Dr. Rajendra Bhimma
Department Of Maternal & Child Health, Nelson R Mandela School Of Medicine, University Of Kwazulu-Natal, Durban, South Africa

Address for Correspondence:
Rajendra Bhimma,
Department Of Maternal & Child Health, Nelson R Mandela School Of Medicine, University Of Kwazulu-Natal, Private Bag 7, Congella, 4013, Durban, South Africa. E-MAIL: bhimma@ukzn.ac.za

Keywords:
immunosuppression, children, kidney, transplantation

An increasing number of immunosuppressive agents are available for use in both adult and paediatric kidney transplants and these target different steps of the immunological response to an allograft. Table 1 lists the most common immunosuppressive agents used in kidney transplantation.

Table 1. Immunosuppressive agents used in solid organ transplantation

Currently, the majority of paediatric transplant recipients are treated with some form of induction antibody [8]. Although presently no ‘universal’ protocol for immunosuppression in paediatric kidney transplant exists, current trends are towards early steroid withdrawal or directed at eliminating either steroids or calcineurin inhibitors, or both [9,10]. Another approach is to use robust induction therapy with alemtuzumab (Campath®), followed by eventual monotherapy with Tacrolimus [11,12].

Thus, although presently there is no clearly defined approach to immunosuppression in kidney transplantation for children, the eventual goal is to permit long-term graft survival and minimize side effects by the use of the fewest possible chronic medications.

A. Induction Therapy
  (a) Polyclonal lymphocyte depleting antibodies.
   The 2 polyclonal antibodies currently in use are Equine gamma globulin and anti thymocyte globulin.
   Equine gamma globulin has to be given through a central catheter because of the sclerosing nature of    the preparation. Calcineurin inhibitors are generally withheld during administration. The dose used is    15mg/kg per day. Thymoglobulin may be given through a peripheral line at a dose of 1.5-2mg/kg per    day. A single centre study has shown that recipients of thymoglobulin have decreased incidence of    acute rejections [13]. However, this result may be the reflection of the overall improved outcomes of    kidney transplants in more recent cohorts of patients [8]. Anti-lymphocyte antibody preparations are    still widely used to treat steroid resistant acute rejection episodes and are effective in 70-96% of    patients [14,5,16,17]. If a second course of polyclonal antibody therapy is required in a patient, it is    advisable to ;use a preparation obtained from a different species because of reduced efficacy    resulting from the development of xeno-specific neutralizing antibodies.

   (b) Monoclonal antibodies
     (i) Monoclonal lymphocyte depleting antibodies
     In comparison to polyclonal antibody preparations, monoclonal antibodies do not contain irrelevant      proteins, are more standardised and have a single well-defined specificity. The two most widely      used monoclonal lymphocyte-depleting antibodies are OKT3 and Alemtuzumab.

     OKT3 is administered as a bolus injection into a peripheral vein daily for 10-14 days at a dose of      5mg per day for children >30kg and 2.5mg per day for children <30kg. Calcineurin inhibitors are      withheld during the use of OKT3. Adverse effects include neurological problems [18] reactivation of      viral infections such as cytomegalovirus and Epstein-Barr virus as well as ‘first-dose      reaction’ [19,20]. Campath has been used in multiple uncontrolled pilot trials mainly in adult renal      transplant recipients. Alemtuzumab was well tolerated, but some children had acute rejection      episodes. This agent has been used more extensively in paediatric small bowel transplants [21].      Presently there is no recommended paediatric dosing for children undergoing kidney transplantation.

    (ii) Monoclonal nondepleting antibodies
    IL2-receptor antibodies

    The two IL2-receptor antibodies presently used are basiliximab and daclizumab. These two high-    affinity chimeric or humanized antibodies act on the inducible a chain of the interleukin-2 receptor     (IL-2r) on the surface of the activated lymphocyte.

    Basiliximab is given on day 0 and 4 post-transplant (generally 10mg for children <40kg and 20mg     for those >40mg) [22]. One study showed that paediatric patients receiving basiliximab as induction     therapy may have elevated cyclosporine levels and therefore would require reduced doses to avoid     toxicity [23]. Induction with basiliximab in adult kidney transplant recipients has been induced to     allow the successful early withdrawal of steroids [24] and even steroid avoidance albeit with a high     incidence of rejection [25].

    Daclizumab is generally given in a regimen of 1mg/kg intravenously on the day of transplantation     and every 14 days thereafter for 5 doses [26]. Higher doses may be required for saturation of IL-2r     in younger children [9].

    Unlike OKT3, these antibodies do not produce a first-dose-reaction and have few side effects.

   (a) Corticosteroids
   Corticosteroids are still widely used as an important component of most immunosuppressive    regimens and are almost universally used as first line treatment for acute rejection. The North    American Pediatric Renal Transplant Collaborative Study (NAPRTCS) reports shows that until recently,    up to 96% of children who underwent kidney transplantation and still have a functioning graft were    maintained in prednisone [8,27]. In most steroid based regimens the dosage is usually high in the    immediate post-transplant period, approximately 2mg/kg//day (maximum 80mg), with a gradual    reduction to approximately 0.2-0.3mg/kg/day within a 6-month to 1-year period.

   Corticosteroids have a variety of anti-inflammatory and immunomodulatory effects [28]. These    include stabilization of lysosomal membranes, suppression of prostaglandin synthesis, reduction of    &histamine and bradykinin release and lowering of capillary permeability. Anti-inflammatory effects    are mediated mainly through induced production of cytokines, including IL-1, IL-2, IL-6, ILN- ß and    TNF–ß and TNF-a. Corticosteroids impair monocyte/macrophage function and decrease the number of    circulating CD4+ T-cells.

   The numerous mechanisms of action of corticosteroids lead to multiple side effects and toxicities. The    major concern in children with respect to its long-term use in children is growth retardation. Studies    have shown that doses in excess of 8.5mg/day will impair normal growth [29].

   Other side effects include increased appetite with weight gain with Cushingoid facies, acne, glucose    intolerance, hypertension, increased susceptibility to infection, impaired wound healing, aseptic    necrosis of bone, cataracts, psychosis and peptic ulceration [30]. Sometimes there are consequences    of the mineralocorticoid activity of these agents leading to fluid retention, hypokalemia and    hypertension.

   In view of these multiple side effects of maintenance steroid therapy, attempts are focused on early    withdrawal or reduction of steroids or steroid avoidance [31]. Unfortunately the majority of these    attempts have failed because of the development of acute rejection episodes [32,33,34]. Alternate    day steroid therapy reduces its impact on growth inhibition and should be encouraged. IL-2r antibody    has been used in steroid avoidance protocols, with low acute rejection rates and striking reduction in    post-transplant complications. The Cooperative Clinical Trial in Paediatric Transplantation aimed at    corticosteroid withdrawal showed acute rejection rates at 6 months to be very low. However the    incidence of post-transplant lymphoproliferative disease was unacceptably high. In comparison, in    the control group receiving chronic low-dose corticosteroids there was no higher rate of late rejection    and long-term graft survival was similar in both groups [35]

   (b) Antiproliferative agents
     (i) Azathioprine
     This was the first immunosuppressive agent approved for organ transplantation use. Azathioprine is      metabolized to 6-mercaptopurine (6-MP) through reduction by glutathiamine, and then converted to      6-thiouric acid, 6-methyl-MP, and 6 thioguanine (6 TG). These compounds are incorporated into      replicating DNA, halt DNA replication, and block the de novo pathway of purine synthesis by puration      of thio-iosinic acid. This latter effect confers specificity of action on lymphocytes that lack a salvage      pathway for purine synthesis.

    For paediatric patients the dosage is 1-2mg/kg/day as a single dose. It can be used in combination     with all other immunosuppressive agents except mycophenolate mofetil (MMF). The most serious side     effects include skin cancers following chronic use, bone marrow suppression that is dose dependent     and occasional liver impairment and cholestatic jaundice. Minor effects include hypersensitivity     reactions manifesting as a rash [36].

    (ii) Mycophenolate mofetil (MMF)
    MMF and mycophenolate sodium (MPS) are rapidly converted in the liver to mycophenolic acid, which    is the active compound. The target of mycophenolic acid is inosine monophosphate dehydrogenase    (IMDPH). This is the rate-limiting enzyme in the de-novo synthesis of guanosine nucleotides,     themselves essential for DNA synthesis. The majority of cells generate guanosine nucleotides by two     pathways, the IMPDH pathway, and a salvage pathway; hence blockade of the IMPDH pathway     results in relatively selective blockage of lymphocyte proliferation [37].

    The recommended dose for paediatric patients is 1200/m2/day, divided in two, three, or four doses     [38]. Although therapeutic monitoring is available, current standards in paediatric patients are not     yet available to guide treatment [39,40,41,42,43,44]. MMF must not be used in combination with     azathioprine.

    The most common dose limiting adverse effects is diarrhoea. Other gastrointestinal side effects     include nausea, vomiting and abdominal pain. Bone marrow suppression also occurs. Some clinical     trials have shown an increased incidence of viral infections (CMV, herpes simplex) and candida [45].

    Analysis of large databases of renal transplant recipients have shown decreased incidence of chronic     allograft nephropathy with improved long-term renal graft function in patients on MMF [46,47].

   (c) Calcineurin inhibitors
     (i) Cyclosporine
     The mechanism of inhibiting T-cell activation by calcineurin inhibitors (CNI) is well-understood [48].      After entering the cytoplasm, CNIs form complexes with their immunophilins. Cyclosporine binds to      cyclophilin and Tacrolimus and Pimecrolimus bind to the 12 kDa FK 506-binding protein (FKBP-12).      The CNI-immunophilin complexes inhibit calcineurin activity, and hence prevent nuclear      translocation of NF-AT and cytokine gene transcription. The net result is that CNIs block the      production of cytokines and as IL-2 and inhibit T cell activation and proliferation.

     For induction purposes, cyclosporine is given intravenously in a dosage of 165mg/m2/kg/day in      children over 6 years of age, and 145mg/kg/day should be given as a continuous infusion over a      24-hour period starting intraoperatively. Induction therapy should be continued only for 48 hours      and then converted to oral cyclosporine. The recommended starting oral dose for children less than      6 years old is 500mg/m2/kg/day, administered in three divided doses. The doses given in children      are much higher than in adults as the drug is metabolized more rapidly in children [5]. Calcium      channel blockers are used concomitantly to reduce nephrotoxicity [49]. The irregular absorption and      inherent nephrotoxicity of the drug makes drug monitoring and adjustment essential. In the first      three months whole blood trough levels measured by high-pressure liquid chromatography should      be maintained between 200-250mg/ml, then between 100-200mg/ml in patients after 3 months.      More recent data suggest that measuring the level 2 hours after receiving the dose may lead to      more accurate dosing, assessing the true area under the curve and avoiding toxicity [50,51,52].

     Many of the side effects of CNIs are dose dependent and relate to the sites where calcineurin      concentrations are highest, notably the brain and kidney [53]. Nephrotoxicity is mainly due to      severe vasoconstriction of the afferent arteriole, with concomitant reduction in renal blood flow and      glomerular filtration rate [54,55,56]. Long-term use of CNIs leads to interstitial fibrosis and      obliterative arteriolar changes due to fibrosis intimal thickening in the kidneys; changes that are      non-reversible [57]. Because of its renal effects, hypertension is a common side effect of CNIs [58].

     The neurotoxicity of CNIs are more common with tacrolimus than cyclosporine and are exacerbated      by hypomagnesaemia [59]. Neurotoxic effects include headaches, tremors, agitation, convulsion,      psychosis, hallucinations, encephalopathy, and impaired consciousness [60].

     CNIs also have metabolic effects that include hyperglycemia, hyperkalemia, hyperuricemia, and      hyperlipidemia. Hyperglycaemia is two to four times more common with Tacrolimus than      cyclosporine, and may also reflect different sensitivity to the diabetogenic effects of corticosteroids      [61,62]. Other side effects of CNIs include hyperplasia and hypertrichosis that are drug specific side      effects of cyclosporin. Alopecia on the other hand may accompany Tacrolimus use [63].

     Cyclosporin has been used in combination with all other immunosuppressive agents except      tacrolimus. However, because of the potential increased risk of post transplant lymphoproliferative      disease (PTLD), the use of a combination of a calcineurin inhibitor, rapamycin and corticosteroids      should probably be avoided, particularly in high-risk children [64].

     (ii) Tacrolimus
     Tacrolimus is presently being increasingly used in paediatric renal transplant [65]. When compared      to cyclosporin, patient and graft survival at 2 years using tacrolimus are equivalent [66]. Tacrolimus      use is associated with a lower incidence of acute rejection and improved graft function. Children      treated with tacrolimus had a lower incidence of rejection (9.7% vs. 18.3%) at 2 years.

     Induction therapy is given as a continuous infusion using a dose of 0.1mg/kg/24 hours, with a switch      to oral therapy within 2-3 days. Sometimes the drug is commenced via nasogastric tube using the      oral preparation because it has very good absorption. Initial oral doses should not exceed      0.15mg/kg twice daily and should not exceed 0.1mg/kg as maintenance dose. Monitoring trough      blood levels is essential because of its nephrotoxicity. Recommended trough levels are between 10-     20 mg/l in the first 3 months and therefore between 7-12mg/l up to 12 months and then maintained      at 5-7mg/l.

     In view of the similar mechanism of action with cyclosporin, the side-effect profile of tacrolimus is      similar to that seen with cyclosporine [67]. Hypertrichosis and the dysmorphic features like gum      hypertrophy seen with cyclosporin use are not seen with tacrolimus [68].

     Nephrotoxicity is seen similar to cyclosporin use [69]. Neurological side effects are common and      may be seen more frequently than with cyclosporine [59,70]. Tacrolimus treated patients have a      higher incidence of post transplant lymphoproliferative disease and hyperglycaemia [71,72].      However, with lower doses the incidence of post transplant lymphoproliferative disease has      significantly decreased [73].

     Tacrolimus can be used in combination with all other immunosuppressants except cyclosporine.      Combination with rapamycin and corticosteroids should be used with caution in children with a      higher risk of developing post transplant lymphoproliferative disease [64].

   (d) Mammalian target of rapamycin (mTOR) inhibitors
   Sirolimus and everolimus are the newest immunosuppressive agents being used for kidney    transplant. Both are macrocyclic lactones, with sirolimus being a naturally occurring fermentation    product of the actinomycete streptomyces hygroscopicus, while everolimus represents a chemical    modification of sirolimus to improve absorption.

   TOR is a cytoscolic enzyme that regulated differentiation and proliferation of lymphocytes. Inhibition    of mTOR has a profound effect on the cell signaling pathway required for cell-cycle progression and    cellular proliferation. The net effect is blockade of T-cell activation by preventing progression of the    cell cycle from the GI to the S phase. The TOR inhibitors bind to the immunophillin

   FKBPI2 inhibits the actions of TOR [74,75,76,77,78,79]. TOR inhibitors may be particular important in    long-term immunosuppression since they stimulate T-cell apoptosis. They inhibit mesenchymal    proliferation, an important factor in graft vascular disease [80,81]. mTOR also inhibits fibroblast    growth factors required for tissue repair thus resulting in impaired wound healing.

   Rapamycin is available as either a solid or a liquid oral preparation. Although in adults a single dose    may suffice to maintain therapeutic levels, in children it has a much shorter half-life and thus    necessitates twice-daily dosaging [6]. Recommended therapeutic levels in children remain speculative    and range from 12-25ng/ml in the early post-transplant period without calcineurin inhibitors and 4-   12ng/ml with calcineurin inhibitors [6,82]. After the early post-transplant period (>3-6months), levels    are maintained between 5-10ng/ml. Lower therapeutic levels are desired when used with calcineurin    inhibitors because of enhanced nephrotoxicity.

   Side effects of mTOR inhibitors include metabolic, haematological, dermatological effects and effects    related to growth factor inhibition [83,84]. The most common side effects of rapamycin include    hyperlipidaemia, thrombocytopenia, leucopenia and delayed wound healing [85]. Dermatological side    effects include acne and mouth ulcers. Another side effect that is being increasingly seen is interstitial    pneumonitis, which appear to be dose related and resolves with drug withdrawal [89]. Peripheral    oedema, diarrhoea and lymphocele formation post renal transplant are also well recognized    complications [87].

   Rapamycin has been found to be effective in combination with calcineurin inhibitors [88,89,85,90,91]    in a calcineurin-inhibitor sparing protocol [83] and in a steroid-free protocol [92].

C. Novel Immunosuppressant Agents
Several new biological agents are in various stages of development for the purposes of replacing maintenance therapy with calcineurin inhibitors and steroids [7]. Table 2 shows the list of some of these new agents and their status in transplantation. Table 2 Biologic agents in the transplant pipeline

Adapted from: Vincenti F, Hirose R. Novel Immunosuppressants. In: Fine RN, Weber SA, Olthoff KM, Kelly DA, Harmon WE, eds. Pediatric Solid Organ Transplantation, 2nd Edition.
Massachusetts USA: Blackwell Publishing Ltd. 2007: 89-94.

Currently, only LEA29YT (belatacept) is in phase III trials. Whilst the co-stimulatory pathway is emerging as an important therapeutic area for immunosuppression therapy, other promising targets include interleukin-15 and adhesion molecules [3].

Costimulation signal is provided by engagement of one or more T-cell surface receptors with their specific ligands on antigen presenting cells. Signaling through the T-cell receptor alone without a costimulatory signal can lead to a prolonged state of T-cell energy [93]. Presently the only agent used in clinical trials in adult kidney transplant recipients, which blocks co-stimulation is belatacept [94]. This agent is typically administered intravenously on a once-per-month schedule. The results showed decreased incidence of acute rejection at 6 months (6-8%), improved glomerular filtration rate at 12 months (62-66ml/1.73m2/min) and decreased incidence of chronic allograft nephropathy. There were 3 episodes of post-transplant lymphoproliferative disease, two of which were related to primary Epstein-Barr virus infection. Thus the concern regarding its use in children is the potentially higher risk of post-transplant lymphoproliferative disease. However, this has to be balanced agents its potential benefit of improving compliance since it is administered monthly, particularly in adolescents.

To date the majority of paediatric renal transplant recipients are treated with triple immunosuppression [95]. The increasing number of agents available has increased the number of combinations and to date there are over 60 possible reported protocols [96]. These large number of protocols bear testimony to the fact that there is no single defined approach to immunosuppression for children. The final common goal is to achieve long-term graft acceptance with the fewest possible chronic medication.