Overexpression of Human CD55 and CD59 Or Treatment with Human CD55 Protects Against Renal Ischemia-Reperfusion Injury in Mice

Overexpression of Human CD55 and CD59 or Treatment with Human CD55 Protects against Renal Ischemia-Reperfusion Injury in Mice This information is current as of October 1, 2021. Anjan K. Bongoni, Bo Lu, Evelyn J. Salvaris, Veena Roberts, Doreen Fang, Jennifer L. McRae, Nella Fisicaro, Karen M. Dwyer and Peter J. Cowan J Immunol published online 12 May 2017

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 12, 2017, doi:10.4049/jimmunol.1601943 The Journal of Immunology

Overexpression of Human CD55 and CD59 or Treatment with Human CD55 Protects against Renal Ischemia-Reperfusion Injury in Mice

Anjan K. Bongoni,*,1 Bo Lu,*,1 Evelyn J. Salvaris,* Veena Roberts,*,† Doreen Fang,*,† Jennifer L. McRae,* Nella Fisicaro,* Karen M. Dwyer,‡ and Peter J. Cowan*,†

Deficiency in the membrane-bound complement regulators CD55 and CD59 exacerbates renal ischemia-reperfusion injury (IRI) in mouse models, but the effect of increasing CD55 and CD59 activity has not been examined. In this study, we investigated the impact of overexpression of human (h) CD55 6 hCD59 or treatment with soluble rhCD55 in a mouse model of renal IRI. Unilaterally nephrectomised mice were subjected to 18 (mild IRI) or 22 min (moderate IRI) warm renal ischemia, and analyzed 24 h after reperfusion for renal function (serum creatinine and urea), complement deposition (C3b/c and C9), and infiltration of neutrophils and macrophages. Transgenic mice expressing hCD55 alone were protected against mild renal IRI, with reduced creatinine and Downloaded from urea levels compared with wild type littermates. However, the renal function of the hCD55 mice was not preserved in the moderate IRI model, despite a reduction in C3b/c and C9 deposition and innate cell infiltration. Mice expressing both hCD55 and hCD59, on the other hand, were protected in the moderate IRI model, with significant reductions in all parameters measured. Wild type mice treated with rhCD55 immediately after reperfusion were also protected in the moderate IRI model. Thus, manipulation of CD55 activity to increase inhibition of the C3 and C5 convertases is protective against renal IRI, and the additional expression of hCD59, which regulates the terminal complement pathway, provides further protection. Therefore, anti-complement therapy using http://www.jimmunol.org/ complement regulatory proteins may provide a potential clinical option for preventing tissue and organ damage in renal IRI. The Journal of Immunology, 2017, 198: 000–000.

schemia-reperfusion injury (IRI) is a common problem oc- potentiates the inﬂammatory response compounding the existing curring in diverse clinical settings such as transplantation, damage (1). IRI signiﬁcantly contributes to morbidity and mor- I vascular surgery trauma, cardiac surgery, and shock. IRI is tality. Therefore, therapeutic prevention and treatments of IRI are encountered when an organ’s circulation is restored after being being actively pursued, particularly in acute renal failure and kidney

transiently deprived of blood flow. IRI is a complex phenomenon transplantation. by guest on October 1, 2021 involving an inflammatory response to cellular injury. Ischemia Complement participates in the pathogenesis of IRI, and acti- results in necrosis and apoptosis; restoration of blood flow (reper- vation of complement is a major component of renal IRI (2). The fusion), although critical to prevent ongoing injury, paradoxically complement system is part of the innate immune response to pathogens and foreign Ags. It comprises a set of circulating proteins that recognize and respond to signals on foreign or injured *Immunology Research Centre, St. Vincent’s Hospital Melbourne, Fitzroy, Victoria 3065, Australia; †Department of Medicine, University of Melbourne, Melbourne, cells (3). A critical stage in the activation process is the formation ‡ Victoria 3065, Australia; and School of Medicine, Faculty of Health, Deakin Uni- of the multisubunit C3 and C5 convertases on the surface of target versity, Waurn Ponds, Victoria 3216, Australia cells. The convertases cleave C3 and C5 into soluble (a) and 1A.K.B. and B.L. have equal first authorship. membrane-bound (b) products. C3a and particularly C5a are po- ORCIDs: 0000-0003-2312-0374 (A.K.B.); 0000-0001-7741-0743 (E.J.S.); 0000-0002- 2487-9019 (N.F.); 0000-0002-4376-9720 (K.M.D.); 0000-0001-9016-4954 (P.J.C.). tent proinflammatory signals; C3b further amplifies complement Received for publication November 15, 2016. Accepted for publication April 14, activation; and C5b forms the scaffold upon which the lytic 2017. membrane attack complex (MAC, C5b-9) is assembled. Com- This work was supported by grants from the Swiss National Science Foundation plement activation on intact self cells is tightly regulated by a (Fellowship P2BEP3_155459) and the National Health and Medical Research Coun- number of circulating and membrane-bound complement regula- cil of Australia. tory proteins (CRPs). Two key membrane-bound CRPs are CD55, A.K.B. and B.L. participated in the research design, writing of the article, performance of the research, and data analysis; E.J.S. participated in performance of the which accelerates decay of the C3 and C5 convertases, and CD59, research and data analysis; V.R. and D.F. participated in the research design, perfor- which prevents assembly of the MACs (3). In pathophysiological mance of the animal surgery and research, and data analysis; J.L.M. and N.F. par- states such as IRI, these regulatory mechanisms are overwhelmed ticipated in performance of the research and data analysis; K.M.D. participated in the research design; P.J.C. participated in the concept and design of the study, perfor- and unable to prevent complement-mediated damage. In addition, mance of experiments, analyzing the data, and writing the manuscript, and carried the there is significant crosstalk between complement and other arms main responsibility for the study. of innate immunity such as the TLR system (3), which has also Address correspondence and reprint requests to Dr. Peter J. Cowan, Immunology Research Centre, St. Vincent’s Hospital Melbourne, P. O. Box 2900, Fitzroy, Victoria been implicated in renal IRI (4). 3065, Australia. E-mail address: [email protected] CD55 in particular appears to be important in determining the Abbreviations used in this article: CRP, complement regulatory protein; h, human; response to renal IR. Expression of CD55 was significantly reduced IRI, ischemia-reperfusion injury; MAC, membrane attack complex; NMS, normal after reperfusion in a rat model of renal warm IRI, whereas the mouse serum; Tg, transgenic; WT, wild type. levels of other membrane-bound CRPs (CD59 and CD46) were Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 unaffected (5). Postconditioning (repeated short cycles of IR after

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601943 2 RENAL ISCHEMIA-REPERFUSION INJURY IN hCD55/hCD59 Tg MICE the initial ischemic challenge) prevented this downregulation of a FLUOstar Omega microplate reader (BMG Labtech, Mornington, Aus- CD55 and resulted in significant protection from IRI (5). Fur- tralia). Supernatants from the assay were collected in 10 mM EDTA and thermore, mice deficient in CD55 exhibited worse renal warm IRI assayed for C5a using the Mouse C5a DuoSet ELISA kit (DY2150; R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. than controls (6). A definitive role for CD59 in renal IRI has been harder to establish in mouse models because mice express two Warm renal IRI model closely linked CD59 genes (CD59a and CD59b), and the relative Mice were anesthetized by i.p. administration of ketamine 85 mg/kg and functional importance of the two isoforms is controversial (7, 8). xylazine 15 mg/kg. After the mice were anesthetized, they were placed on Mice deficient in CD59a were no more susceptible to renal warm a heating pad to maintain their core body temperature at 37˚C during the IRI than controls (6), although this may simply reflect redundancy. surgery. A midline abdominal incision was made and the renal pedicles were bluntly dissected. After right nephrectomy, a microvascular clamp Interestingly, however, IRI was more severe in CD55/CD59a (Roboz, Rockville, MD) was placed on the left renal pedicle for 18 or 22 double-deficient mice than in CD55-deficient mice, suggesting min, while the animal was kept at 37˚C in an incubator and well hydrated. synergy between the CRPs (6). For rhCD55 treatment, 2 mg rhCD55 (R&D Systems) in 0.2 ml PBS was Although it is clear that reducing the expression of CD55 and injected via the penile vein right after starting reperfusion. The clamp was possibly CD59 exacerbates renal warm IRI, the concept that in- removed after ischemia and the kidney observed to confirm complete reperfusion. The surgical wounds were then sutured in two layers with creasing the level of these CRPs may protect against renal IRI has 5-0 silk. Warm normal saline (100 ml/kg) was instilled into the peritoneal not been investigated. This may be particularly pertinent in xeno- cavity during the procedure. The mice were allowed to recover for 2 h transplantation, in which human (h) CRPs have been expressed in under a heating lamp and then kept on a heating pad. The mice were transgenic (Tg) pigs to prevent hyperacute rejection (9) but may euthanized 24 h after reperfusion, and blood and kidney samples obtained. have additional benefit in attenuating IRI. In this study, we used Assessment of renal function mild and moderate models of renal warm IRI to examine Tg Downloaded from 6 Renal function was assessed by measuring serum creatinine and urea using mouse lines that strongly express hCD55 hCD59. Both hCRPs a kinetic colorimetric assay based on the Jaffe method, analyzed on an have been shown to regulate complement activation in mice (10–12). Olympus AU 2700 (Integrated Sciences, Chatswood, Australia) at the Comparison of the degree of renal IRI in these lines and non-Tg Pathology Department, St. Vincent’s Hospital Melbourne. littermate controls has allowed us to determine whether increased Renal histology and scoring endogenous CD55 activity is protective, and whether coexpression of CD59 adds further protection. In addition, we tested whether Kidney tissue sections (4 mm) were 10% formalin fixed and paraffin em- http://www.jimmunol.org/ treatment with rhCD55 protects wild type (WT) mice from renal bedded then stained with H&E. Histology scores were assessed by a IRI. rhCD55 injected into mice prior to reperfusion of the intestine or hind limb has been shown to be deposited on the blood vessel endothelium in several tissues, and can attenuate both local and remote IR-associated injury in these models (11, 12). To our knowledge, this is the first study to suggest that increased activity of the complement regulator hCD55, either through endogenous overexpression or exogenous administration, reduces by guest on October 1, 2021 injury in mouse models of renal IRI. In addition, renal IRI was further attenuated by coexpression of hCD59 with hCD55.

Materials and Methods Mice All animal experiments were conducted in compliance with the Australian Code of Practice for the Care and Use of Laboratory Animals for Scientific Purposes (Eighth edition, 2013) and the Prevention of Cruelty to Animals Act 1986, Victoria, Australia. Mice were bred and housed in an approved animal facility at St. Vincent’s Hospital, Melbourne, Australia, and all experiments were carried out with the approval of the Animal Ethics Committee of St. Vincent’s Hospital Melbourne. Male 10–12-wk old mice from Tg lines hCD55 and hCD55/hCD59, which express hCD55 6 hCD59 from the mouse H-2Kb (MHC class I) promoter, were used with WT littermates as controls. These Tg mice were generated on the CBA 3 C57BL/6 F1 background, and have been back- crossed onto C57BL/6 for more than 10 generations. For experiments with rhCD55, C57BL/6 mice purchased from Animal Resource Centre (Canning Vale, Western Australia) were used. Experimental groups (n =8–12per group) were as follows: 1) hCD55 Tg mice IRI; 2) hCD55/hCD59 Tg mice IRI; 3) WT littermate mice IRI; 4) C57BL/6 WT mice IRI treated with rhCD55; 5) C57BL/6 WT mice IRI treated with rhCD55 vehicle; 6) hCD55 mice sham control; 7) hCD55/hCD59 mice sham control; and 8) WT FIGURE 1. Transgenically expressed hCD55 and hCD59 regulate littermate mice sham control. Sham-operated mice had right nephrectomy mouse complement. (A and B) Flow cytometric analysis of hCD55 (A) and only, without IR. hCD59 (B) expression on PBMC from WT and hCD55 6 hCD59 Tg mice. In vitro mouse complement activation assay (C and D) Regulation of mouse complement activation by mouse PBMC expressing hCD55 6 hCD59. WT normal mouse serum (NMS, 5%) was The activation of complement in mouse serum was measured using a incubated in mouse IgM-coated 96-well plates in the presence or absence modification of the mouse complement ELISA assay kit HIT420 (Hycult Biotech, Uden, the Netherlands). A 5% WT normal mouse serum (NMS) was of PBMC (50,000 cells per well) for 60 min at 37˚C. Complement acti- C incubated in the wells of mouse IgM-coated microplates for 60 min at 37˚C in vation was calculated by measuring deposition of C5b-9 ( ) and generation the presence or absence of mouse PBMCs (50,000 cells per well). Depo- of C5a in the supernatant (D). Data are mean 6 SD (n = 5), and statistical sition of the terminal C5b-9 complex was measured according to the analysis was carried out by one-way ANOVA testing with Bonferroni manufacturer’s protocol, with color development quantified at 450 nm using correction (*p # 0.05, **p # 0.01, ***p # 0.001). The Journal of Immunology 3 semiquantitative method (13). Briefly, three high-power fields in each of complement. First, PBMCs were isolated from WT, hCD55/ the cortex, cortical-medullary junction, and medulla in a minimum of two hCD59, and hCD55 Tg mice, and expression of the transgenes on sections were assessed in a blinded fashion. The degree of tubular ne- the cell surface was demonstrated by flow cytometry (Fig. 1A, crosis was graded and a modified scoring system was used: 0 = normal kidney; 1 = minimal (#10% involvement); 2 = mild (10–25% involve- 1B). Next, WT NMS (5%) was incubated in mouse IgM-coated ment); 3 = moderate (26–50% involvement); 4 = severe (51–75% in- 96-well plates to activate complement, in the presence or absence volvement); and 5 = very severe (.75% involvement). of PBMC. The degree of complement activation was measured Immunofluorescence using two methods: deposition of C5b-9, and generation of C5a. A significant reduction in C5b-9 deposition was observed in the m Snap-frozen biopsy samples were cut into 5- m–thick sections, air dried, presence of hCD55/hCD59 (p , 0.01) or hCD55 (p , 0.05) and either processed immediately or stored at 280˚C until further analysis. After fixation with acetone and hydration, the sections were stained using: PBMC as compared with WT PBMC (Fig. 1C). Similarly, C5a rabbit anti-C3b/c FITC (Dako), rabbit anti-mouse C9 Alexa 488 (Bioss generation was significantly reduced in the presence of hCD55/ Antibodies, Woburn, MA), rat anti-mouse Ly-6G/6C FITC (Hycult Bio- hCD59 or hCD55 PBMC (both p , 0.05) as compared with WT tech), rat anti-mouse F4/80 FITC (Bio-Rad, Raleigh, NC), mouse anti- PBMC (Fig. 1D). These results demonstrated that expression of hCD55 FITC (clone IH4), mouse anti-hCD59 FITC (clone MEM-43) or hCD55 6 hCD59 inhibited activation of mouse complement. FITC-conjugated isotype-matched control Abs: rabbit IgG (Thermo Fisher Scientific, Invitrogen, Waltham, MA), and rat IgG1 (BD Biosciences, San Renal expression of hCD55 and hCD59 in Tg mice Diego, CA). The slides were analyzed using a fluorescence/confocal mi- croscope (Nikon A1R). Quantification of fluorescence intensity as raw Frozen kidney samples were stained to determine the pattern and integrated density was performed using ImageJ software, version 10.2 level of expression of hCD55 and hCD59. As expected, there was (National Institutes of Health) on unmanipulated TIFF images. no expression of hCD55 and hCD59 on WT tissue, whereas Tg Statistical analysis tissue showed strong expression, mainly on tubular (Fig. 2A, Downloaded from 2C) and glomerular epithelium (upper inset panels, denoted as Multiple groups were compared using one-way ANOVA with a posttest Bonferroni correction (GraphPad Prism 5.0). Two groups were compared G), and vascular endothelium (lower inset panels, denoted as using an unpaired Student t test (two-tailed) or a Mann–Whitney U test. V). Quantitative image analysis demonstrated that the level Results are expressed as mean 6 SEM or SD. A p value ,0.05 was of renal hCD55 expression was similar in the two Tg lines considered to be statistically significant. (Fig. 2B, 2D).

Immunoﬂuorescence analysis showed that there was no signif- http://www.jimmunol.org/ Results icant change in the level and distribution of hCD55 and hCD59 Regulation of mouse complement activation in vitro by expression in the kidneys of Tg mice at 24 h postreperfusion transgenically expressed hCD55/hCD59 (Fig. 2B, 2D). The distribution of rhCD55 within the kidney in Before testing the hCD55 6 hCD59 mice in the renal IRI model, treated WT mice was also analyzed post-IRI. rhCD55 was de- an in vitro assay was used to conﬁrm that hCD55 and hCD59 tected on tubules, glomeruli, and vascular endothelium (Fig. 2E), expressed on Tg mouse cells were capable of regulating mouse a similar distribution to that of transgenically expressed hCD55 by guest on October 1, 2021

FIGURE 2. Expression of hCD55 and hCD59 in mouse kidneys. Frozen sections were examined for hCD55 and hCD59 expression by immunoﬂuo- rescence/confocal staining and ImageJ/Prism analysis. Nuclei were stained with DAPI. Scale bar, 50 mm. (A) hCD55 and hCD55/hCD59 Tg mice both showed strong expression of hCD55, mainly on the tubular and glomerular epithelium (inset image, denoted as G) and vascular endothelium (inset, denoted as V). There was no difference in the level of hCD55 expression before and 24 h after moderate IR (B). hCD55/hCD59 Tg mice showed strong expression of hCD59 (C) with no difference 24 h after moderate IR (D). WT mice treated with 2 mg rhCD55 immediately after reperfusion showed tubular and glomerular (inset, G) epithelial and vascular endothelial (inset, V) deposition of hCD55 in the kidney 24 h after moderate IR (E). Data are mean 6 SD (n = 3–5) and statistical analysis was carried out by one-way ANOVA testing with Bonferroni correction (*p # 0.05, **p # 0.01, ***p # 0.001). 4 RENAL ISCHEMIA-REPERFUSION INJURY IN hCD55/hCD59 Tg MICE

creatinine and urea levels than WT littermates (Fig. 3A, 3B; both p , 0.05 versus WT). We next tested the hCD55 6 hCD59 mice in the moderate IRI model, in which the duration of ischemia was extended to 22 min. The longer period of ischemia increased the degree of renal injury in WT littermates, reflected by an increase in the levels of serum creatinine and urea (Fig. 3C, 3D; creatinine: p , 0.01, urea: p , 0.05 versus 18 min model). hCD55/hCD59 mice were protected from moderate renal IRI, as evidenced by significantly reduced serum creatinine and urea levels compared with WT littermates (Fig. 3C, 3D; both p , 0.05 versus WT). hCD55 mice, despite showing protection in the mild renal IRI model, were not protected in the moderate IRI model (Fig. 3C, 3D). The moderate renal IRI model was also used to test the effect of treatment with rhCD55. Vehicle-treated WT C57BL/6 mice showed significant renal injury in this model (Fig. 3E, 3F). In fact, serum creatinine and urea in this group were both higher than in the WT littermate IRI group (Fig. 3C, 3D). Mice injected with rhCD55 immediately postreperfusion showed significantly reduced serum creatinine (p , 0.01) and urea (p , 0.05) compared Downloaded from with vehicle-treated controls, indicating that rhCD55 was protective in the moderate IRI model (Fig. 3E, 3F). Modulation of histopathological damage by Tg hCD55/hCD59 expression or treatment with rhCD55

Hematoxylin and eosin-stained kidney sections were examined for http://www.jimmunol.org/ histopathological changes using a ﬁve-point injury score. Repre- sentative examples of different grades of injury are shown in Fig. 4A. The WT littermate IRI groups in the mild (Fig. 4B) and moderate (Fig. 4C) models showed extensive tubular injury involving 26–75% (moderate to severe) of the renal cortex, cast formation, by guest on October 1, 2021

FIGURE 3. Tg expression of hCD55/hCD59 or treatment with rhCD55 protects mice from renal IRI. After right nephrectomy, the left renal pedicle was clamped for 18 min (mild IRI) or 22 min (moderate IRI); 24 h after reperfusion, renal function was assessed by measuring serum creatinine and urea. (A and B) Both hCD55/hCD59 and hCD55 Tg mice were protected compared with WT littermates in the mild renal IRI model. (C and D) hCD55/hCD59, but not hCD55 Tg mice, were protected compared with WT littermates in the moderate renal IRI model. (E and F) Treatment with 2 mgrhCD55afterstartingreperfusionprotectedWTmiceinthe moderate renal IRI model. Data shown are mean 6 SEM (n = 8–12); signiﬁcance was tested using one-way ANOVA with Bonferroni correction (*p # 0.05, **p # 0.01, ***p # 0.001).

(Fig. 2A, 2B). However, the intensity of staining of rhCD55 was lower at the 24 h time point than that of Tg hCD55. Reduction of renal IRI by Tg hCD55 6 hCD59 expression or FIGURE 4. Tg expression of hCD55/hCD59 or treatment with rhCD55 treatment with rhCD55 reduces tubular injury associated with renal ischemia-reperfusion. H&E- stained kidney samples were assessed for tubular injury in a blinded We first tested Tg hCD55 6 hCD59 mice in the mild IRI model, in manner using a score from 0 to 5 (0 = normal; 1 = ,10% injury, minimal; which the right kidney is removed, the left renal pedicle is 2 = 10–25%, mild; 3 = 26–50%, moderate; 4 = 51–75%, severe; 5 = clamped for 18 min, and serum creatinine and urea are measured .75%, very severe). (A) Representative tissue samples illustrating the 24 h after reperfusion. The levels of creatinine and urea were scoring system. Scale bar, 50 mm. (B and C) hCD55/hCD59, but not significantly increased in WT littermates subjected to IR hCD55 Tg mice, showed reduced tubular injury compared with WT littermates in the mild (B) and moderate (C) renal IRI models. (D) Treatment compared with sham controls (both p , 0.001), reflecting sig- with 2 mg rhCD55 immediately during reperfusion reduced tubular injury nificant deterioration in renal function (Fig. 3A, 3B). Mice in WT mice in the moderate renal IRI model. Data shown are mean 6 SEM; expressing hCD55 alone or in combination with hCD59 were statistical analyses were done by one-way ANOVA with Bonferroni correc- protected from IRI in this model, showing significantly lower tion (*p # 0.05, **p # 0.01, ***p # 0.001); n = 5–8. The Journal of Immunology 5 cell swelling and dilatation, and overall disruption of the renal kidney sections for complement C3b/c and C9 was examined by architecture. hCD55/hCD59 mice subjected to IRI showed a sig- confocal microscopy and quantitated using ImageJ software. nificant reduction in the tubular injury score compared with WT Control (sham) samples exhibited minimal C3b/c and C9 depo- littermates in both the mild (Fig. 4B) and moderate (Fig. 4C) sition. Compared to the sham group, the WT littermate IRI group models (both p , 0.05). The hCD55 IRI group also showed a re- showed significantly higher (p , 0.001) deposition of C3b/c duction in the tubular injury score, but this did not reach statistical (localized to the basement membrane of tubular and glomeru- significance (Fig. 4B, 4C). Treatment of WT C57BL/6 mice with lar epithelium and vascular endothelium) and C9 (localized to rhCD55 significantly reduced tubular injury in the moderate renal IRI the basement membrane of tubular epithelium and vascular en- model compared with vehicle-treated controls (Fig. 4D; p , 0.05). dothelium) in both the mild (Fig. 5A, 5B) and moderate (Fig. 5C, 5D) renal IRI models. Deposition of C3b/c and C9 was signifi- 6 Regulation of complement activation by Tg hCD55 hCD59 cantly reduced in the hCD55/hCD59 IRI group compared with expression or treatment with rhCD55 the WT littermate IRI group, in both models (Fig. 5A–D). For the We next investigated whether renal IRI leads to intrarenal depo- hCD55 mice, C3b/c deposition was reduced in both models, sition of complement factors. Immunofluorescence staining of although this was statistically significant only in the moderate Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Tg expression of hCD55/hCD59 or treatment with rhCD55 reduces renal IR–mediated complement deposition. Frozen kidney sections were analyzed for deposition of mouse complement proteins C3b/c and C9 using immunoﬂuorescence staining/confocal microscopy. Nuclei were stained with DAPI. Fluorescence intensities were quantitatively measured as RawIntDen by ImageJ software. In both the mild and moderate IRI models, C3b/c deposition was observed on tubular and glomerular (inset, G) epithelium and vascular endothelium, and tubular C9 deposition. (A and B) In the mild IRI model, hCD55/hCD59 Tg mice showed reduced deposition of tubular, glomerular and vascular C3b/c (A), and tubular C9 (B), whereas hCD55 Tgs showed reduced deposition of C9 only, compared with WT littermates. (C and D) In the moderate renal IRI model, both hCD55/hCD59 and hCD55 Tg mice showed reduced deposition of C3b/c (C) and C9 (D) compared with WT littermates. In both models, hCD55/hCD59 Tg mice showed further reduced deposition of C9 compared with CD55 Tg mice. (E and F) In the moderate renal IRI model, treatment of WT mice with rhCD55 reduced deposition of C3b/c (E) and C9 (F). Scale bar, 50 mm. Statistical analysis was carried out by one-way ANOVA testing with Bonferroni correction. Data are mean 6 SD (n = 5–9), *p # 0.05, **p # 0.01, ***p # 0.001. 6 RENAL ISCHEMIA-REPERFUSION INJURY IN hCD55/hCD59 Tg MICE

IRI model (Fig. 5C); C9 deposition was significantly reduced in immunofluorescence staining (Fig. 6A–F). Sham-operated kidneys both models (Fig. 5B, 5D). C9 deposition was further reduced in had no or very few infiltrating Ly-6G/6C-positive neutrophils (one the hCD55/hCD59 IRI group compared with the hCD55 IRI or two cells per high-power field). Renal IRI induced significant group in both models (Fig. 5B, 5D). Treatment of WT C57BL/5 neutrophil recruitment within the tubular interstitium at the cor- mice with rhCD55 significantly reduced C3b/c and C9 deposition ticomedullary junction of WT kidneys (p , 0.001 versus re- compared with vehicle-treated mice in the moderate IRI model spective sham). Kidneys from the hCD55/hCD59 IRI group (Fig. 5E, 5F). showed a significant reduction in neutrophil infiltration compared with kidneys from the WT littermate IRI group in the mild 6 Reduction of innate immune cell infiltration by Tg hCD55 (Fig. 6A, 6D) and moderate (Fig. 6B, 6E) IRI models. Kidneys hCD59 expression or treatment with rhCD55 from the hCD55 IRI group showed reduced neutrophil infiltration Kidney sections were examined for the presence of infiltrating in the moderate IRI model (Fig. 6B, 6E), as did kidneys from WT neutrophils, one of the hallmarks of IRI-induced inflammation, by C57BL/6 mice treated with rhCD55 (Fig. 6C, 6F). Downloaded from http://www.jimmunol.org/ FIGURE 6. Tg expression of hCD55/hCD59 or treatment with rhCD55 reduces renal IR–mediated neutrophil (A–F) and macrophage (G–L) infiltration. Frozen kidney sections were analyzed for leukocyte infiltration by immunofluorescence staining/confocal microscopy, using Abs to Ly-6G/6C and F4/80 to iden- by guest on October 1, 2021 tify neutrophils and macrophages, respectively. Nuclei were stained with DAPI. The number of cells present is expressed as count per high-power field (HPF). Compared to WT littermates, hCD55/hCD59 Tg mice showed reduced tubular interstitial infiltration of neutrophils in the mild (A and D) and moderate (B and E) models of renal IRI; macrophage infiltration was also reduced in the mild (G and J)and moderate (H and K) models. Treatment of WT mice with rhCD55 also reduced infiltration of neutrophils (C and F) and macrophages (I and L)in the moderate model. Statistical analysis was carried out by one-way ANOVA testing with Bonferroni correction. Data are mean 6 SD (n =5–9), *p # 0.05, **p # 0.01, ***p # 0.001. The Journal of Immunology 7

Sections were also analyzed for the presence of F4/80-positive of these later studies by examining the effects of Tg expression macrophages (Fig. 6G–L). Macrophage infiltration of WT kidneys of hCD55/hCD59 on inhibition of mouse complement activa- was significantly increased following renal IR, but was reduced in tion. Tg expression of hCD55/hCD59 or hCD55 on PBMC the hCD55/hCD59 and hCD55 IRI groups compared with the WT significantly inhibited Ab-mediated activation of mouse com- littermate IRI group in both the mild (Fig. 6G, 6J) and moderate plement, indicating that the hCD55 and hCD59 activities were (Fig. 6H, 6K) models. Similarly, kidneys from the rhCD55-treated not species restricted. Therefore, inhibition of complement ac- IRI mice showed significantly reduced macrophage infiltration tivation by coexpression of CD59 with CD55 may have bene- compared with vehicle-treated IRI mice in the moderate model ficial effects in anaphylatoxin- and MAC-mediated injury (Fig. 6I, 6L). associated with IRI. The impact of Tg hCD55 6 hCD59 expression or treatment By applying a unilateral in vivo renal IR protocol using two with rhCD55 on all parameters measured is summarized in different IRI models (mild and moderate), we evaluated the role of Table I. hCD55 and hCD59 in renal IRI through the use of Tg mice that overexpress hCD55 alone or in combination with hCD59. Sero- Discussion logical and histological analysis of WT kidney samples showed It has been well documented that complement is strongly asso- significant deterioration of renal function and damage to proximal ciated with the inflammatory response to renal IRI. Therefore, tubular epithelial cells. Additionally, our findings demonstrate that treatments targeted at blocking or attenuating complement acti- the severity of renal injury is influenced by the duration of is- vation have gained increasing attention over the last two decades. chemia. As expected, a longer ischemia time caused more severe The complement activation cascade can be targeted at different damage. These changes were associated with complement C3b/c levels to attenuate complement-mediated injury. In animal models, and C9 deposition, which was mainly localized to the tubular Downloaded from renal IRI can be abrogated by complement inhibitors such as epithelium, and immune cell infiltration of the tubular interstitium, soluble complement receptor 1 (14, 15), small interfering RNAs predominantly at the corticomedullary junction. Our findings are in targeting C3 and C5 (16, 17), anti-C5 Abs (15, 18), anaphylatoxin line with the observation that, in contrast to other organs, the receptor antagonists (19), or manipulation of C3, C5 or C6 in primary site of complement activation in renal IRI is the paren- knockout mice (20, 21). However, despite numerous attempts in chyma (21). This may be related to the fact that CD55 and CD59 recent years, the effective therapy of renal IRI by blocking com- are expressed minimally or not at all on proximal tubules in the http://www.jimmunol.org/ plement activation has not been reported. In this study, to our normal mouse kidney (29, 30). However, ubiquitous expression of knowledge we demonstrated for the first time that inhibition of the hCD55 and hCD59 in Tg mice significantly abrogated both the complement system by Tg overexpression of hCRPs (hCD55 and mild or moderate IRI-induced renal dysfunction by reducing C3b/c hCD59) or treatment with exogenous rhCD55 is protective and C9 deposition. Inhibition of the formation of C3-/C5- against IR-induced renal damage in a mouse model. convertases and MAC in our renal model of IRI also prevented The role of complement in renal IRI has been addressed by extensive secondary cellular damage and inflammation and thus multiple investigators using rodent models. Predominantly, ana- improved renal function. phylatoxin (C3a and C5a), anaphylatoxin-receptor (C3aR and Neutrophils and macrophages are critical early initiators of by guest on October 1, 2021 C5aR)–mediated and MAC-mediated mechanisms were described innate immunity in the kidney and play critical role in inflammation to play a pathogenic role in renal IRI (18, 21–23). The kidney subsequent to IR (31–33). In renal IRI, activated endothelial cells represents a major extrahepatic site for the synthesis of comple- and proximal tubular epithelial cells produce cytokines and che- ment C3 (20), the major protein involved in all three pathways of mokines that induce inflammatory cell infiltration. This upregu- complement. Therefore, complement-focused therapies in the lation of cytokines upon renal IRI is largely attributed to setting of renal IRI should be targeted at blocking anaphylatoxin complement activation, because regulation of the complement functions as well as preventing MAC-mediated injury. Under system inhibits the induction of cytokine secretion (18). In addi- normal conditions, activation of complement on autologous tis- tion, the MAC has been reported to be involved in neutrophil sues is regulated by soluble and cell surface-bound CRPs. In influx and to induce apoptosis and necrosis (21). Our data showed several early studies, membrane CRPs were implicated as ho- significantly increased infiltration of neutrophils and macrophages mologous restriction factors that provided protection specifically in WT kidneys post IRI. The mechanism by which infiltrating against homologous complement (24–27). However, later work cells cause kidney injury is not clear, but likely involves the re- demonstrated that hCRPs are not species specific in that they can lease of cytokines, free radicals, and other inflammatory stimu- inhibit complement from many other species including rodents, lants as well as direct endothelial activation and injury (34). sometimes more efficiently than they regulate homologous com- Furthermore, the appearance of neutrophils and macrophages also plement (28). Our first experiments replicated and extended some mediates Ab-independent innate immunity activation to cause

Table I. Summary of changes in markers of renal IRI in hCD55 6 hCD59 Tg mice, and in WT mice treated with rhCD55

hCD55 Tg hCD55/hCD59 Tg WT + rhCD55 189 Ischemia 229 Ischemia 189 Ischemia 229 Ischemia 229 Ischemia Serum creatinine ↓ (p , 0.05) n.s. ↓ (p , 0.05) ↓ (p , 0.05) ↓ (p , 0.001) Serum urea ↓ (p , 0.01) n.s. ↓ (p , 0.05) ↓ (p , 0.05) ↓ (p , 0.001) Tubular injury n.s. n.s. ↓ (p , 0.05) ↓ (p , 0.05) ↓ (p , 0.05) C3b/c deposition n.s. ↓ (p , 0.01) ↓ (p , 0.01) ↓ (p , 0.001) ↓ (p , 0.001) C9 deposition ↓ (p , 0.01) ↓ (p , 0.05) ↓ (p , 0.001) ↓ (p , 0.001) ↓ (p , 0.01) Neutrophil infiltration n.s. ↓ (p , 0.05) ↓ (p , 0.001) ↓ (p , 0.001) ↓ (p , 0.001) Macrophage infiltration ↓ (p , 0.05) ↓ (p , 0.01) ↓ (p , 0.001) ↓ (p , 0.001) ↓ (p , 0.01) ↓, significantly reduced compared with WT littermate IRI group (hCD55 6 hCD59 IRI) or vehicle-treated IRI group (rhCD55 IRI). n.s., not significant. 8 RENAL ISCHEMIA-REPERFUSION INJURY IN hCD55/hCD59 Tg MICE severe tissue damage (35). In this study, Tg expression of hCD55/ Acknowledgments hCD59 significantly inhibited the tissue infiltration of neutrophils We thank staff at the Immunology Research Centre, St. Vincent’s Hospital and macrophages after IR. These data indicate that activation of Melbourne for technical support and advice and staff at the BioResources complement C3 and C5 is central to the influx of immune cells. Centre for animal care. Moreover, Tg hCD55 6 hCD59-mediated reduction of renal neutrophil and macrophage infiltration may also have contributed to the Disclosures improved renal function by reducing cellular damage. The authors have no financial conflicts of interest. Nevertheless, the fact that hCD55/hCD59 mice were more protected against renal IRI than hCD55 mice raises the possibility that C3-independent MAC formation may play a part in renal IRI References 1. Aydin, Z., A. J. van Zonneveld, J. W. de Fijter, and T. J. Rabelink. 2007. New (36). Recent studies on the crosstalk between complement and horizons in prevention and treatment of ischaemic injury to kidney transplants. coagulation illustrate the ability of certain coagulation enzymes, Nephrol. Dial. Transplant. 22: 342–346. such as thrombin, to directly activate C5 independently of C3 to 2. Damman, J., T. A. Schuurs, R. J. Ploeg, and M. A. Seelen. 2008. Complement and renal transplantation: from donor to recipient. Transplantation 85: 923–927. form the MAC, a mechanism that is not regulated by CD55 (37). 3. Ricklin, D., G. Hajishengallis, K. Yang, and J. D. Lambris. 2010. Complement: a These complement–coagulation interactions contribute to the de- key system for immune surveillance and homeostasis. Nat. Immunol. 11: 785–797. velopment of life-threatening complications in the context of 4. Wu, H., G. Chen, K. R. Wyburn, J. Yin, P. Bertolino, J. M. Eris, S. I. Alexander, A. F. Sharland, and S. J. Chadban. 2007. TLR4 activation mediates kidney diseases with an inflammatory pathogenesis (38). It would there- ischemia/reperfusion injury. J. Clin. Invest. 117: 2847–2859. fore seem logical to combine expression of CD59 with expression 5. Wang, W., T. Tang, P. Zhang, and H. Bu. 2010. Postconditioning attenuates renal of CD55 and our results emphasize the potential benefit of using ischemia-reperfusion injury by preventing DAF down-regulation. J. Urol. 183:

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