Kidney transplantation is the best therapeutic option for end-stage kidney disease and offers the best survival, quality of life, and cost-effectiveness compared with dialysis. The waiting list for kidney transplantation continues to grow, however, with more than 100 000 patients currently listed for a kidney transplant in the United States, but only about 17 000 transplants performed annually ; extending the lifespan of an individual transplant is therefore of paramount importance. Although short-term allograft outcomes have improved with potent immunosuppressive strategies, long-term allograft failure rates remain unacceptably high, predominantly because of chronic antibody-mediated rejection, infections, and/or toxicity of immunosuppressive regimens . These complications often result from our inability to accurately gauge the alloimmune response, leading to the infamous ‘over’ or ‘under’ immunosuppression. The limited panel of clinical testing currently available includes qualitative or semiquantitative measurement of alloantibodies, in addition to drug-level monitoring and markers of allograft injuries, but clearly, this repertoire requires expansion. In this review, we will highlight the commonly used biomarkers in kidney transplantation alongside those under investigation, outlining their advantages and limitations.
WHAT IS A GOOD BIOMARKER?
Skin transplants were perhaps the earliest biomarker in kidney transplantation: weeks before the first kidney transplant performed by Joseph Murray at the Peter Bent Brigham in Boston in 1954, full thickness skin grafts were exchanged between the donor–recipient twin brothers. One month later, when the potential recipient was hospitalized for uremia and was supported by ‘the artificial kidney’, there was no evidence of rejection of the skin grafts, providing proof of tissue compatibility . Once transplanted, the kidney allograft functioned immediately and had a prolonged survival. A later but equally pivotal milestone in the identification of kidney transplantation biomarkers was the development of the microcytotoxicity assay by Paul Terasaki in Los Angeles: donor cells were mixed with recipient serum to detect cytotoxicity, which predicted donor–recipient pairs at risk for ‘immediate failure’ . Almost half a century later, the quest for ideal biomarkers in kidney transplantation continues, reflecting the complexity of the immune response.
According to the NIH Biomarkers Definition Working Group in 2001 , a biomarker is a biological marker with ‘a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention’. An ideal biomarker is one that can be tested cheaply and rapidly using easily obtained samples, with high positive and negative predictive values. The identification of biomarkers is a multiphase process that starts with a scientific discovery based on experimentation. The candidate biomarker is then verified within a small sample, followed typically by internal single-center then external multicenter validation phases. Once validity is established, the standardization phase refines the technique, before reaching the commercialization phase. These development phases are often intertwined, each providing feedback and optimization potential . This is an arduous path, however, and there are few biomarkers that have successfully navigated the stringent requirements to reach commercialization.
CURRENT BIOMARKERS IN KIDNEY TRANSPLANTATION
Efforts to identify clinically relevant transplant biomarkers have extended to focus on the alloimmune pathways of antigen recognition, gene transcription and translation, posttranscription regulation of gene expression, antigen expression, cell activation, cytokine secretion, and antibody secretion (Fig. 1).
Antigen recognition biomarkers
As mentioned above, a positive microcytoxicity assay heralds the presence of circulating alloreactive antibodies, and, consequently, the likelihood of hyperacute or acute rejection. T and B cell flow cytometry crossmatches are more sensitive assays, and can detect circulating donor-specific antibodies that are alloreactive but insufficient to result in a positive microcytotoxic assay. Nevertheless, a positive T-cell flow crossmatch is associated with early graft loss, subclinical rejection, and antibody-mediated rejection . Although the crossmatch remains the cornerstone of clinical transplantation, it has several limitations, including the need for donor cells, fresh recipient serum to capture recent sensitizing events, and high false-positive rates, particularly with the B-cell flow crossmatch.
T-cell alloreactivity assays
Although antidonor memory T-cell activity is not commonly assessed before transplantation, it can be determined by measurement of interferon-γ (IFN-γ) secretion by recipient T cells when exposed to donor antigens. The IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay can quantify donor-reactive memory T cells before and after kidney transplantation; early studies showed that higher levels of cellular alloreactivity are associated with acute rejection, delayed graft function, and chronic allograft dysfunction . However, in the multicenter Clinical Trials in Organ Transplantation-01 (CTOT-01), a similar pretransplant correlation was not seen universally, thought because of the effects of variable use of depleting induction immunotherapy [9▪]. Although serial monitoring of T-cell alloreactivity with this assay may be very helpful, the need for donor cells and the labor-intensive nature of ELISPOT makes this a less than an ideal biomarker.
Adenosine triphosphate secretion
Increased adenosine triphosphate (ATP) production is thought to be one of the earliest steps that follow antigen recognition. The Immuknow assay, whereby CD4+ T cells are magnetically isolated from mitogen-stimulated peripheral blood mononuclear cells and then lyzed to facilitate measurement of ATP release, received regulatory approval following a multicenter trial that analyzed the immune response by measuring ATP release in clinically stable transplant patients, compared to those with confirmed rejection episodes or documented infections . Low ATP levels were associated with infections, whereas high levels were associated with increased likelihood to develop rejection. A subsequent large retrospective study failed to show a similar association at 90 days after one measurement , whereas a meta-analysis showed poor predictive value of this assay for infection or rejection after kidney transplantation . The widespread clinical application of this test has been stymied by the need for a short interval between collection and sample storage, the ‘time after transplant’ effect on ATP values , and a limited ability to interpret single results.
Gene expression biomarkers
T-cell cytotoxicity: perforin/granzyme B
Activated cytotoxic T lymphocytes (CD8+) induce target cell apoptosis by producing perforin (PERF) and granyme B (GrzB) and lead to tubular epithelial cell injury in kidney transplant rejection. Indeed, mRNA levels of PRF and GrzB in urinary cells have been shown to correlate with acute rejection . Furthermore, in several studies, intra-graft GrzB transcripts showed significant accumulation in acute rejection compared to nonrejection [15–17]. A meta-analysis encompassing 680 patients from studies looking at quantitative detection of GrzB and PRF mRNA showed that combined elevation of these markers was associated with higher probability of acute rejection [18▪]. Stability of mRNA in decaying cells may limit the precision of these assays, which are yet to be validated in large trials.
Regulatory molecules: FOXP3
Regulatory T cells are mainly CD4+ T cells that are CD25 and FOXP3 positive, and which exert suppressive action in an activated T-cell milieu. Their activity is mediated by cytokine deprivation, direct inhibition, or even cytotoxicity of neighboring activated T cells. Increased FOXP3 mRNA in urinary cells has been associated with reversibility of allograft rejection when studied in kidney transplant recipients with or without recovery from acute rejection . Furthermore, peripheral blood FOXP3 transcripts were significantly lower in kidney transplant recipients with chronic rejection compared to ‘clinically tolerant recipients’ . Conversely, intragraft FOXP3 mRNA levels correlated with the severity of cellular rejection ; indeed, a higher density of intragraft FOXP3+ T cells as detected by immunohistochemistry was noted in patients with acute cellular rejection and was associated with worse graft survival . Among patients with delayed graft function, levels of FOXP3 mRNA in kidney tissue, urinary cells, and peripheral leucocytes were also higher in those patients who transpired to have acute rejection compared to those with acute tubular injury or no rejection . The type of induction agent used, the temporal relation of FOXP3 mRNA measurement to the rejection episode, and the lack of dynamic measurements could explain these differences, particularly when FOXP3 mRNA levels are interpreted in isolation.
The ‘molecular microscope diagnostic system’ is a microarray-based system that evaluates gene expression on kidney allograft biopsies to predict T-cell-mediated or antibody-mediated rejection before changes are visible by standard pathological methods . Using microarray testing with ‘pathogenesis-based transcript sets’ (PBTs) that associate with T-cell-mediated rejection, antibody-mediated rejection, interstitial fibrosis/tubular atrophy, or acute kidney injury phenotypes, in addition to machine learning, a biopsy result is plotted in relation to a reference set of samples with established diagnoses [25▪]. This allows diagnostic and prognostic stratification in comparison to the reference set. A recent large international multicenter study validated this method, showing that with a 29-h workflow, microarray analysis of allograft biopsy correlated better with clinical judgment than did histology [26▪▪]. Advantages of this assay include the smaller sample requirement, reproducibility of the results, and the probabilistic readout of the result; the main disadvantage is the need for a kidney biopsy to obtain a sample for analysis.
The ‘kidney solid organ response test’ (kSORT) is a less invasive microarray-based test, performed using peripheral blood, that identifies a set of 17 genes involved in leukocyte trafficking, activation, adhesion, and cytolysis . The test could predict rejection with high sensitivity and specificity, but could not distinguish between T-cell and antibody-mediated rejection. A subsequent study by the same group further validated this assay, and showed that combining it with the antidonor IFN-γ ELISPOT assay may help distinguish the rejection phenotype [28▪▪]. Advantages of the kSORT assay include the high accuracy for predicting rejection using peripheral blood; its main limitation is the inability to discern the nature of the rejection when used alone.
Genomics of chronic allograft rejection score
The prospective multicentre study, genomics of chronic allograft rejection (GoCAR), aimed to generate a gene set that could predict chronic failure due to fibrosis rather than focus on predicting allograft rejection. The study identified and externally validated a set of 13 genes that was superior to standard clinical and histological variables in predicting progression to fibrosis and early graft loss after transplantation [29▪]. The genes were involved in cell growth pathways, tumor growth and suppression, and membrane repair. However, the gene signature was obtained 3 months after transplantation, which is likely to reflect a very different alloimmune milieu than that present long after kidney transplant and is associated with the development of de-novo donor-specific antibodies and shorter allograft survival, and is again limited by the requirement for an invasive biopsy.
Based on the single center work at Cornell to predict allograft rejection via a gene signature in urinary cells , a multicenter clinical trials in organ transplantation (CTOT-04) took place, investigating thousands of samples collected from hundreds of patients at varying time-points after kidney transplantation, wherein measured mRNA levels were correlated with the occurrence of acute allograft rejection. A 3-gene signature of CD3ε and interferon-inducible protein 10 (IP-10) mRNAs, in addition to18S rRNA, could predict the occurrence of rejection, and could distinguish acute cellular rejection from acute antibody-mediated rejection. Furthermore, neither urinary tract infection nor the type of induction immunosuppression used affected the diagnostic capacity of the gene signature . The main advantage of this assay is the noninvasive nature of the sample required (Fig. 2), yet, processing urine for mRNA extraction could be technically challenging especially in peripheral laboratories.
Gene translation and protein synthesis biomarkers
The CXCR3 chemokines CXCL9 [monokine induced by interferon-γ (MIG)] and CXCL10 [interferon-induced protein-10 (IP-10)] have been of significant interest in the search for reliable transplant biomarkers, and studies to date indicate they are among the most promising candidates [32,33]. In the multicenter CTOT-01 trial, urinary levels of CXCL9 mRNA and protein were shown to robustly diagnose acute rejection, with levels rising up to 30 days prior to clinical detection . Furthermore, low levels of urinary CXCL9 protein at 6 months were associated with low likelihood of future development of acute rejection or reduction in GFR up to 2 years posttransplant . A further study showed that, compared to stable individuals and those with calcineurin inhibitor toxicity or interstitial fibrosis/tubular atrophy, levels of CXCL9 and CXCL10 were elevated in both acute rejection and polyoma (BK-virus) virus infection, although were unable to distinguish between these entities . CXCL10 has also been shown to sensitively detect acute rejection, with levels rising prior to any clinical changes, and in particular, in a cohort of patients with both indication and protocol biopsies, has been shown to be capable of detecting borderline and subclinical tubulitis, and overt acute rejection . More recent work has shown that the combination of urinary CXCL10, normalized to urinary creatinine levels, and donor-specific antibody levels, improved the diagnosis of antibody-mediated rejection compared to donor-specific antibodies alone; furthermore, the urinary CXCL10:Cr ratio at the time of antibody-mediated rejection was independently associated with graft loss [35▪]. In addition to proven efficacy, testing for urinary CXCL9 and CXCL10 is available on an ELISA or Luminex-based platform, is simple, noninvasive and cost-effective, and is therefore ideally suited to longitudinal monitoring.
Humoral response biomarkers
Accumulating data support that preexisting or de-novo donor-specific anti-HLA and non-HLA antibodies often precede the development of proteinuria, rapid decline in allograft function, and allograft failure [36–39]. Techniques for alloantibody detection have evolved over the last five decades, and although the complement-dependent cytotoxicity crossmatch remains in widespread use, a significant increase in the sensitivity of this assay has been provided by the flow cytometry crossmatch and the advent of solid phase assays (i.e. Luminex), which are FDA-approved for qualitative use, but in practice, are often interpreted per their quantitative strength. They are used in generating calculated panel reactive antibody titers and virtual crossmatch testing, and for longitudinal screening. The high sensitivity of the Luminex assay results in significant inter and intra-laboratory variability  which may hamper interpretation; furthermore, the lack of a cellular element in this platform compared to the crossmatch assays poses a challenge in establishing the relevance of the detected antibody when clinical allograft injury is absent. Attempts to address this pitfall were made by measuring the complement binding ability of Luminex-detected donor-specific anti-HLA antibodies; such ability was found to be a predictor of aggressive antibody-mediated disorder including allograft loss . A subsequent report confirmed this observation but also highlighted a comparable negative effect of noncomplement binding donor-specific antibodies [42▪▪].
Cellular antigen expression profiling
Among the unique features of lymphocytes is their ability to undergo somatic rearrangement that allows them to express T-cell and B-cell receptors that respond to specific antigens, conferring the immune system with immense diversity. This diversity is made possible by the random somatic recombination of DNA from genes coding for the variable, diversity, and joining segments of the receptors, and endows each T-cell or B-cell with the ability to detect one or very few antigens . Immunosequencing allows the profiling of each T-cell and B-cell clone by multiplex PCR combined with high-throughput bioinformatics, generating a repertoire of specific lymphocyte clones that can be followed longitudinally, the expansion of which may correlate with rejection, infection, or tolerance after kidney transplantation [44▪▪,45,46]. This approach is only in the very early stages of biomarkers discovery but may prove to be a promising tool to measure the alloimmune response.
Proteomics and mass spectrometry
Mass spectrometry offers a nonbiased high throughput approach to identify one or more markers of rejection. Although rejection is a dynamic heterogeneous process, the proteome can theoretically mirror these changes and offers an ideal platform to identify biomarkers for rejection . Most of the published studies have focused on the urine proteome, with identification of multiple molecules, although only a few have been reproduced by different groups, including α1-antichymotrypsin, uromodulin, β2-microglobulin, and fragments of collagen . It is important to note that differing MS methods may influence the proteome signature and markers identified which may complicate its translation to the clinic.
Because of the complex and redundant nature of the immune response, the path towards the identification of an ideal biomarker in transplantation remains long. Array-based and/or targeted assays that aim to predict rejection, alloantibody formation, infection, fibrosis, or allograft loss by utilizing kidney tissue, peripheral blood or urine are under investigation. Thus far, very few tests have gained regulatory approval for commercial clinical use in kidney transplantation, but many are being validated in the hope that they will facilitate improved longitudinal monitoring, allowing more tailored immunosuppression, and, in turn, improved long-term allograft survival.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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