Transplantation Reviews 28 (2014) 15–20

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Transplantation Reviews journal homepage: www.elsevier.com/locate/trre

Urine proteomics in kidney transplantation☆,☆☆ Steven C. Kim, Eugenia K. Page, Stuart J. Knechtle ⁎ Emory University Hospital, Department of Surgery, Atlanta, GA, 30322

a b s t r a c t The transplanted kidney, through its urinary output, provides a medium through which the molecular constitution can provide insight into either the healthy function or developing dysfunction of a newly transplanted organ. An assay that would detect the aberration of early biomarkers of allograft injury using only urine samples from patients would provide many advantages over the current use of creatinine and tissue biopsies, as these means are either relatively non-specific or very invasive. Several urine biomarkers have been correlated with allograft injury, including CXCL9, CXCL10, CCL2, NGAL, IL-18, cystatin C, KIM-1 and Tim-3. The recent results of the CTOT-01 trial serve to validate the predictive value of the CXCL9 biomarker as a non-invasive biomarker for rejection and a prognostic indicator of graft function. There is now a preponderance of evidence showing a value of urinary monitoring of CXCL9 and CXCL10 with respect to detection of acute kidney allograft rejection. The value of the assay has been validated as a means of reducing the need for kidney transplant biopsy and applying biopsy in a more targeted manner. Additional goals for non-invasive monitoring would include predictive value prior to creatinine elevation that in turn would permit earlier, preemptive treatment of rejection. © 2014 Published by Elsevier Inc.

1. Background Effective monitoring for complications of kidney transplantation currently depends on frequent invasive procedures. While clinical judgment and serum laboratory values may alert the physician of acute injury to the newly transplanted kidney, a rising creatinine does not differentiate the many etiologies of post-transplant allograft injury. BK viral nephropathy, for example, reflects overimmunosuppression whereas acute rejection indicates at least a transient need for more immunosuppression; this difference in treatment necessitates a quick and accurate assessment of what is causing injury. The gold standard for diagnosis remains kidney biopsy, as only histological examination of the affected renal tissue can differentiate acute rejection, BK virus infection, and acute tubular necrosis, from other types of injury. The need for repeated tissue biopsy is not only resource consuming, but it also poses an additional post-operative risk to patients. The role of non-invasive monitoring through biomarkers has been a subject of interest for many years [1]. Not only would the identification of sensitive and specific biomarkers for post-surgical kidney injury obviate the risk of repeated invasive monitoring, but a rapid and accurate diagnosis of postoperative complications in

☆ Contributions: All authors contributed to the design and writing of this review paper. ☆☆ Funding: No funding. ⁎ Corresponding author at: 101 Woodruff Circle, 5105-WMB, Atlanta, GA 30322. Tel.: +1 404 712 9910; fax: +1 404 727 3660. E-mail address: [email protected] (S.J. Knechtle). 0955-470X/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.trre.2013.10.004

transplant patients would provide the best chance for long term graft survival for these patients [2]. Additionally, the use of highthroughput methods would allow for a rapid assay of many potential biomarkers of clinical disease with a relatively minute biological fluid sample. The difficulty that investigators have run into with the development of these assays and identification of useful clinical biomarkers has been the lack of clinical validation studies for any potential biomarkers that are identified. Due to the untargeted approach that is taken when attempting to identify biomarkers or correlate –omics findings with clinical pictures, it is difficult to develop robust studies that can be implemented in a clinical setting with complex patients [3]. Nevertheless, ongoing biomarker discovery studies in the fields of proteomics and metabolomics are resulting in the identification of many exciting potential markers for specific injury processes as well as guiding future research that may shed better understanding on the disease processes.

2. Candidate and potential biomarkers Many studies looking for potential biomarkers for acute rejection and kidney injury in post-transplant patients have searched for noninvasive sampling means to identify elevated markers of damage. Assays comparing the significantly differing levels of markers between patients with healthy grafts and those with grafts of biopsy-proven signs of acute rejection have yielded a multitude of potential candidates for clinical markers of early organ injury either due to drug toxicity, infection, or rejection.

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2.1. Untargeted Discovery of Chemokines and Cytokines A common approach to identifying previously unrecognized biomarkers is to use screening assays of urine samples from patients demonstrating evidence of acute rejection. One study by Hu et al. [4] utilized a multiplex assay to identify further chemokines and cytokines that would supplement previously identified markers of interferon gamma (IFN-γ)-induced protein 10 kDa (IP-10, also known as CXCL10) and monokine induced by IFN-γ (Mig, also known as CXCL9) in the recognition of early renal injury. For this prospective study, renal transplant patients were selected based on an elevated serum creatinine and clinical indication for a diagnostic biopsy. Urinary samples were collected before biopsy from 84 patients who had an elevated creatinine of 20% or above baseline, and 29 renal transplant patients who were prospectively selected with stable serum creatinines were used as controls. 19 additional healthy non-transplanted patients were used as additional controls. An initial antibody array including 120 cytokines and chemokines was performed on the urine samples from three renal transplant patients and three healthy controls as an initial screen; 23 cytokines and chemokines were found elevated in the rejection patients. A targeted multiplex assay on these 23 cytokines/chemokines was then performed using the Luminex Multi-Analyte Profiling (xMAP) platform. Among the 113 renal transplant patients recruited for the study, diagnoses of acute rejection, antibody-mediated acute rejection, borderline rejection, BK viral nephropathy, acute tubular necrosis, chronic allograft nephropathy, and stable graft function were made using biopsy as the gold standard. This study found urinary IP-10, Mig, macrophage inflammatory protein (MIP-1δ), and osteoprotogerin (OPG) are early indicators of renal allograft injury. Renal transplant patients with elevated IP-10 and Mig more often had acute rejection, acute tubular necrosis, BK viral nephropathy, and antibody-mediated rejection, whereas patients with chronic allograft nephropathy and stable graft function had lower levels of IP-10 and Mig. Importantly, MIP-1δ and OPG were found to be elevated in patients with borderline rejection or chronic nephropathy, but normal in patients with stable allograft function. This study provides an early basis for the development of a non-invasive assay that could differentiate stable graft function from either chronic damage or other means of acute damage in the immediate postoperative setting of kidney transplant. A group in China [5] performed a similar protein expression assay comparing serum samples from transplant recipients. This was a retrospective study of 526 kidney transplant patients across 5 transplantation centers in China. Patients included in the analysis consisted of those with biopsy-confirmed acute cellular rejection, stable renal allograft controls, delayed graft function, and those with pulmonary infection. Similar to the study by Hu et al., the Luminex assay system was used to evaluate sera from these patients. Ninetyfive proteins were included in the initial screen, and 11 markers specific to acute cellular rejection were identified and quantified. Results of the study showed the greatest differential expression of interleukin (IL)-1Ra, IL-20, and sCD40L among the study populations. In 95.2% of patients that went on to develop acute cellular rejection, these three chemokines were significantly reduced in the serum. A validation cohort of 24 patients consisted of 12 patients with stable graft function and 12 patients with acute cellular rejection; 22 of the 24 samples were correctly assigned when using a split-point scoring system based on the three markers identified in this study: IL-1Ra, IL20, and sCD40L. However, as with most untargeted studies the lack of further clinical validation raises concerns of these markers’ specificity for cellular rejection. Despite the use of validation cohorts, the clinical implementation of these detection assays is difficult because of their lack of specificity for disease process. A cytokine-based screen developed from De Serres et al. identified six cytokines as markers of acute rejection: IL-

1β, IL-6, tumor necrosis factor (TNF-α), IL-4, GM-CSF, and monocyte chemoattractant protein-1 (MCP-1). Sixty-five patients were prospectively recruited over the course of 1 year; recruitment criteria included an admission at least 2 weeks out from transplant surgery to undergo graft biopsy for an acute rise in serum creatinine. Follow up blood samples were provided from 33 of the patients 3 months after the initial presentation. The blood samples collected on the day of biopsy were analyzed using a multiplex cytokine panel. After candidate cytokines were identified, further regression modeling and use of a validation cohort identified IL-6 as the most accurate cytokine marker for clinical rejection. While a cutoff value of IL-6 could serve to identify clinical rejection, it was widely variable in patients with borderline rejection. Blood samples drawn at 3 month follow up demonstrated that IL-6 levels were lower with acute rejection. Given the sensitivity of IL-6 in identifying early allograft dysfunction, a prospective trial using this marker assay in the setting of subacute and early acute rejection may be particularly useful. 2.2. CXCL9/CXCL10 Much of the progress in targeted identification of biomarker validation has been made with urinary CXC-receptor 3 chemokines. CXC chemokine receptors are G protein-linked membrane proteins. CXCR3 is a receptor in the CXC family that is expressed predominantly on T lymphocytes and induced upon immune cellular activation. The related ligands for this receptor that have been most investigated in the realm of transplant immunology are CXCL9 and CXCL10. These chemokines, predominantly secreted by leukocytes in transplanted allografts and tubular epithelial cells, are important for cell signaling toward sites of inflammation [6,7]. Therefore, these chemokines are prime biomarker candidates for early detection of inflammation in new kidney transplant patients. In a study by Schaub et al. [8], CXCR3 chemokine levels were correlated with the extent of allograft inflammation to evaluate their potential use as an early detector of graft dysfunction. Urine samples were obtained just prior to either a protocol graft biopsy or a clinically indicated biopsy. The biopsies were graded according to the Banff 2007 criteria; 88 patients were included in the study and 5 groups were identified: normal tubular histology group (n = 24), subclinical borderline tubulitis group (n = 15), subclinical tubulitis Ia/Ib group (n = 22), clinical tubulitis Ia/Ib group (n = 17), and interstitial fibrosis/tubular atrophy (IFTA) group (n = 10). CXCL9 and CXCL10 urinary levels were found significantly higher in subclinical tubulitis Ia/Ib group than in the subclinical borderline tubulitis and the normal tubular histology groups. This correlation was not seen with CXCL11 or the control chemokines CXCL4 and CCL2, suggesting that CXCL9 and CXCL10 serve as urinary surrogates for the extent of subclinical graft inflammation. In the urine of patients with clinical tubulitis Ia/Ib compared to those patients with no evidence of inflammation on histology, all urinary chemokine levels were elevated. In patients with IFTA, however, the urinary chemokine levels were not significantly elevated compared to the patients with normal tubular histology, suggesting that these markers indicate an acute inflammatory process. Two markers of tubular injury, urinary α1-microglobulin and neutrophil gelatinase-associated lipocalin (NGAL), were also analyzed in the urine samples from the patients in the 5 clinical inflammation groups. These markers did not differ significantly between the subclinical borderline and subclinical tubulitis groups, but they were elevated when compared to the normal tubular histology group. Both markers were also significantly elevated in patients with clinical tubulitis when compared to normal tubular group patients. Interestingly, unlike the chemokine markers CXCL9 and CXCL10, α1-m and NGAL were increased in the urine of patients with IFTA. This study showed that elevated levels of urine CXCL9 and CXCL10 were good indicators of early graft dysfunction and rejection,

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as levels were normal in normal histology and chronic inflammation (IFTA) groups. Ho et al. [9] published a validation study to address the capability of CXCL9 and CXCL10 level measurement to detect early graft inflammation. This study attempted to validate the findings of CXCL9 and CXCL10 elevation in subclinical tubulitis in an independent cohort. 102 urine samples were collected and analyzed from 91 patients undergoing either protocol or for-cause biopsies. Six clinical groups were identified based on renal biopsy: normal histology (n = 22), IFTA (n = 20), IFTA and borderline tubulitis (n = 13), borderline tubulitis (n = 13), subclinical tubulitis (n = 17), and clinical tubulitis (n = 17). CXCL10 accurately distinguished between the various degrees of tubulitis as well as normal graft histology. This study also utilized the CXCL10:Cr cutoff of 5.3 ng/mmol used in the study by Schaub et al., and found similar capability of CXCL10 in distinguishing borderline, subclinical, and clinical tubulitis from the normal histology group. The authors went further by analyzing CXCL10 levels as a continuous variable in order to determine an ideal cut-off ratio of CXCL10:Cr which maximized both sensitivity and specificity. When differentiating normal histology from borderline and subclinical tubulitis, a cut-off value of 1.97 ng CXCL10/mmol Cr provided maximal sensitivity and specificity; when differentiating normal histology from subclinical and clinical tubulitis, a cut-off value of 2.87 ng CXCL10/mmol Cr was determined ideal. This study expands upon the previous finding of CXCL10 as a sensitive marker for graft inflammation and demonstrates that its elevation prior to elevated creatinine levels serves as an early marker of prognostic significance with regards to graft inflammation and long-term survival. Extending this validation then into a real clinical application, HirtMinkowski et al. [10] conducted a clinical trial with an independent patient cohort. 213 consecutive patients were selected and had 362 surveillance biopsies at 3 and 6 months post transplantation; 80 indication biopsies were also performed within the first year of transplantation. All biopsies were graded and assigned to five groups according to Banff criteria: acute score zero, interstitial infiltrates only, tubulitis t1 with any inflammation, tubulitis t2-3 with any inflammation, and isolated vascular compartment inflammation. Urinary CXCL10 levels were measured retrospectively using a sandwich ELISA and measured in relation to creatinine to account for differing urine dilutions. Among the five groups defined by histologic inflammation grade, CXCL10 to creatinine ratios were found to be significantly variable among the groups, even when excluding patients who had biopsies with concomitant infection with BK virus or urinary tract infection. With each increasing level of inflammation on biopsy, there was an increase in the CXCL10:Cr ratio. In an effort to find a CXCL10:Cr cutoff sensitive and specific for subclinical tubular inflammation, the five inflammation groups found on biopsy were separated into two groups: the acute score zero and interstitial infiltrates in one group, and the biopsies in the remaining three inflammation grades in the second group. Urinary CXCL10:Cr levels were significantly higher in the second group consisting of the tubulitis t1, t2-3, and isolated vascular compartment inflammation patients. When comparing subclinical and clinical tubulitis groups with respect to CXCL10:Cr levels, there was a significant increase in level in the clinical tubulitis group versus the subclinical tubulitis group t2-3. This study was significant in that it validated the previous studies with a separate patient population in showing that CXCL10 was incrementally elevated in the progression of tubulointerstitial inflammation. Given the many etiologies of tubulointerstitial inflammation, a study by Jackson et al. [11] investigated CXCL9 and CXCL10 level variations among different causes of graft inflammation including acute rejection, BK virus infection, calcineurin inhibitor toxicity, or interstitial fibrosis. This study was conducted in a pediatric transplant population (n = 46) as well as adult patients (n = 79) and healthy controls (n = 31). Biopsies from each patient revealed 25 patients

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with acute rejection, 50 clinically stable transplant patients, 24 patients with BK virus infection, 17 patients with calcineurin inhibitor toxicity, and 9 patients with IFTA; of note, only patients with a definitive, exclusive diagnosis per biopsy Banff score were included in the analysis. When comparing all adult and pediatric patients, this study found that no significant difference in CXCL9 and CXCL10 levels existed between healthy controls and histologically stable transplant patients. Chemokine levels were elevated in patients with acute rejection and BK infection, and were more sensitive and specific for detecting graft injury from these etiologies than serum creatinine alone. Because patients with subclinical rejection were included in this analysis, a subset analysis was conducted, which found elevated CXCL9 and CXCL10 levels in patients with subclinical rejection as well as subclinical BK infection; these findings are similar to what previous studies have reported about these chemokines’ potential roles as early markers for graft inflammation. Similar to adults, the pediatric transplant patients with acute rejection and BK infection also had elevated levels of urine CXCL9 and CXCL10 compared to the healthy controls and stable graft function transplant patients. While urine creatinine was increased in those patients with acute rejection, there was no significant elevation for those patients whose graft inflammation was due to BK viremia. This study demonstrates, along with other prior analyses, that the potential for CXCL9 and CXCL10 as a noninvasive screening tool for the detection of subclinical graft inflammation may be realized with further prospective clinical trials. Recently, a prospective, multicenter validation study was conducted through the Clinical Trials in Organ Transplantation-01 protocol [12], which included over 2000 urine samples from 258 patients. Real-time PCR was performed on urine sediment RNA querying for a number of genes previously reported to be elevated during acute rejection. Additionally, ELISA assays were run on urine supernatant samples to compare values of CXCL9 and CXCL10; this study found that CXCL10 proteins were similar during acute rejection and infection, but CXCL9 could discriminate acute rejection over other diagnoses. Combining CXCL9 protein data with mRNA showed a negative predictive value of 92.5% at ruling out rejection, with elevated CXCL9 levels found up to 30 days before clinical evidence of graft dysfunction. Furthermore, low urine CXCL9 levels in surveillance samples at 6 months corresponded to patients at low risk for future acute rejection, and those with high levels corresponded with biopsyproven tubulitis. Another recently published prospective, multicenter clinical trial investigated mRNA levels in 4300 urine samples from 485 kidney recipients, through the Clinical Trials in Organ Transplantation 04 Study [13]. The investigators formulated a three-gene signature of CD3ε mRNA, CXCL10 mRNA, and 18S ribosomal RNA to distinguish acute cellular rejection from antibody-mediated and borderline rejection. A receiver-operating-characteristic (ROC) curve analysis showed an area under the curve of 0.85, giving a 79% sensitivity and 78% specificity using its designated cutoff point. Patients who developed biopsy-proven rejection had a sharp rise in their gene signature during the weeks prior to rejection. 2.3. CCL2 Another chemokine that serves as a chemoattractant for inflammatory populations of cells is CCL2 – a substrate for CCR2 receptor. Ho et al. [14] analyzed urinary levels of CCL2 along with urinary α1M, CXCL9, and CXCL10 at 6 and 24 months after transplant to evaluate variations in chemokine levels and their correlations with protocol biopsies. Samples from 111 patients were analyzed, and the degree of IFTA determined by protocol biopsy. This study found that levels of CCL2 at 1 month after transplantation was associated with moderate IFTA at 6 months on protocol biopsy, but not impaired renal function. To assess long-term outcomes correlating with markers at 6 months,

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additional protocol biopsies at 24 months were performed. CCL2 at 6 months was found to be positively correlated with the development of severe grade IFTA and graft dysfunction at 24 months, unlike other chemokines that were analyzed in this study. This raises the implications of its use as an early detectable urinary marker for long-term graft survival and function. A study by Ho et al. [15] then extended the findings of CCL2 as a marker for IFTA development at 24 months and applied it to longterm graft loss. While IFTA may be a marker for late graft loss, the correlation between CCL2 and long term graft loss had not been studied in an independent cohort of transplant patients until this study. 231 patients were included in the study and only patients with detectable urine CCL2 at 6 months after surgery were compared. The groups consisted of patients with normal graft function (n = 116), graft dysfunction (n = 75), death with function (n = 20), and graft loss (n = 20). Elevated CCL2 levels in urine at 6 months after surgery were associated with late graft loss but not with stable graft function or graft dysfunction not resulting in graft loss. This analysis showed that in addition to certain patient factors such as delayed graft function, recipient age, and early acute rejection, the level of urinary CCL2 6 months from surgery was an independent predictor for death-censored graft loss. This held true even when transplant patients with donor specific antibody present before operation and those with delayed graft function after operation were included in the analysis. Interestingly, while patients with graft loss at 24 months had elevated CCL2 levels, patients with only graft dysfunction at 6 months did not have a noted increase in urinary CCL2 levels. The significance of this study then lies in the demonstration of CCL2 levels as an independent predictor of eventual graft loss, which could lead to increased monitoring and tailored immunosuppression protocols in those patients immediately post-transplant who exhibit elevated levels of this chemokine.

2.4. Urine Neutrophil Gelatinase-Associated Lipocalin (NGAL) and IL-18 In addition to cellular rejection, attention has also been turned to identifying biomarkers that could be markers for delayed graft function after transplantation. While many scoring methods and algorithms for predicting delayed graft function have been proposed, a study in 2006 by Parikh et al. [16] sought to identify early markers for the onset of delayed graft function using a genome assay for biomarker discovery and identified NGAL as being markedly elevated soon after ischemic kidney injury. Delayed graft function was defined in this study as requiring dialysis within the first week after transplantation. 53 patients undergoing living related or deceased donor kidney transplantation were prospectively enrolled, and spot urine samples were collected in the first 24 hours after transplantation and daily postoperatively. An ELISA assay was then performed on the urine samples and NGAL levels were quantified and reported as a ratio to creatinine. In patients with delayed graft function, serum creatinine peaked 2–4 days after the operation, while NGAL and IL-18 levels were markedly elevated in patients even within 24 hours after surgery; levels were significantly higher in deceased donors with delayed graft function compared to both living donors and deceased donor transplants who had prompt graft function. Hall et al. [17] in 2010 performed a multicenter prospective study of patients receiving deceased donor kidney transplants to assess urine levels of NGAL and IL-18 and their correlation to delayed graft function. 91 patients were included in the study and 34 required dialysis within 1 week of transplantation. This prospective cohort study showed similar results as previous studies – namely that levels of NGAL and IL-18 were significantly elevated in urine of deceased donor kidney transplants within 24 hours of the operation who subsequently required dialysis within 1 week of transplantation. Furthermore, NGAL levels successfully differentiated between kidney

recipients who had immediate graft function, slow graft function, and delayed graft function. In applying this elevation of urinary NGAL to acute allograft rejection in addition to delayed graft function, investigations have also attempted to describe the uses of NGAL monitoring after transplant [18]. A study by Heyne et al. [19] took samples from 182 patients following kidney transplantation and analyzed the distribution of patient characteristics with variations in NGAL levels. In patients with stable allograft function, median urinary NGAL concentrations were 7.8 ng/mL. In contrast, patients with acute kidney injury (AKI) from all other causes other than acute rejection had elevated NGAL levels at a median concentration of 59.1 ng/mL, while patients with AKI from biopsy proven allograft rejection had a marked elevation of NGAL concentrations with a median value of 339 ng/mL. ROC analyses were also performed to assess cutoff values of NGAL to predict AKI (30 ng/ mL, sensitivity = 0.86, specificity = 0.86) and acute allograft rejection (100 ng/mL, sensitivity = 1.00, specificity = 0.93). 2.5. Cystatin C Cystatin C (CyC), a cysteine protease inhibitor, is freely filtered by glomeruli [20] and has been shown to be a potent inhibitor of neutrophil chemotaxis [21]. In healthy kidneys, this low molecular weight protein is effectively degraded by proximal tubular cells and is detected in only low concentrations in urine. Abnormal reabsorption due to tubular cell injury results in elevated urine CyC levels. It has thus been considered as reflecting proximal tubular function, and several groups have reported the superiority of using serum CyCbased estimations of glomerular filtration over serum creatinine [22]. Others have also used urine CyC as a biomarker for acute kidney injury following cardiac surgery. Hall et al. conducted a prospective, multicenter observational cohort study of 91 kidney transplant recipients grouped into delayed graft function (hemodialysis within first week after transplant), slow graft function (serum creatinine reduction b 70% within first week), and intermediate graft function (creatinine reduction ≥70% within first week). Urine CyC levels were highest for delayed graft function, with elevations as early as the 0 hour timepoint that were sustained until the second postoperative day. ROC analysis showed that urine CyC were moderately accurate in predicting delayed graft function, with areas under the curve ranging from 0.57 – 0.74. Lower urine CyC/ creatinine ratios and larger decreases in urine CyC in the early postoperative days correlated with higher GFR at 3 months. Fluctuations in serum cystatin C have been documented with high dose steroids post-transplantation [23], so future studies controlling for the effects of immunosuppression on urine CyC should be performed. Also, animal studies have shown that proteinuria (induced by albumin injection in rats) was accompanied by increased CyC and NGAL excretion but not IL-18 [24], perhaps through competitive inhibition of low molecular weight protein reabsorption. This study suggests that proteinuria should be considered when interpreting elevated levels of uring CyC and NGAL. 2.6. Kidney Injury Molecular-1 (KIM-1) KIM-1, a type 1 transmembrane protein with an immunoglobulin and mucin domain, has been described as a biomarker for proximal tubule injury [25]. Nogare et al. analyzed KIM-1 mRNA in for-cause biopsy tissues and found that the molecule is expressed on T helper type 2 cells and not normal renal cells; it is also expressed on dedifferentiated cells in damaged proximal tubule areas from ischemic and toxic injuries [26]. The same group followed this study with a review of RNA from kidney biopsies and urine samples from 77 renal transplant patients. Four groups were identified: acute tubular necrosis (n = 9), acute rejection (49), calcineurin inhibitor nephrotoxicity (10) and IFTA (29). Higher levels of KIM-1 mRNA were

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detected in biopsies and urinary sediment cells of patients with IFTA than other groups [27]. Van Timmeren et al. followed 145 renal transplant patients for a median of 4 years, and used a Luminex-based assay to evaluate urine KIM-1 levels with long-term outcomes. Urine excretion of KIM-1 was found to be an independent predictor of graft loss, independent of creatinine clearance, proteinuria or donor age [28]. 2.7. T-cell immunoglobulin domain, mucin domain (Tim-3) Tim-3, a type 1 cell membrane protein expressed on terminally differentiated T helper cells also with an immunoglobulin and mucin domain, is increased in renal biopsies of rejecting allografts [29]. Manfro et al. evaluated whether Tim-3 expression could diagnose acute rejection in renal transplant recipients with acute graft dysfunction or delayed graft function [30]. Fifty patients with delayed graft function, 50 with acute graft dysfunction, and 15 with stable graft function were included, each with kidney biopsy, urine and peripheral blood samples. Real-time PCR was used to detect Tim-3 mRNA. Significantly elevated levels were detected in patients with biopsy-proven acute rejection, compared to calcineurin-inhibitor nephrotoxicity, IFTA, and normal tissues. Tim-3 levels also decreased significantly in kidney, blood and urine in the patients treated for acute rejection. Renesto et al. in a prior study also noted that urine Tim-3 mRNA levels were significantly higher in patients with acute rejection compared to those with biopsies showing no acute rejection or with stable graft function [31]. Tim-3 mRNA levels were over 10-fold higher than interferon gamma mRNA in those with acute rejection, which makes it an attractive noninvasive biomarker to study. 3. Discussion Kidney transplant recipients represent a unique subset of the transplant population in that these patients lend themselves to potentially easier means of allograft monitoring. The transplanted kidney, through its urinary output, provides a medium through which the molecular constitution can provide insight into either the healthy function or developing dysfunction of a newly transplanted organ. The development of an assay that would detect the aberration of sensitive and specific early biomarkers of allograft injury using only urine samples from patients would provide many advantages over the current use of creatinine and tissue biopsies, as these means are either relatively non-specific or very invasive. The challenge, as discussed above, remains in developing assays that are clinically validated in a robust population of patients and controls. While there have been many candidate biomarkers of early rejection or allograft injury identified using untargeted high throughput means of discovery, validation trials in patients with long term follow-up permit conclusions about the clinical use of these assays, and such validation trials have not been performed for most candidate biomarkers. The recent results of the CTOT-01 trial serve to validate the predictive value of the CXCL9 biomarker as a non-invasive biomarker for kidney transplant rejection and as a prognostic indicator of graft function. Taken together, there is now a preponderance of evidence showing a value of urinary monitoring of CXCL9 and CXCL10 with respect to detection of acute kidney allograft rejection. The value of the assay has been validated as a means of reducing the need for kidney transplant biopsy and applying biopsy in a more targeted manner. Additional goals for non-invasive monitoring would include predictive value prior to creatinine elevation that in turn would permit earlier, preemptive treatment of rejection. The early detection of acute rejection through noninvasive means is not only important in confirming cellular rejection without placing patients through repeated tissue biopsies, but it would also serve to detect subclinical rejection in patients. The impact that multiple

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subclinical rejections can have on long-term renal allograft survival has been described [32], and the ability to detect these episodes of rejection that do not manifest clinically would ultimately be protective for the recipients of new kidneys. Proper identification and treatment of subclinical rejection would minimize the inflammatory injury on a molecular level of these newly transplanted organs in order to offer newly transplanted patients the maximal benefit of allograft survival. Importantly, the application of a sensitive and specific noninvasive assay would also benefit a population of transplant patients who do not tolerate invasive monitoring well – pediatrics. Children who receive transplanted kidneys face a number of challenges in follow up and close monitoring of their allografts. The challenges of effective communication, education about their new transplanted organs, and the difficulty of tolerating multiple tissue biopsies are all barriers that face the clinician when dealing with the pediatric transplant population. The ease of allograft assessment with a urine assay that the pediatric patient could easily provide during a clinic visit would greatly help in closely monitoring allograft function after surgery. Ultimately, the goals in the development of noninvasive monitoring assays for kidney transplant patients lie in providing the best chance for long-term allograft survival. The more sensitive and accessible the means of surveillance are, the greater the chance that patients will be more compliant, invested, and vigilant about monitoring. This vigilance and rapid detection of allograft injury is what would result in the identification of patients who will require more intensive monitoring to ensure that their new allografts function well for the longest possible time. Ultimately, the potential for patients to monitor their own allograft function using home monitoring assays remains to be seen, as this would provide the means for patients to be vigilant about the health of their new organs without requiring repeated lab draws, invasive tissue biopsies, and repeated clinical visits which can provide practical barriers to providing the best care for a clinically demanding patient population. Disclosures: The authors have no financial or conflict of interest disclosures.

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