Accommodation in ABO-Incompatible Kidney Allografts: Graft Self-Protection
via Downregulation of Genes
Joseph P. Grande, M.D., Ph.D.Mark D. Stegall, M.D.
Walter D. ParkMayo Clinic - Rochester, MN USA
METHODS
• 16 ABO-incompatible allografts studied at 3 and 12 months
RESULTS
• Circulating anti-blood group antibody and target blood group antigen demonstrated in all patients
• 13/16 grafts had normal renal function and histology
• 3 grafts with prior humoral rejection demonstrated significant glomerulopathy
METHODS• Compared five one-year protocol ABO-
compatible biopsies to four accommodated ABO-incompatible graft biopsies
• Alterations in gene expression in 440 probe sets identified– Smads– Protein tyrosine kinase– TNF– Mucin 1
• Alterations in gene expression verified by RT-PCR and/or immunohistochemistry
RESULTS
• Genes not increased in ABO-incompatible grafts– Heme oxygenase 1– Bcl-2– Bcl-XL
CONCLUSIONS
• Accommodation is present in well-functioning ABO-incompatible renal allografts
• Accommodation may involve several novel mechanisms including perturbation of signal transduction, alterations in cellular adhesion, and prevention of apoptosis
INTRODUCTION
• ABO-incompatible allografts have been used to meet donor shortage
• Refinements in immunosuppression and patient selection have increased survival of ABO-incompatible renal allografts
• Anti-donor blood group antibody usually returns and persists despite chronic immunosuppression
INTRODUCTION
• In most patients, the graft continues to function well despite the presence of antibody
• Mechanisms underlying “accommodation” are unclear
METHODS
• 16 ABO-incompatible living donor renal allografts performed between May 1999-January 2001
• Immunosuppression– Thymoglobulin antibody induction (1.5 mg/kg/d
x 10 d)– Tacrolimus (target 15 ng/dl)– Mycophenolate mofetil (2 g/d)– Prednisone (500 mg taper to 10 mg/d by 3 months)
METHODS
• Recipients of non-A2 kidneys received pre-transplant plasmaphoresis (daily x4) and splenectomy at time of transplant
• Controls consisting of 5 ABO-compatible patients, with normal three-month and one year protocol biopsies and stable function
METHODS
• Antibody titers– A1 or B blood group red cells suspended in
dilutions of recipient serum– Immediate spin assay represents IgM activity– Specimens incubated at 37° and with anti-
human globulin represents IgG activity
METHODS
• Accommodation, definition1. Detectible antidonor antibody in recipient
serum
2. Normal histology by light microscopy
3. Persistence of A or B antigen in the kidney
4. GFR >45 mL/min/1.73 m2
MICROARRAY ANALYSIS
• 16 gauge biopsies placed in RNA later (Ambien, Inc.)
• RNA extracted with TRIzol reagent (Invitrogen) core
• RNA purified using RNeasy Mini Kit (Qiagen, Inc.)
MICROARRAY ANALYSIS
• Sample quality assessed with Agilent 2100 Bioanalyzer for 18 and 28 s at RNA peaks
• Biotinylated target RNA prepared from total RNA and hybridization of cRNA to Affymetrix test 3 and U95Av2 microarrays performed in microarray core facility
STATISTICAL ANALYSIS
• Log average ratio calculated by gene shift microarray suite v4.01 (Affymetrix)
• Hybridization index: average LAR for a transcript within a group of samples HI = HIaccommodation – HIABO compatible
• Gene expression verified by RT-PCR
RESULTS
• Patient and graft survival 100% at one year
• No hyperacute or acute cellular rejection identified
• Four patients had episode of humor rejection in first month after transplant
• Responded to corticosteroids and plasmapheresis
RESULTS
• All 16 patients showed persistence of donor blood group antigen in the graft and anti-blood group antibody in circulation
• 13 patients had normal renal function and normal kidney biopsy – 7 recipients of A2 kidneys– 6 recipients of non-A2 kidneys who had
undergone splenectomy
RESULTS
• Anti-donor blood group antibody levels lower than pre-transplant levels
• Accommodated group had less IgM at 3 and 12 months than pre-transplant levels
RESULTS
• Of 12,600 genes examined by U95Av2– 4933 had HI values <1 or exhibited small
changes in expression– 440 probe sets had significant changes in
expression• 404 downregulated
• 33 upregulated
RESULTS
• Upregulated genes– Protein tyrosine kinase GFRA1– Immunoregulator MUC1
• Downregulated genes– TNF– Smad5
RESULTS
• Unable to detect– HO-1– Bcl-2– Bcl-XL– Bax
• MUC1 expression strongly positive along glomerular capillary wall
RESULTS
• Accommodation can occur over a wide range of anti-blood group antibody titers– 6 of 13 patients with anti-A/B anti-titers >1:32
had excellent graft function at one year
• Protocol biopsies of these patients were unremarkable.
MECHANISMS OF GRAFT INJURY
• Complement-mediated vascular thrombosis
• Antibody-dependent cellular cytotoxicity
• Binding of antibody to endothelial cell antigen– Endothelial cell apoptosis
ACCOMMODATION
• Recent studies suggest that some forms of accommodation are associated with induction of anti-apoptotic genes – HO-1– Bcl-XL
RESULTS
• TGF- signaling is reduced in accommodated grafts
• Smad4 HI -1.06, P = 0.022
• Smad5 HI -1.68, P = 0.014
• EGFR HI +0.63, P = 0.010
RESULTS
• Protein tyrosine kinases
• GFRA1 HI +1.45, P = 0.018– Receptor which mediates binding and
activation of RET
• PRKB HI -2.04, P = 0.003– PRKB binds cAMP
TNF FAMILY
• TNF HI -0.82, P = 0.033
• TACE HI +1.16, P = 0.044– Cleaves precursor TNF to its mature form
• TRAF6 HI -0.97, P = 0.030
TNF FAMILY
• MUC1 HI 1.18, P = 0.016– Transmembrane protein expressed on the apical
surface of ductal epithelial cells
• Involved in– Cell adhesion– Cell signaling– Immunoregulation
LIMITATIONS, MICROARRAY ANALYSIS
• Decreased sensitivity of microarrays
• Heterogeneous cell populations
• Reproducibility
SUMMARY
• Accommodation is associated with alterations in genes related to – Signal transduction– Cell-cell adhesion– T-cell activation– Prevention of apoptosis (pro-survival pathways)
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