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Accommodation in ABO-Incompatible Kidney Allografts: Graft Self-Protection via Downregulation of Genes PowerPoint Presentation
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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

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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
METHODS
  • 16 ABO-incompatible allografts studied at 3 and 12 months
results
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
methods4
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
    • TNFa
    • Mucin 1
  • Alterations in gene expression verified by RT-PCR and/or immunohistochemistry
results5
RESULTS
  • Genes not increased in ABO-incompatible grafts
    • Heme oxygenase 1
    • Bcl-2
    • Bcl-XL
conclusions
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
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
introduction8
INTRODUCTION
  • In most patients, the graft continues to function well despite the presence of antibody
  • Mechanisms underlying “accommodation” are unclear
methods9
METHODS
  • 16 ABO-incompatible living donor renal allografts performed between May 1999-January 2001
  • Immunosuppression
    • Thymoglobulin antibody induction (1.5 mg/kg/dx 10 d)
    • Tacrolimus (target 15 ng/dl)
    • Mycophenolate mofetil (2 g/d)
    • Prednisone (500 mg taper to 10 mg/d by 3 months)
methods10
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
methods11
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
methods12
METHODS
  • Accommodation, definition
    • Detectible antidonor antibody in recipient serum
    • Normal histology by light microscopy
    • Persistence of A or B antigen in the kidney
    • GFR >45 mL/min/1.73 m2
microarray analysis
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 analysis14
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
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
results16
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
results17
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
results18
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
results19
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
results20
RESULTS
  • Upregulated genes
    • Protein tyrosine kinase GFRA1
    • Immunoregulator MUC1
  • Downregulated genes
    • TNF
    • Smad5
results21
RESULTS
  • Unable to detect
    • HO-1
    • Bcl-2
    • Bcl-XL
    • Bax
  • MUC1 expression strongly positive along glomerular capillary wall
results22
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
MECHANISMS OF GRAFT INJURY
  • Complement-mediated vascular thrombosis
  • Antibody-dependent cellular cytotoxicity
  • Binding of antibody to endothelial cell antigen
    • Endothelial cell apoptosis
accommodation
ACCOMMODATION
  • Recent studies suggest that some forms of accommodation are associated with induction of anti-apoptotic genes
    • HO-1
    • Bcl-XL
results25
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
results26
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
tnf family
TNF FAMILY
  • TNF D HI -0.82, P = 0.033
  • TACE D HI +1.16, P = 0.044
    • Cleaves precursor TNF to its mature form
  • TRAF6 D HI -0.97, P = 0.030
tnf family28
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
LIMITATIONS, MICROARRAY ANALYSIS
  • Decreased sensitivity of microarrays
  • Heterogeneous cell populations
  • Reproducibility
summary
SUMMARY
  • Accommodation is associated with alterations in genes related to
    • Signal transduction
    • Cell-cell adhesion
    • T-cell activation
    • Prevention of apoptosis (pro-survival pathways)