figure 2B). As the difference in present study, western blot analysis showed upregulation of renal function between Bsgϩ/ϩ and BsgϪ/Ϫ mice became ap- E-selectin in the kidney 12 h after ischemia/reperfusion in both parent 2 d after ischemia/reperfusion (Figure 1C), it is conceiv- Bsgϩ/ϩ and BsgϪ/Ϫ mice, but the expression was not signifi- able that neutrophil influx, rather than macrophage influx, was cantly different between the two genotypes (Figure 2D). The crucial for the role of Bsg in renal damage in our model. expression of ICAM-1 in the kidney at 24 h postischemia was It is widely accepted that chemokines are generated by isch- also comparable between the two genotypes (Figure 2E). E- emic tubular epithelial cells in the early phase of renal ischemic injury.20 In addition, the tubular epithelial cells express Bsg.21 Accordingly, we next examined whether the difference in in- flux of neutrophils was mediated through differences in che- mokine levels.