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

1. 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

2. METHODS • 16 ABO-incompatible allografts studied at 3 and 12 months

3. 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

4. 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

5. RESULTS • Genes not increased in ABO-incompatible grafts • Heme oxygenase 1 • Bcl-2 • Bcl-XL

6. 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

7. 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

8. INTRODUCTION • In most patients, the graft continues to function well despite the presence of antibody • Mechanisms underlying “accommodation” are unclear

9. 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)

10. 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

11. 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

12. 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

13. 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.)

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. RESULTS • Upregulated genes • Protein tyrosine kinase GFRA1 • Immunoregulator MUC1 • Downregulated genes • TNF • Smad5

21. RESULTS • Unable to detect • HO-1 • Bcl-2 • Bcl-XL • Bax • MUC1 expression strongly positive along glomerular capillary wall

22. 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.

23. MECHANISMS OF GRAFT INJURY • Complement-mediated vascular thrombosis • Antibody-dependent cellular cytotoxicity • Binding of antibody to endothelial cell antigen • Endothelial cell apoptosis

24. ACCOMMODATION • Recent studies suggest that some forms of accommodation are associated with induction of anti-apoptotic genes • HO-1 • Bcl-XL

25. 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

26. 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

27. 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

28. 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

29. LIMITATIONS, MICROARRAY ANALYSIS • Decreased sensitivity of microarrays • Heterogeneous cell populations • Reproducibility

30. SUMMARY • Accommodation is associated with alterations in genes related to • Signal transduction • Cell-cell adhesion • T-cell activation • Prevention of apoptosis (pro-survival pathways)