Assessment and clinical implications of functional vitamin B6 deficiency
Minovic, Isidor
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Pathway, Systemic
Inflammation, and
Long-Term Outcome
after Kidney Transplantation
Laura V. de Vries1 Isidor Minović1,2,3 Casper F.M. Franssen1 Martijn van Faassen2 Jan-Stephan F. Sanders1
Stefan P. Berger1 Gerjan Navis1 Ido P. Kema2 Stephan J.L. Bakker1,3
1Department of Internal Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 2Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 3Top Institute Food and Nutrition, Wageningen, The Netherlands.
Abstract
Background: Tryptophan is metabolized along the kynurenine pathway,
initially to kynurenine, and subsequently to cytotoxic 3-hydroxykynurenine. There is increasing interest in this pathway, because of its pro-inflammatory nature, and drugs interfering in it have received increasing attention. We aimed to investigate whether serum and urinary parameters of the tryptophan-kynurenine pathway, and particularly cytotoxic 3-hydroxytryptophan-kynurenine, are associated with systemic inflammation and long-term outcome in renal transplant recipients (RTR).
Methods: Data were collected in outpatient RTR with a functioning graft for
>1 year. Tryptophan, kynurenine and 3-hydroxykynurenine in serum and urine were measured using LC-MS/MS.
Results: A total of 561 RTR (age 51±12 years; 56% male) were included at
median 6.0 [2.6-11.6] years post-transplantation. Baseline median serum tryptophan was 40.0 [34.5-46.0] µmol/l; serum kynurenine was 1.8 [1.4-2.2] µmol/l; serum 3-hydroxykynurenine was 42.2 [31.0-61.7] nmol/l. Serum kynurenine and 3-hydroxykynurenine were strongly associated with parameters of systemic inflammation. During follow-up for 7.0 [6.2-7.5] years, 51 RTR (9%) developed graft failure and 120 RTR (21%) died. Both serum kynurenine and 3-hydroxykynurenine were independently associated with graft failure (HR 1.72 [1.23-2.41], P=0.002 and HR 2.03 [1.42-2.90], P<0.001). Serum 3-hydroxykynurenine was also independently associated with mortality (HR 1.37 [1.08-1.73], P=0.01), while serum kynurenine was not. Urinary tryptophan-kynurenine pathway parameters were not associated with outcome.
Conclusion: Of tryptophan metabolites, serum 3-hydroxykynurenine is
cross-sectionally most strongly and consistently associated with systemic inflammation and prospectively with adverse long-term outcome after kidney transplantation. Serum 3-hydroxykynurenine may be an interesting biomarker and target for the evaluation of drugs interfering in the tryptophan-kynurenine pathway.
Introduction
The kynurenine pathway is the major metabolic pathway of the essential amino-acid tryptophan. Under physiological conditions, tryptophan is metabolized by tryptophan 2,3-dioxygenase (TDO) in the liver. However, under inflammatory conditions, extra-hepatic indoleamine 2,3-dioxygenase (IDO) is activated (1, 2). Originally, it was believed that TDO and IDO were the two enzymes that metabolize tryptophan to kynurenine (3). Recently, however, a third enzyme able to metabolize tryptophan to kynurenine has been described (3, 4). It is referred to as indoleamine 2,3-dioxygenase-2 (IDO2) and the enzyme formerly named IDO is now known as indoleamine 2,3-dioxygenase-1 (IDO1) (3, 5).
In the next step of the pathway, the enzyme kynurenine 3-monooxygenase (KMO) metabolizes kynurenine to cytotoxic 3-hydroxykynurenine (1). Both IDO1 and KMO enzymes are activated by pro-inflammatory stimuli and are expressed in a variety of tissues and immune cells (6), whereas IDO2 is less responsive to pro-inflammatory stimuli, and its expression is limited to a lower number of cell types, of which expression in dendritic cells may be physiologically the most relevant (5).
Kynurenine and particularly down-stream cytotoxic 3-hydroxykynurenine are thought to play an important role in systemic inflammation and oxidative stress (7-10). As such, accumulation of these metabolites has been linked to many health problems in which systemic inflammation is present, including obesity (11, 12), rheumatoid arthritis (13-15), and neuro-inflammatory diseases (16, 17). Accumulation of kynurenine metabolites has also been linked to the development of atherosclerosis and cardiovascular disease (18-23), particularly in patients with impaired kidney function (7, 24, 25). In renal transplant recipients (RTR), activation of the tryptophan-kynurenine pathway has been linked to the occurrence of acute rejection (26-28). Because the tryptophan-kynurenine pathway is thought to play a role in the pathophysiology of many inflammation-related diseases, there is great interest in drugs interfering in this pathway. Initially, drugs capable of inhibiting the enzyme IDO1 gained the most interest, because this enzyme catalyzes the first and rate-limiting step of the pathway (29, 30). Recently, however, inhibition of the enzyme KMO gained more interest, because this would more directly block production of cytotoxic 3-hydroxykynurenine (31). Consequently, KMO inhibitors are currently being evaluated as therapeutic strategies in experimental models of various inflammation-related diseases (29, 30, 32).
Systemic inflammation is also thought to play an important role in long-term complications after kidney transplantation (33). Whether activation of the tryptophan-kynurenine pathway is linked to systemic inflammation and related to long-term complications after kidney transplantation is yet unknown. Therefore, we aimed to investigate whether serum and urinary parameters of the tryptophan-kynurenine pathway are associated with systemic inflammation in a large cohort of stable outpatient RTR. In addition, we aimed to investigate whether these tryptophan-kynurenine pathway parameters, and particularly cytotoxic 3-hydroxykynurenine, are associated with long-term patient and graft survival in these patients.
Materials and methods
Research design and subjects
In this observational single-center cohort study with longitudinal follow-up, all adult, stable RTR who visited our outpatient transplant clinic between August 2001 and July 2003 and had a functioning graft for at least 1 year were invited. RTR with overt congestive heart failure or cancer other than cured skin cancer were considered ineligible for the study. A total of 606 of 847 eligible RTR (72%) gave written, informed consent. The group that did not give informed, written consent was comparable with the group that gave written consent with respect to age, sex, body mass index (BMI), serum creatinine, creatinine clearance and proteinuria. For this post-hoc analysis, tryptophan, kynurenine, and 3-hydroxykynurenine were measured in 561 RTR (92.6%). Additional details of this study have been published previously (34-38). The Institutional Review Board of the University Medical Center Groningen (Groningen, the Netherlands) approved the study protocol (METc 2001/039), which adhered to the Declaration of Helsinki.
Patient characteristics
The Groningen Renal Transplant Database contains information on all kidney transplantations performed at our center since 1968. Relevant transplantation-related characteristics, such as donor age and sex, human leukocyte antigen mismatches, and date of transplantation were extracted from this database. Current medication was taken from the medical record. Standard immunosuppression was as described previously (35). Smoking status and cardiovascular history were obtained using a self-report
questionnaire at baseline visits. Cardiovascular history was defined as a history of myocardial infarction, percutaneous transluminal angioplasty or stenting of the coronary or peripheral arteries, bypass operation of coronary or peripheral arteries, intermittent claudication, amputation for vascular reasons, transient ischemic attack, or an ischemic cerebrovascular accident. BMI, hip and waist circumference, and blood pressure were measured as described previously (35). Diabetes mellitus was diagnosed if the fasting plasma glucose was at least 7.0 mmol/l (≥ 126 mg/dl) or if antidiabetic medication was used.
End points
The primary end points of the study were death-censored graft failure and recipient mortality. Death-censored graft failure was defined as return to dialysis or need for a re-transplantation. If a variable is associated with death-censored graft failure, and there would be a relatively high risk of mortality after graft failure, variables could be seemingly associated with mortality because of their association with graft failure. Therefore, we also analyzed mortality limited to death before development of graft failure as a secondary end point. The continuous surveillance system of the outpatient program ensures up-to-date information on patient status and cause of death. General practitioners or referring nephrologists were contacted in case the status of a patient was unknown. End points were recorded until May 2009; median [interquartile range] follow-up was 7.0 [6.2-7.5] years. Cause of death was obtained by linking the number of the death certificate to the primary cause of death, as coded by a physician from the Central Bureau of Statistics. There was no loss due to loss of follow-up.
Laboratory measurements
At baseline visit, blood was drawn after an 8- to 12-hour overnight fasting period. Serum and urine samples were stored at -80°C until assessment of biochemical measures for this study. Serum and urinary concentration of tryptophan, kynurenine, and 3-hydroxykynurenine were measured using automated high-throughput on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) (39). Urinary excretion of tryptophan, kynurenine, and 3-hydroxykynurenine was calculated by multiplying urinary concentration with 24-hour urinary volume. Serum and urinary kynurenine-to-tryptophan ratios were calculated by
dividing kynurenine concentration by tryptophan concentration, expressed in nanomoles per micromole. Serum and urinary 3-hydroxykynurenine-to-kynurenine ratios were calculated by dividing 3-hydroxy-kynurenine concentration by kynurenine concentration, expressed in nanomoles per micromole. Plasma creatinine concentrations were measured using a modified version of the Jaffé method (MEGA AU 510; Merck Diagnostics). Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation (40). Glucose, insulin, hemoglobin A1c (HbA1c), total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, high-sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecule type 1 (sICAM-1), soluble vascular cellular adhesion molecule type 1 (sVCAM-1), procalcitonin, albumin, kidney injury molecule-1 (KIM-1), N-acetyl-beta-D-glucosaminidase (NAG), neutrophil gelatinase associated lipocalin (NGAL), and heart fatty acid binding protein (H-FABP) were measured as described previously (36, 38, 41, 42).
Statistical analysis
Data were analyzed with SPSS statistics version 22.0 for Windows (SPSS Inc.) and GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA). Normally distributed data are presented as mean ± standard deviation, non-normally distributed data as median [interquartile range (IQR)], and nominal data as number (percentage). Hazard ratios (HR) are reported with 95% confidence intervals (CI). A two-sided P-value < 0.05 was considered to indicate statistical significance. Variable distribution was tested with histograms and probability plots. Differences in baseline serum and urinary tryptophan, kynurenine, 3-hydroxykynurenine, kynurenine-to-tryptophan ratio, and 3-hydroxykynurenine-to-kynurenine ratio, between RTR who experienced graft failure and RTR who did not, and between RTR who survived and RTR who did not, were tested using a Mann-Whitney U-test. Linear regression analyses were performed to identify age- and sex-adjusted associations of serum and urinary tryptophan, kynurenine, 3-hydroxykynurenine, kynurenine-to-tryptophan ratio, and 3-hydroxykynurenine-to-kynurenine ratio with clinical and biochemical parameters. Subsequently, multivariable linear regression analyses were performed to identify independent associates of these serum and urinary tryptophan-kynurenine pathway parameters. Multivariable regression models were constructed using backward selection (Pout > 0.05), including variables that were significantly associated with serum
and urinary tryptophan-kynurenine pathway parameters in univariable analysis. Non-normally distributed variables were log-transformed to fulfill criteria for linear regression analyses.
Associations of serum and urinary tryptophan, kynurenine, 3-hydroxykynurenine, kynurenine-to-tryptophan ratio, and 3-hydroxy-kynurenine-to-kynurenine ratio with graft failure, overall mortality, and mortality before development of graft failure were assessed using Cox proportional hazards regression analysis, with stepwise adjustment for recipient age and sex, metabolic parameters, inflammation parameters, and kidney function, respectively. Finally, associations of serum kynurenine with graft failure and mortality were adjusted for serum 3-hydroxykynurenine, and vice versa. Continuous variables were entered as continuous variables in the models. Cox regression models were built stepwise to avoid overfitting and to keep the number of covariates accurate in relationship to the number of events (43).
Results
Study population
A total of 561 RTR (56% men; mean age 51±12 years) were included in the study at a median time of 6.0 [2.6-11.6] years after transplantation. At baseline, they had stable kidney function with a mean eGFR of 47±16 ml/ min/1.73 m2 and median urinary protein excretion of 0.2 [0.0-0.5] g/24h
(Table 1). All RTR used prednisolone with a median daily dose of 10.0 [7.5-10.0] mg/day; 79% of RTR used a calcineurin inhibitor (65% cyclosporine, 14% tacrolimus), and 74% used a proliferation inhibitor (33% mycophenolate mofetil, 41% azathioprine). Other baseline characteristics are presented in Table 1.
Tryptophan-kynurenine pathway parameter concentrations
Median baseline serum tryptophan concentration was 40.0 [34.5-46.0] µmol/l, which was below the lower reference limit for the general population (Figure 1) (39). Median serum kynurenine concentration was 1.8 [1.4-2.2] µmol/l and 3-hydroxykynurenine concentration was 42.2 [31.0-61.7] nmol/l, both of which fell within the reference range for the general population (Figure 1) (39). Median serum kynurenine-to-tryptophan ratio was 44.2 [35.0-57.9] nmol/µmol and 3-hydroxykynurenine-to-kynurenine ratio was 23.9 [19.0-30.5] nmol/µmol (Figure 1).
Table 1. Baseline characteristics of the study population
Variable (n=561) Distribution MIN MAX
Recipient demographics Age (yrs) 51 ± 12 21 80 Male sex, n (%) 312 (56) Weight (kg) 77 ± 14 35 128 Waist (cm) 97 ± 14 62 142 BMI (kg/m2) 26 ± 4 15 43 Blood pressure SBP (mmHg) 153 ± 23 108 235 DBP (mmHg) 90 ± 10 61 122 No. of antihypertensives (n) 1.9 ± 1.2 0 5 Lipids
Total cholesterol (mmol/l) 5.6 ± 1.1 2.3 15.2
HDL cholesterol (mmol/l) 1.1 ± 0.3 0.4 2.7
LDL cholesterol (mmol/l) 3.5 ± 1.0 0.6 12.0
Triglycerides (mmol/l) 1.9 [1.4-2.6] 0.4 12.3
Lipid-lowering drug use, n (%) 281 (50)
Diabetes
Glucose (mmol/l) 4.5 [4.1-5.0] 2.9 16.6
Insulin (µU/ml) 11.0 [8.0-16.1] 2.0 53.4
HbA1c (%) 6.5 ± 1.1 3.9 11.4
Anti-diabetic drug use, n (%) 73 (13)
Inflammation hsCRP (mg/l) 2.0 [0.8-4.8] 0.1 159 sICAM-1 (ng/l) 603 [516-718] 229 2,462 sVCAM-1 (ng/l) 952 [772-1,196] 386 5,048 Procalcitonin (ng/l) 23 [17-35] 1 134 Kidney function
Serum creatinine (µmol/l) 134 [112-166] 63 525
eGFR (ml·min-1·1.73 m2) 47 ± 16 15 108
Creatinine clearance (ml/min) 61 ± 22 19 166
Tubular damage and proteinuria
Urinary protein excretion (g/24h) 0.2 [0.0-0.5] 0 13.8
KIM-1 (ng/24h) 1.2 [0.6-2.2] 0.01 13.1
NAG (U/24h) 9.0 [5.1-14.7] 0.4 62.5
NGAL (ng/24h) 321 [254-447] 62 3,490
H-FABP (µg/24h) 8.5 [7.8-9.3] 6.0 13.0
Primary kidney disease, n (%)
Primary glomerular disease 158 (28)
Table 1. Baseline characteristics of the study population (Continued)
Variable (n=561) Distribution MIN MAX
Tubular interstitial disease 86 (15)
Polycystic kidney disease 97 (17)
Dysplasia and hyperplasia 21 (4)
Renovascular disease 31 (6)
Diabetes mellitus 22 (4)
Other or unknown cause 109 (19)
Transplantation
Transplant vintage (yrs) 6.0 [2.6-11.6] 1.0 31.6
Living donor, n (%) 80 (14)
Acute rejection, n (%) 256 (46)
Previous dialysis duration (mo) 27 [13-48] 0 398
HLA mismatches (n) 1.7 ± 1.4 0 6
Warm ischemia times (min) 35 [30-45] 0 157
Cold ischemia times (h) 21 [14-27] 1 50
Immunosuppression
Prednisolone dose (mg/24h) 10.0 [7.5-10.0] 7.5 10.0
Calcineurin inhibitors
Cyclosporine use, n (%) 363 (65)
Cyclosporine trough levels (µg/l) 108 [80-139] 25 302
Tacrolimus use, n (%) 80 (14)
Tacrolimus trough levels (µg/l) 8.7 [6.0-10.2] 2 16
Proliferation inhibitors
Mycophenolate mofetil use, n (%) 228 (41)
Azathioprine use, n (%) 185 (33)
mTOR inhibitors use, n (%) 1 (0.2)
Nominal data are presented as absolute number (percentage), normally distributed data as mean ± standard deviation, and non-normally distributed data as median [interquartile range]. For continuous variables minimum and maximum values are also displayed. Abbreviations: BMI, body mass index; DBP, diastolic blood pressure, HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; H-FABP, heart fatty acid binding protein; HLA, human leukocyte antigen; hsCRP, high sensitivity C-reactive protein; CV, cardiovascular; KIM-1, kidney injury molecule-1; LDL, low-density lipoprotein; mTOR, mechanistic target of rapamycin; NAG, N-acetyl-beta-D-glucosaminidase; NGAL, neutrophil gelatinase associated lipocalin; SBP, systolic blood pressure, sICAM-1, soluble intercellular adhesion molecule 1; sVCAM-1, soluble vascular cell adhesion molecule 1.
Serum tryptophan concentration was significantly lower in RTR who experienced graft failure compared with those who did not, whereas serum kynurenine and 3-hydroxykynurenine concentrations, and kynurenine-to-tryptophan and 3-hydroxykynurenine-to-kynurenine ratios were significantly higher (Figure 1). Serum tryptophan concentration was
also lower in non-survivors compared with survivors, but this was not significant. Serum kynurenine and 3-hydroxykynurenine concentrations, and kynurenine-to-tryptophan and 3-hydroxykynurenine-to-kynurenine ratios were significantly higher in non-survivors compared with survivors (Figure 1). Median 24-hour urinary tryptophan excretion was 44.1 [24.8-75.1] µmol/24h, median urinary kynurenine excretion was 3.8 [2.1-7.2] µmol/24h, and median urinary 3-hydroxykynurenine excretion was 1.2 [0.66-2.1] µmol/24h. Median urinary kynurenine-to-tryptophan ratio was 88.6 [61.2-133.3] nmol/µmol and 3-hydroxykynurenine-to-kynurenine ratio was 304 [220-430] nmol/µmol (Figure 2). There was no significant difference in urinary tryptophan, kynurenine, 3-hydroxykynurenine excretion, urinary kynurenine-to-tryptophan ratio, or 3-hydroxykynurenine-to-kynurenine ratio in RTR who experienced graft failure compared with those who did not, nor between non-survivors and survivors (Figure 2).
Associations with tryptophan-kynurenine pathway parameters
In age- and sex-adjusted linear regression analyses, higher serum tryptophan concentration was associated with lower blood pressure, lower HbA1c, lower inflammation parameters (i.e. serum hsCRP, sVCAM-1, and procalcitonin), better kidney function (i.e. lower serum creatinine and higher eGFR), less urinary excretion of protein and tubular damage markers, and lower daily prednisolone dose (Table 2). Serum tryptophan concentration was positively associated with serum kynurenine concentration (β=0.12, P<0.01; Table 2) and inversely associated with serum 3-hydroxykynurenine concentration (β=-0.17, P<0.001; Table 2). Higher serum kynurenine concentration was associated with being overweight (i.e. higher weight, waist circumference, and BMI), a more unfavorable metabolic profile (i.e. lower HDL cholesterol, higher triglycerides, higher HbA1c), higher inflammation parameters (i.e. serum hsCRP, sVCAM-1, sICAM-1, and procalcitonin), worse kidney function (i.e. higher serum creatinine and lower eGFR), and a higher urinary excretion of protein and tubular damage markers (Table 2). Associations with serum 3-hydroxykynurenine concentration showed directions similar to those with serum kynurenine concentration, but were generally stronger (Table 2). The associations of the serum kynurenine-to-tryptophan ratio showed directions similar to those of serum kynurenine, but were generally slightly weaker. The associations of 3-hydroxykynurenine-to-kynurenine ratio showed directions similar to those of 3-hydroxykynurenine, but were also generally slightly weaker.
Fi gur e 1. B ox a nd w hi sk er p lo ts o f b as elin e s er um t ry pt op ha n ( TRP), k yn ur en in e (KYN), a nd 3-h ydr oxy ky nur en in e (3HK) c on ce nt ra tio ns , ky nur en in e-t o-t ry pt op ha n r at io , a nd 3-h ydr oxy ky nur en in e-t o-k yn ur en in e r at io , in R TR w ho di d n ot d ev el op g ra ft f ai lur e (GF , n=510) c om pa re d to R TR w ho d ev el op ed GF (n=51) [p an el A], a nd in R TR w ho s ur viv ed (n=441) c om pa re d t o R TR w ho di d n ot s ur viv e (n=120) [p an el B] St at ist ic al sig nific an ce wa s t es te d u sin g a M ann-W hi tn ey U-t es t. H or izo nt al lin es in b ox es r ep res en t 1s t q ua rt ile , m edi an, a nd 3r d q ua rt ile; w hi sk er s r ep res en t 2.5t h t o 97.5t h p er cen tile . R ef er en ce va lues f or s er um t ryp to ph an, k yn ur enin e, a nd 3-h ydr oxy ky nur enin e a re dep ic te d b y g ra y s hade d a re as in g ra ph s 39. N o r ef er en ce va lues f or ra tios a re a va ila ble .
Fi gur e 2. B ox a nd w hi sk er p lo ts o f b as elin e ur in ar y e xcr et io n o f t ry pt op ha n ( TRP), k yn ur en in e (KYN), a nd 3-h ydr oxy ky nur en in e (3HK), a nd ur in ar y k yn ur en in e-t o-t ry pt op ha n a nd 3-h ydr oxy ky nur en in e-t o-k yn ur en in e r at io s, in R TR w ho di d n ot d ev el op g ra ft f ai lur e (GF , n=510) co m pa re d t o R TR w ho d ev el op ed GF (n=51) [p an el A], an d in R TR w ho s ur viv ed (n=441) c om pa re d t o R TR w ho di d n ot s ur viv e (n=120) [p an el B] St at ist ic al sig nific an ce wa s t es te d u sin g a M ann-W hi tn ey U -tes t. H or izo nt al lin es in b ox es r ep res en t 1s t q ua rt ile , m edi an, a nd 3r d q ua rt ile; w hi sk er s r ep res en t 2.5 th to 97.5t h p er cen tile . N o r ef er en ces va lues a re a va ila ble .
There was a strong positive association of serum kynurenine with serum 3-hydroxykynurenine concentration (β=0.72, P<0.001; Table 2). Of the urinary parameters, higher urinary tryptophan excretion was weakly associated with lower plasma glucose, higher creatinine clearance, and higher urinary protein excretion, and strongly associated with higher urinary NGAL excretion. Higher urinary kynurenine and 3-hydroxykynurenine excretion was weakly associated with lower total cholesterol and LDL cholesterol, lower plasma glucose, worse kidney function (i.e. higher serum creatinine, lower eGFR), and higher daily prednisolone dose, and strongly associated with higher urinary NGAL excretion. High urinary kynurenine-to-tryptophan ratio was weakly associated with worse kidney function and higher daily prednisolone dose. High urinary 3-hydroxykynurenine-to-kynurenine ratio was weakly associated with lower HDL-cholesterol concentrations. All of the urinary parameters showed strong positive associations with each other (Table 2). There were no significant associations of serum tryptophan-kynurenine pathway parameters with urinary tryptophan-tryptophan-kynurenine pathway parameters. In addition, there was no significant association of use of either azathioprine, mycophenolate mofetil, cyclosporine, or tacrolimus, or trough levels of cyclosporine or tacrolimus with serum or urinary parameters of the tryptophan-kynurenine pathway (Table 2). Inhibitors of mechanistic target of rapamycin (mTOR) were used too infrequently (n = 1) to allow for meaningful analyses.
Independent associates of tryptophan-kynurenine pathway parameters
Using multivariable linear regression analysis with backward elimination, we found that male sex and BMI were positively associated with serum tryptophan concentration, whereas hsCRP, serum creatinine, and daily prednisolone dose were inversely associated (Table 3). Age, waist circumference, hsCRP, sVCAM-1, and serum creatinine were positively associated with serum kynurenine concentration and kynurenine-to-tryptophan ratio, whereas male sex and HDL cholesterol were inversely associated. Waist circumference, HbA1c, hsCRP, sVCAM-1, and serum creatinine were positively associated with serum 3-hydroxykynurenine concentration, whereas male sex and HDL cholesterol were inversely associated. Independent associations with serum 3-hydroxykynurenine-to-kynurenine ratio were similar to those with serum 3-hydroxykynurenine, except that serum 3-hydroxykynurenine-to-kynurenine ratio was not independently associated with sVCAM-1 (Table 3). Independent associates of urinary tryptophan, kynurenine,
3-hydroxykynurenine excretion, kynurenine-to-tryptophan ratio, and 3-hydroxykynurenine-to-kynurenine ratio are also presented in Table 3.
Tryptophan-kynurenine pathway parameters and graft failure
During a median follow-up of 6.9 [6.1-7.4] years, 51 of 561 (9%) RTR developed graft failure, of which one case was due to acute rejection (2%), one was due to vascular occlusion (2%), one was due to recurrence of primary renal disease (2%), and the remaining 48 were classified as due to chronic transplant dysfunction (94%). Of these latter 48 cases, a biopsy was performed in 21 (41%) cases, with all showing interstitial fibrosis and tubular atrophy (IF/TA), and six cases (12%) showing additional signs of potential calcineurin inhibitor toxicity. Of the 51 RTR who developed graft failure, 49 RTR (96%) returned to dialysis and two RTR (4%) were pre-emptively re-transplanted. In age- and sex-adjusted Cox regression analyses, serum tryptophan concentration was inversely associated with graft failure (HR 0.62 [95% CI, 0.48-0.70]; P<0.001 per SD increase), whereas serum kynurenine concentration (HR 2.95 [95% CI, 2.26-3.85]; P<0.001), serum 3-hydroxykynurenine concentration (HR 2.54 [95% CI, 2.04-3.16]; P<0.001), serum kynurenine-to-tryptophan ratio (HR 3.45 [95% CI, 2.65-4.49]; P<0.001), and serum 3-hydroxykynurenine-to-kynurenine ratio (HR 1.69 [95% CI, 1.37-2.07]; P<0.001) were positively associated with graft failure (Table 4). The positive associations of serum kynurenine, serum 3-hydroxykynurenine, serum kynurenine-to-tryptophan ratio, and serum 3-hydroxykynurenine-to-kynurenine ratio with graft failure remained significant after adjustment for potential confounders including age, sex, waist circumference, HDL cholesterol, sVCAM-1, hsCRP, and serum creatinine, whereas the association of serum tryptophan with graft failure lost significance after adjustment for kidney function (Table 4). The association of serum kynurenine with graft failure lost significance after further adjustment for serum 3-hydroxykynurenine (HR 1.26 [95% CI, 0.84-1.90]; P=0.3), whereas the association of serum 3-hydroxykynurenine with graft failure was only slightly attenuated by further adjustment for serum kynurenine (HR 1.74 [95% CI, 1.11-2.73]; P=0.01). In contrast to the significant independent associations observed for serum parameters, associations of urine parameters, including urinary excretion of tryptophan, kynurenine, and 3-hydroxykynurenine, and urinary kynurenine-to-tryptophan ratio and 3-hydroxykynurenine-to-kynurenine ratio, with graft failure lost significance after adjustment for kidney function reflected by serum creatinine (Table 5).
Ta bl e 2. A ge- a nd s ex-a dj us te d a ss oci at io ns o f c lin ic al a nd b io ch em ic al p ar am et ers w ith s er um a nd ur in ar y t ry pt op ha n-k yn ur en in e p at hwa y par am et er s Va ri ab le (n=561) Se ru m Ur in e TRP KYN 3HK KYN/TRP 3HK/KYN TRP KYN 3HK KYN/TRP 3HK/KYN Re cip ien t d em og ra ph ic s W eig ht (kg) 0.07 0.13 ** 0.19 *** 0.08 0.16 *** 0.04 0.03 0.05 - 0.00 0.03 W ai st (cm) 0.05 0.20 *** 0.29 *** 0.14 ** 0.24 *** 0.00 - 0.03 0.01 - 0.04 0.00 BMI (kg/m 2) 0.10 * 0.12 ** 0.18 *** 0.04 0.15 *** 0.00 - 0.03 0.01 - 0.04 0.06 Bl oo d p re ss ure SB P (mmH g) - 0.11 ** 0.08 0.11 * 0.14 ** 0.08 0.03 0.08 0.10 * 0.07 0.03 D BP (mmH g) - 0.07 0.03 0.06 0.07 0.06 0.03 0.04 0.05 0.02 0.01 N o. o f a nt ih yp er ten siv es ( n) - 0.06 0.10 * 0.20 *** 0.12 ** 0.20 *** 0.05 0.07 0.07 0.02 0.00 Li pi ds Total c ho les ter ol (mm ol/l) 0.06 - 0.05 - 0.06 - 0.09 * - 0.03 - 0.04 - 0.06 - 0.10 * - 0.02 - 0.07 HD L c ho les ter ol (mm ol/l) 0.07 - 0.27 *** - 0.32 *** - 0.28 *** - 0.23 *** 0.08 0.07 - 0.01 - 0.02 - 0.12 * LD L c ho les ter ol (mm ol/l) 0.02 - 0.04 - 0.05 - 0.04 - 0.04 - 0.06 - 0.06 - 0.09 * - 0.01 - 0.05 Tr ig ly cer ides (mm ol/l) 0.06 0.15 *** 0.17 *** 0.09 * 0.12 ** - 0.05 - 0.04 - 0.01 0.01 0.04 Li pid-lo w er in g dr ug u se (y es) 0.03 - 0.03 - 0.02 - 0.05 0.00 0.04 0.10 * 0.10 * 0.08 0.02 D ia be te s G lucos e (mm ol/l) 0.01 0.07 0.16 *** 0.06 0.17 *** - 0.10 * - 0.13 ** - 0.09 * - 0.04 0.05 In su lin (µU/m l) - 0.01 0.04 0.09 * 0.04 0.10 * - 0.05 - 0.07 - 0.01 - 0.03 0.09 * HbA 1c (%) - 0.09 * 0.10 0.24 *** 0.14 ** 0.26 *** - 0.03 - 0.03 0.02 - 0.01 0.08 A nt i-di ab et ic dr ug u se (y es) 0.11 * 0.02 0.05 0.05 0.05 - 0.04 - 0.05 - 0.04 - 0.01 - 0.00
Ta bl e 2. A ge- an d s ex-a dj us te d a ss oci at io ns o f clin ic al a nd bi oc he m ic al pa ra m et ers w ith s er um a nd ur in ar y tr yp to ph an-k yn ur en in e pa th wa y pa ra m et ers (C on tin ue d) Va ri ab le (n=561) Se ru m Ur in e TRP KYN 3HK KYN/TRP 3HK/KYN TRP KYN 3HK KYN/TRP 3HK/KYN Infl amm at io n hsCRP (m g/l) - 0.14 ** 0.24 *** 0.39 *** 0.29 *** 0.34 *** - 0.06 - 0.03 - 0.00 0.03 0.04 sI CAM-1 (n g/l) - 0.02 0.24 *** 0.26 *** 0.22 *** 0.15 *** - 0.08 - 0.07 - 0.05 0.01 0.02 sV CAM-1 (n g/l) - 0.15 *** 0.30 *** 0.30 *** 0.35 *** 0.17 *** - 0.01 0.04 0.03 0.05 - 0.03 Pr oc alci to nin (n g/l) - 0.23 *** 0.40 *** 0.50 *** 0.49 *** 0.36 *** - 0.13 ** - 0.06 - 0.05 0.08 - 0.01 K idn ey f un ct io n Ser um cr ea tinin e (µm ol/l) - 0.30 *** 0.60 *** 0.64 *** 0.71 *** 0.39 *** 0.01 0.12 * 0.09 * 0.14 ** - 0.04 eGFR (m l·min -1·1.73 m 2) 0.26 *** - 0.56 *** - 0.60 *** - 0.65 *** - 0.37 *** - 0.02 - 0.13 ** - 0.10 * - 0.15 ** 0.05 Cr ea tinin e c le ara nce (m l/min) 0.25 *** - 0.45 *** - 0.45 *** - 0.54 *** - 0.25 *** 0.10 * - 0.01 0.02 - 0.13 ** 0.05 Tu bu la r d am ag e a nd p ro te in ur ia U rin ar y p ro tein ex cr et io n (g/24h) - 0.15 *** 0.27 *** 0.32 *** 0.33 *** 0.22 *** 0.09 * 0.08 0.06 - 0.00 - 0.03 KIM-1 (n g/24h) - 0.08 0.27 *** 0.31 *** 0.28 *** 0.21 *** - 0.03 - 0.01 0.00 0.03 0.02 N AG (U/24h) - 0.14 *** 0.24 *** 0.34 *** 0.30 *** 0.27 *** 0.05 0.02 0.01 - 0.03 - 0.01 N GAL (n g/24h) - 0.12 ** 0.15 *** 0.21 *** 0.21 *** 0.17 *** 0.31 *** 0.24 *** 0.20 *** - 0.07 - 0.04 H-F AB P (µg/24h) - 0.04 0.16 *** 0.20 *** 0.17 *** 0.15 *** 0.05 0.08 0.08 0.04 0.01 Tr an sp lan ta tio n Tra ns pl an t v in ta ge (y rs) 0.11 ** - 0.02 - 0.07 - 0.09 * - 0.09 * - 0.02 - 0.03 - 0.03 - 0.02 - 0.01 Li vin g do no r (y es) 0.05 0.04 - 0.01 0.00 - 0.04 - 0.06 - 0.10 * - 0.06 - 0.06 0.06 Ac ut e r ej ec tio n (y es) - 0.00 0.10 * 0.05 0.09 * - 0.02 0.05 0.03 - 0.01 - 0.01 - 0.08
Ta bl e 2. A ge- an d s ex-a dj us te d a ss oci at io ns o f clin ic al a nd bi oc he m ic al pa ra m et ers w ith s er um a nd ur in ar y tr yp to ph an-k yn ur en in e pa th wa y pa ra m et ers (C on tin ue d) Va ri ab le (n=561) Se ru m Ur in e TRP KYN 3HK KYN/TRP 3HK/KYN TRP KYN 3HK KYN/TRP 3HK/KYN Pr ev io us di al ysi s d ura tio n (m o) - 0.08 0.01 0.05 0.06 0.06 0.04 0.06 0.05 0.02 - 0.02 HL A mi sm at ch es (n) - 0.03 0.01 0.02 0.03 0.01 0.00 0.02 - 0.01 0.03 - 0.05 W ar m i sc hemi a t im es (min) - 0.06 - 0.02 0.01 0.02 0.03 - 0.12 ** - 0.08 - 0.07 0.04 0.01 C old i sc hemi a t im es (h) - 0.05 - 0.02 - 0.03 0.01 - 0.02 0.04 0.13 ** 0.09 0.10 * - 0.07 Im mu no su pp re ss io n Pr edni so lo ne dos e (m g/24h) - 0.14 ** - 0.03 0.03 0.06 0.06 0.02 0.15 *** 0.11 * 0.17 *** - 0.07 Ca lcin eur in in hi bi to rs C yc los po rin e u se (y es) - 0.08 - 0.08 - 0.03 - 0.08 0.08 0.08 0.08 0.08 0.01 0.01 Cy clos po rin e t ro ug h le ve ls (µg/l) - 0.08 - 0.08 - 0.03 - 0.05 0.08 0.04 0.06 0.03 0.03 - 0.06 T acr olim us u se (y es) 0.01 0.08 0.05 0.08 - 0.02 - 0.04 - 0.02 - 0.00 0.02 0.03 Tacr olim us t ro ug h le ve ls (µg/l) 0.08 - 0.07 - 0.05 - 0.07 0.09 0.02 - 0.02 - 0.05 - 0.04 - 0.05 Pr olif era tio n in hi bi to rs M yco ph en ol at e m of et il u se (y es) - 0.09 - 0.02 0.00 0.06 0.02 0.04 0.02 0.01 - 0.01 - 0.02 Aza thio pr in e u se (y es) 0.09 0.07 0.03 - 0.02 - 0.02 - 0.06 - 0.08 - 0.07 - 0.03 0.01 Tr ypt op ha n m et ab ol is m Ser um co ncen tra tio n TRP (µm ol/l) -0.12 ** - 0.17 *** - 0.52 *** - 0.35 *** - 0.03 - 0.06 - 0.05 - 0.04 0.01 KYN (µm ol/l) 0.12 ** -0.74 *** 0.79 *** 0.18 *** - 0.05 - 0.03 - 0.03 0.03 - 0.01 3HK (nm ol/l) - 0.17 *** 0.72 *** -0.73 *** 0.80 *** - 0.04 - 0.01 - 0.01 0.03 0.00
Ta bl e 2. A ge- an d s ex-a dj us te d a ss oci at io ns o f clin ic al a nd bi oc he m ic al pa ra m et ers w ith s er um a nd ur in ar y tr yp to ph an-k yn ur en in e pa th wa y pa ra m et ers (C on tin ue d) Va ri ab le (n=561) Se ru m Ur in e TRP KYN 3HK KYN/TRP 3HK/KYN TRP KYN 3HK KYN/TRP 3HK/KYN KYN/TRP (nm ol/µm ol) - 0.53 *** 0.77 *** 0.73 *** -0.37 *** - 0.02 0.02 0.01 0.05 - 0.02 3HK/KYN (nm ol/µm ol) - 0.35 *** 0.17 *** 0.81 *** 0.37 *** -- 0.01 0.00 0.01 0.01 0.01 U rin ar y ex cr et io n TRP (µm ol/24h) - 0.03 - 0.05 - 0.03 - 0.02 - 0.01 -0.66 *** 0.52 *** - 0.35 *** - 0.19 *** KYN (µm ol/24h) - 0.06 - 0.02 - 0.01 0.02 0.00 0.66 *** -0.82 *** 0.42 *** - 0.22 *** 3HK (nm ol/24h) - 0.06 - 0.03 - 0.01 0.01 0.01 0.52 *** 0.83 *** -0.47 *** 0.37 *** KYN/TRP (nm ol/µm ol) - 0.04 0.03 0.03 0.05 0.01 - 0.35 *** 0.47 *** 0.41 *** -- 0.05 3HK/KYN (nm ol/µm ol) 0.01 - 0.01 0.00 - 0.02 0.01 - 0.19 *** - 0.22 *** 0.37 *** - 0.05 -Ab br ev ia tio ns: 3HK, 3-h ydr oxy ky nur enin e; BMI, b od y m as s in dex; D BP , di as to lic b lo od p res sur e, HbA1c, h em og lo bin A1c; HD L, hig h-den sit y li po pr ot ein; H-F AB P, he ar t fa tty acid b in din g p ro tein; HL A, h um an leu ko cyt e a nt ig en; h sCRP , hig h s en sit iv ity C-r eac tiv e p ro tein; KIM-1, k idn ey in jur y m ole cu le-1; KYN, k yn ur enin e, LD L, lo w-den sit y li po pr ot ein; N AG, N-acet yl-b et a-D-g lucos aminid as e; N GAL, n eu tro phi l g el at in as e a ss oci at ed li po ca lin; S BP , sys to lic b lo od p res sur e, sI CAM-1, s ol ub le in ter ce llu la r ad hesio n m ole cu le 1; sV CAM-1, s ol ub le va sc ul ar ce ll ad hesio n m ole cu le 1; TRP , t ryp to ph an. R es ul ts o f lin ea r r eg res sio n a na lys es w ith t ryp to ph an-k yn ur enin e pa th wa y p ara m et er s a s dep en den t va ria ble . S ta nd ar dize d b et a-r eg res sio n co efficien ts o f a ge- a nd s ex-ad ju ste d a ss oci at io ns a re s ho w n. N on-n or m al ly di str ib ut ed va ria bles w er e log t ra nsf or m ed b ef or e en ter in g r eg res sio n a na lysi s. F or dic ho to m ou s va ria bles, 0=n o a nd 1=y es. *P<0.05, **P<0.01, a nd ***P<0.001, le ve l o f sig nific an ce .
Ta bl e 3. In de pe nd en t a ss oci at es o f s er um a nd ur in ar y t ry pt op ha n-k yn ur en in e p at hwa y p ar am et ers Va ri ab le (n=561) Se ru m Ur in e TRP KYN 3HK KYN/TRP 3HK/KYN TRP KYN 3HK KYN/TRP 3HK/KYN Pa tie nt de m ogr ap hic s A ge (y rs) -0.10 ** -0.10 ** -M ale s ex (y es) 0.13 ** - 0.08 * - 0.22 *** - 0.11 ** - 0.25 *** 0.12 ** -M et ab oli c p ar am et ers Bo dy m as s in dex (kg/m 2) 0.13 ** -W ai st cir cumf er en ce (cm) -0.08 * 0.11 ** 0.09 * 0.09 * -- 0.13 ** HD L c ho les ter ol (mm ol/l) -- 0.08 * - 0.11 ** - 0.08 * - 0.08 * -Li pid-lo w er in g dr ug u se (y es) -0.11 * 0.12 ** 0.09 * -G lucos e (mm ol/l) -- 0.14 ** -HbA 1c (%) -0.08 * -0.11 * -In flam m at io n p ar am et er s hsCRP (m g/d l) - 0.11 ** 0.08 * 0.23 *** 0.14 *** 0.21 *** -sV CAM-1 (n g/l) -0.16 *** 0.12 *** 0.17 *** -Pr oc alci to nin (n g/l) -- 0.17 *** -K idn ey f un ct io n Ser um cr ea tinin e (µm ol/l) - 0.29 *** 0.49 *** 0.48 *** 0.59 *** 0.23 *** -0.12 ** -Tu bu la r d am ag e a nd p ro te in ur ia U rin ar y p ro tein ex cr et io n (g/24h) -N GAL ex cr et io n (pg/24h) -0.33 *** 0.22 *** 0.21 *** 0.09 *
-Ta bl e 3. In de pe nd en t a ss oci at es o f s er um a nd ur in ar y t ry pt op ha n-k yn ur en in e p at hwa y p ar am et ers Va ri ab le (n=561) Se ru m Ur in e TRP KYN 3HK KYN/TRP 3HK/KYN TRP KYN 3HK KYN/TRP 3HK/KYN Tr an sp lan ta tio n-re la te d Pr edni so lo ne dos e (m g) - 0.09 ** -0.13 ** 0.14 ** -Li vin g do no r (y es) -- 0.12 ** -Ab br ev ia tio ns: 3HK, 3-h ydr oxy ky nur enin e; HbA1c, hem og lo bin A1c; HD L, hig h-den sit y lip op ro tein; hsCRP , hig h sen sit iv ity C-r eac tiv e pr ot ein; KYN, ky nur enin e; N GAL, n eu tro phi l g el at in as e a ss oci at ed li po ca lin; TRP , t ryp to ph an; sV CAM-1, so lu ble va sc ul ar ce ll ad hesio n m ole cu le t yp e 1. R es ul ts o f lin ea r r eg res sio n a na lys es w ith bac kwa rd e limin at io n, w ith t ryp to ph an-k yn ur enin e p at hwa y p ara m et er s a s dep en den t va ria ble . On ly s ta nd ar dize d b et a-r eg res sio n co efficien ts o f sig nific an t co va ria tes in th e fin al m ode l f or e ac h p ara m et er a re s ho w n. T ot al m ode l g oo dn es s o f fi t: f or s er um t ryp to ph an, R2=0.14; f or s er um k yn ur enin e, R2=0.42; f or s er um 3-h ydr oxy ky nur enin e, R2=0.49; f or s er um k yn ur enin e-t ryp to ph an ra tio , R2=0.53; f or s er um 3-h ydr oxy ky nur enin e-k yn ur enin e ra tio R2=0.29; f or ur in ar y t ryp to ph an, R2=0.12; f or ur in ar y ky nur enin e, R2=0.12; f or ur in ar y 3-h ydr oxy ky nur enin e, R2=0.10; f or ur in ar y k yn ur enin e-t ryp to ph an ra tio , R2=0.08; a nd f or ur in ar y 3-h ydr oxy ky nur enin e-k yn ur enin e ra tio , R2=0.05. *P<0.05, **P<0.01, a nd ***P<0.001, le ve l o f sig nific an ce .
Tryptophan-kynurenine pathway parameters and mortality
During a median follow-up of 7.0 [6.2-7.5] years, 120 of 561 (21%) RTR died, of which 104 (19%) died before development of graft failure. In age- and sex-adjusted Cox regression analyses, serum tryptophan concentration was inversely associated with mortality (HR 0.79 [95% CI, 0.66-0.95]; P=0.01 per SD increase), whereas serum kynurenine concentration (HR 1.45 [95% CI, 1.21-1.75]; P<0.001), serum 3-hydroxykynurenine concentration (HR 1.66 [95% CI, 1.39–1.97]; P<0.001), serum kynurenine-to-tryptophan ratio (HR 1.56 [95% CI, 1.31-1.86]; P<0.001), and serum 3-hydroxykynurenine-to-kynurenine ratio (HR 1.45 [95% CI, 1.23-1.72]; P<0.001) were positively associated with mortality (Table 4). The positive association of serum 3-hydroxykynurenine concentration and serum 3-hydroxykynurenine-to-kynurenine ratio with mortality remained significant after adjustment for potential confounders, whereas the associations of serum tryptophan and kynurenine concentration, and serum kynurenine-to-tryptophan ratio with mortality lost significance after adjustment for kidney function (Table 4). An association of serum kynurenine with mortality remained absent after further adjustment for serum 3-hydroxykynurenine (HR 0.93 [95% CI, 0.71-1.21]; P=0.6), whereas the association of serum 3-hydroxykynurenine with mortality was unaffected by adjustment for serum kynurenine (HR 1.42 [95% CI, 1.08-1.87]; P=0.01). In contrast to the significant, independent associations observed for serum parameters, associations of urine parameters, including urinary excretion of tryptophan, kynurenine, and 3-hydroxykynurenine, and urinary kynurenine-to-tryptophan ratio and 3-hydroxykynurenine-to-kynurenine ratio with mortality lost significance after adjustment for kidney function reflected by serum creatinine (Table 5). When we limited analyses to RTR who died before development of graft failure, associations of serum tryptophan, kynurenine, 3-hydroxykynurenine, kynurenine-to-tryptophan ratio, and 3-hydroxykynurenine-to-kynurenine ratio with mortality were generally slightly weaker than in analyses with overall mortality (Table 4), consistent with the notion that the stronger independent associations of the tryptophan-kynurenine parameters with graft failure add to the strength of the associations of the tryptophan-kynurenine pathway parameters with overall mortality. Associations of urinary tryptophan-kynurenine pathway parameters with mortality before development of graft failure were absent and did not materially differ from those with overall mortality (Table 5).
Table 4. Associations of serum tryptophan, kynurenine, and 3-hydroxykynurenine concentrations, and serum kynurenine-to-tryptophan and 3-hydroxykynurenine-to-kynurenine ratios with graft failure, mortality, and mortality before development of graft failure
Graft Failure (n=51/561) Mortality (n=120/561) Graft Failure (n=104/561)Mortality Before HR [95% CI] P value HR [95% CI] P value HR [95% CI] P value
Tryptophan Model 1 0.62 [0.48-0.79] < 0.001 0.79 [0.66-0.95] 0.01 0.83 [0.68-1.01] 0.06 Model 2 0.65 [0.51-0.83] 0.001 0.79 [0.66-0.95] 0.01 0.82 [0.68-1.01] 0.05 Model 3 0.71 [0.55-0.92] 0.01 0.86 [0.71-1.03] 0.11 0.89 [0.72-1.10] 0.3 Model 4 1.06 [0.81-1.39] 0.7 0.91 [0.75-1.12] 0.4 0.89 [0.72-1.11] 0.3 Kynurenine Model 1 2.95 [2.26-3.85] < 0.001 1.45 [1.21-1.75] < 0.001 1.29 [1.05-1.57] 0.01 Model 2 3.05 [2.30-4.04] < 0.001 1.38 [1.14-1.67] 0.001 1.23 [1.00-1.51] 0.05 Model 3 2.90 [2.17-3.88] < 0.001 1.22 [1.00-1.49] 0.06 1.09 [0.87-1.35] 0.5 Model 4 1.72 [1.23-2.41] 0.002 1.10 [0.97-1.39] 0.4 1.09 [0.85-1.41] 0.5 3-hydroxykynurenine Model 1 2.54 [2.04-3.16] < 0.001 1.66 [1.39-1.97] < 0.001 1.45 [1.20-1.77] < 0.001 Model 2 2.71 [2.11-3.49] < 0.001 1.59 [1.33-1.91] < 0.001 1.39 [1.14-1.71] 0.001 Model 3 2.83 [2.11-3.80] < 0.001 1.44 [1.17-1.77] 0.001 1.23 [0.98-1.55] 0.07 Model 4 2.03 [1.42-2.90] < 0.001 1.37 [1.08-1.73] 0.01 1.29 [1.00-1.67] 0.05 KYN/TRP ratio Model 1 3.45 [2.65-4.49] < 0.001 1.56 [1.31-1.86] < 0.001 1.38 [1.14-1.68] 0.001 Model 2 3.33 [2.53-4.38] < 0.001 1.51 [1.26-1.80] < 0.001 1.34 [1.10-1.64] 0.004 Model 3 3.44 [2.54-4.68] < 0.001 1.34 [1.09-1.64] 0.005 1.17 [0.94-1.47] 0.17 Model 4 1.63 [1.11-2.39] 0.01 1.23 [0.94-1.63] 0.14 1.27 [0.95-1.73] 0.11 3HK/KYN ratio Model 1 1.69 [1.37-2.07] < 0.001 1.45 [1.23-1.72] < 0.001 1.34 [1.11-1.61] 0.003 Model 2 1.68 [1.34-2.11] < 0.001 1.41 [1.19-1.67] < 0.001 1.30 [1.07-1.57] 0.008 Model 3 1.56 [1.21-2.02] 0.001 1.31 [1.09-1.57] 0.004 1.19 [0.97-1.46] 0.09 Model 4 1.39 [1.01-1.89] 0.03 1.26 [1.04-1.52] 0.02 1.20 [0.97-1.47] 0.09
Data are presented as hazard ratio (HR) per standard deviation increase in log transformed serum tryptophan, kynurenine, 3-hydroxykynurenine, kynurenine-to-tryptophan ratio, and 3-hydroxykynurenine-to-kynurenine ratio, plus 95% confidence interval (CI). Model 1: age- and sex-adjusted associations; model 2: model 1 + additional adjustment for metabolic parameters (waist, HDL cholesterol); model 3: model 2 + additional adjustment for inflammation parameters (sVCAM-1, hsCRP); model 4: model 3 + additional adjustment for kidney function (serum creatinine).
Table 5. Associations of urinary excretion of tryptophan, kynurenine, and 3-hydroxykynurenine, and urinary kynurenine-to-tryptophan and 3-hydroxykynurenine-to-kynurenine ratios with graft failure, mortality, and mortality before development of graft failure
Graft failure (n=51/561) Mortality (n=120/561) Mortality Before Graft Failure (n=104/561) HR [95% CI] P value HR [95% CI] P value HR [95% CI] P value
Tryptophan Model 1 0.83 [0.64-1.10] 0.2 1.03 [0.85-1.25] 0.7 1.06 [0.86-1.31] 0.6 Model 2 0.97 [0.73-1.28] 0.8 1.06 [0.87-1.28] 0.6 1.07 [0.86-1.32] 0.6 Kynurenine Model 1 1.02 [0.77-1.37] 0.9 1.17 [0.97-1.40] 0.10 1.15 [0.95-1.40] 0.15 Model 2 0.98 [0.72-1.34] 0.9 1.15 [0.96-1.38] 0.14 1.14 [0.94-1.39] 0.2 3-hydroxykynurenine Model 1 1.07 [0.80-1.42] 0.6 1.14 [0.96-1.36] 0.15 1.14 [0.94-1.37] 0.2 Model 2 1.06 [0.79-1.44] 0.7 1.13 [0.95-1.35] 0.2 1.13 [0.94-1.37] 0.2 KYN/TRP ratio Model 1 1.24 [0.97-1.58] 0.08 1.21 [1.00-1.46] 0.05 1.16 [0.94-1.43] 0.2 Model 2 1.03 [0.73-1.45] 0.9 1.16 [0.94-1.43] 0.2 1.13 [0.91-1.41] 0.3 3HK/KYN ratio Model 1 1.08 [0.81-1.42] 0.6 0.96 [1.06-1.11] 0.7 0.98 [0.80-1.19] 0.8 Model 2 1.15 [0.85-1.60] 0.4 0.98 [0.81-1.18] 0.8 0.98 [0.81-1.20] 0.9
Data are presented as hazard ratio (HR) per standard deviation increase in log transformed urinary tryptophan, kynurenine, 3-hydroxykynurenine excretion, kynurenine-to-tryptophan ratio, and 3-hydroxykynurenine-to-kynurenine ratio, plus 95% confidence interval (CI). Model 1: age- and sex-adjusted associations; model 2: model 1 + additional adjustment for serum creatinine.
Discussion
In this large prospective cohort of 561 stable RTR, we studied tryptophan-kynurenine pathway activation and its association with systemic inflammation and long-term outcome after kidney transplantation. We found that serum kynurenine and particularly cytotoxic serum 3-hydroxykynurenine, and also serum kynurenine-to-tryptophan and 3-hydroxykynurenine-to-kynurenine ratios are strongly associated with parameters of systemic inflammation. In addition, they are associated with increased risk of graft failure long-term after kidney transplantation, independent of potential confounders such as kidney function. Serum 3-hydroxykynurenine and 3-hydroxykynurenine-to-kynurenine ratio were also independently associated with mortality, whereas serum kynurenine and kynurenine-to-tryptophan ratio were not. There were no independent associations of urinary parameters of the tryptophan-kynurenine pathway with graft failure or mortality.
In the field of kidney transplantation, the tryptophan-kynurenine pathway has gained interest, because of its role in predicting acute rejection (44). Therefore, studies on the tryptophan-kynurenine pathway in RTR have typically focused on tryptophan and kynurenine, and their ratio, and the association of these parameters with short-term outcome after transplantation (26-28,45). We hypothesized that concerning longer-term complications related to systemic inflammation, tryptophan and kynurenine may also be relevant markers. In addition, we hypothesized that down-stream 3-hydroxykynurenine may be an even more interesting marker, because of its cytotoxicity. Indeed, we found that both serum kynurenine and serum 3-hydroxykynurenine, and serum kynurenine-to-tryptophan and 3-hydroxykynurenine-to-kynurenine ratios, were associated with long-term outcome after kidney transplantation. Interestingly, associations of serum 3-hydroxykynurenine with outcome were much more robust than those of serum kynurenine, and cross-sectional associations of serum 3-hydroxykynurenine with inflammation parameters, metabolic parameters (i.e. obesity, dyslipidemia, diabetes), and kidney function were also stronger. In contrast to the strong associations we found of serum parameters of the tryptophan-kynurenine pathway with systemic inflammation and long-term outcome after kidney transplantation, we did not find a significant association of urinary parameters with either systemic inflammation or long-term outcome. Therefore, our data suggest that serum parameters of the tryptophan-kynurenine pathway are of better use in predicting long-term outcome in RTR than urinary parameters. Our data also suggest that serum tryptophan-kynurenine parameter evaluation better fits evaluation of the chronic low-grade inflammatory status in stable outpatient RTR.
We could not find any previous clinical studies on 3-hydroxykynurenine in RTR, but there is evidence from other populations that 3-hydroxykynurenine might be a better marker for adverse outcome than other tryptophan-kynurenine pathway metabolites. For example, in the general population, serum 3-hydroxykynurenine had a stronger association with cardiovascular morbidity and mortality than serum kynurenine (21,23). Similarly, in patients with end-stage kidney disease, the association of serum 3-hydroxykynurenine with the occurrence of cardiovascular disease was stronger than that of serum kynurenine (7). In addition, 3-hydroxykynurenine showed the strongest association with risk of acute myocardial infarction of all down-stream tryptophan-kynurenine pathway metabolites measured in patients with stable coronary artery disease (20).
The pathophysiology of 3-hydroxykynurenine-related damage has been studied scarcely in the context of kidney disease, but more elaborately in the context of neuro-inflammatory disease (46). In the brain, 3-hydroxykynurenine is known to induce oxidative damage and cell death (8-10), which are most likely caused by free radical formation (8,9) and impairment of cellular energy metabolism (47). There is one observational study in patients with end-stage kidney disease that also showed a strong association of serum 3-hydroxykynurenine with parameters of systemic oxidative stress (7).
Our study and other observational studies in humans consistently demonstrate tryptophan-kynurenine pathway activation to be associated with reduced allograft survival (26,27,45). However, previous studies in experimental transplantation models showed that activation of the tryptophan-kynurenine pathway may benefit allograft survival (44,48). Namely, tryptophan-kynurenine pathway activation was shown to induce immune tolerance (49,50), supposedly by cytotoxic effects of kynurenine tryptophan-pathway metabolites on regulatory T-cells (51,52). In a review article on this subject, Löb and Königsrainer (21) give a possible explanation for this discrepancy. They suggest that, in human RTR, metabolites of the tryptophan-kynurenine pathway increase as a compensatory response that counteracts ongoing allograft rejection and may, therefore, serve more as markers of ongoing inflammation or immune activation, rather than actively act as immunosuppressive compounds like they do in experimental settings. In our study, serum kynurenine and 3-hydroxykynurenine, and their ratio, were strongly associated with parameters of systemic inflammation. Indeed, the tryptophan-kynurenine pathway has been implicated in many disease states in which systemic inflammation is present (53,54). Via its role in systemic inflammation, it has also been implicated in the development of atherosclerosis and cardiovascular disease (55,56), especially in patients with impaired kidney function (7,24,57,58). In these patients, tryptophan-kynurenine pathway metabolites are known to accumulate (59-63). It has been hypothesized that the increase in serum kynurenine metabolites in patients with impaired kidney function is caused not only by reduced renal clearance of the metabolites themselves (64), but also by increased tryptophan break-down to kynurenine by the pro-inflammatory uremic environment(61,63). In line with this, we found serum tryptophan to be the lowest and serum kynurenine and 3-hydroxykynurenine to be the highest in RTR with the worst kidney function.
The tryptophan-kynurenine pathway plays a role in many pathophysiological processes. Interfering in it by blocking different enzymatic steps is currently being evaluated as potential therapeutic strategy (29,30). Inhibitors of IDO1 gained most interest, because this enzyme catalyzes the first and rate-limiting step in the pathway. IDO2 was recently identified as another enzyme able to metabolize tryptophan to kynurenine. To date, little is known about the effects of blocking of IDO2. This may in part be due to its relatively new discovery, but its relatively low substrate binding affinity and lower turnover rates compared with IDO1 may also play a role (29). Specific inhibition of IDO2, however, holds promising perspectives in the field of immune tolerance, particularly because of its expression in dendritic cells. Considering inhibition of enzymatic steps in the tryptophan-kynurenine pathway, it recently became apparent that the IDO1/IDO2 product kynurenine is not only metabolized to cytotoxic 3-hydroxykynurenine, but also to (neuro)protective kynurenic acid (29,30). Therefore, inhibitors of the down-stream enzyme KMO were developed with the aim of reducing production of 3-hydroxykynurenine and shifting the pathway toward production of kynurenic acid (29-31). Interestingly, we found that associations of serum kynurenine with adverse outcome were greatly weakened by adjustment for serum 3-hydroxykynurenine. In contrast, associations of serum 3-hydroxykynurenine were almost unaffected by adjustment for serum kynurenine. In addition, the two metabolites showed very strong correlations with each other. Therefore, the associations of the “intermediate” metabolite kynurenine with adverse outcome that we found in our study may be in large part explained by its strong association with the down-stream cytotoxic metabolite 3-hydroxykynurenine. In our study, we chose to focus on 3-hydroxykynurenine, because of its cytotoxic properties, and did not measure kynurenic acid. For future studies in RTR, it might be interesting to also measure kynurenic acid to see whether this compound also has protective properties in RTR.
Whereas inhibition of enzymatic steps in the tryptophan-kynurenine pathway may directly alter concentrations of its metabolites, it has been suggested that treatment with immunosuppressive drugs may also influence concentrations of these metabolites (65,66). Despite this, we did not find any association of use of azathioprine, mycophenolate mofetil, cyclosporine, tacrolimus, or trough levels of cyclosporine or tacrolimus with serum or urinary parameters of the tryptophan-kynurenine pathway. The two available in vitro studies in this field showed contradictory results (65,66). One study
found that cyclosporine, tacrolimus, and mycophenolate mofetil decreased IDO1 activity in a dose-dependent manner in peripheral blood mononuclear cells (65). The other study found that these drugs increased IDO1 protein expression in mesangial cells (66). Therefore, it remains to be elucidated whether, and under what circumstances, immunosuppressive drugs affect metabolites of the tryptophan-kynurenine pathway.
Several limitations of our study warrant consideration. First, our study was observational in nature. Although we adjusted for several potential confounding variables, including parameters of kidney function, the possibility of residual confounding cannot be excluded. Our study design did not allow us to investigate the mechanisms through which tryptophan-kynurenine pathway activation led to increased mortality and graft failure risk. Therefore, we could only speculate on the causative mechanisms. Second, tryptophan-kynurenine pathway metabolites were measured at a single time point only, and therefore, we could not take potential changes over time into account. However, it was shown in other studies that when intra-individual variability is taken into account, the association of a parameter with outcome is only strengthened (41,67). The main strength of this study is that it is, to our knowledge, the first study to address the association of tryptophan-kynurenine pathway activation and long-term outcome in stable RTR. Moreover, it is the first study that specifically addresses 3-hydroxykynurenine as marker for adverse outcome in RTR. Other strengths of our study include measurement of tryptophan-kynurenine pathway parameters in both serum and urine, the well-characterized patient population, the relatively large sample size, and the long-term and complete follow-up.
In conclusion, we are the first to show that activation of the tryptophan-kynurenine pathway is associated with adverse long-term outcome after kidney transplantation in a large cohort of stable RTR. This is reflected particularly by the strong associations of 3-hydroxykynurenine with long-term graft failure and mortality. Therefore, serum 3-hydroxykynurenine may be an interesting biomarker and target for the evaluation of drugs interfering in the tryptophan-kynurenine pathway.
Acknowledgements
The authors kindly thank Claude van der Ley and Bettine Haandrikman for their valuable technical assistance. This work was supported by a grant from the Top Institute Food and Nutrition (Grant CH-003).
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