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.

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)