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. Schematic of MHC 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).

7. Comparison of MHC Class I vs II
Comparison of MHC class I and class II molecules
Class I MHC
Class II MHC
Structure
MHC encoded chain, beta-2 microglobulin
MHC encoded chain, MHC encoded beta-chain
Antigen binding groove*
Closed
Open
Peptide length
9 to 10 amino acids
12 to 28 amino acids
Source of antigen
Intracellular
Extracellular
Expression
All nucleated cells
B cells, macrophages, dendritic cells
Antigen presented to:
CD8+ T cells
CD4+ T cells

8. Direct and Indirect Allorecognition

9. The Processing of Antigens and Presentation to T Cells

10. T Cell Recognition of Alloantigen: Indirect Pathways
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.

11. T Cell Recognition of Alloantigen: Direct Pathways
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.

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

13. T Cell Receptor and APC Interaction: Signal 1
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

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. B, T and NK Cells and Effect: Rejection
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.

18. Immune Activation and Rejection
Immune System
Encounters the
Transplant
Hyperacute
Rejection
Stimulus
T Lymphocytes
Are Activated
And Make
Cytokines
Activation
T Cells
Divide
Plasma
Cells
Make
Antibody
Expansion
Effector
T Cells
Attack the
Transplant
Humoral
Rejection
Immune
System
Develops
T and B
cell memory
Memory
The development of successful immunosuppressive drug regimens has been based on an understanding of the immune mechanisms leading to allograft rejection. The T-lymphocyte plays a key role in the early immune events occurring after exposure to an allograft. Ultimately, T-cells regulate B-cell activity leading to a combination of cellular and humoral events that lead to acute rejection and/or chronic allograft nephropathy over a well-delineated course of time
… 24 hours… days… 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 Cytotoxicity CDC 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. The Phases of Immunosuppression
Pre-Transplant Therapy
Antibody Suppression
Late Acute Rejection
Early Acute Rejection
Acute Immune
Desensitization
Induction
Therapy
Acute Post-Transplant
Immunosuppression
Immune
Accommodation
Maintenance
Immunosuppression
Chronic Allograft
Dysfunction
Immunosuppression is delivered to organ transplant recipients in serial phases. For highly sensitized patients, this may begin prior to transplantation with treatments designed to reduce anti-HLA antibody titers (e.g., plasmapheresis, IVIg, anti-CD20 antibodies). Induction therapy, consisting of treatment with anti-lymphocyte antibodies, is administered immediately after transplantation in selected patients. Maintenance immunosuppression is generally required for the life of the allograft, including phases characterized by early acute rejection, immune accommodation, and, in some cases, late graft failure.
Graft Failure

27. Immunosuppressant Action Overview
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.
This slide depicts the immune cascade as related to a transplanted organ and shows the various points within the cascade where immunosuppressive agents affect the immunologic process.

28. Categories of Agents Induction agents Desensitization 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
The categories of immunosuppressive agents include:
Induction agents—Monoclonal or polyclonal antibodies are administered intravenously in the perioperative period to induce an acute, powerful, short-lived immunosuppression before initiation of long-term maintenance immunosuppression.
Primary immunosuppressants—CNIs remain the cornerstones of immunosuppressive therapy.
Adjuvant agents—One or more medications, such as corticosteroids, MMF, AZA, or SRL, are prescribed in addition to a CNI.

29. Induction Agents Non Depleting Antibodies Depleting Antibodies
Basiliximab
Daclizumab
Depleting Antibodies
Alemtuzumab
Muromonab-CD3
Equine polyclonal IgG antibody
Rabbit polyclonal IgG antibody
This slide lists the induction agents currently used in transplantation in the United States. In addition to their role in delivering early, acute, short-lived immunosuppression, these agents can be used to delay administration of CNIs until after a transplanted kidney has recovered function. Several of these agents are also used to treat severe acute cellular rejection.

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

33. Is Tacrolimus Just a Clone of Cyclosporine?
Merten et al asked the question: “Is NaHco3 just a NS clone or is there more to it wrt preventing CN”

34. CNI Side Effects Event1 Comments Hepatotoxicity Tacrolimus = CsA
Cardiovascular
Hypertension
Hypercholesterolemia
Less HTN and Hyperlipidemia with Tacrolimus
Post Transplant DM
Tacrolimus causes more post transplant Diabetes Mellitus
Neurotoxicity
Tremor
Headache
Insomnia
Paresthesia
Seen more often with TAC and generally improve with dose reduction
TAC and CsA have similar mechanisms of action and, therefore, share a number of adverse events. The common side effects of the CNIs are discussed in the table presented on this slide and the next.
1. Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35.
2. Johnson C, et al. Transplantation. 2000;69:
3. Margreiter R. Lancet. 2002;359:
1. Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35.
2. Johnson C, et al. Transplantation. 2000;69:
3. Margreiter R. Lancet. 2002;359:

35. CNI Side Effects Event Comments Cosmetic Gingival hypertrophy
Hirsutism
Alopecia
Use of corticosteroids may exaggerate development
Gingival hypertrophy and hirsutism are associated with CsA
Alopecia can occur with TAC
Malignancy
Skin cancers
Cervical cancer
Lymphoproliferative disorders
Incidence appears to be a function of overall amount and duration of immunosuppression rather than any specific agent
(CNI Side-Effects, continued from previous slide.)
Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35.
Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35.

36. Metabolic Interactions That Increase CNI Levels
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
Frequently, drug interactions involve altered drug metabolism, often as mediated by the cytochrome P-450 isoenzyme system. Concomitant administration of a CNI and other agents that compete for this metabolic pathway can result in altered CNI drug levels. This slide lists concomitant medications (and foods) that can increase CNI blood levels.

37. Metabolic Interactions That Decrease CNI Levels
Antituberculosis drugs
Rifampin
Rifabutin
Isoniazid
Anticonvulsants
Barbiturates
Phenytoin
Carbamazepine
Herbal preparations
St John’s wort
This slide lists concomitant medications and herbal preparations that can decrease CNI blood 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. Azathioprine
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

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

42. Sirolimus/Everolimus
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

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. Corticosteroids Side Effects
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

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

51. Post Surgical Complications
Urine Leaks
Occur within the first few days
Due to technical or as a result of ureteral slough due to disruption or ureteral blood supply. Blood supply to distal ureter is the most endangered during harvesting
Ureteral Obstruction
Technical
Blood clots
Ureteral slough
Ureteral fibrosis
Ischemia, rejection or polyoma virus BK
Extrinsic compression
Kinking or periureteral fibrosis from lymphoceles or graft rejection

52. Post Surgical Complications
Wound
Infections
Healing becomes a problem in obese recipients
GI Complications
Bowel obstruction, colonic perforation, gastric ulcer

53. Delayed Graft Function
Poor definition:
Need for dialysis in the 1st week post transplant
Etiology
Immune
Hyperacute rejection
Non immune
ATN, intravascular volume contraction
Technical: arterial, venous or ureteric occlusion, urine leak
Recurrent disease (HUS/TTP)

54. Delayed Graft Function
Factors promoting ischemic injury in Deceased donor renal transplantation
Donor Factors (age, h/o HTN, DM, ARF)
Donor Management (brain-death stress, ionotropes, nephrotoxins, cardiac arrest)
Procurement surgery (Hypotension, traction on renal vessels, flushing solution)
Kidney storage (pump vs cold storage, prolonged time)
Recipient status (volume status, body habitus, pelvic atherosclerosis, poor cardiac output, hypotension in OR, preformed antibodies, prolonged warm ischemia time)

55. Renal Transplant Program at McMaster University
All forms of transplantation
Standard Criteria
Donation after Cardiac Death
Extended Criteria (donors > 60yo, donors 50-59yo with 2 additional RF (h/o HTN, death as a result of a stroke, elevated terminal serum Cr > 132umols/L)
Cross Match Positive Transplants
ABO incompatible Transplants

56. Rejection and Treatment
Acute Cellular Rejection
Steroids, Thymoglobulin, CNI
Antibody Mediated Rejection
PLEX
IVIG
Rituximab
Bortezomib
Urgent splenectomy

57. Rejection and Treatment
And of course prayer!
Surgical resident ?

58. Longterm Complications
Allograft Failure
On average 50% of DDRT allografts are functioning at the year mark
On average 50% of living donor allografts are functioning at the year mark
Etiology of Allograft Failure
Immune: Rejection, HLA mismatch, prior sensitization history, medication non compliance
Non Immune: donor factors (age, ECD, etc), CNI toxicity, HTN, Hyperlipidemia, Infection (CMV), Proteinuria, renal size mismatch, recurrent disease, BK Nephropathy

59. Longterm Complications

60. Longterm Complications
Renal Tx patients are at high risk of dying with a functioning allograft
40% from CVD
25% infection
10% malignancy
25% other

61. Longterm Complications
CVD Risk Factors
20% of renal transplant recipients will develop impaired fasting glucose post tx and 10% will develop T2DM
Prior ESRD pts
HTN
Hyperlipidemia
Cigarette smoking
Inflammation
Infections (CMV)

62. Longterm Complications
Infections
CMV BK
Hepatitis C
Influenza
Fungal

63. Infection Timeline Post Transplant
Fishman NEJM 2007

64. Malignancy Skin Cancer: 3.5 fold increase
Post Transplant Lymphoproliferative disorder
GU cancers ~HPV related vulva, vagina, cervical cancers
Liver cancer (Hep B, C pts)
Cumulative incidence of malignant tumors over 30 years is 33%

65. Summary Overview of Immunology Immunosuppressive Medications Rejection
Know pharmacology and adverse effects
Rejection
Antibody Mediated Rejection
Acute Cellular Rejection
Causes of Allograft Failure: differentiate immune vs non immune
Surgical Complications
Longterm Medical Complications

66. Thank You For Your Attention

68. Causes of Elevated Resistive Index Parenchymal
Acute Rejection
Acute Tubular Necrosis
Pyelonephritis
Vascular
Renal Vein Thrombosis
Hypotension
Urological
Ureteral Obstruction
Technical
Graft Compression