Transcript Accommodation in ABO-Incompatible Kidney Allografts: Graft

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. Park
Mayo 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 ABOcompatible biopsies to four accommodated
ABO-incompatible graft biopsies
• Alterations in gene expression in 440 probe
sets identified
–
–
–
–
Smads
Protein tyrosine kinase
TNFa
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 1999January 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 pretransplant 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 antihuman globulin represents IgG activity
METHODS
•
Accommodation, definition
1. 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
D 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 antiblood 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
– TNFa
– 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 D HI -1.06, P = 0.022
• Smad5 D HI -1.68, P = 0.014
• EGFR D HI +0.63, P = 0.010
RESULTS
• Protein tyrosine kinases
• GFRA1 D HI +1.45, P = 0.018
– Receptor which mediates binding and
activation of RET
• PRKB D HI -2.04, P = 0.003
– PRKB binds cAMP
TNFa FAMILY
• TNFa D HI -0.82, P = 0.033
• TACE D HI +1.16, P = 0.044
– Cleaves precursor TNFa to its mature form
• TRAF6 D HI -0.97, P = 0.030
TNF FAMILY
• MUC1 D 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)