• Blood group incompatibility;
  • gene expression;
  • immunohistochemistry;
  • kidney transplantation;
  • oligonucleotide microarray


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. References

To elucidate the mechanism of self-protection against anti-donor blood-group antibody known as accommodation, we studied 16 human ABO-incompatible living-donor kidney transplant recipients at 3 and 12 months post transplantation. Both circulating anti-blood-group antibody and the target blood-group antigen in the graft were demonstrable in all patients after transplantation. Thirteen of 16 grafts had normal renal function and histology, while three grafts with prior humoral rejection demonstrated significant glomerulopathy and thus did not meet the criterion for accommodation. Using microarrays, we compared five 1-year protocol ABO-compatible renal graft biopsies to four accommodated ABO-incompatible graft biopsies. Significant alterations in gene expression in 440 probe sets, including SMADs, protein tyrosine kinases, TNF-α and Mucin 1 were identified. We verified these changes in gene expression using RT-PCR and immunohistochemistry. Heme oxygenase-1, Bcl-2 and Bcl-xl were not increased in ABO-incompatible grafts at any time-point. We conclude that accommodation is always present in well-functioning, long-surviving ABO-incompatible kidney transplants. This self-protection against antibody-mediated damage may involve several novel mechanisms including the disruption of normal signal transduction, attenuation of cellular adhesion and the prevention of apoptosis.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. References

An ever-growing gap between the number of patients requiring kidney transplantation and the number of donor organs available has become a major problem throughout the world (1). To overcome this, kidney transplantations across ABO blood-group barriers, especially those involving living donors, are being performed with increasing frequency (2,3). Recent refinements in immunosuppression and patient selection have increased both short- and long-term graft survival of ABO-incompatible kidney allografts with success approaching that of cadaveric kidney transplants at 5 years (2).

The major immunologic barrier to the successful transplantation of ABO-incompatible kidneys is the presence of antibody against the donor blood-group. Pre-transplant removal of antibody with plasmapheresis prevents the occurrence of antibody-mediated hyperacute rejection (4). However, despite chronic immunosuppression, anti-donor blood-group antibody usually returns and persists after successful ABO-incompatible transplantation (5,6). In most patients the graft continues to function well despite the continued presence of this antibody and the persistence of the target antigen in the kidney – a situation termed accommodation (7). The mechanism by which the kidney graft can protect itself from these antibodies is unclear. The aim of the current study was to assess the presence of accommodation in human ABO-incompatible living-donor kidney-transplant recipients, and to investigate its possible mechanisms via analysis of intragraft gene expression and immunohistochemistry.

Materials and Methods

ABO-incompatible living-donor kidney transplants and subsequent analyses were performed using protocols approved by the Institutional Review Board of the Mayo Foundation and Clinic.

ABO-incompatible kidney transplants

We performed 16 ABO-incompatible living-donor kidney transplants at our institution between May 1999 and January 2001. All patients received conventional immunosuppression from the time of transplantation, including thymoglobulin antibody induction (1.5 mg/kg/day × 10 days), tacrolimus (target level 15 ng/dL), mycophenolate mofetil (2 g daily in divided doses) and prednisone (500 mg initial intraoperative bolus, tapered to 10 mg/day by 3 months). Based on previous studies suggesting that A2 donor kidneys express lower levels of blood-group antigen and are less likely to evoke antibody-mediated damage than A1 kidneys (8), recipients of A2 donor kidneys (n = 8) received no additional therapy to remove antibody prior to transplantation. Recipients of non-A2 kidneys (A1 and B donors, n = 8) received pretransplant plasmapheresis (daily × 4) and splenectomy at the time of transplantation in an effort to decrease anti-donor blood-group antibody in accordance with protocols reported by others (2).

A group of five ABO-compatible patients, maintained with similar immunosuppression, served as the control group for the microarray experiments. Between 3 months and 1 year post transplantation these patients had normal protocol biopsies, stable creatinine levels, good graft function and no episodes of rejection requiring clinical intervention.

Antibody titers by isoagglutination

Using direct and indirect isoagglutination assays (9), anti-donor blood-group antibody levels were determined pre-transplant (prior to any therapy), at 3 months and at 1 year after transplantation. A1 or B blood-group red blood cells were suspended in serial doubling dilutions of recipient serum, centrifuged and analyzed for agglutination. This immediate spin assay was defined as representing IgM activity. Specimens were then incubated at 37 °C for 30 min, washed with normal saline and anti-human globulin (Coombs) antiserum was added. The specimen was then centrifuged and analyzed for agglutination. This anti-globulin assay was defined as representing IgG activity. By standard red-cell agglutination readings, the most dilute unequivocally positive reaction was defined as the anti-A/B IgM or IgG antibody titer value in each assay. Anti-A or B antibody titers were determined by indirect isoagglutination assay with the anti-A/B IgG antibody titer value defined as the most dilute positive reaction.

Glomerular filtration rate and histology

At 1 year post transplantation, the glomerular filtration rate (GFR) was determined in all patients by iothalamate clearance (10). An ultrasound-guided protocol kidney biopsy was performed and three 16-gauge tissue cores were obtained. Two cores were formalin-fixed and processed for light microscopy and immunohistochemistry. Chronicity indices were assessed by the Banff criteria (11).


Immunohistochemistry was performed on formalin-fixed biopsy tissue to verify the presence of A and B blood-group antigens, Mucin1 (Muc1), heme oxygenase (HO-1), Bcl-2, Bcl-xl and Bax within the ABO-incompatible graft. Formalin-fixed, paraffin-embedded tissue was cut at 4 μm. The slides were de-paraffinized, blocked for endogenous peroxidase activity and subjected to antigen retrieval by treatment with 1 mM EDTA, pH 8.0 in a steamer. The slides were incubated with 5% blocking serum from the species of the secondary antibody for 30 min, and primary antibodies were added in appropriate dilutions. After 30 min of incubation in a humidified chamber the slides were washed in PBS-0.05% Tween 20, and HRP-labeled secondary antibodies were added in appropriate dilutions and incubated for 30 min. The reaction was developed by peroxidase substrate DAB kit (Vector Laboratories, Burlingame, CA, USA) and the slides were counterstained with hematoxylin. For control sections, the primary antibodies were substituted with normal immunoglobulins of the appropriate class and isotype. Antibodies to the following antigens were used: mouse anti-human blood-group antigen A (clone B45.1) and mouse anti-human blood-group antigen B (clone B460) (Biomeda Corp., Foster City, CA, USA); mouse monoclonal anti-MUC1 (clone HMPV), mouse monoclonal anti-Bcl-2 (clone Bcl-2/100), rabbit polyclonal anti-Bax (BD Biosciences, San Diego, CA, USA); rabbit polyclonal anti-Bcl-xl (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit polyclonal anti-HO-1 (Stressgen, Victoria, BC). Secondary antibodies were anti-mouse IgG-HRP and anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Assessment of accommodation

Accommodation was defined at 1 year post transplantation when recipients met all four of the following criteria: 1) detectable anti-donor antibody in the recipient's serum; 2) normal histology by light microscopy; 3) persistence of A or B antigen in the kidney; and 4) GFR > 45 mL/min/1.73 m2 (mean GFR in 107 ABO-compatible kidney transplants at our institution using the same baseline immunosuppression was 54.5 ± 17.9 mL/min/1.73 m2).

Microarray analysis

Histologically normal 3-month and 1-year protocol 16-gauge biopsies from 4 ABO-incompatible kidneys (2–3 month A1 to O and 2–12 month A2 to O) with accommodation and 5 ABO-compatible kidneys with good graft function were placed in RNAlater® (Ambion Inc., Austin, TX) and stored at −80 °C. For each sample, total RNA was extracted from the complete biopsy with TRIzol® Reagent (Invitrogen Corp, Carlsbad, CA) and further purified using the RNeasy Mini Kit® (Qiagen Inc., Valencia, CA). Sample quality was assessed with an Agilent 2100 Bioanalyzer® (Agilent Technologies, Palo Alto, CA). All samples possessed 18S and 28S rRNA peaks with no signs of RNA degradation. The minimum total RNA quantity used for fragmentation and labeling was 2.0 μg.

Preparation of biotinylated target RNA from total RNA and subsequent hybridization of cRNA to the Affymetrix Test3 and U95Av2 (Affymetrix, Inc., Santa Clara, CA) probe array cartridges was accomplished in the Mayo Cancer Center Microarray Core Facility (Mayo Foundation, Rochester, MN) using protocols described previously (12,13). For each sample, data points from approximately 12 600 probe sets were organized into a spreadsheet program for further comparative and statistical analyses.

Statistical analyses

The log average ratio (LAR) was calculated by the GeneChip® Microarray Suite v4.01 (Affymetrix, Inc) for each probe set and serves as the raw measure of gene expression (12). The LAR was derived from raw fluorescence data reflecting the average difference in fluorescence between the perfectly matched oligonucleotides and the mismatched oligonucleotides for a given probe set on the U95Av2. The average LAR for a transcript within a group of samples reflects the hybridization index (HI) for that probe set. For all probe sets (genes), the HI of the accommodated grafts was compared to the HI of the ABO-compatible grafts using a two-tailed Student's t-test. Changes in gene expression for all significant probe sets was then calculated by: ΔHI = HIaccommodation– HIABO-compatible. No correction was made for multiple comparisons and probe sets with HI values below the level of detection were omitted from follow-up analysis. The statistical software package SAS (SAS Institute Inc, Cary, NC) was used to perform all analyses with the assistance of the Transplant Center Statistician.

Gene expression by RT-PCR

Semi-quantitative RT-PCR was performed on 18 significant transcripts for validation of the microarray data. Probe sequences from the U95Av2 were used to create primers for two-step RT-PCR. The transcripts investigated were Muc1, TNF-α, SMAD4, SMAD5, cAMP-dependent protein kinase subunit RII-β (PRKB), chemokine C-C motif receptor 6, neurotrophic tyrosine kinase receptor 2, GPI-linked anchor protein alpha (GFRA1), KREV interaction trapped 1, proliferation potential-related protein, synaptotagmin, transcription factor TFIID, BRCA2, voltage-gated sodium channel, epidermal growth factor receptor (EGFR), Midline 1 and EPH-like tyrosine kinase 2 (HEK2). Additionally the starting amount of cDNA was normalized using primers for GAPDH. Reverse transcription was performed using 1 μg of total RNA with the ProSTARTM First-Strand RT-PCR kit (Stratagene, La Jolla, CA) to a final reaction volume of 50 μL. PCR was performed with 1.0 μL of cDNA, 1.5 units of Taq-Gold DNA polymerase (PE Biosystems, Foster City, CA), 2.5 μL PCR Buffer II (PE Biosystems), 2.0 mm MgCl2, 0.2 mm each dNTP (Roche, Indianapolis, IN) and 0.4 μm of each primer. PCR products were electrophoresed through 2% agarose, stained with ethidium bromide and visualized by Gel Doc 2000® (Bio-Rad, Hercules, CA). For each primer pair the linear range of amplification was determined visually and relative quantitation done by densitometry.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. References

Graft survival and the development of accommodation

Patient and graft survival was 100% at 1 year. No hyperacute or acute cellular rejection episodes occurred. Four patients had an episode of humoral rejection in the first month after transplantation. These episodes presented as an increase in serum creatinine accompanied by histologic changes associated with humoral rejection. All humoral rejections responded to treatment with corticosteroids and plasmapheresis.

One year after transplantation, all 16 patients showed persistence of donor blood-group antigen in the graft and anti-blood-group antibody in the circulation. Thirteen patients (12 without a prior humoral rejection episode and one with prior humoral rejection) had normal renal function and a normal kidney biopsy, thus meeting all of the criteria of accommodation (Table 1). These included seven recipients of A2 kidneys and six recipients of non-A2 kidneys who had undergone splenectomy. Anti-donor blood-group antibody was present in the 13 accommodated patients at 3 and 12 months after transplantation although generally lower than the pretransplant titers (p-value 0.0503 at 3 months and 0.0033 at 12 months by paired t-test, respectively). The accommodated group had significantly less IgM at 3 and 12 months than pretransplant levels (p-value 0.0027 and 0.0057). Finally, the mean GFR of accommodated kidneys was similar to a control group of 107 ABO-compatible kidneys receiving similar immunosuppression 1 year after transplantation (data not shown).

Table 1.  Accommodation 1 year after ABO-incompatible living-donor kidney transplantation
Donor/RecipientTotal titerIgMGFR-12 m
 Pre1–3 m12 mPre1–3 m12 m 
  • A

    Titer 64 = positive at 1 : 64 dilution by isoagglutination assay.

  • B

    Titer not done.

  • C

    GFR measured at 4 months post-transplant.

  • D

    Biopsy of this patient was used for microarray experiments.

  • E

    The patient had an episode of humoral rejection on postoperative day 7.

1. A1/O64A864164841
2. A1/O1288nd82nd68D
3. A1/O164244254D
4. B/A6482324268
5. B/O644241184C
6. B/O3244162283
No Splenectomy
7. A2/O6416321641684
8. A2/OE1283232164850
9. A2/O25612864164456
10. A2/O12864166416852D
11. A2/O64128641616845D
12. A2/O64ndB3232nd1660
13. A2/B32128163216863
No Accommodation
1. A1/O51288162220
2. A2/O1644164223
No Splenectomy
3. A2/O256646444211

Three patients with persistent antibody and antigen demonstrated low GFR (all < 30 mL/min/1.73 m2) and were not included in further analyses. All three of these non-accommodated patients had experienced a prior humoral rejection episode, and their renal function never returned to normal.

All 13 accommodated patients had normal-appearing 1-year protocol biopsies (Figure 1a) and the expression of blood-group antigen on endothelial cells was present in all kidneys in roughly the same distribution (Figure 1b). In the three non-accommodated patients the protocol biopsies showed extensive glomerular sclerosis and interstitial fibrosis consistent with chronic allograft nephropathy (Figure 1c).


Figure 1. Histologic evidence of the outcomes of ABO-incompatible transplantation. Panel A is an H&E showing the normal graft histology of an accommodated patient 1 year after transplantation in a blood-group O recipient of an A1 living unrelated renal allograft. Panel B demonstrates the presence of A blood-group antigen in the same graft. Panel C illustrates the glomerular endothelial cell swelling and interstitial fibrosis evident in the biopsy of a non-accommodated graft. 100× magnification.

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Changes in intragraft gene expression in accommodated ABO-incompatible kidneys

To investigate the mechanism of accommodation in more detail, we compared intragraft gene expression in 4 ABO-incompatible kidney transplants to 5–12-month ABO-compatible kidney transplants. Of the 12 600 genes examined by the U95Av2, 4933 had HI values below the level of detection (HI < 1) or exhibited very small changes in expression. This left 7667 probe sets for further analyses by a two-sided Student's t-test. Of these, 440 probe sets had significant changes in expression with 404 being downregulated and 33 being upregulated. As ranked by p-value, the 20 most up/downregulated transcripts are shown in Table 2. Overall, our data show gene expression changes in accommodation appear to involve alterations in several genes involved in cellular metabolism, transcription, translation and signal transduction. In addition, we have performed separate microarray analyses of 5–3-month A1 to O biopsies and 5–3-month ABO-compatible biopsies, which showed similar patterns of gene expression (data not shown).

Table 2.  Most upregulated (A) and downregulated (B) genes in ABO-incompatible kidney biopsies (ranked by P value)
(A)P valueΔHI ± SEDescription of gene
1.0.00180.43 ± 0.09Clone 23803 mRNA
2.0.00260.45 ± 0.10Ig heavy chain variable region (V3−31) gene
3.0.00370.56 ± 0.13Rac protein kinase β
4.0.00510.91 ± 0.23KIAA1045 protein
5.0.00540.37 ± 0.09Ubiquitin mRNA
6.0.00960.63 ± 0.18Epidermal growth factor receptor
7.0.01360.30 ± 0.09β-actin mRNA
8.0.01631.18 ± 0.37Mucin 1 mRNA
9.0.01791.45 ± 0.47GFRA1 mRNA
10.0.01871.43 ± 0.47DKFZP586G011 protein
11.0.01941.30 ± 0.43Neurotrophic tyrosine kinase receptor 2
12.0.02050.90 ± 0.30Sjogren sydrome antigen A2
13.0.02170.56 ± 0.19Cell division cycle 25C mRNA
14.0.02311.89 ± 0.65Protein tyrosine kinase receptor HEK2
15.0.02390.42 ± 0.15Dishevelled 1 mRNA
16.0.02440.39 ± 0.14EST AF050078
17.0.02570.90 ± 0.32Epithelial tumor mucin antigen
18.0.02662.11 ± 0.75DKFZp564D036 protein
19.0.02740.89 ± 0.32Fas anitgen (M. musculus)
20.0.02760.84 ± 0.30Polymorphic epithelial mucin
(B)P valueΔHI ± SEDescription of gene
1.0.0000−1.85 ± 0.14DKFZp761B2423 protein
2.0.0002−1.68 ± 0.24Synaptotagmin I
3.0.00030.90 ± 0.13Transcription factor TFIID subunit TAFII28
4.0.0004−1.51 ± 0.24BRCA2 mRNA
5.0.0005−1.07 ± 0.17MID mRNA
6.0.0006−0.81 ± 0.14Chymotrypsin-like mRNA
7.0.0008−1.62 ± 0.28Proliferation-associated 2G4 mRNA
8.0.0008−1.12 ± 0.20Thryoid hormone receptor interactor 7
9.0.0008−1.12 ± 0.20Voltage-gated sodium channel type VIα
10.0.0009−0.45 ± 0.08Ig heavy chain V region recursor (similar)
11.0.0010−1.24 ± 0.23Solute carrier family 12 (Na/K/CI transporters)
12.0.0010−1.15 ± 0.21Alcohol dehydrogenase 1A (class I)
13.0.0013−1.61 ± 0.31RAP2A, member of RAS oncogene family
14.0.0016−1.50 ± 0.30Dynactin 4 mRNA
15.0.0017−1.16 ± 0.24Ring finger protein (C3H2C3 type) 6
16.0.0019−1.93 ± 0.40RAR-related orphan receptor B
17.0.0021−1.01 ± .021Adenylate kinase 3
18.0.0025−0.89 ± 0.19Tre oncogene
19.0.0026−2.04 ± 0.45cAMP-dependent regulatory kinase IIb
20.0.0028−1.09 ± 0.24Palmitoyl-protein thioestrase 2

The expression pattern detected by the microarray was confirmed using semi-quantitative RT-PCR on 18 transcripts. These transcripts were selected based on the level of significance and involvement in important immune-related pathways. For example, the cytokine TNF-α and TGF-β1 regulating protein SMAD5 were downregulated, whereas the protein tyrosine kinase GFRA1 and immunoregulator Muc1 were upregulated (Figure 2). In general, the microarray correctly predicted the direction of change observed by RT-PCR. The magnitudes of the changes were not as correlative, but this was not surprising given the nature of the LAR and limited amount of sample available for each RT-PCR.


Figure 2. RT-PCR of ABO-compatible and ABO-incompatible mRNA. After determining the linear range of PCR amplification for significant transcripts, a comparison of gene expression between ABO-compatible (lane 1) and -incompatible (lane 2) samples was made. As predicted by the microarray, TNF-α and SMAD5 appear down-regulated in ABO-incompatible grafts, whereas Muc1 and GFRA1 are up-regulated. For each panel, lane 3 represents a negative control.

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Intragraft Muc1, HO-1, Bcl-xl and BAX in accommodation

We used immunohistochemistry to detect proteins of interest in biopsy tissue from histologically normal ABO-incompatible protocol biopsies at 1 year and 3 months after transplantation. We tested for the presence of Muc1, HO-1, Bcl-2, Bcl-xl and Bax proteins within the graft. Muc1 expression was strongly positive along the glomerular capillary wall (Figure 3).


Figure 3. Immunohistochemistry of Muc1 protein in ABO-incompatible grafts. Muc1 was identified in the proximal convoluted tubules (Panel B), the distal convoluted tubules and in the collecting ducts (Panel C). Panel A is the IgG negative control. 100× magnification.

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We were unable to detect HO-1 protein in ABO-incompatible grafts (Figure 4). Similarly, Bcl-2 and Bcl-xl, putative anti-apoptotic molecules demonstrated in accommodation, and Bax, a pro-apoptotic marker, were not detectable in accommodated ABO-incompatible kidneys (data not shown).


Figure 4. Immunohistochemistry of HO-1 protein in ABO-incompatible grafts. HO-1 expression was undetectable in the kidney of an accommodated patient (Panel A). In contrast, kidneys from rats with glycerol-induced, heme protein mediated renal injury demonstrated the presence of HO-1 and served as positive control (Panel B). 100× magnification.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. References

Accommodation was first observed in ABO-incompatible renal allografts (7). The term was later applied to xenografts (14) and in ABO-compatible grafts in which alloantibody is present (15). The current study defines several unique features of accommodation in ABO-incompatible kidney transplants including: 1) detectable anti-donor antibody; 2) normal graft histology; 3) presence of A or B antigen in the graft; and 4) renal function similar to ABO-compatible patients. In addition, these data demonstrate that anti-AB antibodies return to detectable levels in all patients (accommodated and non-accommodated), even in the absence of humoral rejection or chronic graft damage. Furthermore, blood-group antigen is detectable in all recipients months after transplantation, making it likely that some form of accommodation must occur for enduring graft survival in ABO-incompatible kidney transplants.

Three patients had low GFR values and protocol biopsies showing chronic changes including interstitial fibrosis and glomerulopathy. It is unknown if these observations were secondary to the prior humoral rejection or due to a lack of protection from persistent antibody-mediated damage, i.e. lack of accommodation. One patient who had an episode of humoral rejection now has an accommodated graft at 1 year. It remains questionable whether or not the presence of subsequent accommodation will protect the graft from future chronic damage.

In contrast to previous studies suggesting that high-titer patients do not develop accommodation easily, the current data show that accommodation can occur over a wide range of anti-blood-group antibody titers. Ishida et al. found that all patients with anti-AB antibody titers <1 : 8 maintained excellent graft function, but 11 of 16 patients (69%) with anti-AB antibody titers persisting >1 : 32 lost their grafts within 12 months after transplantation (16). In a study of ABO-compatible kidney transplants the presence of anti-MHC alloantibody appeared to be associated with chronic allograft nephropathy, even when many criteria for accommodation were met (17). In our experience, 6 of 13 patients with anti-AB antibody titers ≥1 : 32 demonstrated excellent graft function at 1 year. Protocol biopsies of these grafts did not show chronic changes, suggesting that accommodation is protective in these patients. Given these issues, continued monitoring of graft histology for patients with high antibody titers will be important to determine whether chronic allograft nephropathy will become a problem in long-term graft survival.

The mechanism by which circulating antibody damages the graft at different time-points after transplantation remains unclear. Possible mechanisms include complement-mediated vascular thrombosis, antibody-dependent cellular cytotoxicity or direct signaling via binding to endothelial cell antigens. This binding might deliver a signal that causes endothelial cell activation leading to apoptosis, possibly in the absence of complement (18). Recent data suggest that some forms of accommodation may occur as the result of the increased intragraft expression of anti-apoptotic ‘protective’ genes by the graft which blunt the response to antibody. Genes such as HO-1, Bcl-xl and A20 have been associated with accommodation in rodent xenograft models (19–22). Similarly, Bcl-xl expression was increased in ABO-compatible kidney-transplant recipients with apparent accommodation to circulating anti-graft alloantibody (15). No microarray studies of these different models of accommodation have been described.

Data from this study suggest that HO-1, Bcl-2, Bcl-xl and Bax are not associated with accommodation in ABO-incompatible kidney transplants. However, it is possible that these molecules may be important in protection against antibody-mediated injury early after transplantation – a time-point not assessed in these studies. Interestingly, although HO-1 expression has been associated with humoral rejection, we did not find its expression in grafts experiencing humoral rejection (data not shown). Our data indicate that a significant alteration in intragraft physiology occurs in accommodated kidneys and some of the pathways appear to have significant implications for protection against antibody-mediated injury. Using microarrays to perform an unbiased survey of changes in gene expression, we found that 440 transcripts related to numerous biological pathways were significantly up-/down-regulated. Specifically, our data suggest that the accommodated graft is in a pro-survival environment through modifications in the expression of pathways involving SMAD, protein tyrosine kinases, TNF-α and Muc1.

The SMAD proteins form a complex pathway regulating the signaling effects of Transforming Growth Factor-β (TGF-β) and Bone Morphogenetic Protein (BMP), both of which are involved in regulating cell growth, differentiation and apoptosis (23). Mediating the effects of these intracellular signals appears very important in the accommodated graft, as three significant transcripts observed in this study were related to this pathway: EGFR (ΔHI + 0.63, p-value 0.010), SMAD5 (ΔHI − 1.68, p-value 0.014) and SMAD4 (ΔHI − 1.06, p-value 0.022). The SMADs are a family of receptor substrates that, with the aid of coactivators/repressors, translocate to the nucleus where they act as transcription factors (23). SMAD2 is a receptor-regulated SMAD (R-SMAD) whose corepressor is TGIF. When activated, EGFR triggers a cascade of events involving the Ras-Mek signaling pathways leading to the phosphorylation and stabilization of TGIF (24). The presence of TGIF within the cell will promote the formation of SMAD2/TGIF corepressor complexes in response to TGF-β (23), thereby prohibiting the binding of SMAD2 coactivators and repressing the effects of TGF-β. Similarly SMAD5, an R-SMAD whose ligand is BMP (23), was significantly down-regulated, thereby promoting a cell survival environment. After binding to coactivator molecules, each R-SMAD couples with a Co-SMAD and translocates to the nucleus as a functional transcriptional complex (23). SMAD4 is a Co-SMAD whose deletion has been shown experimentally to abrogate/abolish the signaling effects of TGF-β (25). Taken together, these effects may attenuate intracellular signaling via TGF-β and BMP, promoting cell survival.

Protein tyrosine kinases (PTK) represent a large class of molecules that we found significantly altered in accommodation. Several molecules, including GFRA1, HEK2 and PRKB, are associated with PTK and are found among our most statistically significant transcripts. GFRA1 (ΔHI + 1.45, p-value 0.018) is a receptor which mediates binding and activation of the PTK RET, leading to a decrease in apoptosis and an increase in cell survival (26). In contrast to GFRA1, PRKB was very down-regulated (ΔHI − 2.04, p-value 0.003). PRKB binds cAMP, a potent inhibitor of T-cell activation (27). In vitro studies have shown that mutation of PRKB markedly decreases expression of leptin (27), a known regulator of proliferation of naïve and memory T cells (28). A reduction in leptin will decrease pro-inflammatory Th1 cells and increase Th2 cells. These changes in various PTK molecules provide insights into the mechanism by which accommodation affects signal transduction.

The microarray also identified changes in gene expression in the TNF-α family of molecules. The accommodated patients had significantly decreased levels of TNF-α (ΔHI − 0.82, p-value 0.033) and the enzyme that cleaves precursor TNF-α to its mature form, TACE (ΔHI − 1.16, p-value 0.044). Mature TNF-α is a cytokine that causes leukocyte recruitment to the site of inflammation during early immune response (29). TNF-α binds the TNF-R1 receptor, leading to activation of NF-kB and c-Jun N-terminal protein kinase (29). NF-kB is a transcription factor that mediates gene expression for cytokines and other genes related to inflammation and leads to cell death via apoptosis (30). Interestingly, the TNF-α receptor-associated factor TRAF6, a signal transducer in the NF-kB pathway (31), is also significantly down-regulated (ΔHI − 0.97, p-value 0.030). Taken together, these changes implicate the TNF-α family in accommodation.

Finally, three significantly up-regulated probe sets were related to Muc1. Muc1 is a large transmembrane protein normally expressed on the apical surface of ductal epithelia (32). It contains a very large extracellular domain consisting mostly of tandem repeats, a membrane-spanning domain and a small phosphorylated cytoplasmic tail (33). Several in vitro studies have characterized the protein, revealing several functions, including cell adhesion, cell signaling and immunoregulation (33–37). Cell adhesion has been explored through the interactions of Muc1 with E-selectin and intercellular adhesion molecule 1 (ICAM-1), two well-characterized mediators of leukocyte adhesion to endothelial cells during the inflammatory process. The interaction between ICAM-1 and E-selectin with Muc1 has been localized to the tandem repeat region of the Muc1 extracellular domain. Overexpression of Muc1 can promote cell–cell adhesion through binding of exogenous ICAM-1 (38). Conversely, overexpression of Muc1 can abrogate adhesion by blocking the binding sites of endogenous E-selectin, thereby protecting the cell against immune surveillance mechanisms (33). Further, when phosphorylated, the cytoplasmic tail of Muc1 can bind β-catenin, causing a reduction in the amount available for binding with E-cadherin and promoting anti-adhesion between endothelial cells (39). Adding to the functional complexities of Muc1, the cytoplasmic tail has also been shown to adversely affect signal transduction via Grb2/SOS (37) which mediate the signals for several receptor tyrosine kinases, including EGFR (35).

In total, the observed changes in gene expression provide insight into novel mechanisms of self-protection against antibody-mediated injury via alterations in signal transduction, cell–cell adhesion, T-cell activation pathways and the prevention of apoptosis (pro-survival). It should be noted that when using microarrays to analyze such elaborate processes, it is possible that some important transcripts/pathways may not be easily resolved. This is due in part to the decreased sensitivity of microarrays which prohibit the identification of cytokines and other potentially important markers that are expressed at undetectable levels. In addition, mRNA derived from heterogenous cell populations (i.e. biopsies) may mask relevant changes to markers within an individual cell type. These limitations add to the complexities of working with microarrays and necessitate the need to study important identified markers by some other means such as RT-PCR or immunohistochemistry.

The use of ABO-incompatible living-donor kidneys provides a new source of kidneys for transplantation. However, the unique situation involving the presence and persistence of anti-donor blood-group antibodies presents a complex set of immunologic problems that are poorly understood. In this study, we investigated antibody titers, presence of antigen, GFR and graft histology to determine which patients had accommodation. In addition, we monitored intragraft changes in gene expression and found dysregulation of signal transduction machinery and immune surveillance that is consistent with the promotion of cell survival. These results provide evidence that the graft plays a role in its own survival in accommodation. Interestingly, the situation in which circulating antibody and its target antigen coexist in an organ without requiring clinical intervention occurs in numerous human disease states, both renal and nonrenal including Goodpasture's syndrome (40), p-ANCA positive vasculitis (41) and autoimmune diabetes (42). Thus, it is likely that some form of self-protection against antibody-mediated injury exists normally in humans and is not just a phenomenon observed in transplantation.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. References
  • 1
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  • 2
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