The short-term outcome of renal transplantation has improved during the past 20 years. The introduction of cyclosporine and the use of newer and combination of immunosuppressive drugs has substantially increased the rate of short-term graft survival.1 However, long-term graft survival has not noticeably improved.2 Among all causes of long-term graft failure, chronic transplant dysfunction (CTD) is the most common indication for retransplantation.1 The cause of CTD is multifactorial, with alloresponsiveness, diabetes mellitus, hypertension, and nephrotoxicity of immunosuppressants having a role herein.3-5 Clinically, hypertension, increased levels of serum creatinine, and proteinuria are found in the patients with signs of CTD.6 The definite diagnosis requires histological analysis of a kidney biopsy in which interstitial fibrosis, tubular atrophy, and glomerulosclerosis are indicators.7 Although needle biopsy of the graft is the most sensitive diagnostic method, there is a 5% to 10% risk of biopsy-associated complications,8 such as hematoma, infections (including sepsis and perinephritic abscess), and even loss of the renal graft.9
Because currently no single test is available to accurately predict the risk for CTD, the search for a suitable biomarker for CTD is still ongoing. Such a marker would allow close monitoring of the development of CTD, especially in high-risk patients. Consequently, the use of renal graft biopsy for graft monitoring may be minimized. Additionally, immunosuppressive therapy could be tailored according to the individual risk profile.10
Monitoring of tryptophan (trp) metabolism through the enzyme indoleamine 2.3-dioxygenase (IDO) has been previously proposed to predict acute rejection in renal transplant patients.11 Indoleamine 2.3-dioxygenase12 catalyzes the initial and rate-limiting step of trp oxidative catabolism with ormation of several intermediaries, collectively referred to as kynurenine (kyn).13,14 The rate of trp degradation, expressed as the kyn/trp ratio, has been used as a good estimate of enzymatic activity of IDO.15,16
Indoleamine 2.3-dioxygenase has been documented to be critically involved in establishing immune tolerance against paternal antigens in pregnant mice17 and in inducing T-cell unresponsiveness.12,18,19 Moreover, several studies indicate IDO activity or levels of its substrate and/or metabolites to associate with or predict acute rejection. Brandacher et al20 documented elevated serum and urinary kyn/trp ratios during acute rejection of human kidney transplants. Further, Lahdou et al11 found that increased pre-transplantation serum kyn levels predict acute kidney allograft rejection in humans. Additionally, trp metabolism correlates with the severity of chronic kidney disease.14 There is yet no data regarding IDO activity or trp metabolism in relation to the development of CTD or in long-term, uncomplicated renal transplantation. As a first step toward assessing the potential use of IDO activity as a biomarker in patients with CTD, we analyzed the levels of trp and kyn in both serum and urine samples during a follow-up period of 2 years in kidney transplant patients. Moreover, level of rejection (Banff score) and IDO expression in renal biopsies were assessed using (immuno)histochemistry and correlation, and prediction analyses were performed between the parameters of trp metabolism and the 2-year outcome of renal transplantation.
MATERIALS AND METHODS
Study Design and Patient Population
Forty-eight patients (between 18 and 70 years), receiving a kidney transplant were included in a 24-month, prospective, randomized trial. The immunosuppressive treatment before the transplantation and during the follow-up was previously described.2 Briefly, the perioperative immunosuppressive regimen consisted of 20 mg badiliximab intravenous before transplantation and on day 4 and 2 doses of 50 mg prednisolone intravenous during the first 48 hours. During the first 6 months after transplantation, all patients received oral prednisolone (P) and triple-drug therapy consisting of cyclosporine A (CsA), mycophenolic sodium and everolimus (Table 1). After 6 months, the patients were randomized to double therapy with P/CsA, P/mycophenolic sodium, or P/everolimus. Patients with histological features of rejection continued on the triple-drug medication. Immunosuppressive drug exposure was closely monitored, and its level was adjusted when necessary. Scheduled renal biopsies were performed at 6 months and 2 years after transplantation.
Serum and urine were collected at 2 weeks, 6 months, and 2 years after transplantation. Clinical parameters as well as serum creatinine and albuminuria were measured at each timepoint. Estimated creatinine clearance was calculated according to the Cockroft-Gault formula: (140 − age) × body weight × gender coefficient / serum creatinine.14
Tryptophan and Kynurenine Measurements
The concentration of trp and kyn in serum and urine was measured by a high-throughput on-line solid-phase extraction-liquid chromatographic-tandem mass spectrometer, as described earlier.21 Briefly, 50 μL of serum or urine were prepurified by automated on-line solid-phase extraction, using strong cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogues occurred by C18 reversed phase chromatography. Mass spectrometric detection was performed in the multiple reaction-monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Detection limit was 30 nmol/L for trp and 1 nmol/L for kyn. Finally, kyn/trp ratio was calculated as an indirect estimate of IDO activity.
Histology and Immunohistochemistry
Periodic acid schiff staining was performed on biopsies taken at 6 months and at 2-year follow-up, according to the standard protocol.
Immunohistochemistry for IDO was performed on biopsies taken at 6 months and at 2 years after transplantation. Normal kidneys were used as controls. Biopsies were dewaxed and subjected to antigen retrieval by 15 minutes incubation in 0.1 M Tris/HCl buffer, pH 9.0, at 80 °C. Monoclonal antibody 4.16H1 recognizing human IDO was produced in the laboratory of Van den Eynde and Théate (unpublished data). Specificity controls and validation of the monoclonal antibody for immunohistochemistry were described in Jacquemier et al.22 For immunohistochemistry, a 3-step immunoperoxidase technique was used, according to standard techniques. Peroxidase activity was developed using diaminobenzidine and H2O2. The cortical staining was measured using an Aperio-Image Scope based protocol, and these values were further used in the correlation analysis.
Data are presented as mean ± SEM in case of normal distribution and as median (interquartile range) in case of skewed distribution. Significance was tested with the analysis of variance for repeated measures followed by a least significant difference post hoc test, or with the Friedman test, as appropriate (Statistical Package for the Social Sciences [SPSS]; IBM Corporation, Armonk, NY). The potential relationships between trp and kyn concentrations and kyn/trp ratio and clinical parameters were analyzed using Pearson parametric correlation test or Spearman nonparametric test as appropriate (SPSS). Multivariate linear regression analyses with forward stepwise procedure were performed to identify significant predictors of long-term renal outcome (plasma creatinine and albuminuria as dependent variables) among the concentrations of trp, kyn, and kyn/trp ratio 2 weeks and 6 months after transplantation (independent variables). To avoid multicollinearity, the independent variables which were in correlation were excluded from data set and included to another data set where no intervariable correlation occurred. The predictive value of serum creatinine as renal outcome was calculated by receiver operating characteristics curve analysis. The cutoff point for serum creatinine was 110 μmol/L for men and 95 μmol/L for women.23 Differences were considered significant at P less than 0.05.
Patients, Assessment of the Biopsies, and Clinical Follow-up
Table 1 gives an overview of the demographic characteristics of the patients.
Body weight, blood pressure, serum creatinine, and albuminuria were monitored during the 2-year follow-up (Table 2). Body weight increased significantly at 2 years as compared to 2-week values. There were no significant changes in systolic blood pressure between all 3 timepoints. However, diastolic blood pressure was significantly elevated at 6 months and 2 years after transplantation, compared to the values at 2 weeks after transplantation. Serum creatinine decreased and estimated creatinine clearance increased at 6 months after transplantation and remained at this level until the end of the follow-up. A small but significant increase in albuminuria was seen at 6 months and 2 years after transplantation, compared to the second posttransplantational week. There were no significant differences between the 4 arms of immunosuppressive treatments (data not shown).
Based on histological analysis of the renal biopsy at 6 months follow-up, from the 48 patients studied, 3 patients suffered from acute rejection (Banff score 1A), 5 patients were on the borderline of acute rejection, 15 patients showed slight reactive changes, and 15 patients did not show any significant histological changes. For 6 patients no biopsy was performed. Four biopsies showed no adequate or no kidney tissue.
Table 2 depicts the results of the histological analysis at 2-year follow-up. Two patients showed clear signs of acute rejection (Banff 1A), 25 patients showed a borderline acute rejection pattern, and 23 patients showed various degrees of interstitial fibrosis and tubular atrophy (IFTA). In 15 patients, both signs of borderline acute rejection as well as IFTA were found. No significant changes were found in 10 biopsies. For 3 patients, no biopsy was available or the biopsy was inadequate.
Serum trp, kyn, and kyn/trp Ratio
In comparison with the 2-week values, the serum level of trp was increased at 6 months and 2 years after transplantation and slightly decreased at 2 years in comparison with 6 months (Figure 1A, 44.03 ± 1.97, 53.20 ± 1.54 and 49.17 ± 1.88 μmol/L, respectively, P < 0.05). Serum level of kyn increased at 6 months as compared to 2 weeks and returned to the 2-week level by 2 years after transplantation (Figure 1B, 2.22 ± 0.08, 2.64 ± 0.12, and 2.08 ± 0.08 μmol/L, respectively, P < 0.05). Consequently, the kyn/trp ratio was not changed at 6 months after transplantation as compared to the 2-week level and decreased significantly at 2 years (Figure 1C, 0.06 ± 0.00, 0.05 ± 0.00, and 0.04 ± 0.00, respectively, P < 0.05). For comparison, serum trp, kyn, and kyn/trp ratio in healthy subjects was 64.86 ± 2.64, 1.87 ± 0.1, and 0.03 ± 0.00 μmol/L, respectively (n = 16).24 No significant differences were found in serum trp, kyn, and kyn/trp ratio between the 4 immunosuppressive treatments (data not shown). Because early acute rejection is an important risk factor for the late graft dysfunction, it is very important to clarify if the high level of kyn/trp is correlated with acute rejection or inadequate immunosuppressants. No significant differences were found in both 6 months and 2 years serum trp, kyn, and kyn/trp ratio when patients were divided into subgroups according to the histopathological features of rejection (acute rejection + borderline acute rejection, IFTA, borderline acute rejection + IFTA, and no changes). In summary, trp degradation along the kyn pathway in serum is decreased at 2 years after transplantation as compared to 2-week levels.
Urine trp, kyn, and kyn/trp Ratio
We also determined urine concentrations of trp, kyn, and kyn/trp ratio in our patients. The level of trp at month 6 decreased significantly compared to week 2 (Figure 1D, 63.77 ± 7.36 vs 42.17 ± 4.16 μmol/L, P < 0.05) and remained stable at 2 years after transplantation (34.12 ± 5.63 μmol/L). The urine level of kyn decreased significantly at 6 months compared to week 2 (Figure 1E, 5.99 ± 0.72 vs 3.41 ± 0.37 μmol/L, P < 0.05). Consequently, no change was observed in kyn/trp ratio from week 2 to 6 months. However, the kyn/trp ratio did significantly increase at 2 years in comparison with both 2 weeks and 6 months (Figure 1F, 0.10 ± 0.01, 0.08 ± 0.02 and 0.12 ± 0.01, respectively, P < 0.05). No significant differences were found in urine trp, kyn, and kyn/trp ratio between the 4 immunosuppressive treatments (data not shown). Also, no significant differences were found in both 6 month and 2 years urine trp, kyn, and kyn/trp ratio when patients were divided into subgroups according to the histopathological features of rejection. When adjusted for urine creatinine, the values of trp, kyn, and kyn/trp ratio showed the same pattern across the time as the uncorrected values. In conclusion, trp degradation along the kyn pathway in urine is increased at 2 years after transplantation as compared to 2-week and 6-month levels.
We further investigated the expression of IDO protein in the 6-month and 2-year biopsies using immunohistochemistry. In normal kidneys, a very limited expression of IDO was found in some distal tubular epithelial cells (Figure 2A). In the 6-month biopsies of the patients with acute rejection, a clear expression of IDO was found in the infiltrating inflammatory cells that morphologically resembled macrophages and dendritic cells (Figure 2B). There was also some expression in glomerular (mostly endothelial) cells (Figure 2C). In the 2-year biopsies of the patients with signs of chronic damage (IFTA), IDO expression was found around the atrophic tubules (Figure 2D). There was also a variable amount of glomerular staining, with some glomeruli showing strong expression of IDO in cells morphologically resembling endothelial cells and mesangial cells (Figure 2E). A very limited expression was found in the tubular epithelial cells (Figure 2F).
The amount of IDO staining, as assessed by morphometric analysis (data not shown), did not correlate with the histological damage in the renal biopsies, nor with the levels of trp, kyn, and kyn/trp ratio in serum and urine.
The Serum Level of kyn 6 Months After Transplantation Predicted the Serum Creatinine at 2 Years After Transplantation
Both the serum kyn and the kyn/trp ratio at 6 months after transplantation correlated with the serum creatinine at 2 years after transplantation (Figure 3A, R = 0.444, Figure 3B, R = 0.410, P < 0.05, respectively). Multiple regression analyses identified serum kyn level at 6 months as the only independent predictor for serum creatinine at 2 years (best fitting model: serum creatinine 2 years = 85.3 + 19.8 × kyn 6 months; R = 0.336; P < 0.05), when serum trp 2 weeks, kyn 2 weeks, and kyn 6 months were included as independent factors. The area under the curve of the receiver operating curves, a measure of diagnostic accuracy, was for the kyn at 6 months 0.76 (95% confidence interval, 0.600-0.913; P < 0.05, Figure 3C). Thus, higher serum kyn level at 6 months significantly predicted the higher serum creatinine at 2 years. We did not find correlations between trp or trp metabolites levels and the extent of the transplant damage, as assessed by the renal biopsies (data not shown).
The Urine Level of trp 2 Weeks After Transplantation Predicted the Serum Creatinine at 6 Months and the Estimated Creatinine Clearance 2 Years After Transplantation
We found that the urine trp level and kyn/trp ratio at week 2 after transplantation correlated with the serum creatinine at 6 months and 2 years after transplantation, in the case of trp the correlation being negative (Figure 3D, R = −0.281, Figure 3E, R = 0.341, Figure 3F, R = −0.319, Figure 3G, R = 0.326, P < 0.05, respectively). Also, the urine trp level at week 2 correlated with the estimated creatinine clearance at 2 years after transplantation (R = 0.403, P < 0.05). Moreover, both urine trp and kyn levels at week 2 correlated with the albuminuria at 2 years after transplantation (Figure 3H, R = 0.285, Figure 3I, R = 0.365, P < 0.05, respectively). Additionally, urine kyn/trp ratio at 6 months correlated with the albuminuria at 2 years after transplantation (Figure 3J, R = 0.342, P < 0.05). Multiple regression analyses identified urine trp level at 2 weeks as the only independent predictor for serum creatinine at 6 months and for the estimated creatinine clearance at 2 years (best fitting model: serum creatinine 6 months = 116.2 − 0.49 × trp 2 weeks; R = 0.530; P < 0.05; best fitting model: estimated creatinine clearance 2 years = 52.4 + 0.36 × trp 2 weeks; R = 0.497; P < 0.05), when urine trp 2 weeks, kyn/trp ratio 2 weeks, trp 6 months, and kyn/trp ratio 6 months were included as independent factors. However, the receiver operating curves for trp at 2 weeks did not show diagnostic accuracy (area under the curve = 0.44, P > 0.05).
The current study demonstrates that the serum and urine levels of trp and kyn measured early after renal transplantation (i.e., at 2 weeks and 6 months) predict the 2-year renal outcome after transplantation, as assessed by serum creatinine and albuminuria, in patients without overt CTD.
The progressive decline in the renal function and the development of proteinuria and hypertension, are indicators alerting the clinician to the presence of CTD.6 A biomarker which could predict these changes years before may allow early identification of patients at high risk for development of CTD. Our data indicate that the analysis of trp metabolism early after transplantation may contribute to early detection of these patients.
Several studies suggested the changes in trp, kyn, and/or IDO activity to reflect the short-term outcome of kidney transplantation. Serum kyn/trp ratio is higher in nonrejecting renal allograft recipients in comparison with healthy volunteers.20 Our data confirm this previous funding, as serum kyn/trp ratio at all timepoints was higher in the allograft recipients compared to healthy subjects. Moreover, kyn/trp is rapidly increasing in recipients with acute rejection compared to nonrejectors as early as day one post-transplantation. Additionally, IDO-positive cells were detected in renal biopsy of rejecting patients but not of those who did not reject the graft.20 It has also been shown that serum trp and kyn levels predict acute rejection of the renal graft in humans. The pretransplant levels of serum kyn and trp were increased in patients who went on to develop acute renal rejection compared to those who did not.11 These and other findings25 indicate that IDO activity is induced early during acute rejection. In our study, there was no difference in the level of both 6 months and 2 years serum and urine trp, kyn, and kyn/trp ratio when patients were divided into subgroups according to the histopathological features of rejection (e.g., acute rejection, IFTA, etc). However, previous studies showed increased kyn/trp levels in acute rejection only before11 or early after transplantation,20 possibly offering the explanation for this apparent discrepancy. So far, there is no information published regarding IDO expression and activity in the long-term posttransplant period. Schefold et al14 analyzed trp catabolism and estimated IDO activity in a cohort of forty patients with chronic kidney disease with various backgrounds, such as diabetic and hypertensive nephropathy and glomerulonephritis. The serum trp level was relatively unchanged, but the estimated IDO activity and serum levels of trp catabolites (such as kyn, kynurenic acid, and quinolinic acid) increased with the severity of chronic kidney disease. Furthermore, the levels of kynurenic and quinolinic acid correlated with serum creatinine, creatinine clearance and, in the case of kynurenic acid, also with eGFR.14 The induction of IDO in chronic kidney diseases may primarily be a reflection of chronic inflammation.14
In our study, serum kyn/trp decreased, whereas urine kyn/trp increased at 2 years after transplantation, as compared to 2 week values. It is possible that these alterations might partly occur due to the switch in immunosuppressive therapy at 6 months after transplantation. Incubation of human peripheral blood mononuclear cells with immunosuppressive agents (such as tacrolimus, CsA, sirolimus, and corticosteroids) resulted in a dose-dependent suppression of trp degradation, whereas mycophenolate-mofetil inhibited the trp metabolism only at the highest concentration tested. At the lowest dose, there was even an increase in the kyn/trp ratio.26 It is questionable, however, if the concentrations found to be effective in vitro are in fact comparable to the human in vivo situation. Also, IDO activity was found altered at the maternal interface in pregnant transplant recipients, likely modulated by immunosuppressive agents.27 On the other side, there is no change in trp metabolism in patients with rheumatoid arthritis treated with prednisolone.28 In our study, there were no significant differences in serum and urine trp, kyn, and kyn/trp between all 4 therapeutical arms at any timepoint, making a strong influence of the immunosuppressive switch at 6 months on the trp metabolism less plausible.
In line with the increased urine kyn/trp, strong IDO expression in the biopsies was found in our study at 2 years after grafting. This suggests that the increased kyn/trp in urine at 2 years reflects the increased expression of IDO in the transplanted kidney. However, no correlation was found between the amount of IDO staining and the kyn/trp ratio in urine, possible because not all the IDO protein in the kidney is enzymatically active. Earlier, Brandacher et al20 documented IDO expression in acutely rejected renal grafts. Indoleamine 2.3-dioxygenase–positive cells were identified in the mononuclear cell infiltrates and the tubular epithelial cells. In our study, the strongest expression was also found in the interstitial inflammatory infiltrates. Only weak expression was found in tubular epithelial cells and, as opposed to the study of Brandacher et al,20 glomerular staining in (mostly) endothelial cells was also documented. The reason for this difference is unclear; it may rely for instance on the time to biopsy (6 months in our study vs weeks in the study of Brandacher), the differences in the anti-IDO antibody or on the differences in the (induction) immunosuppressive medication in these 2 studies. In the 2-year biopsies IDO expression followed a similar pattern, however, stronger expression was noticed in (both endothelial and mesangial) glomerular cells. This pattern of IDO expression is not unexpected. Indoleamine 2.3-dioxygenase is known to be up-regulated in immunologically active cells (i.e., infiltrating mononuclear cells). Also, expression of IDO in endothelial cells at the fetomaternal interface has been earlier documented.29 Expression of IDO in mesangial cells has, to the best of our knowledge, not been reported so far.
In summary, in this study, we describe for the first time the changes of the trp metabolism along the kyn pathway and renal IDO expression during a 2-year follow-up after kidney transplantation in humans. Moreover, we document that the levels of trp and its metabolite kyn at week 2 and month 6 correlate with the 2-year values of serum creatinine and albuminuria. Hence, analyzing the trp, kyn, and kyn/trp ratio early after the transplantation may assist the clinician to define a subgroup of patients more likely to develop CTD, with consequences for the intensity of the follow-up and the treatment of these patients.
The authors thank Claude P. Ley for technical assistance.
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