Renal Transplantation

1. Renal Transplantation Azim Gangji MD FRCPC FACP McMaster University

2. Objectives • Overview of Immunology • Cross match test • Immunosuppressive Medications • Rejection • Antibody Mediated Rejection • Acute Cellular Rejection • Causes of Allograft Failure • Surgical Complications • Longterm Medical Complications

3. What is MHC and What is its Role • Human Leukocyte Antigen (HLA) system is synonymous with the human Major Histocompatibility Complex (MHC) • In humans, it’s called HLA due to expression of gene products on surface of WBC • These terms describe a group of genes on chromosome 6 that encode a variety of cell surface markers, antigen presenting molecules and other proteins involved in immune function • Inheritance ~ a haplotype from each parent

4. What is MHC and What is its Role • HLA region is subdivided into 3 regions: • Class I • encodes “classical antigens” HLA A, B, C • Expressed on all nucleated cells • Class I alpha genes are coded by the MHC whereas the B microglobulin gene is coded on chromosome 15 (non MHC) • Class II • Encodes HLA- DR, DQ, DP • Has both alpha and beta genes both of which are coded by MHC genes • DR genes all share the same alpha chain but vary in the beta chain whereas DQ and DP can have polymorphic alpha and beta chains • In addition, the number of DR genes can vary among individuals. For example, 2 DR molecules are expressed on a single haplotype: both dimers use the same alpha chain but one uses a beta chain encoded by DRB1 and the other uses a beta chain encoded by a second DR locus called DRB3, DRB4, DRB5, etc. Alleles at this locus are usually expressed at much lower levels on the cell surface. • Class III • Contains genes for complement, TNF, Heat shock protein

5. MHC Molecules 3D structure of MHC Class II Molecule

6. The structure of class I MHC and class II MHC molecules. The schematic diagrams and models of the crystal structures of class I MHC and class II MHC molecules Both types of MHC molecules contain peptide-binding clefts and invariant portions that bind CD8 (the 3 domain of class I) or CD4 (the 4 domain of class II). Schematic of MHC

7. Comparison of MHC Class I vs II

8. Direct and Indirect Allorecognition

9. The Processing of Antigens and Presentation to T Cells

10. Intact donor MHC molecules are also known to be continuously shed into the circulation where they may then be endocytosed by the recipient's antigen-presenting cells. Within the acidic endosomal compartment, the MHC proteins are fragmented into their constituent peptides. They are then transferred into the endoplasmic reticulum, loaded into the antigen binding cleft of MHC class II of the recipient and finally expressed on the cell surface of recipients APCs. This mechanism has been referred to as the "indirect allorecognition.“ However, this is of course the normal route by which T cells normally encounter antigen ie in context of self MHC. T Cell Recognition of Alloantigen: Indirect Pathways

11. T cell activation by the "direct" pathway occurs when T cells encounter intact donor MHC on the surface of donor antigen-presenting cells (APC). Since MHC molecules w/o a bound peptide are unstable & unrecognizable by T cells, endogenous proteins bound to the donor MHC may play a role. Direct allorecognition of intact surface MHC molecules has only been demonstrated in allogenic transplantation This pathway is thought to be of dominant importance during early acute rejection as engrafted organs contain a large number of passenger leukocytes. T Cell Recognition of Alloantigen: Direct Pathways

12. The Processing of Antigens and Presentation to T Cells C L A S S II C L A S S I

13. HLA peptide presentation by APC to T Cell Triggering of the TCR by antigen initiates a signaling cascade started by the signaling complex made up of CD3 T Cell Receptor and APC Interaction: Signal 1

14. APC and T Cell Interaction:Role of Costimulation: Signal 2

15. Role of Costimulation: Signal 2 • Naïve T cells require both signal 1 and • Memory T cells do not require costimulation through CD28 to become activated. Costimulation through ICOS is sufficient for memory T cell activation. c, Absence of B7 or blocking of B7 with CTLA-4 Ig leads to anergy of the T cell.

16. Allorecognition and Stimulation of T Cells and Effect

17. Fig 2. Classes of lymphocytes. Different classes of lymphocytes recognize distinct types of antigens and differentiate into effector cells whose function is to eliminate the antigens. B lymphocytes recognize soluble or cell surface antigens and differentiate into antibody-secreting cells. Helper T lymphocytes recognize antigens on the surfaces of APCs and secrete cytokines, which simulate different mechanisms of immunity and inflammation. Cytolytic T lymphocytes recognize antigens on infected cells and kill these cells. (Note that lymphocytes recognize peptides that are displayed by MHC molecules.) Natural killer cells recognize changes on the surface of infected cells and kill these cells. It should be emphasized that native T cells (CD4 or CD8) are activated by professional APCs. Effector CD8 T cells, not native T cells, can kill and infected cell expressing the specific peptide-class I complex. B, T and NK Cells and Effect: Rejection

18. Stimulus Activation Expansion Rejection Memory T Cells Divide Plasma Cells Make Antibody Effector T Cells Attack the Transplant Humoral Rejection Immune System Encounters the Transplant Hyperacute Rejection T Lymphocytes Are Activated And Make Cytokines Immune System Develops T and B cell memory Immune Activation and Rejection 0 0.5 1 2… 24 hours…. 3-4 days… 7 days...

19. Acute Cellular Rejection

20. T Cell Mediated Rejection • T cell receptor (TCR) of alloreactive cytotoxic lymphocytes (CTL) recognize allogeneic major histocompatibility complex (MHC) on target cells. CTL mobilize cytotoxic granules containing perforin and granzyme B (Gz B) toward the target cell releasing the cytotoxic molecules into the intercellular space. Perforin inserts into the target cell membrane and Gz B binds to its receptor and both are internalized to induce apoptosis. TCR stimulation increases expression of Fas ligand (FasL) on the CTL surface and binds the Fas receptor, triggering the apoptotic cascade. CTL can produce the cytotoxic cytokine tumor necrosis factor α (TNF-α), which binds the TNF-R on the target cell leading to apoptosis. CTL can also release interferon (IFN)-γ, which will activate the macrophage to release proinflammatory substances. NO, nitric oxide; ROS, reactive oxygen species.

21. T Cell Mediated Rejection on Renal Biopsy • Tubulitis • Infiltration of tubular epithelium by T lymphocytes (arrows) that have crossed the basement membrane. • Endothelialitis • Invasion of the endothelium of a large artery by graft-infiltrating lymphocytes (arrow).

22. Antibody and Complement Mediated Cytotoxicity ie AMR

23. Antibody Mediated Rejection on Renal Transplant Biopsy • Evidence of peritubular capillary C4d staining c/w AMR

24. Complement Dependent CytotoxicityCDC Crossmatch Recipient’s serum + Donor lymphocytes (with defined HLA) + Complement, Incubate, add Eosin; remember here lymphocytes are just serving as cells that can be lysed; nil to do with the immune process/function

25. Complement Dependent Crossmatch

26. Pre-Transplant Therapy Antibody Suppression Late Acute Rejection Early Acute Rejection Acute Immune Desensitization Immune Accommodation Induction Therapy Acute Post-Transplant Immunosuppression The Phases of Immunosuppression Chronic Allograft Dysfunction Maintenance Immunosuppression Graft Failure

27. Three events in T cell activation Signal 1: Engagement of the T cell receptor with the antigen peptide in the context of self major histocompatibility complex (MHC) class II molecule leads to the activation of the calcineurin pathway and results in the induction of cytokine genes (e.g., interleukin [IL]-2). Signal 2: The costimulatory signal, involves the engagement of CD28 with members of the B7 family. This synergizes with signal 1 to induce cytokine production. Signal 3: Interaction between cytokine production and its corresponding receptor leads to induction of cell division, probably through the target of rapamycin pathway. This constitutes signal 3. Immunosuppressant Action Overview

28. Categories of Agents • Induction agents • Monoclonal or polyclonal antibodies • Administered intravenously in the perioperative period • Induce acute, powerful, short-lived immunosuppression • Desensitization agents • Pretransplant IVIg can desensitize patients’ immunity to HLA • Rituximab is gaining interest as a desensitization agent • Primary immunosuppressants • CNIs are the cornerstones of immunosuppressive therapy • Adjuvant agents • 1 or more medications prescribed in addition to a CNI

29. Induction Agents • Non Depleting Antibodies • Basiliximab • Daclizumab • Depleting Antibodies • Alemtuzumab • Muromonab-CD3 • Equine polyclonal IgG antibody • Rabbit polyclonal IgG antibody

31. Primary ImmunosuppressantsCalcineurin Inhibitors (CNIs) • Calcineurin inhibition prevents nuclear factor (NFAT) activation. • In the absence of cyclosporine, calcium activates calcineurin by exposing its phosphatase site, which, in turn, activates NFAT. • Cyclosporine forms a complex with cyclophilin (CP), which binds to calcineurin (CN) and sterically hinders the phosphatase site.

32. Calcium-Calmodulin-Calcineurin form a complex to dephosphorylate NFAT Tacrolimus binds to FK binding protein which binds to calcineurin and inhibits this process Mechanism of Action: Tacrolimus

33. Is Tacrolimus Just a Clone of Cyclosporine?

34. CNI Side Effects 1. Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35. 2. Johnson C, et al. Transplantation. 2000;69:834-841. 3. Margreiter R. Lancet. 2002;359:741-746.

35. CNI Side Effects Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35.

36. Calcium channel blockers Verapamil Diltiazem Nicardipine Antifungal agents Ketoconazole Fluconazole Itraconazole Clotrimazole Metronidazole Immunosuppressants Sirolimus increases CsA levels Antibiotics Erythromycin Clarithromycin Azithromycin Protease Inhibitors Saquinavir Indinavir Nelfinavir Ritonavir Foods Grapefruit Grapefruit juice Metabolic Interactions That Increase CNI Levels

37. Antituberculosis drugs Rifampin Rifabutin Isoniazid Anticonvulsants Barbiturates Phenytoin Carbamazepine Herbal preparations St John’s wort Metabolic Interactions That Decrease CNI Levels

38. Mechanism of action of mycophenolic acid (MPA) • Lymphocytes use the do novo pathway for generation of purines (guanine). • Mycophenolate mofetil (MMF) is converted in the liver by ester hydrolysis to mycophenolic acid which in turn non-competitively and reversibly inhibits IMPDH activity during DNA synthesis in the S phase of the cell cycle. • In the salvage pathway, guanine is converted to GMP by the enzyme hypoxanthine-guanine phosphoribosyltransferase

39. MPA Adverse Reactions • Bone marrow suppression • n/v/d • Oral and colonic ulcerations • Colitis

40. Is an antimetabolite that is a purine analogue that is incorporated into DNA and halts synthesis Inhibits proliferation of T and B cells and effect is mediated by AZA metabolites, 6-MP, 6-TU, 6-MMP, 6-TGN Azathioprine

41. Azathioprine Adverse Effects • Bone marrow suppression • Hepatotoxicity • Alopecia • Drug Interactions: • Allopurinol

42. Sirolimus binds to FK binding protein but does NOT inhibit calcineurin. Instead Sirolimus inhibits mTOR and blocks IL-2 mediated cell proliferation mTOR activates protein that trigger cell cylcle G1 to S progression Sirolimus/Everolimus

43. Sirolimus Adverse Effects • Hypercholesterolemia • Hypertriglyceridemia • Edema • Hypertension • Rash • Bone marrow suppression • Interstitial pneumonitis • Delayed wound healing • Mouth ulcers • Myalgia/weakness • Drug fever • Proteinuria

44. Anti inflammatory and immunosuppressive effects Suppress production of numerous cytokines (IL-1, TNF, IL-2, chemokines, prostaglandins, proteases, NFK-B) Also affect chemotaxis (neutrophilia) Corticosteroids Side Effects Acne Cushingoid facial appearance Hirsutism Mood disorders Hypertension Glucose intolerance Cataracts Osteoporosis Growth retardation in children Corticosteroids

45. Done by your Local Urologist I always recommend drinking at least 6-8 glasses of fluid for the procedure!”

46. Transplant Surgery

47. Transplant Surgery

48. Post Surgical Complications • Vascular complications • Bleeding from vessels in the hilum • Anastamosis hemorrhage (more common with multiple arteries) • Renal artery thrombosis (due to hypotension in the OR, technical complications, antiphosholipid ab syndrome) • Renal vein thrombosis (usually 3-9 days post op and related to technical complications, antiphosholipid ab syndrome)

49. Post Surgical Complications • Vascular complications • Transplant Renal Artery Stenosis • Atherosclerosis of recipient vessel • Clamp injury to donor or recipient vascular endothelium • Faulty suture technique (primarily seen with end-to-end anastomosis • Angulation due to diproportionate length between graft artery and iliac artery • Kinking of the renal artery

50. Post Surgical Complications • Lymphocele • Collection of lymph caused by leakage from several lymphatics that overlie the iliac vessels • Usually present weeks after transplantation • Can lead to ureteral obstruction, compression of iliac vein and swelling/DVT of leg, incontinence due to bladder compression, scrotal masses • Can be avoided by minimizing dissection of iliac vessels and ligating lymphatics • Sirolimus increased the incidence of lymphoceles from 18% to 38% therefore not used as an induction agent