Activation of Parenchymal CD47 Promotes Renal Ischemia-Reperfusion Injury

BASIC RESEARCH www.jasn.org

† † Natasha M. Rogers,* Angus W. Thomson and Jeffrey S. Isenberg*

*Division of Pulmonary Allergy and Critical Care Medicine, Vascular Medicine Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and †Starzl Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

ABSTRACT Ischemia-reperfusion injury (IRI) contributes to decreased allograft function and allograft rejection in transplanted kidneys. Thrombospondin-1 is a stress protein typically secreted in response to hypoxia and the ligand activator for the ubiquitously expressed receptor CD47. The function of activated CD47 in IRI remains completely unknown. Here, we found that both CD47 and its ligand thrombospondin-1 were upregulated after renal IRI in mice. CD47-knockout mice were protected against renal dysfunction and tubular damage, suggesting that the development of IRI requires intact CD47 signaling. Chimeric CD47- knockout mice engrafted with wild-type hematopoietic cells had signiﬁcantly lower serum creatinine and less tubular damage than wild-type controls after IRI, suggesting that CD47 signaling in parenchymal cells predominantly mediates renal damage. Treatment with a CD47-blocking antibody protected mice from renal dysfunction and tubular damage compared with an isotype control. Taken together, these data imply that CD47 on parenchymal cells promotes injury after renal ischemia and reperfusion. Therefore, CD47 blockade may have therapeutic potential to prevent or suppress ischemia-reperfusion–mediated damage.

J Am Soc Nephrol 23: 1538–1550, 2012. doi: 10.1681/ASN.2012020137

Renal ischemia-reperfusion injury (IRI) is a major guanylate cyclase activation.8–10 Organs subjected cause of ARF resulting from tubular dysfunction, and to IRI demonstrate suppression of NO,11 and thera- contributes to morbidity and mortality. In the context pies that supplement NO have been shown to mitigate of transplantation, IRI promotes delayed graft function IRI.12–15 and acute rejection, and subsequently negatively influ- In this study, we tested the hypothesis that renal ences long-term allograft outcome.1–3 The cascade of tissue–expressed CD47 promotes IRI. We demon- events leading to cellular injury and activation is estab- strate for the first time that TSP1 and CD47 are lished by initial oxygen depravation that leads to ATP significantly induced in IRI. In null mice, absence depletion and mitochondrial dysfunction (reviewed of activated CD47 leads to profound cytoprotection by Murillo et al.4). After reperfusion, proinflammatory from renal IRI with significant abrogation of cell conditions and a coordinated release of cytokines, re- death and ROS production, suppressed cytokine active oxygen species (ROS), and adhesion molecules production/secretion, and robust reperfusion, as promote the innate immune response to IRI. Thrombospondin-1 (TSP1) is a matricellular glycoprotein maintained predominantly within Received March 3, 2012. Accepted June 20, 2012. platelets and present in nanomolar concentrations 5 Published online ahead of print. Publication date available at in the blood. It is secreted by multiple cell types, www.jasn.org. including inflammatory and vascular cells, typically in response to hypoxia.6 It is also produced by renal Correspondence: Dr. Jeffrey S. Isenberg, Division of Pulmonary Allergy and Critical Care Medicine, Vascular Medicine Institute, 7 tubular epithelial cells (RTECs) after IRI. TSP1, University of Pittsburgh School of Medicine, E1240 Biomedical through binding the ubiquitously expressed cell Science Tower, Room E1258, 200 Lothrop Street, Pittsburgh, PA surface receptor CD47, regulates the canonical nitric 15261. Email: [email protected] oxide (NO) pathway via limitation of soluble Copyright © 2012 by the American Society of Nephrology

1538 ISSN : 1046-6673/2309-1538 JAmSocNephrol23: 1538–1550, 2012 www.jasn.org BASIC RESEARCH assessed by laser Doppler analysis. This protection is independent of inﬂammatory cell activation because null mice transplanted with wild-type (WT) bone marrow, and hence bearing CD47+/+ inﬂammatory cells, continue to experience substantial protection from renal IRI. Furthermore, we show that limiting CD47 activation with an antibody that prevents TSP1 binding mitigates complications of renal IRI in mice, providing a potential, clinically relevant, therapeutic intervention.

RESULTS

CD47 Is Expressed by RTECs and the TSP1-CD47 Signaling Axis Is Upregulated in Cells Exposed to Hypoxia-Reoxygenation A single previous study reported upregulation of TSP1 protein after renal IRI,7 although expression of cellular TSP1 and CD47 in the kidney has not been defined. Staining cultured human RTECs for CD47 demonstrated significant cell surface expression (Figure 1A). RTECs challenged with hypoxia (FiO2 1%) and then 24 hours of reoxygenation (as a mimic of IRI) showed a significant induction of TSP1 protein coinci- dent with persistence of CD47 protein, and persistence of both TSP1 and CD47 mRNA, compared with cells treated with normoxia (Figure 1, B and C).

Renal IRI Upregulates Cell Receptor CD47 and its Ligand TSP1 Extending upon results obtained with cultured RTECs, we Figure 1. RTECs express CD47 and upregulate TSP1 in response found robust induction of both TSP1 mRNA and protein to hypoxia. (A) Human RTECs were stained with CD47 antibody or expression in WT mice 24 hours after reperfusion, compared isotype control (CTRL) and visualized by confocal microscopy. with sham-operated animals (Figure 2A). There are no data on RTECs were exposed to 30-minute hypoxia (FiO2 1%) and then expression of its cognate receptor CD47 in response to acute 24-hour reoxygenation or normoxia alone and TSP1 and CD47 (B) protein and (C) mRNA assessed. Data shown are means 6 SD, injury, let alone in renal IRI. We now show for the first time and representative Western blots with relative densities are cal- concurrent upregulation of CD47 mRNA and protein expres- , # 2 2 culated from n=4 experiments. *P 0.01; **P=0.49; P=0.88; / d sion (Figure 2B) in WT mice after IRI. Interestingly, CD47 P=0.38 normoxia versus hypoxia. mice showed decreased TSP1 expression compared with WT mice after IRI (Figure 2A). CD47 axis has been documented to inhibit tissue blood flow, we 2 2 CD47 Limits Reperfusion after Renal IRI hypothesized that CD47 / mice would experience less tissue CD47 activation by its ligand TSP1 blocks NO and soluble injury post-IRI. To further test the importance of CD47 in renal 2 2 guanylate cyclase signaling in multiple cell types.9,16,17 NO IRI, we compared serum creatinine levels in CD47 / versus directly promotes tissue perfusion and deletion of TSP1 dramat- congenic WT (CD47+/+) mice. Knockout of CD47 resulted in ically increases blood flow after NO challenge.8 Using real-time less functional renal impairment after 24 hours of reperfusion as laser Doppler analysis of blood flow to the left kidney (Figure 3A), assessed by serum creatinine levels (Figure 4A), indicating paren- 2 2 WT and CD47 / mice demonstrated comparable basal blood chymal cytoprotection. Clinically, WT mice were grossly uremic flow and subsequently underwent renal IRI. Initial reperfusion and failed to survive past 36–48 hours after reperfusion (data not 2 2 (within 30 minutes) was similar between both groups of animals; shown). In contrast, CD47 / mice did not demonstrate these 2 2 however, blood flow was significantly greater in CD47 / mice at findings and survived long term (up to 7 days; n=4 per group). To 24 hours after IRI (Figure 3B). further confirm the presence of renal injury in our model, we evaluated renal histology. After 24 hours of reperfusion, kidneys 2 2 CD47 / Mice Experience Less Renal Dysfunction and of WT mice showed widespread and substantial tubular necro- Acute Tubular Damage in a Model of Severe Renal IRI sis, tubular dilation, and cast formation. The injury, although Activation of proinflammatory pathways and oxidative stress are being predominantly corticomedullary, also typically extended 2 2 major contributors to tissue injury after IRI.18 Because the TSP1- to the renal cortex. In contrast, kidneys from CD47 / mice

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studies.19,20 These data suggest an important role for CD47 in the induction of a cellular inﬂammatory response during IRI.

Absence of Activated CD47 Reduces Proinflammatory Cytokine and Chemokine Expression after Renal IRI We next investigated whether CD47 deficiency also influenced expression of cytokines and chemokines critical to the inflammatory response after IRI. mRNA transcript profiles of IL-6, TNFa, IL-1b, monocyte chemoattractant protein-1 (CCL2), and macrophage inflammatory protein-1 (CXCL2) were examined in kid- 2 2 neys from WT and CD47 / mice after sham operation or IRI (Figure 6). mRNA expression in WT mice subjected to renal IRI was uniformly increased compared with sham-operated controls. Overall, cytokine expression increased to a much lesser extent 2 2 in CD47 / mice and both TNFa and IL-1b transcript levels were not elevated above baseline values.

2 2 CD47 / Mice Show Reduced Evidence of Programmed Renal Cell Death after IRI Renal IRI is also characterized by RTEC apoptosis.21 CD47 ligation by TSP1 regulates cellular apoptotic machinery by modulating Fas signaling22 and has been linked through Bcl-2 homology 3-only pro- Figure 2. Renal IRI upregulates CD47 and its ligand TSP1. Kidney lysate from WT tein 19 kD interacting protein-3 (BNIP3) C57BL/6 male mice 24 hours after IRI compared with sham-operated controls was activation to programmed cell death.23 prepared and (A) TSP1 mRNA and protein (TSP1 is detected at 150 kD) or (B) CD47 We assessed tubular cell apoptosis by mRNA and protein expression (CD47 is detected at 47–52 kD) assessed. mRNA data terminal deoxynucleotidyl transferase– shown are mean 6 SD, n=8 mice per group with the WT sham-operated animals as the mediated digoxigenin-deoxyuridine nick-end reference group; representative Western blots with relative densities calculated from labeling (TUNEL) staining and expression 2/2 n=4 mice per group. *P,0.001 CD47 after IRI versus WT after IRI; **P,0.001 WT of caspase-3, a transducer of programmed sham versus WT after IRI. cell death. Compared with sham-operated animals, WT mice subjected to IRI demonstrate significant RTEC apoptosis at 24 displayed significantly less tubular damage 24 hours after IRI hours (Figure 7, A and B). This correlated with detection of (Figure 4B). Thus, CD47 deficiency had a protective effect on activated caspase-3 on Western blot (Figure 7, C and D). Nota- renal function and damage after induction of severe IRI. bly, the number of apoptotic cells in kidneys and caspase-3 2 2 expression were markedly lower in CD47 / mice at 24 hours 2 2 Inflammatory Cell Influx Is Decreased in CD47 / Mice after reperfusion. after IRI Leukocyte influx is a hallmark of renal IRI. We found Absence of Activated CD47 Mitigates Renal IRI significantly fewer interstitial neutrophils (Figure 5A) and through a Reduction in ROS 2 2 macrophages (Figure 5B) in CD47 / mice at 24 hours after IRI is associated with increased production of ROS, partic- 2 reperfusion compared with WTmice. The number of infiltrat- ularly superoxide (O2 ), which is a major mediator of cellu- ing macrophages (in terms of cell number per high-power lar injury.24 To assess whether the cytoprotective effect of a field) was also significantly less than reported in other lack of CD47 expression could be attributed to a reduction

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blot analysis of kidney tissue indicated the presence of nitrated protein bands at 27 and approximately 47 kD (only bands at 47 kD are shown in Figure 8B) in WT mice after 24-hour reperfusion, which 2 2 was reduced in CD47 / mice. Interest- ingly, basal tissue nitration trended lower 2 2 in CD47 / kidneys compared with WT. Oxidative stress also induces nitric oxide synthase (iNOS) expression, which aggra- vates tubular injury by supplying addi- 2 tional NO for ONOO formation in the presence of excess ROS.26,27 mRNA expression for renal iNOS was increased in WT mice but signiﬁcantly attenuated in 2 2 CD47 / mice (Figure 8C).

CD47-Mediated Kidney IRI Requires Functional CD47 Signaling on Renal Parenchymal Cells CD47 is expressed on all circulating cells, including leukocytes, platelets, and red blood cells.28 Thus, it is not clear if results 2 2 in CD47 / mice are secondary to decreased signaling among tissue parenchymal cells or circulating and interstitial leukocytes. To test this, we used bone marrow (BM) transplantation in irradiated CD47+/+ (WT) and 2 2 CD47 / mice to generate chimeric mice expressing CD47 on renal-resident leukocytes but not on renal parenchymal cells 2 2 (WTBM-CD47 / ), as well as appropriate 2 2 control animals. The inability of CD47 / BM to engraft WTmice has been documented previously,29 putatively from augmented recipient phagocytosis of donor BM cells Figure 3. Renal reperfusion is limited by CD47. Eight-week-old male C57BL/6 WT and 2 2 in vivo. Recapitulating this observation, we CD47 / mice underwent 22 minutes of left-sided renal pedicle occlusion followed by were not able to generate chimeric mice ex- reperfusion. (A) Representative laser Doppler color images of renal blood flow at baseline, ischemia, 30 minutes, and 24 hours of reperfusion. Red coloration of images pressing an absence of leukocyte CD47 2/2 n indicates maximum blood flow and blue coloration minimum blood flow. (B) Analysis (CD47 BM-WT) because all mice ( =5) of tissue blood flow was performed. Changes in renal perfusion at indicated time died in the early post-transplant period (me- points are presented as flux normalized to baseline values (% control). Results rep- dian survival time 19 days). In other BM resent the mean 6 SD of four measurements at each time point from n=5 WT and n=5 chimeras, we determined the expression of 2 2 2 2 CD47 / mice. *P,0.001 CD47 / versus WT at 24 hours of reperfusion. CD47 on whole blood by flow cytometric analysis 8 weeks after transplantation, dem- in the production of ROS, intracellular generation of onstrating complete engraftment in all animals (n=7) (Figure 2 the ROS moiety O2 was visualized with the fluoroprobe 9A). WTBM-WT mice showed significant kidney dysfunction 2 2 2 2 dihydroethidium (DHE). Using confocal microscopy, sec- and injury 24 hours after IRI, whereas CD47 / BM-CD47 / tions of kidney from WT mice showed a widespread and chimeric mice were substantially protected from kidney IRI as marked increase in DHE fluorescence compared with sham- measured by serum creatinine and tubular damage (Figure 9, operated controls (Figure 8A), and this signal was dramati- B and C, respectively). The difference in serum creatinine levels 2 2 cally abrogated in CD47 / mice at 24 hours of reperfusion. and tubular injury scores recapitulated the results seen previ- 2 2/2 The combination of O2 with resident NO forms perox- ously (see Figure 3). In addition, WTBM-CD47 chimeras 2 2 2 ynitrite (ONOO ), which is capable of aggravating cellular were protected from renal IRI to the same degree as CD47 / 2 2 damage by nitration of protein tyrosine residues.25 Western BM-CD47 / mice. These results suggest that functional

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Figure 5. Inflammatory cell infiltration is significantly decreased 2 2 in CD47 / mice subjected to renal IRI. Representative photomicrographs and quantitative analysis of kidney tissue sections 2/2 Figure 4. CD47 mice are protected against severe renal IRI. stained by immunohistochemistry for (A) neutrophils and (B) (A) Quantification of serum creatinine after bilateral renal ische- macrophages (total cell number per 10 hpf for both parameters). 2/2 mia and 24 hours reperfusion, in CD47 mice (white bars), WT Data shown are mean 6 SD, n=8–10 per group. *P=0.001 and 2 2 controls (black bars), and sham-operated mice (gray bars). Data **P,0.05 CD47 / after IRI versus WT after IRI. Original magni- 2/2 are mean 6SD, n=8–10 per group. *P,0.001 CD47 versus fication, 3400. WT. (B) Representative renal tissue sections and quantitative analysis of tubular damage of corticomedulla from WT and 2 2 2 2 2 2 CD47 / mice 24 hours after sham operation or ischemia- decreased in CD47 / BM-CD47 / mice after IRI (Figure 9E). reperfusion (periodic acid–Schiff stained) are shown. Data shown Renal mRNA levels of TSP1 and CD47 were suppressed in 2/2 2 2 are mean 6 SD, n=8–10 per group, *P,0.001 CD47 after IRI CD47 / mice after IRI regardless of BM genotype. versus WT after IRI. Original magnification, 3200; inset, 3400 fi magni cation insert. Blockade of CD47 Using mAb In Vivo Abrogates Renal IRI We have reported soft tissue protection in mice treated with a CD47 on renal parenchymal cells makes the greatest contribu- CD47 blocking antibody.30 However, it was not clear if sys- tion to tissue damage. temic treatment results in significant tissue localization of the Interestingly, TSP1proteinexpressionwasdecreasedafter IRI CD47 antibody. To test this, we treated uninjured animals 2 2 in WTBM- CD47 / chimeras and even further suppressed in with a CD47 mAb (0.4 mg/gbodyweightinsterilesaline) 2 2 2 2 CD47 / BM-CD47 / mice (Figure 9D) suggesting that acti- via a single intraperitoneal injection. Analysis of tissue sec- vated leukocytes are a significant source of tissue TSP1 after tions obtained from animals 90 minutes after treatment dem- injury. Conversely, CD47 protein expression was significantly onstrated the presence of CD47 antibody on RTECs (Figure

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lower mRNA levels of renal IL-1b,IL-6, CCL2, CXCL2, and iNOS observed on quantitative PCR (Figure 10, E and F). In- terestingly, TNF-a mRNA levels were not decreased in CD47 antibody-treated mice after IRI.

DISCUSSION

This study has several major findings . First, we provide evidence of strong expression of cell receptor CD47 on human RTECs. In response to the IRI-like conditions of hypoxia and reoxygenation, RTECs demonstrate persistent upregulation of the TSP1-CD47 signaling axis. Results obtained with cultured RTECs were repro- duced by results in animals challenged with renal IRI. In WT mice there is rapid upregulation of activated CD47 after renal IRI, providing the first in vivo evidence of injury-mediated CD47 induction. Fur- ther support for an instigating role for CD47 in the pathophysiology of renal 2 2 IRI is provided by studies in CD47 / mice that are protected from severe renal IRI. Although we have reported increased tissue survival after injury in CD47 null mice,30–32 the exact mechanism involved has remained unclear. We herein demon- Figure 6. Proinflammatory cytokine and chemokine mRNA profiles are reduced in strate that, in renal IRI, cytoprotection is 2 2 CD47 / mice subjected to renal IRI. mRNA expression of proinflammatory cytokines conferred predominantly by absence of 2 2 IL-6, TNFa,andIL-1b and CCL2 and CXCL2 in kidneys from WT and CD47 / mice CD47 expression on RTECs and identify subjected to IRI. Results have been normalized to the housekeeping gene (HPRT1) RTECs as major targets of and responders and WT sham-operated animals used as the reference group. Data shown are 2 2 to CD47-mediated IRI. Finally, in transla- means 6 SD, n=6–10 per group. *P,0.001 CD47 / after IRI versus WT after IRI; # 2/2 tional studies we show that therapeutic P=0.01 CD47 after IRI versus WT after IRI. blockade of CD47 activation in the kidney decreases renal dysfunction after IRI. To- 10A). Extending these studies, we evaluated whether blockade gether, these data suggest an important role for tissue-resident of CD47 activation could influence clinical outcome of renal activated CD47 in the pathophysiology of renal IRI. IRI, treating mice with the same dose of the CD47 blocking TSP1 is a secreted glycoprotein made by vascular cells, antibody as above 90 minutes before induction of IRI. Western inflammatory cells, and platelets in response to injury, al- blot analysis of renal tissue demonstrates downregulation of though it is detectable at low concentrations in plasma of both CD47 and TSP1 protein expression in WTmice receiving healthy individuals.5 In contrast, cell membrane CD47 is ex- the CD47 blocking antibody compared with mice that received pressed ubiquitously6 and TSP1 binding to and activation of an isotype control antibody (Figure 10B). Mice treated with a CD47 serves an important physiologic function in terms of CD47 blocking antibody had markedly improved renal func- regulating cardiovascular responses.33 However, nothing is tion at 24 hours of reperfusion, as reflected by lower serum known about the role of CD47 in renal disease. We are the creatinine levels and decreased tubular damage by light mi- first to show increased CD47 mRNA and protein expression croscopy assessment of tissue sections (Figure 10, C and D), in kidneys subjected to IRI, which occurs concurrently with 2 2 2 2 with results recapitulating those achieved in CD47 / mice upregulation of TSP1. Intriguingly, data from CD47 / and after IRI. Moreover, CD47 blockade resulted in global sup- chimeric mice demonstrated that, in the absence of parenchy- pression of inflammation after IRI, with significantly fewer tis- mal CD47, IRI-mediated gene induction of both TSP1 and sue neutrophils observed via immunohistology and markedly CD47 did not occur, and are in agreement with our recent report

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(preservation of tubular integrity assessed by light microscopy) compared with WT mice. Programmed cell death remains an important feature of IRI and both apoptosis, assessed by TUNEL staining, and expression of activated caspase-3 were elevated in 2 2 WT compared with CD47 / mice after 24 hours of reperfusion. The difference between WT and knockout mice may have been greater if observed at an earlier time point (3–6 hours of reperfusion) that re- flects the early phase development of apoptosis in IRI,37 but apoptotic figures are typically evident at 24 hours of reperfusion.38,39 The role of CD47 in programmed cell death has been defined in T cells through an interaction with BNIP3.23 The involvement of CD47 in promoting apoptosis in other cell types has not been investigated, but the decreased RTEC apo- 2 2 ptosis observed in CD47 / mice may be explained by the proapoptotic function of CD47. Recruitment of leukocytes, particularly 2 2 Figure 7. Programmed cell death and apoptosis are reduced in the kidney in CD47 / neutrophils and macrophages, is a hallmark – mice after IRI. (A) Representative renal tissue sections from IRI and sham-operated of IRI40 42 and both cell types are actively mice (n=6 per group) were stained by TUNEL assay and visualized by confocal mi- involved in tissue damage in ischemia- 2 2 croscopy. (B) Apoptotic cells/hpf were counted in 10 successive fields in CD47 / related ARF.41,43 The impaired leukocyte 2 2 2 2 mice (white bars) and WT mice (black bars). *P,0.001 CD47 / after IRI versus wild- influx seen in kidneys from both CD47 / 2/2 type after IRI. (C) Kidney tissue lysate was prepared from WT or CD47 mice sub- mice (and WT mice treated with a CD47 jected to sham operation or IRI, resolved by SDS-PAGE and probed for caspase-3 blocking antibody) may be in part as a con- (activated caspase-3 is detected at 17 and 19 kD). Densitometry represents mean 6SD 2 2 sequence of reduced proinflammatory cyto- of n=6 samples per group (D). *P,0.001 CD47 / after IRI versus WT after IRI. kine and chemokine levels (particularly Original magnification, 3200. CXCL2), and may account for the lower degree of renal dysfunction and tissue damage compared with WT controls. of linked TSP1-CD47 gene regulation in hypoxic pulmonary Arguing against a primary role for inflammatory cells in endothelial cells.34 These data suggest an active role of the CD47 promotion of renal IRI are results obtained in chimera 2 2 CD47 receptor in affecting pathophysiology in acute renal in- mice. Mice with CD47 / parenchymal cells, but CD47+/+ jury and the possible existence of a common pathway control- circulating leukocytes, were protected from renal dysfunction 2 2 ling the gene expression of these proteins. and damage to the same degree as fully CD47 / mice, sug- Experimental data suggest that IRI rapidly activates multiple gesting that the absence of CD47 on tissue-resident cells is pathways involving renal hemodynamics, proinflammatory most protective against injury. Due to the inability to engraft 2 2 mediators, oxidative stress, and the innate immune system CD47 / leukocytes into WT mice, this model remains in- (reviewed in Eltzschig and Eckle35 and Snoeijs et al.36). From our complete and further work is required to fully characterize the 2 2 data, it is clear that an absence of CD47, despite the presence of contribution of CD47 / myeloid cells after renal IRI. its soluble ligand TSP1, significantly abrogates the induction of Lastly, to assess the clinical implications of these findings, many of these pathophysiological pathways. WT mice demon- we tested whether CD47 blockade could mimic the improved strate substantial ongoing renal perfusion defects at 24 hours outcome seen in null mice after renal IRI. Immunofluorescent 2 2 after IRI, whereas CD47 / mice have enhanced blood flow histology provided preliminary evidence that treating via sys- recovery approaching baseline. Improved blood flow may be temic injection with CD47 mAb results in renal cellular and 2 2 part of the reparative repertoire seen in CD47 / mice and re- tissue-specific localization of the antibody. These data are im- flect significant normalization of renal dysfunction (measured portant in that the results were obtained in the absence of renal by serum creatinine) and abrogation of tissue damage IRI, and suggest the potential to obtain adequate receptor

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In summary, our results document a new and important role for activated CD47 in mediating RTEC injury in IRI and that inhibition of this pathway is central to cytoprotection from inﬂammatory and oxidative stress. Signiﬁcantly, renal IRI appears to be mediated predominantly by tissue-expressed CD47. Elimination of activated CD47 signaling, whether in knockout animals or via antibody blockade, provided robust protection from renal IRI, suggesting a role for CD47 blockade strategies to improve outcomes after pro- cedures and disease processes that effect organ perfusion.

CONCISE METHODS

A detailed methods section can be found in the Supplemental Material.

Animals 2 2 Male C57BL/6 WTand CD47 / mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All studies were performed using protocols approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and in accordance with National Institutes of Health guidelines.

Cell-Based Experiments Human RTECs (Lonza, Basel, Switzerland) were 2 2 Figure 8. Renal oxidative stress is reduced in CD47 / mice after IRI. (A) Represen- grown in appropriate media. Cells were serum tative renal tissue sections from IRI and sham-operated mice (n=6–8 per group) starvedat80%confluence,subjectedto30-minute stained with DHE and visualized by confocal microscopy. Intensity of staining was hypoxia (FiO2 1%) and then 24-hour reoxygena- 2 2 quantified using Image J. *P,0.001 CD47 / after IRI versus WT after IRI. (B) Kidney tion and collected for RNA or protein (as de- 2/2 tissue lysate was prepared from WT or CD47 mice subjected to sham operation or scribed below). For immunofluorescence, cells IRI, resolved by SDS-PAGE, and probed for 3-nitrotyrosine (3NT). Densitometry rep- a 2 2 were subjected to cytospin, stained with CD47 resents mean 6 SD of n=4 samples per group. *P,0.001 CD47 / after IRI versus WT 2 2 antibody (clone B6H12; Santa Cruz Biotechnol- after IRI. (C) mRNA expression profile of iNOS in renal tissue in WT and CD47 / mice ogy, Santa Cruz, CA) or IgG1 isotype control, and subjected to IRI or sham operation. Results have been normalized to the house- then AlexaFluor 555 (Molecular Probes, Eugene, keeping gene (HPRT1) and WT sham-operated animals used as the reference group. 2 2 9 Data shown are mean 6 SD, n=6–10 per group. *P,0.001 CD47 / after IRI versus OR) and 4 ,6-diamidino-2-phenylindole (Molec- WT after IRI. Original magnification, 3200. ular Probes). targeting as a pretreatment to renal transplant. WTmice treated IRI with a CD47 antibody that blocks its activation30,44 de- Mice were anesthetized using isoflurane and oxygen titrated to effect, monstrated downregulation of tissue expression of both cell and body temperature maintained at 37°C. Microaneurysm clips were receptor CD47 and ligand TSP1, concurrent with decreased placed to occlude both renal pedicles for 22 minutes. The abdomen renal damage and pan-suppression of inflammatory cell re- was closed with 5/0 monofilament suture. Mice were euthanized 24 sponse and cytokine mRNA after IRI. Results for a single hours after reperfusion; blood was collected and kidney tissue snap intraperitoneal dose of antibody recapitulated the null pheno- frozen, placed in RNAlater, embedded in optimal cutting temperature typic response after renal IRI and suggest that this therapeutic compound (OCT; Sakura Finetek, Torrance, CA), or fixed in 10% approach may be efficacious as a mitigant for transplant IRI. neutral buffered formalin.

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intraperitoneally in 100 mlsterilePBS)oran IgG2a isotype-matched control antibody (Santa Cruz Biotechnology) injected 90 minutes before surgery. Measurement of creatinine and histology are as described below. Control mice received antibody treatment but did not undergo IRI, and kidneys were embedded in optimal cutting temperature compund. Sections were ﬁxed in ethanol and subsequently stainedwithAF-555and4’,6-diamidino-2- phenylindole.

Assessment of Renal Function after IRI Renal function was assessed by measurement of serum creatinine at 24 hours after IRI using a Jaffe creatinine picric acid reaction (OSR 6178; Beckman Coulter, Brea, CA) analyzed on an Olympus AU640 analyzer (Beckman Coulter).

Kidney Histology Kidneys embedded in paraffin were sectioned at 3 mm and stained with periodic acid–Schiff stain by standard methods. Markers of tubular damage (tubular dilation, cell necrosis, infarction, and cast formation) were scored by calculation ofthepercentageoftubulesinthecortico- medullary junction that displayed such features. The scores were as follows: 0, none; 1, 1%–10%; 2, 11%–25%; 3, 26%–45%; 4, 46%–75%; and 5, Figure 9. Expression of parenchymal activated CD47 determines kidney damage .75%. Histologic examination was performed after IRI. (A) Donor recipient chimerism of the hematopoietic system 8 weeks after blinded on six randomly selected corticomedul- 2/2 BM transplantation in wild-type and CD47 mice. Whole blood collected from fi fi 3 2 2 lary elds (magni cation, 200). CD47 / mice transplanted with WT BM were subjected to FACS analysis of CD47 expression and compared with relevant control groups. Analysis of (B) serum creatinine and (C) tubular damage in respective BM chimeric mice at 24 hours re- Immunostaining perfusion after bilateral renal ischemia. Data are mean 6 SEM, n=6–7 per group. Formalin-fixed, paraffin-embedded sections of 2 2 2 2 2 2 *P,0.001 WTBM→WT versus WTBM→CD47 / and CD47 / BM→CD47 / .Kid- 4 mm thickness were deparaffinized and boiled ney tissue lysate was prepared from chimeric mice and (D) TSP1 and (E) CD47 for 30 minutes in 10 mM sodium citrate buffer protein and mRNA assessed. Data are mean 6 SEM, n=6pergroup.*P,0.01 2 2 2 2 2 2 (pH 6.0). Immunohistochemistry was performed WTBM→WT versus WTBM→CD47 / ;**P=0.001 WTBM→CD47 / versus CD47 / 2 2 d using the following primary antibodies: rat anti- BM→CD47 / ; #P,0.0001 compared with WTBM→WT; P=0.45 WTBM→WT versus 2 2 mouse neutrophil (clone Ly6B.2) and macro- WTBM→CD47 / . phage (clone F4/80, both AbDSerotec, Oxford,

UK). Sections were exposed to 3% H2O2 in meth- Laser Doppler Blood Flow Analysis anol to quench endogenous peroxidases and blocked with 10% normal Renal perfusion was measured using laser Doppler imaging goat serum and Avidin/Biotin block (Vector Laboratories, Burlingame, fl (MoorLDI-2%; Moor Instruments, Devon, UK). Brie y, animals CA). The primary antibody was incubated for 2 hours at room tem- were anesthetized and core temperature maintained at 37°C. perature, sections were washed with PBS, and then biotinylated goat fl Renal blood ow was assessed at baseline, in response to ischemia and anti-rat secondary antibody was added in combination with a Vec- reperfusion at 30 minutes and 24 hours. Results are expressed as the tastain ABC kit (Vector Laboratories) and a metal enhanced 3,39 percent change from baseline control of the region of interest. diaminobenzidine substrate kit (Thermo Scientific, Waltham, MA). Sections were counterstained for hematoxylin, dehydrated, CD47 Antibody Treatment and covered. Quantification of the cellular infiltrate was performed WT C57BL/6 mice were randomized to receive either anti-mouse in a blinded manner by assessing 20 consecutive high-power fields CD47 mAb (clone 301, Santa Cruz Biotechnology; 0.4 mg/g (magnification, 3400).

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primers and probe on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Thermal cycling conditions were 50°C for 2 minutes, 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Data were analyzed using the DDCt method with expression normalized to the housekeeping gene and WT sham-operated animals used as the referent control.

Western Blotting Kidney tissue was homogenized in ice-cold lysis buffer. Supernatants were collected and lysates quantiﬁed using a Bradford assay (BioRad, Hercules, CA). Thirty micrograms of total protein was resolved by SDS-PAGE and transferred onto nitrocellulose (BioRad). In blots for CD47, nonreducing Laemmli buffer was used with 8% SDS-PAGE. Blots were probed with primary antibody to the respective proteins and visualized on an Odyssey Imaging System (Licor, Lincoln, NE). The intensity of the bands was quantiﬁed using Image J.

Generation of BM Chimeric Mice 2 2 BM was collected from WTor CD47 / mice by flushing femurs and tibiae with HBSS. Recipient mice were lethally irradiated with two doses of 5 Gy from a 137Cs source using a Mark1 irradiator Figure 9. Continued. (JL Shepherd & Associates, San Fernando, CA). Six hours after irradiation, recipient mice were given 13107 BM cells retro-orbitally. The ani- Renal Parenchymal Cellular Apoptosis mals were allowed to recover for 8 weeks to ensure stable engraftment TUNEL staining was used to detect apoptosis with a commercially before being subjected to renal IRI. Full chimerism of each mouse was available in situ cell death detection kit (Roche, Basel, Switzerland), confirmed by flow cytometry of whole blood. performed according to the manufacturer’sinstructions. Statistical Analyses Renal Oxidative Stress The data are presented as mean 6 SD unless otherwise stated. Data DHE (Molecular Probes) was used to evaluate in situ production of were analyzed with the t test (parametric variables) or Mann–Whitney superoxide. DHE (10 mM) was applied to unfixed frozen sections, U test (nonparametric variables) for means between two groups or incubated in a light-protected humidified chamber at 37°C for 30 ANOVA between multiple (.2) groups using STATA software (version minutes, washed with PBS, and mounted with fluorescent mounting 11.0; STATA Corporation, College Station, TX). In all cases, P,0.05 medium (DAKO). Images were quantified using Image J software was deemed significant. (http://rsbweb.nih.gov/ij/).

RNA Extraction and Quantification by Real-Time PCR ACKNOWLEDGMENTS Total RNA was extracted using Qiagen RNeasy Mini Kits (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. RNA was This work was supported by the National Heart Lung and quantified using the Take3 Gen5 spectrophotometer (BioTek, Blood Institute (R01 HL-108954 to J.S.I.), the National Institutes Winooski, VT). One microgram of RNA was treated with DNase I of Health (1P01HL103455-01), the American Heart Association (amplification grade; Invitrogen) and then reverse-transcribed using (11BGIA7210001 to J.S.I.), the Institute for Transfusion Medicine the Superscript III First Strand Synthesis SuperMix (Invitrogen). cDNA and the Hemophilia Center of Western Pennsylvania (to J.S.I.), and was amplified using Platinum Quantitative PCR SuperMix-UDG the Australian National Health and Medical Research Council (Invitrogen) in 20-ml volumes in quadruplicate with gene-specific (APP1016276 C.J. Martin Award to N.M.R.).

J Am Soc Nephrol 23: 1538–1550, 2012 Tissue CD47 Promotes Renal IRI 1547 BASIC RESEARCH www.jasn.org

DISCLOSURES J.S.I. is chair of the scientiﬁc advisory board of Vasculox Inc. (St. Louis, MO) and Radiation Control Technologies Inc. (Rockville, MD).

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