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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.

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Presentation on theme: "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."— Presentation transcript:

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 –TNF  –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/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)

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 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 m 2

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  HI = HI accommodation – HI ABO 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  HI -1.06, P = 0.022 Smad5  HI -1.68, P = 0.014 EGFR  HI +0.63, P = 0.010

26 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

27 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

28 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

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)





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