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Basic and Experimental Research

Blood Group Antigen-Targeting Peptide Suppresses Anti-Blood Group Antibody Binding to Antigen in Renal Glomerular Capillaries After ABO-Incompatible Blood Reperfusion

Yoneyama, Tohru1; Hatakeyama, Shingo1; Tobisawa, Yuki1; Yamamoto, Hayato2; Imanishi, Kengo2; Okamoto, Teppei2; Tokui, Noriko2; Sugiyama, Naoki2; Suzuki, Yuichiro2; Kudo, Shigemasa2; Yoneyama, Takahiro2; Hashimoto, Yasuhiro2; Koie, Takuya2; Kamimura, Noritaka2; Fukuda, Michiko N.3; Ohyama, Chikara1,2,4

Author Information
doi: 10.1097/TP.0b013e3182795b9c
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Abstract

Kidney transplantation (KTx) is the best treatment for chronic kidney failure. However, the donor population in Japan is tenfold smaller than that in the United States and European countries, and the lack of donors is a problem even in Western countries (1–3). To address this chronic shortage of kidney donors, it has been necessary to greatly increase the number of potential donor–recipient combinations with ABO-incompatible (ABO-I) KTx. Although the use of ABO-I donor kidneys is a possible solution to the shortage of donor kidneys for transplantation, natural antibodies and de novo antibodies against ABO histo-blood group antigens are a major barrier to successful ABO-I KTx. Recently, effective antibody removal and several immunosuppressive treatments have improved graft survival, especially in KTx (4–8). However, graft failure caused by antibody-mediated rejection (AMR) or serious infections due to overimmunosuppression still occurs in some patients.

Montgomery et al. (9) reported that long-term survival of ABO-I recipients was not significantly different from that of ABO-compatible matched controls. However, graft loss was significantly higher, particularly in the first 14 days after transplantation, although little to no difference was observed between the transplant groups after day 14. With ABO-I liver transplantation, recipients with high anti-blood group antibody titers were also at very high risk of graft failure (10, 11). Therefore, ABO incompatibility in organ transplantation remains a high risk factor for AMR despite the progress in effective treatments. Because ABO incompatibility has not been completely overcome, development of a novel treatment for AMR is of considerable importance.

AMR results from complement activation and procoagulation after an antigen–antibody reaction. AMR is associated with ABO histo-blood group antigens and antibodies as well as with complement and coagulation factors. Clinical treatments have been developed to control antibodies and complement as well as coagulation. These treatments include antibody removal by plasmapheresis, use of antimetabolites and anti-CD20 monoclonal antibodies (mAbs) for the suppression of antibody production, use of intravenous immunoglobulin for complement inhibition, and use of anticoagulant and antiplatelet drugs for the suppression of microthrombi formation. However, the option of direct modification or blocking of ABO blood group antigens in grafts or neutralization of anti-A/B antibodies is not yet available for clinical use and has been reported in only a few articles. These reported methods include neutralization of blood group A antigen by a monoclonal anti-A antibody F(ab) fragment (12), removal of A/B blood group antigens from baboon kidney by in vivo and ex vivo administration of endo-β-galactosidase (13), and neutralization of preformed anti-A/B antigen antibodies in baboons by intravenous infusion of an ABO blood group trisaccharide carbohydrate epitope (14).

Because antibody drugs are expensive and sometimes cause serious side effects, a specific peptide that functions as an antibody mimic could provide a practical biological tool and a potent alternative to antibody-based therapies. Peptide-displaying phage technology provides a method for identifying short peptide sequences that are specific for a target. This technology may provide a means for identifying peptide sequences that mimic specific carbohydrate or antibody epitopes. Several peptide sequences that inhibit interactions between carbohydrate-binding proteins and their ligands have been identified using peptide-displaying phage technology (15–18). In this study, we have used a peptide-displaying phage system to screen for an ABO histo-blood group antigen-targeting peptide (BATP) that has the ability to suppress AMR.

RESULTS

Screening of Blood Group Antigen-Targeting Peptides

We identified a blood group A/B antigen-targeting peptide using T7 phage-displayed 7-mer random peptide library. After the fourth round of screening, we achieved significant enrichment of A and B trisaccharide-binding phage clones (Fig. 1A,B). We picked 12 phage clones from the A and B trisaccharide-binding phage pools after the fourth round of screening and determined six peptide sequences (Fig. 1C) that contained two common sequences (RPRNPNK and SPARRPR) identified in both A and B trisaccharide-binding phage clones. Three of the peptide sequences (ASNKRPR, RPRNPNK, and SPARRPR) contained an RPR (Arg-Pro-Arg) motif. We therefore suspected that the RPR motif was important for recognizing both A and B trisaccharide structures.

F1-3
FIGURE 1:
Screening of A and B trisaccharide BSA-binding peptides and characterization of BATP. A, four rounds of screening of A trisaccharide BSA-binding phage. B, four rounds of screening of B trisaccharide BSA-binding phage. C, peptide sequences of enriched phage clones identified from the A and B phage clone pools after the fourth round of screening. *,** indicate identical sequences identified from both phage clone pools. D, HA inhibition assay. HA assays were performed by the microtitration method. Each titer was determined after a 2-hr incubation of anti-A/B antibodies with 500 μg/mL BATP blocked HA of A/B RBCs. RBCs without BATP exhibited the maximum titer of 211 (type A RBCs) and 210 (type B RBCs). Data were obtained from a representative experiment repeated three times. E, dissociation constant of BATP with A and B trisaccharide BSA. F, inhibitory effect of BATP on the binding of anti-A/B antibodies to A/B antigens. Blood group A or B trisaccharide BSA-coated wells were blocked with BATP at the indicated concentrations for 1 hr at RT. Anti-A/B antibodies (1:1000 dilution in TBST) were added to each well and incubated for 1 hr at RT. Using HRP-conjugated anti-rabbit IgM, changes in antigen–antibody interactions were monitored using a microplate reader (Multiskan FC; Thermo Scientific, Waltham, MA). The level of antibody binding without BATP was considered 100%. The results are expressed as mean±standard deviation of three independent experiments. G, cytotoxicity of BATP with blood type O HGMEC. Blood type O HGMEC was cultured near confluent. Then, cells were incubated with 1 mg/mL control (Ctrl) peptide or BATP and 5 μM CellEvent caspase-3/7 detection reagent at 37°C. Caspase-3/7–positive cells were counted by fluorescent microscope. BSA, bovine serum albumin; BATP, blood group antigen-targeting peptide; HA, hemagglutination; HGMEC, human kidney glomerular capillary microvascular endothelial cells; HRP, horseradish peroxidase; RT, room temperature; TBST, TBS containing 0.5% Tween 20.

Characterization of Blood Group Antigen-Targeting Peptides

A hemagglutination (HA) inhibition assay was used to analyze the ABO blood group antigen-blocking effect of the peptide (Fig. 1D). Red blood cells (RBC) treated with each of the six peptides exhibited decreased HA activity (22–3 of the control for blood group A RBC and 21–3 of the control for blood group B RBC). Although these peptides had only a weak inhibitory effect on HA activity, we found that the RPRNPNK peptide had the strongest inhibitory effect of all the peptides tested, suggesting that it may bind to A and B blood group antigens on RBCs and inhibit antigen–antibody interactions. We therefore selected RPRNPNK (designated as BATP) for use in subsequent experiments.

Enzyme-linked immunosorbent assay (ELISA) was used to analyze the specificity and affinity of BATP for A and B trisaccharide bovine serum albumin (BSA). We first investigated the binding affinity of BATP for A and B trisaccharide BSA (Fig. 1E). The dissociation constant (Kd) of BATP was determined to be 79.24 μM for A trisaccharide BSA and 79.48 μM for B trisaccharide BSA. We next investigated the dose-dependent inhibitory effects of BATP on anti-A/B antibody binding to A and B trisaccharide BSA (Fig. 1F). The addition of more than 200 μg/mL BATP decreased the binding levels of anti-A/B antibodies to A and B trisaccharide BSA to approximately 20% of the levels observed in the controls. The IC50 value of BATP was found to be 91.70 μM for anti-A antibody and 92.88 μM for anti-B antibody. To investigate the cytotoxicity of BATP, we added BATP (1 mg/mL) to the blood type O human kidney glomerular capillary microvascular endothelial cells (HGMEC) and then incubated for 24 hr. After 0 to 24 hr of treatment with BATP, caspase-3/7 activities were directly assessed by the CellEvent caspase-3/7 green detection reagent, which is a nucleic acid binding dye that harbors the caspase-3/7 cleavage sequence, DEVD, and is fluorescent after being cleaved and bound to DNA. On microscopic observations, activated caspase-3/7 signals increase after 24 hr BATP treatment (Fig. 1G). However, control peptide and CellEvent reagent also increase caspase-3/7 signals. Therefore, this effect was caused by CellEvent reagent toxicity. More than 90% of cells were survived. This suggests that the toxic effect of BATP was very low level.

To determine the BATP recognition site, we tested BATP binding in A, B, and O normal human kidney tissues (Fig. 2). Figure 2 shows that BATP bound to peritubular and microvascular endothelia in type A and B kidney tissues but not in type O kidney tissues. This indicated that BATP might recognize A antigen [GalNAc(α1–3)(Fuc(α1–2))Gal-] and B antigen [Gal(α1–3)(Fuc(α1–2))Gal-] structures but not the H antigen [Fuc(α1–2)Gal] structure.

F2-3
FIGURE 2:
Binding patterns of the control (Ctrl) peptide or BATP with blood type A, B, and O kidney tissues. Paraffin sections of type A, B, and O kidneys were stained with 200 μg/mL biotinylated control peptide or biotinylated BATP overnight at RT and then incubated for 45 min at RT with HRP-conjugated streptavidin. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride for 1 min, and the sections were counterstained with hematoxylin. BATP, blood group antigen-targeting peptide; HRP, horseradish peroxidase; RT, room temperature. Scale bar, 100μm.

Blocking of ABO Histo-Blood Group Antigens in Human Kidney Tissue by Blood Group Antigen-Targeting Peptides

To examine whether BATP inhibited anti-A/B antibody binding to A and B histo-blood group antigens in normal kidney tissue, we tested its inhibitory effect on anti-blood group antibody binding in A and B normal human kidney tissues (Fig. 3). The staining intensity of glomerular capillaries as well as peritubular and microvascular endothelia in type A and B kidney sections was markedly reduced by BATP treatment. This suggested that BATP masked A and B histo-blood group antigens on glomerular capillaries as well as peritubular and microvascular endothelia.

F3-3
FIGURE 3:
Immunohistochemical analyses of human kidney tissues with A/B blood group antigens and the control (Ctrl) peptide (A–C) or BATP (D–F). The control peptide or BATP was added to kidney sections of individuals with A/B blood group at a concentration of 200 μg/mL for 30 min at RT. Subsequently, anti-A/B antibodies (diluted 1:1000) were added and incubated for 1 hr at RT followed by incubation with HRP-conjugated anti-mouse IgM for 45 min at RT. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride for 1 min, and the sections were counterstained with hematoxylin. BATP, blood group antigen-targeting peptide; HRP, horseradish peroxidase; RT, room temperature. Scale bar, 100μm.

Blocking of ABO Histo-Blood Group Antigens in Human Kidney by Ex Vivo Perfusion With Blood Group Antigen-Targeting Peptides

We next investigated whether the inhibitory effect of BATP on antibody binding observed in kidney tissue also occurred in excised kidneys perfused ex vivo with BATP. To assess this, we examined RBC coagulation in glomerular capillaries after ABO-I blood reperfusion. We subjected A and B kidney samples excised from renal cell carcinoma patients to ex vivo perfusion with BATP. Ex vivo perfusion of BATP in excised type A (Fig. 4A, C) and B (Fig. 4B, D) kidneys resulted in significantly decreased numbers of RBCs in glomerular capillaries after ABO-I blood reperfusion as well as significantly decreased deposition of human IgM and IgG in the glomerular capillaries (Fig. 5A,B).

F4-3
FIGURE 4:
Blood clotting formation in blood type A (A) and blood type B (B) glomerular capillaries after ABO-I blood reperfusion. To remove the donor RBCs, blood type A and B kidneys were perfused with normal saline. After perfusion with normal saline, the ABO blood group antigens in kidney tissues were masked by ex vivo perfusion with the control (Ctrl) peptide or BATP. After perfusion with the peptide, the blood type A kidneys were perfused with blood of type B (A and C) and the blood type B kidneys were perfused with blood of type A (B and D). Finally, each kidney was perfused with normalsaline. Paraffin sections of the perfused kidneys were stained with hematoxylin–eosin (HE). To confirm incompatible blood perfusion, RBCs of A and B blood groups were stained with anti-A/B antibodies and Alexa 488–conjugated mouse IgM (A and B). ABO-I, ABO-incompatible; BATP, blood group antigen-targeting peptide; RBC, red blood cells. Scale bar, 100 μm.
F5-3
FIGURE 5:
Human IgG and IgM deposition in blood type A (A) and blood type B (B) glomerular capillaries after ABO-I blood reperfusion. To remove the donor RBCs, blood type A and B kidneys were perfused with normal saline. After perfusion with normal saline, the ABO blood group antigens in kidney tissues were masked by ex vivo perfusion with the control (Ctrl) peptide or BATP. After perfusion with the peptide, the blood type A kidneys were perfused with blood of type B (A) and the blood-type B kidneys were perfused with blood of type A (B). Finally, each kidney was perfused with normal saline. Paraffin sections of the perfused kidneys were stained with Alexa 594–labeled anti-human IgG (red) or Alexa 488–labeled anti-human IgM (green). ABO-I, ABO-incompatible; BATP, blood group antigen-targeting peptide; RBC, red blood cells. Scale bar, 100μm.

DISCUSSION

In this study, we screened for a 7-mer BATP, RPRNPNK, for use in developing a novel therapeutic strategy for ABO-I KTx. It is well known that the major barrier to ABO-I KTx is humoral rejection triggered by antigen–antibody reactions (19–21). Serum antibody reduction and antibody production suppressive treatments have been used to overcome ABO-I KTx (22–24). However, blocking of ABO histo-blood group antigens has never been tested clinically because ABO histo-blood group antigen-blocking tests are very difficult to perform in vivo. To the best of our knowledge, this study is the first use of an ex vivo human model that mimics ABO-I KTx.

We performed ABO blood group antigen-neutralizing experiments with BATP using ELISA, HA assays, and human kidney tissues. ELISA and HA assays were performed in ex vivo experiments that mimic ABO-I KTx. Human kidney tissues were used for assessing possible clinical applications. A and B trisaccharide BSA on plates and A and B blood group antigens on RBCs were significantly blocked by BATP (Fig. 1F). BATP bound to peritubular and microvascular endothelia in type A and B kidney tissues but not in type O kidney tissues (Fig. 2), suggesting that BATP specifically recognizes both the A antigen [GalNAc(α–3)(Fuc(α1–2))Gal-] and the B antigen [Gal(α1–3)(Fuc(α1–2))Gal-] structures but not the H antigen [Fuc(α1–2)Gal] structure. Immunohistochemical analyses showed that A and B histo-blood group antigens are highly expressed in glomerular capillaries and peritubular and microvascular endothelia in type A and B kidney tissues. BATP significantly inhibited the reactivity of anti-blood group antibodies against ABO histo-blood group antigens expressed in glomerular capillaries as well as peritubular and microvascular endothelia (Fig. 3). Takasi et al. (25) reported that the ABO histo-blood group antigens in kidney tissues are different from those on RBCs because of differences in carrier proteins. Therefore, we speculate that BATP may recognize A and B trisaccharide epitopes and inhibit ABO histo-blood group antigen–antibody interactions by masking their trisaccharide structures.

Because in vivo experiments using human kidney tissues are ethically difficult, we performed an ex vivo study using normal tissue sections of kidneys excised from renal cell carcinoma patients. The results of ex vivo perfusion with BATP strongly suggested that BATP binds to ABO blood group antigens as an anti-blood group A/B antibody epitope mimetic that acts like an anti-A/B antibody and significantly suppressed IgM and IgG deposition in the glomerular capillaries. The A/B blood group antigens on RBC and in kidney tissues may be neutralized by BATP. These findings imply a lack of reaction between ABO histo-blood group antigens and de novo synthesized anti-A/B histo-blood group antibodies, which may contribute to long-term graft survival without rejection. There is evidence that this accommodation state is established within 2 weeks after transplantation (26–28). We therefore speculate that blocking of A and B antigens in donor organs by BATP administration during the first 2 weeks after transplantation (i.e., until accommodation has most likely occurred) is a reasonable and practical strategy. Ex vivo perfusion of the donor organ during cold storage and before transplantation is also possible. Neutralization of blood group antigens by BATP may represent one strategy for overcoming the challenges of ABO-I KTx. This alternative approach using a peptide may also be useful for minimizing antibody removal and anti–B-cell immunosuppression as adjuvant therapies in ABO-I KTx. We do not yet have data on in vivo peptide stability and systemic side effects. Further studies on ABO-I KTx using an animal model such as baboon (13, 14) are required to establish ABO antigen-blocking therapy using BATP.

MATERIALS AND METHODS

Construction of T7 Phage-Displayed 7-Mer Peptide (X7) Library

Seven-mer peptide (X7) libraries were constructed using the T7Select 415-1b vector as described by Krumpe et al. (29). In brief, random oligonucleotide insert DNA was synthesized as 5′-AACTGCAAGCTTTTA-(MNN)7-ACCACCACCAGAATTCGGATCCCCGAGCAT-3′, where N was a mixed equimolar ratio of each nucleotide and M was a mixed equimolar ratio of adenine and cytosine nucleotides. Amino acid translation of the complementary nucleotide sequence was MLGDPNSGGGX7. The insert DNA was incubated with a complementary extension primer (5′-ATGCTCGGGGATCCGAATTCTGGT-3′), Klenow enzyme (Takara Bio, Shiga, Japan), and deoxyribonucleotide triphosphates to form the complementary DNA strand. The mixed product was digested with EcoRI and HindIII (New England Biolabs, Ipswich, MA) followed by phenol/chloroform extraction and ethanol precipitation using standard techniques. The purified fragments were ligated into the predigested T7Select 415-1b vector using the DNA Ligation Kit “Mighty Mix” containing T4 DNA ligase (Takara Bio). This method inserts the randomized oligonucleotide library DNA in-frame following amino acid 348 of the capsid 10B gene. The ligation reaction mixture was incubated for 16 hr at 16°C and then subjected to in vitro packaging immediately followed by phage titration in a plaque assay. The remaining in vitro packaging solution was amplified once using BL21 cells until lysis. The lysate was centrifuged, titered, and frozen at −80°C in 0.5 M NaCl as a glycerol stock.

Library Biopanning Procedure

Aliquots (100 μL) of Tris-buffered saline (TBS) containing 10 μg blood group A/B trisaccharide BSA (V-Labs, Covington, LA) were added to wells with high-binding capacity (BD Falcon, Franklin Lakes, NJ) and incubated at 4°C overnight. The wells were washed three times with 300 μL TBS and blocked for 1 hr at 4°C with 5% BSA and 5% normal goat serum (NGS) in TBS. The BSA/NGS-blocked blood group A/B trisaccharide BSA-coated wells were washed three times with 200 μL TBS containing 0.5% Tween 20 (TBST); then, 100 μL library phage was applied to each well. The plates were incubated at room temperature (RT) for 30 min with orbital shaking at 250g followed by washing 10 times with 200 μL TBST. Bound phages were eluted by incubation with 100 μL TBS containing 100 mM blood group A/B trisaccharide per well at RT for 20 min, with orbital shaking at 250g. The eluate was collected, and 10 μL were removed for titering by a plaque assay. The remaining eluted phages were amplified in 20 mL freshly prepared BL21 cells in baffled shaker flasks by incubating at 37°C for 3 hr. After centrifuging the inoculated phages at 3400g at 4°C for 15 min, 100 μL of the supernatant were subjected to the next round of biopanning. The second, third, and fourth rounds of biopanning were conducted in the same manner. For each round, the plaque-forming units (PFU) input was held constant (as determined by the plaque-forming assay) to keep the ratio of phage particles to target molecules approximately constant at 3.0×1010 PFU throughout biopanning.

Clone Isolation and DNA Sequence Analysis

In the plaque-forming assay, the eluted clones were plated at a concentration of approximately 75 PFU per 100 mm plate to ensure well-isolated plaques. Each plaque was lifted with an inoculation needle and placed into 500 μL BL21 cells in a plastic test tube. The tubes were incubated at 37°C with orbital shaking at 250g for 3 hr. The NaCl concentration of the lysate was adjusted to 0.5 M followed by centrifugation at 3400g at 4°C for 15 min. A 450 μL aliquot of clarified lysate was transferred to a 1.5 mL tube and stored at 4°C. The following components were used for PCR: Phire Hot Start DNA Polymerase (Finnzymes, Vantaa, Finland), sterile molecular biology grade water, T7 Up primer (10 μM in TE buffer, pH 8.0), and T7 Down primer (10 μM in TE buffer, pH 8.0). The primers were synthesized on a 1 μM scale and cartridge purified. The sequences were as follows: T7 Up, 5′-AGCGGACCAGATTATCGCTAA-3′ and T7 Down, 5′-AACCCCTCAAGACCCGTTTA-3′. Clarified phage lysate (1 μL) was added to each tube, and the T7 insert was amplified in a 30-cycle PCR. The PCR product was electrophoresed and purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced using the T7 Up primer at the Sigma Genosys facility. The DNA and translated amino acid sequences were analyzed using Geneious Pro version 5.0.3 (Biomatters, Auckland, New Zealand).

Inhibitory Effect of Blood Group Antigen-Targeting Peptides on Hemagglutination of Human Red Blood Cells

RBCs of A and B blood groups were isolated from healthy volunteers. Anti-A (Z2A), anti-B (Z5H-2), and anti-H (87-N) mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HA assays were performed by the following microtitration method. Anti-A/B antibodies (100 μg/mL, 35 μL) were diluted in two serial steps and mixed with an equal volume of 0.5% human RBCs preincubated for 30 min at RT with 500 μg/mL control peptide or BATPs (ARARKTG, ASNKRPR, RPRNPNK, SPARRPR, RMSRKLP, GGKTRSK, or SRSSTRK). After incubation at RT for 2 hr, the reciprocal HA titer was determined as 2n.

Inhibitory Effect of Blood Group Antigen-Targeting Peptides on the Binding of Anti-A/B Antibodies to A/B Antigens Measured by Enzyme-Linked Immunosorbent Assay

Blood group A/B trisaccharide BSA-coated wells (5 μg/well) were washed three times with 300 μL TBS and blocked overnight at 4°C with 5% BSA/NGS in TBS. The wells were then treated with 100 μL of 0 to 500 μg/mL BATP (RPRNPNK) and incubated at RT for 1 hr. The wells were washed with 300 μL TBST followed by the addition of 100 μL anti-A (Z2A) or anti-B (Z5H-2) antibodies (1:1000 dilution in TBST) and incubated at RT for 1 hr. The wells were washed with 300 μL TBST, and 100 μL horseradish peroxidase (HRP)–labeled goat anti-mouse IgM antibody (1:10,000 dilution in TBST) was added followed by incubation at RT for 30 min. After the secondary antibody reaction, 100 μL tetramethylbenzidine peroxidase substrate (Funakoshi) was added followed by incubation at RT for approximately 3 min. The reaction was stopped with 100 μL of 1 N HCl, and optical density was measured at 450 nm.

Cytotoxicity of Blood Group Antigen-Targeting Peptides With Human Kidney Glomerular Capillary Microvascular Endothelial Cells

The blood type O HGMEC was obtained from the Applied Cell Biology Research Institute (Kirkland, WA) and grown in CSC complete serum-free medium with penicillin, streptomycin, and culture boost R at 37°C with 5% CO2. Blood type O HGMEC was cultured until near confluent and then cells were incubated with 1 mg/mL control peptide or BATP at 37°C with 5% CO2 for 24 hr. After treatment with BATP, cells were labeled with 5 μM CellEvent caspase-3/7 green detection reagent (Life Technologies, Carlsbad, CA) and with Hoechst 33342 (1 μg/mL in phosphate-buffered saline) in complete medium at 37°C. Stained cells were observed under fluorescence microscopy (floid cell imaging station, Life Technologies) at each time point.

Human Kidney Tissues

Normal human kidney tissues were obtained from renal tumor patients who underwent radical nephrectomy at the Department of Urology, Hirosaki University Hospital (Hirosaki, Japan). After routine radical nephrectomy for renal tumors, small sections of normal kidneys were removed and subjected to ex vivo perfusion for the BATP experiment. Informed consent was obtained from all patients before the initiation of the study. This study was approved by the Ethics Committee of the Faculty of Medicine, Hirosaki University. The study was performed in accordance with the Guidelines of the Declaration of Helsinki.

Immunohistochemistry

The normal kidney sections were fixed in formalin for hematoxylin–eosin and immunohistochemical staining. The deparaffinized sections were then exposed to 3% hydrogen peroxidase for 5 min. After washing with phosphate-buffered saline, expression of ABO histo-blood group antigens was examined using anti-A (Z2A), anti-B (Z5H-2), or anti-H (87-N) mAbs and HRP-labeled anti-mouse IgM antibody or Alexa 488–labeled anti-mouse IgM antibody. Binding of BATP (RPRNPNK) was examined using biotin-labeled BATP (200 μg/mL). Anti-blood group antigen antibodies and BATP were added to the kidney sections and incubated overnight at RT. The sections were then counterstained with hematoxylin and appropriately mounted. To examine the blood group antigen-blocking effect, 200 μg/mL control peptide or BATP (RPRNPNK) was added to the kidney sections followed by incubation for 30 min at 25°C before the first antibody staining. Stained tissues were observed and images collected by fluorescence microscopy (EZ-9000, Keyence, Osaka, Japan).

Ex vivo Perfusion With Blood Group Antigen-Targeting Peptides

The normal excised type A and B kidney tissues were perfused by gravity flow with normal saline and subsequently with 200 μg/mL control peptide or BATP and washed with normal saline. Next, the type A and B kidney tissues were perfused with 1 mL type B and A blood, respectively, followed by perfusion with normal saline. Ex vivo blood reperfusion performed within 1 min after blood withdrawal. Blood clotting formation in the glomeruli after ABO-I blood reperfusion was analyzed by immunohistochemistry and immunofluorescence. Ex vivo perfusion experiments using both blood type A and B kidneys were repeated in triplicate.

Statistical Analyses

Results are expressed as mean±standard deviation. Student’s t test was used to determine the significance of differences between the groups. P<0.05 was considered statistically significant.

ACKNOWLEDGMENTS

The authors thank Drs. Kazuyuki Mori and Shigeru Tsuboi for useful suggestions and comments.

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Keywords:

ABO-incompatible kidney transplantation; Blood group antigen; Blood group antigen-targeting peptide; Random peptide library-displaying T7 phage

© 2013 Lippincott Williams & Wilkins, Inc.