Kidney transplantation successfully treats many patients with end-stage renal failure but many kidneys are still lost to immunological rejection processes. Research is focused on finding predictive laboratory tests that would be useful for identifying patients at high risk of rejection to then adjust their immunosuppression and hopefully extend the life of their kidney transplants.
by Ruth Platt and Richard Battle
Renal transplantation is the treatment of choice for patients with end stage renal failure (ESRF). A successful transplant will free patients from the morbidity and mortality associated with debilitating dialysis routines that replace only around 15% of normal kidney function. However, kidney transplantation is not available to all patients as deceased donor kidneys are a scarce resource and not all patients have a suitable live donor. Despite good short term results, the half-life of a renal allograft at our centre is approximately 8-10 years, so some patients could need three or four kidney transplants during their lifetime.
The process of transplantation requires input from various laboratory specialties, including histocompatibility and immunogenetics (H&I), biochemistry, haematology, immunology and virology. Laboratory results aid selection of the best patient to receive a particular donor’s kidney, provide information about the risk level of the transplant and monitor the patient post-transplant for any developing problems. This review aims to give an overview of laboratory tests currently used, mainly those performed by the H&I laboratory, and also introduces newer areas of research into ‘biomarkers’ for kidney transplant rejection.
Kidney transplant rejection
Recent investigations into causes of kidney transplant loss found that both cellular and antibody mediated immunological mechanisms pose a major threat to the renal allograft [1]. Rejection of the transplanted kidney can occur at different time points post-transplant via different alloantigen presentation and immune mechanisms, as summarised in Table 1 and Figure 1a and 1b. The cellular and molecular mechanisms involved in kidney allograft rejection have been thoroughly reviewed by Cornell et al [2].
The search for a biomarker
A non-invasive means of identifying patients at risk of or currently undergoing an episode of rejection would be a valuable addition to current patient monitoring. The ideal laboratory biomarker for use in kidney transplantation would:
1. Be measurable in urine, serum or peripheral blood samples
2. Aid estimation of risk of rejection as part of pre-transplant testing
3. Precede the onset of acute rejection and chronic allograft nephropathy (CAN) in time to treat and prevent damage to the graft
4. Indicate kidney damage from infection, resurgence of original condition or the nephrotoxic effects of commonly used immunosuppressive drugs including the calcineurin inhibitors cyclosporine and tacrolimus
5. Measure response to rejection treatments
This additional information would enable customisation of immunosuppressive drug regimens to achieve the fine balance between susceptibility to infection and greater risk of rejection. As well as drug nephrotoxicity, over-suppression of the immune system increases susceptibility to infection, malignancies, diabetes and osteoporosis.
Pre-transplant laboratory testing
Virology testing of donor and recipient for infectious diseases, including cytomegalovirus (CMV), will indicate any need for prophylactic antiviral treatment to prevent post-transplant infections which increase the proinflammatory environment and risk of rejection. Kidneys are allocated to ABO matched or compatible patients in all but exceptional circumstances. H&I laboratories measure various aspects of compatibility between donor and recipient including determining the degree of human leukocyte antigen (HLA) matching. Traditionally this was performed using the complement-dependant cytotoxicity (CDC) method but with the development of PCR- based techniques HLA genotypes can be matched more accurately. Allocation of kidney grafts in the UK and mainland Europe incorporates laboratory assessment of matching at HLA-A, B and DR loci. Despite improvements in immunosuppression increasing graft survival overall, transplants with zero HLA mismatch at HLA-ABDR still have the best survival rates [3].
Patients can develop antibodies against foreign HLA to which they are exposed by pregnancy, blood transfusion or a previous transplant. Hyperacute rejection is now very rare, with regular HLA antibody screening of patients used to avoid allocating kidneys with unacceptable HLA mismatches to specifically sensitised patients. HLA-antibody screening can be performed by various methods, the most recently developed Luminex bead-based flow cytometry assay providing very sensitive and specific results [4]. The evolution of antibody screening methods and the problem of determining which antibodies are clinically relevant are thoroughly reviewed in the literature [5]. The pre-transplant crossmatch involves mixing donor T and B cells with patient serum then assessing antibody binding and cell death by the CDC assay or more sensitive flow cytometry methods [6]. A positive crossmatch result was traditionally a contraindication to transplantation after a study in 1969 correlated positive CDC crossmatches with hyperacute rejection [7]. However, some centres now use crossmatch results for assessing the risks of performing a particular transplant and to establish if extra immunosuppression may be indicated to counter the increased risk of rejection.
Cellular assays
Non donor-specific immune parameters have also been associated with kidney transplant rejection. Soluble CD30 (sCD30) has been considered as a marker of the activation of T cells which are involved in acute rejection. CD30 is a 120 kDa transmembrane glycoprotein expressed on T cells secreting Th2 cytokines and is suggested to have a co-stimulatory role regulating the balance between Th1/Th2 immune responses. The soluble form is released from the activated T cell surface and is detectable in serum using an ELISA assay. Pre-transplant sera from kidney transplant recipients contain higher levels of sCD30 than healthy controls and many studies have reported the association between high levels of sCD30 in pre-transplant sera and increased frequency of rejection [8]. However, sCD30 is not a biomarker for rejection as the assay lacks sensitivity and specificity in predicting who will develop rejection. Some patients with high sCD30 do not develop clinically diagnosed rejection whilst some with low sCD30 levels do. As such, despite a strong statistical association at a cohort level, confidence in sCD30 as a predictive biomarker would not be sufficient for its use in adjusting a patient’s levels of immunosuppression [9].
The ‘Immuknow’ assay measures levels of intracellular adenosine triphosphate synthesis (iATP) as produced by CD4+ T cells in response to non-specific stimulation. Reinsmoen et al suggest it gives an indication of the level of the patient’s general immune response and potential for acute rejection [10]. Patients with very low iATP levels have also been shown to be at greater risk of severe post-transplant infections but these results have not always been reproduced by other studies into this marker.
Genetic polymorphism and pre-transplant risk
There is growing interest in the innate immune system’s contribution to the overall immune activity of the patient and potential for rejection. During the transplant procedure, the graft undergoes ischaemia-reperfusion injury, and activation of Toll-like receptors (TLR) of the innate immune system stimulates release of proinflammatory cytokines including TNFα and IL-1 to attract cells to the graft. TLR activation also affects dendritic cells, part of the transition to the donor antigen-specific phase of transplantation alloimmunity. Polymorphisms in genes encoding innate immune system components can be determined by PCR to add to the immunological profile of the patient and aid risk assessment. A polymorphism in the gene encoding TLR4 has been associated with reduced response to microbial pathogens and also associated with reduced risk of acute rejection in kidney transplantation [11].
Polymorphisms in cytokine genes can lead to measurable differences in the amounts of cytokine protein produced. Many pro- and anti-inflammatory cytokines have been investigated for associations with acute rejection and kidney graft survival, including TNFα, IFNγ and IL-1. However, despite some studies suggesting these factors have a clinical influence, most studies report lack of significant association once multivariate statistical analysis is used. Polymorphisms should be considered in both the recipient and donor’s genotype, as the transplanted kidney tissue itself will also produce these cytokines. The controversial role of cytokine polymorphisms in genetic risk of kidney transplant rejection is unsurprising when considering the complex networks and overlapping roles of cytokines in the inflammatory processes of rejection.
Minor histocompatibility antigens (mHAgs) potentially have a role in rejection of well-HLA matched kidneys, for example those from live sibling donors. Increased rejection and worse graft survival was seen in female patients receiving HLA-matched grafts from male donors, the alloreactivity attributed to the female immune system’s response to the male H-Y antigen. Where a choice of donor is available, male patients may benefit from the larger nephron mass of a male kidney and female patients benefit from female donor kidneys removing H-Y as a potential source of alloreactivity [12].
Polymorphisms in the MHC class I chain-related genes A and B (MICA and B) have been examined in autoimmune diseases and haematopoietic stem cell transplantation (HSCT) but have not been thoroughly investigated in kidney transplantation. However, antibodies to MICA have been detected in kidney transplant recipients and are thought to influence the length of graft
survival [13].
Investigation into killer-cell immunoglobulin receptors (KIR) and their HLA ligands in kidney transplantation has provided mixed results. Self-HLA binding to KIR provides activating and inhibitory signals, the balance of which influence natural killer (NK) cell activation. Therefore, a combination of KIR and HLA mismatching can lead to NK cell activation which may contribute to rejection. Whilst KIR genotype has been significantly linked to graft-versus-host disease following HSCT, the most positive correlations with kidney transplant cohorts have been between the patient’s KIR repertoire and their likelihood of acute CMV infection post-transplant [14].
Current post-transplant monitoring:
Immunosuppressive drug levels in patients’ blood are regularly monitored to ensure correct dosage is achieving the narrow therapeutic window and to detect any patient non-compliance with the drug regimen once they have left hospital. Patients can wish to avoid the unpleasant side effects of these drugs but non-compliance is a major risk factor for acute rejection. This monitoring is performed using mass-spectrometry or immunoassay methods [15].
Serum creatinine is currently measured as an indicator of kidney function post-transplant. A very large study by Hariharan et al. showed that serum creatinine levels measured at six months and 1 year post transplant were highly predictive of the half-life of the transplanted kidney [16]. However, creatinine has limited specificity and sensitivity in allograft rejection and is used as an indicator of when to perform a biopsy, which is the gold standard used to identify rejection. The Banff criteria were developed in the early 1990s to standardise histological scoring of kidney biopsies, and were updated in 2007 to include C4d amongst other developments [17]. C4d is an inactive fragment of the C4b component of the classical complement pathway. When activated by the patient’s antibody and C1, it binds to proteins in the peritubular capillaries of the kidney and can be detected by immunofluorescence microscopy or immunohistochemistry. Anti-HLA IgG is detected in around 95% of patients with C4d staining at biopsy. However, biopsy cannot be used for frequent monitoring as it is invasive and due to the ‘patchy’ nature of alloreactive kidney damage, tissue of diagnostic relevance may not be sampled.
Additional post-transplant markers
Effector molecules released by cytotoxic T cells have been investigated as markers for acute kidney rejection. Activated cytotoxic T cells release cytotoxins into the intracellular space when in contact with target cells, including perforins and granzymes. Increased expression of the gene encoding granzyme B intragraft and in peripheral blood has been associated with acute rejection in renal transplantation. Granzyme B is a proapoptotic serine protease and apoptosis occurs upon granzyme uptake in the target cell. A granzyme B ELISPOT assay measured the release of the cytolytic protein which correlated with numbers of cytotoxic T
lymphocytes after HLA-identical live kidney transplantation [18].
The search for a non-invasive biomarker led to study of the urine proteome as this biofluid is available in large volumes and is relatively stable. Β2-microglobulin (B2-m) is an 11.7 kDa protein which is noncovalently bound to the HLA class I antigens found on the surface of all nucleated cells. B2-m is shed from the cell surface and circulates in serum until it is filtered by the glomeruli and reabsorbed by proximal tubular cells. Tubulointerstitial injury from rejection or immunosuppressive nephrotoxicity reduces reabsorption of B2-m, increasing levels detected in the urine and indicating a biopsy to assess graft damage. Many additional proteins markers have been analysed in urine using mass-spectrometry methods but frequently they are also markers for other renal diseases reducing their usefulness in predicting
rejection [19].
In the past few years interest around the association of HLA-antibodies and CAN has grown. Terasaki’s group reported that patients who developed HLA antibodies had a decreased graft survival rate of 58% compared to 81% in HLA antibody negative patients at four years post-transplant. They conclude that testing for HLA-antibody should be routine post-transplant [13].
Conclusions
There is currently no ideal biomarker predictive of kidney transplant rejection. Although current pre- and post-transplant tests allow a degree of prediction of risk, this is not enough to predict when additional immunosuppression is needed and who may be potentially tolerant of their graft with reduced immunosuppression. Achieving a tolerant state without the need for maintenance immunosuppression is the ‘holy grail’ for current transplant research [20]. Occasional cases of patients removed from immunosuppression either accidentally or purposefully have provided rare examples of genuinely tolerant patients with long-term graft function. The search for predictive biomarkers to stratify risk of rejection in kidney transplant patients is an important part of working towards tolerance and improvement in the long-term outcomes of kidney transplantation.
References
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2. Cornell LD et al. Kidney Transplantation: Mechanisms of Rejection and Acceptance. Annu Rev Pathol Mech Dis 2008; 3: 189–220
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13. Terasaki PI et al. Four-year follow up of a prospective trial of HLA and MICA antibodies on kidney graft survival. Am J Transplant 2007; 7: 408.14. Hadaya K et al. Natural killer cell receptor repertoire and their ligands, and the risk of CMV infection after kidney transplantation. Am J Transplant. 2008; 8(12): 2674-83.
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16. Hariharan S et al. Post-transplant renal function in the first year predicts long-term kidney transplant survival. Kidney Int 2002; 62(1): 311-318.
17. Solez K et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008; 8(4): 753-6.
18. Nowacki TM et al. Granzyme B production distinguishes recently activated CD8 + memory cells from resting memory cells. Cell Immunol 2007; 247: 36.
19. Qunitana L et al. Urine Proteomics Biomarkers in Renal Transplantation: An Overview. Transplantation 2009; 88: S45-S49.
20. Goldman M, Wood K. Translating transplantation tolerance in the clinic: where are we, where do we go? Clin Exp Immunol 2009; 156(2): 185-8.
The authors
Ruth E. Platt, BSc (Hons), MRes, MSc &
Richard Battle, BSc (Hons), MSc
Clinical Scientists
Transplant Immunology Laboratory
St James’ Hospital, Leeds, UK
Correspondence to:
Ruth Platt
Transplant Immunology Laboratory,
Gledhow Wing, St James’ Hospital.
e-mail: ruth.platt@leedsth.nhs.uk
Tel: +44 113 2064579
