Dietary protein intake and long-term outcomes after kidney transplantation
Said, M.Yusof
DOI:
10.33612/diss.170755325
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Publication date: 2021
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Said, M. Y. (2021). Dietary protein intake and long-term outcomes after kidney transplantation. University of Groningen. https://doi.org/10.33612/diss.170755325
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homeostasis of asymmetric
dimethylarginine (ADMA):
studies in donors and
recipients of renal transplants
Said MY, Douwes RM, van Londen M, Minovíc I, Frenay AR, de Borst MH, van den Berg E, Heiner-Fokkema MR, Kayacelebi AA, Bollenbach A, van Goor H, Navis G, Tsikas D, Bakker SJL
Abstract
Asymmetric dimethylarginine (ADMA) is a methylated form of arginine and an endogenous nitric oxide synthase inhibitor. Renal function decline is associated with increase of plasma ADMA in chronic kidney disease populations. It is yet unknown how isolated renal function impairment affects ADMA homeostasis in healthy humans. Here, we measured plasma concentrations and urinary excretion of ADMA using GC–MS/MS in 130 living kidney donors before and at 1.6 (1.6–1.9) months after donation. We additionally analyzed 201 stable renal transplant recipients (RTR) that were included > 1 year after transplantation, as a model for kidney disease in the context of single kidney state. We measured true glomerular filtration rate (mGFR) using 125I-iothalamate. To study enzymatic metabolism of ADMA, we also measured L-citrulline as primary metabolite. Mean age was 52 ± 10 years in donors and 54 ± 12 years in RTR. Renal function was significantly reduced from pre- to post-donation (mGFR: 104 ± 17 vs. 66 ± 10 ml/min per 1.73 m2 BSA, − 36 ± 7%, P < 0.001). Urinary ADMA excretion strongly and significantly decreased from pre- to post-donation (60.6 ± 16.0 vs. 40.5 ± 11.5 µmol/24h, − 31.5 ± 21.5%, P < 0.001), while plasma ADMA increased only slightly (0.53 ± 0.08 vs. 0.58 ± 0.09 µM, 11.1 ± 20.1%, P < 0.001). Compared to donors post-donation, RTR had significantly worse renal function (mGFR: 49 ± 18 ml/min/1.73 m2, − 25 ± 2%, P < 0.001) and lower urinary ADMA excretion (30.9 ± 12.4 µmol/24h, − 23.9 ± 3.4%, P < 0.001). Plasma ADMA in RTR (0.60 ± 0.11 µM) did not significantly differ from donors post-donation (2.9 ± 1.9%, P = 0.13). Plasma citrulline was inversely associated with mGFR (st. β: − 0.23, P < 0.001), consistent with increased ADMA metabolism to citrulline with lower GFR. In both groups, the response of urinary ADMA excretion to renal function loss was much larger than that of plasma ADMA. As citrulline was associated with GFR, our data indicate that with renal function impairment, a decrease in urinary ADMA excretion does not lead to a corresponding increase in plasma ADMA, likely due to enhanced metabolism, thus allowing for lower renal excretion of ADMA.
Introduction
Circulatory asymmetric dimethylarginine (ADMA), a methylated form of the amino acid arginine, is an important cardiovascular risk marker that has been associated with mortality in renal transplant recipients (RTR) and increased risk of end-stage renal disease in chronic kidney disease (CKD) populations (1,2). High plasma ADMA concentrations are considered a risk factor for cardiovascular disease (2,3), given that ADMA is an inhibitor of the vasodilator nitric oxide (NO) synthase activity (4). The dynamics of circulating ADMA levels are complex and dependent on ADMA release from the breakdown of methylated arginine-containing proteins and on ADMA elimination by metabolism, predominantly by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) and to lesser extent by alanine-glyoxylate aminotransferase 2 (AGXT2), and partially by renal excretion in unaltered form (5–7). ADMA is predominately metabolized to L-citrulline and dimethylamine (DMA) by DDAH. Studies in CKD populations show that renal function impairment is accompanied by increasing plasma ADMA concentrations (8). Whether this is due to renal function impairment per se or also due to the common associates of renal disease, i.e., cardiovascular damage or low grade inflammation, is unclear.
To assess the effect of renal function as such on ADMA homeostasis in a healthy human study population, we measured plasma concentration and 24h urine excretion of ADMA, true glomerular filtration rate (GFR), estimated GFR (eGFR), and fractional excretion (FE) of ADMA in living, healthy kidney donors before and after unilateral kidney donation. To document metabolism of ADMA, we also measured plasma citrulline as one of the metabolites of ADMA breakdown. We compared the findings to a population of RTR, to study renal function impairment in the context of renal disease combined with a single kidney state.
Methods and materials
Study population
This study is part of a larger prospective cohort study of RTR and healthy donors in the northern regions of the Netherlands (Transplantlines Food and Nutrition cohort, Clinicaltrials.gov no. NCT02811835). Between November 2008 and March
2011, 300 adult (> 18 years), healthy potential donors were invited, of which 297 signed informed consent and of which 196 actually donated a kidney. Exclusion criteria were insufficient understanding of the Dutch language, a history of alcohol or drug abuse, diabetes mellitus (according to American Diabetes Association criteria of 2008 (9)), disease that would make them unsuitable for donation, a medical history of cardiovascular events, and usage of more than two antihypertensives. Of the donors who donated a kidney, 130 had data on measured GFR, plasma ADMA, and 24h-urinary ADMA excretion, and were included in the current study. Next to healthy donors, 817 adult RTR with a functioning graft that were transplanted at least 1 year before and who visited the outpatient clinic of the University Medical Center Groningen, The Netherlands, were invited. Insufficient understanding of the Dutch language, overt congestive heart failure (NYHA class 3–4), a history of alcohol or drug abuse, and a medical history of malignancy other than cured skin cancer were exclusion criteria. Out of 817 RTR, 706 signed written informed consent. Of these, 201 had data available on measured GFR, plasma and urinary ADMA, and were included in the current study. At both visits for laboratory and physical measurements, all subjects were at steady state, meaning without an acute illness (e.g., infection) and were biochemically stable. The study protocol has been conducted in accordance with the declaration of Helsinki and was approved by the institutional ethical review board (METc 2008/186). Clinical measurements
Anthropomorphic measurements were performed on the same day for each participant. Body height was measured without shoes. Body surface area (BSA) was calculated using the Dubois–Dubois formula (10). Body mass index (BMI) was calculated as weight divided by squared height (m). Blood pressure was measured automatically (Dinamap® 1846 monitor. Critikom, Tampa, FL, USA) in half-sitting position. The average of the last three of 15 successive measurements was recorded. Laboratory measurements
Samples from baseline and after donation were stored at − 80 °C. Plasma and urinary ADMA were measured by a validated GC–MS/MS method (Thermoquest TSQ 700, Finnigan MAT, San Jose, CA, USA) as described in detail earlier (2,11). Plasma arginine, a potential source of citrulline (resulting from the conversion of arginine to L-citrulline and nitric oxide), was also measured by this method (11). The plasma
arginine-to-ADMA ratio was calculated as a marker of NO-availability (12). We measured plasma citrulline with a validated UHPLC–MS/MS method using a LC30 UHPLC (Shimadzu, Japan) coupled to a triple quadrupole mass spectrometer (API 4500 QTRAP, Sciex, Canada) with an electrospray ionization source (Sciex). To a mix of 10 µl plasma, quality control samples or calibrators and 10 µl internal standard solution (25 μM L-citrulline-4,4,5,5-D4), 60 μl formic acid in methanol (0.1%) was added and mixed. To 10 µl of this solution, 70 µl borate buffer solution (AccQ-Tag™Ultra, Waters Chromatography B.V., The Netherlands) and 20 µl AccQTag derivatization reagent (AccQ-Tag™Ultra, Waters Chromatography B.V., The Netherlands) were added and allowed to stand for 15 min at 55 °C. After cooling down, 1200 µl formic acid in methanol (0.1%) was added and placed in the autosampler of analyses. Other urine and blood analyses have been performed using routine laboratory methods as described earlier (13,14). GFR was measured for 130 healthy donors and 201 RTR by infusion of low-dose 125I-iothalamate as previously described (15). We calculated the estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula with serum creatinine and cystatin C (16). We defined proteinuria as a urinary protein excretion of ≥ 0.5 g/24h.
Statistical analyses
Basic characteristics of donors were compared before and after donation and were also compared with RTR. The paired differences between pre- and post-donation values were presented as percentage change compared to pre-donation. The population differences between RTR and donor post-donation were presented as percentage change compared to post-donation. The standard deviation (SD) of the absolute population differences (SDd) was calculated as follows:
The SD of the percentage population differences (SDp) were consequently calculated as follows:
Normally distributed variables were tested with either paired (donors pre- vs. post-donation) or non-paired (donors post-donation vs. RTR) t tests. Variables with a skewed distribution were tested with either paired Wilcoxon Signed Rank test (donors pre- vs. post-donation) or Mann-Whitney U test (donors post-donation vs. RTR). We used FE and tubular resorption proportion as renal tubular function parameters. FE of ADMA was calculated as:
Tubular resorption proportion (%) of ADMA was calculated as (1 − FE). mGFR per 1.73 m2 BSA was used as the primary measure of renal function. Although being an inferior measure of true renal function compared to mGFR, we additionally used eGFR in our analyses for comparison, since eGFR is used more often than mGFR in the clinical setting (17). The urine–plasma ADMA ratio was compared to mGFR and eGFR using Pearson’s correlation. We performed multivariable linear regression of plasma citrulline with mGFR, adjusted for age, sex, BSA, and plasma arginine. We additionally adjusted the association for plasma ADMA and finally for urinary ADMA excretion.
Analyses were performed with ibm spss statistics version 23 (2015, IBM corp., Armonk, NY, USA). Figures were made with graphpad prism version 5.04 for Windows (2010, GraphPad Software, La Jolla, CA, USA). Normally distributed variables are presented as mean ± SD, skewed data as median (interquartile range). Regression coefficients are presented as standardized values. We regarded a P value of ≤ 0.05 as statistically significant.
Results
Renal function impairment, plasma ADMA concentration, and urinary ADMA excretion Baseline characteristics for healthy kidney donors pre- and post-donation are presented in Table 1. Renal function was reduced significantly at a median of 1.64 (1.61–1.87) months after donation (mean paired difference: mGFR: − 37.9 ± 11.8 ml/ min/1.73 m2, − 35.9 ± 6.9%, P < 0.001; eGFR: − 46.7 ± 11.3 ml/min/1.73 m2, − 48.5 ± 8.8%, P < 0.001). Mean urinary ADMA excretion in donors was significantly decreased
post-donation compared to pre-donation (mean paired difference: − 20.6 ± 14.9 µmol/24h, − 31.5 ± 21.5%, P < 0.001). Plasma ADMA concentrations increased only moderately with renal function impairment compared to the steep decrease in urinary ADMA excretion after donation (mean paired difference: 0.05 ± 0.10 µM, 11.1 ± 20.1%, P < 0.001). FE of ADMA decreased after donation (mean paired difference 24.7 ± 160.8%, P = 0.02). Plasma citrulline was higher in donors post-donation compared to pre-donation (mean paired difference: 31.9 ± 29.8%, P < 0.001), while plasma arginine was not significantly different (mean paired difference: 16.6 ± 54.9%, P = 0.37). The same was true for the plasma arginine-to-ADMA ratio in donors post-donation compared to pre-donation (P = 0.29). Twenty-four-hour urinary urea excretion and 24h urinary creatinine excretion, reflecting amino acid turnover and muscle mass turnover, respectively (18–20), were slightly lower, yet not significantly different from pre- to post-donation (P = 0.06 and P = 0.07, respectively).
We also studied the difference between donors post-donation and RTR. Compared to donors, RTR had slightly higher BMI, higher blood pressure, higher HbA1c, lower creatinine excretion, and more proteinuria (Table 1). RTR had lowest mGFR (49.2 ± 18.3 ml/min/1.73 m2), significantly lower compared to donors post-donation (− 25.4 ± 2.4%, P < 0.001). However, mean eGFR of RTR (46.4 ± 17.7 ml/min/1.73 m2) was not significantly different from donors post-donation (− 6.1 ± 3.3%, P = 0.06). Urinary ADMA excretion was lower in RTR (30.9 ± 0.11 µmol/24h), compared to donors post-donation (− 23.9 ± 3.3%, P < 0.001). Plasma ADMA concentrations in RTR (0.60 ± 0.11 µM) were not significantly different from donors post-donation (2.9 ± 1.9%, P = 0.13). Similarly to plasma ADMA, fractional ADMA excretion in RTR (56.4 ± 13.6%) was modestly lower compared to donors post-donation (− 4.7 ± 2.4%, P = 0.05). In RTR, mean plasma citrulline (36.5 ± 12.3 µM) was slightly higher than in donors post-donation (8.8 ± 3.3%, P = 0.01). Plasma arginine in RTR [89 (75–106) µM] was not significantly different compared to donors post-donation (4.1 ± 3.7%, P = 0.47). Similarly, the plasma arginine-to-ADMA ratio was not significantly different compared to donors post-donation (P = 0.50).
Ta bl e 1 . B as ic c ha ra ct er is tic s a nd A D M A h ome os ta si s i n h ea lt hy d on or s a nd i n R TR H ea lt hy d on or s ( n=1 30 ) RT R ( n= 20 1) Pa ir ed d iff er en ce o f d on or s po st -d on at ion a nd p re -d on at ion Po pu la tio n d iff er en ce o f R TR an d d on or s p os t-d on at io n Va ri ab le s Pr e-don at ion Po st -d on at io n Perc en ta ge di ffe re nc e P v al ue M ea n p er ce nt ag e di ffe re nc e P v al ue D em og ra ph ic s A ge 52 .1 ± 9 .9 52 .7 ± 1 0. 0 54. 3 ± 1 2. 0 1. 21 ± 0 .8 0 <0 .0 01 3. 11 ± 2 .3 1 0.1 8 M ale , n (%) 59 (4 5. 4) N /A 10 3 (5 1. 2) n/a n/a n/a Ti me a fte r d on at io n ( mo nt hs ) o r tr an sp la nt at io n ( ye ar s) t o v is it n/a 1. 64 [1. 61 –1. 87 ] 4. 91 [ 1. 00 –1 0. 0] n/a n/a n/a n/a Bo dy p ro por tion s W ei ght , k g 79 .8 ± 1 2. 4 79 .2 ± 1 3. 0 80 .7 ± 1 6. 1 -0 .9 2 ± 3 .9 0 0. 01 1. 87 ± 2 .0 3 0. 38 BM I, k g/ m 2 26 .1 ± 3 .4 25 .9 ± 3 .3 26 .8 ± 4. 8 -0 .5 1 ± 5 .0 4 0. 14 3. 44 ± 1 .7 1 0.0 5 BSA , m 2 1. 95 ± 0 .19 1. 94 ± 0 .2 0 1. 94 ± 0 .2 1 -0 .4 0 ± 1 .8 1 0.0 2 0. 12 ± 1 .1 6 0.9 2 Ca rd io va sc ul ar p ar am et er s SB P, m m H g 12 8 ± 1 4 12 2 ± 1 2 13 6 ± 1 8 -4. 0 ± 8 .2 <0 .0 01 11 .7 ± 1 .3 <0 .0 01 D BP , m m H g 78 ± 8 75 ± 9 81 ± 1 1 -3 .1 ± 1 0. 8 <0 .0 01 8. 46 ± 1 .4 8 <0 .0 01 Bl oo d p ar ame te rs A DM A , μ M 0. 53 ± 0 .0 8 0. 58 ± 0 .0 9 0. 60 ± 0 .1 1 11 .1 ± 2 0. 1 <0 .0 01 2. 9 ± 1 .9 0.1 3 Ci tr ul lin e, μM 26 .2 ± 6 .3 33 .6 ± 7 .5 36 .5 ± 1 2. 3 31 .9 ± 2 9. 8 <0 .0 01 8. 8 ± 3 .3 0. 01 A rg in in e, μM 83 [ 69 –1 02 ] 84 [ 74 –1 05 ] 89 [ 75 –1 06 ] 16 .6 ± 5 4. 9 0. 37 4. 1 ± 3 .7 0. 47 A rg in in e/ A D M A r at io , μ M / μM 16 4 ± 5 9 15 9 ± 5 0 16 4 ± 6 3 -6 .3 ± 6 7. 0 0. 29 2. 8 ± 3 .9 5 0. 50 H bA 1c, % 5. 5 ± 0 .3 5. 5 ± 1 .0 6. 1 ± 0 .8 0. 3 ± 5 .3 0. 82 9. 3 ± 1 .2 <0 .0 01
Ta bl e 1 . (c on ti nu ed ) H ea lt hy d on or s ( n=1 30 ) RT R ( n= 20 1) Pa ir ed d iff er en ce o f d on or s po st -d on at ion a nd p re -d on at ion Po pu la tio n d iff er en ce o f R TR an d d on or s p os t-d on at io n Va ri ab le s Pr e-don at ion Po st -d on at io n Perc en ta ge di ffe re nc e P v al ue M ea n p er ce nt ag e di ffe re nc e P v al ue U ri na ry e xc re tio n A D M A , μ mo l/2 4h 60 .6 ± 1 6. 0 40 .5 ± 1 1. 5 30 .9 ± 1 2. 4 -3 1. 5 ± 2 1. 5 <0 .0 01 -2 3. 9 ± 3 .4 <0 .0 01 U re a, m mo l/2 4h 41 7 ± 1 26 39 4 ± 1 13 39 1 ± 1 15 0. 8 ± 3 9. 1 0.0 6 -0 .5 4 ± 3 .2 7 0. 87 Cr ea ti ni ne , m mo l/2 4h 13 .5 ± 4. 4 12 .8 ± 4. 1 11 .5 ± 3 .6 -1 .1 ± 2 7. 9 0. 07 -1 0. 1 ± 3 .4 0.0 03 Pr ot ei n, g /2 4h 0.0 2 [0.0 2– 0. 11 ] 0.0 2 [0.0 2– 0. 18 ] 0. 18 [0.0 2– 0. 35 ] <0 .0 01 [< 0. 001 –4 43 ]* 0.1 8 35 3 ± 6 0 <0 .0 01 Re na l f un ct io n m GF R , m l/m in 11 7. 2 ± 2 4. 4 74. 2 ± 1 4. 4 55 .1 ± 2 0. 5 -3 6. 2 ± 7 .0 <0 .0 01 -2 5. 7 ± 2 .6 <0 .0 01 m G FR , m l/m in /1 .7 3 m 2 10 3. 9 ± 1 7. 4 66 .0 ± 9 .9 49 .2 ± 1 8. 3 -3 5. 9 ± 6 .9 <0 .0 01 -2 5. 4 ± 2 .4 <0 .0 01 eG FR , m l/m in /1 .7 3 m 2 96 .5 ± 1 3. 9 49 .5 ± 1 1. 1 46 .4 ± 1 7. 7 -4 8. 5 ± 8 .8 <0 .0 01 -6 .1 ± 3 .3 0.0 6 Re na l A D M A h an dl in g FE o f A D M A , % 64. 8 ± 2 2. 1 59 .2 ± 1 2. 0 56 .4 ± 1 3. 6 -2 4. 7 ± 1 60 .8 0.0 2 -4. 7 ± 2 .4 0.0 5 Tu bu la r r es or pt io n o f A DM A , % 35 .2 ± 2 2. 1 40 .8 ± 1 2. 0 43 .6 ± 1 3. 6 24. 7 ± 1 60 .8 0.0 2 6. 9 ± 3 .5 0.0 5 A bb re vi at io ns : A D M A : a sy m me tr ic d ime th yl ar gi ni ne ; R TR : r en al t ra ns pl ant r ec ip ie nt s; BM I: b od y m as s i nd ex ; B SA : b od y s ur fa ce a re a; S BP : s ys to lic bl oo d pr es su re ; D BP : d ia st ol ic bl oo d pr es su re ; m G FR : me as ur ed gl ome ru la r fi ltr at io n ra te ; e G FR : e st im at ed gl ome ru la r fi ltr at io n ra te ; F E: fr ac tio na l exc re tion . * M ed ia n p ai re d d iff er en ce
6
Figure 1 shows correlations of plasma ADMA concentration with renal function. In donors pre-donation, higher mGFR was not associated with lower plasma ADMA (Pearson’s r = − 0.05, P = 0.59). After donation, the correlation becomes slightly stronger, yet remains statistically insignificant. Figure 2 shows the association of the ratio of urine-to-plasma ADMA concentration, reflecting ADMA clearance, with renal function. The urine-to-plasma ADMA ratio is strongly correlated with mGFR pre-donation (Pearson’s r = 0.44, P < 0.001) and even stronger post-donation (Pearson’s r = 0.53, P < 0.001). In RTR, higher plasma ADMA concentration is correlated with lower mGFR (Figure 1: Pearson’s r = − 0.29, P < 0.001). The urine-to-plasma ADMA ratio in RTR is strongly correlated to mGFR (Figure 2: Pearson’s r = 0.74, P < 0.001).
Figure 1. Plasma ADMA concentration and renal function.
Unstandardized coefficients of regression lines of donors: mGFR pre-donation (a): β = − 0.0002, P = 0.59; mGFR post-donation (b): β = − 0.001, P = 0.16; eGFR pre-donation (c): β = 0.0002, P = 0.64; eGFR post-donation (d): β = − 0.001, P = 0.10. Unstandardized coefficients of regression lines of RTR: mGFR (a, b): β = − 0.002, P < 0.001, eGFR (c, d): β = − 0.002, P = 0.001
Figure 2. Urine ADMA/plasma ADMA ratio and renal function.
Unstandardized coefficients of regression lines of donors: mGFR pre-donation (a): β = 0.90, P < 0.001; mGFR post-donation (b): β = 1.20, P < 0.001; eGFR pre-donation (c): β = 0.78, P < 0.001; eGFR post-donation (d): β = 0.57, P < 0.001. Unstandardized coefficients of regression lines of RTR: mGFR (a, b): β = 0.99, P < 0.001, eGFR (c, d): β = 1.01, P < 0.001
Association of plasma citrulline concentration with renal function
To study the effect of renal function on ADMA metabolites, we studied the association of plasma citrulline concentrations with mGFR in linear regression analyses (Table 2). In donors pre- and post-donation, there was no significant association of plasma citrulline concentration with mGFR. In the RTR population, however, there was a strong and significant, negative association of plasma citrulline concentration with mGFR (st. β − 0.29, P < 0.001), which remained independent of adjustment for potential confounders (model 1). The association also remained independent of adjustment for plasma ADMA concentration (model 3). When adjusted for urinary ADMA excretion, the association weakened drastically, yet remained statistically significant (model 4). To study whether the difference in associations of plasma citrulline concentration with mGFR between donors post-donation and RTR was
explained by more than the difference in mGFR alone, we pooled the RTR and post-donation groups together and additionally adjusted the linear regression models for RTR status (donor = 0, RTR = 1). In Table 3, the association of plasma citrulline concentration with mGFR is presented, including the coefficient of the RTR status variable. For all models, there was a strong association of plasma citrulline concentration with mGFR despite adjustment for RTR status. RTR status itself was also associated strongly with mGFR (Table 3). Also in this analysis, the association of plasma citrulline concentration with mGFR weakened drastically after adjustment for urinary ADMA excretion. To test whether plasma citrulline concentration is associated differently in RTR compared to donors post-donation, we added an interaction term composed of the product of plasma citrulline concentration with RTR status. The interaction was not significant (crude model: β of interaction: − 0.19, P = 0.42). Thus, the association of plasma citrulline concentration with mGFR is explained by renal function rather than difference in study populations.
Table 2. Association of plasma citrulline concentration with mGFR
Healthy donors RTR
Pre-donation Post-donation
st.β P value st.β P value st.β P value
Crude 0.01 0.95 -0.15 0.08 -0.29 <0.001
Model 1 -0.06 0.38 -0.10 0.11 -0.32 <0.001
Model 2 -0.08 0.23 -0.09 0.16 -0.33 <0.001
Model 3 -0.09 0.21 -0.09 0.16 -0.28 <0.001
Model 4 -0.09 0.14 -0.07 0.24 -0.16 0.001
Model 1 Crude + age, sex, and BSA
Model 2 Model 1 + plasma arginine
Model 3 Model 2 + plasma ADMA
Model 4 Model 2 + urinary ADMA excretion
Abbreviations: mGFR: measured glomerular filtration rate; RTR: renal transplant recipients; BSA: body surface area; ADMA: asymmetric dimethylarginine
Table 3. The association of plasma citrulline concentration with mGFR, pooled for donors
post-donation and RTR
Coefficient plasma citrulline Coefficient RTR status
st.β P value st.β P value Crude -0.23 <0.001 -0.42 <0.001 Model 1 -0.27 <0.001 -0.42 <0.001 Model 2 -0.27 <0.001 -0.42 <0.001 Model 3 -0.25 <0.001 -0.41 <0.001 Model 4 -0.16 <0.001 -0.21 <0.001
Crude Plasma citrulline + RTR status
Model 1 Crude + age, sex, and BSA
Model 2 Model 1 + plasma arginine
Model 3 Model 2 + plasma ADMA
Model 4 Model 2 + urinary ADMA excretion
Abbreviations: mGFR: measured glomerular filtration rate; RTR: renal transplant recipients; BSA: body surface area; ADMA: asymmetric dimethylarginine.
Plasma citrulline and mGFR data of donors post-donation and RTR are pooled for this analysis.
Discussion
In the current study, we have investigated the dynamics of ADMA homeostasis with regard to renal function from a unique perspective of healthy subjects before and after a unilateral nephrectomy as a human model of isolated renal function impairment. Additionally, we compared the donors with RTR as a model of renal disease in combination with a single kidney state. We present a pronounced decrease in urinary ADMA excretion and a relatively modest increase in plasma ADMA after kidney donation, indicating that ADMA homeostasis changes dynamically as a consequence of renal function impairment in the healthy donors. In RTR, there is an association similar to that of donors post-donation, further supporting the effect of renal function per se on ADMA homeostasis. Furthermore, we present an inverse association of plasma citrulline concentration with mGFR, independent of patient status and plasma arginine concentration. Our findings suggest that there is increased metabolism of plasma ADMA to L-citrulline when renal function is impaired, as proposed in Figure 3.
Figure 3. ADMA buildup, breakdown, and excretion.
Arginine moieties of proteins are methylated post-translationally by protein arginine methyltransferases (PRMT). ADMA is released from these proteins by regular proteolysis. In healthy individuals, the largest part of ADMA is metabolized enzymatically by dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and dimethylamine (DMA). ADMA and DMA are then excreted in the urine. In renal function impairment, there is a hypothesized increase in enzymatic metabolism of ADMA, while renal excretion is reduced, as denoted by the plus and minus symbols as well as thickness of the arrows. The contributions of organs that provide methylated arginine proteins (notably brain, lung, and liver) are considered unchanged despite renal function impairment.
Several studies in the general population and the CKD population have observed that high plasma ADMA concentrations are associated with worse cardiovascular, renal and overall outcomes (1,2,21,22). Only few groups have studied urinary ADMA excretion in humans (3). A relatively small fraction of circulating ADMA is excreted unchanged in the urine (estimated to be 17% on average (22,23). It is, therefore, generally hypothesized that reduced metabolism of ADMA to L-citrulline and DMA by dimethylarginine dimethylaminohydrolase (DDAH), a key enzyme in metabolizing ADMA, but not reduced renal clearance of ADMA, is the main reason for high circulating plasma ADMA concentrations, as demonstrated in mice (24) and in small-scale human studies in children and lean or obese men (25). Apart from reduced activity of DDAH, it is also possible that reduced activity of AGXT2 contributes to increased circulating plasma ADMA in subjects with renal disease. Reason is that AGXT2, which has both ADMA and its isomer symmetric dimethylarginine (SDMA) as substrates (26), is primarily expressed in the kidneys (27,28). Indeed, Caplin et al. (2012) found in a small cohort of RTR (n = 35) that allograft tissue AGXT2 expression is inversely associated with plasma ADMA levels independent of DDAH activity, ethnicity, and eGFR (29). In the present study, renal clearance of ADMA became reduced in the context of isolated impairment of renal function. Yet, despite increased tubular resorption, plasma ADMA concentrations remained relatively similar. Tomlinson et al. (2015) performed a study in mice where the authors specifically targeted proximal tubular cell types, the main renal cell type expressing DDAH (30), to delete the DDAH1 gene and to study its effect on systemic and renal ADMA and NO homeostasis (31). They found that renal tubular ADMA concentration was more than sixfold higher and renal tubular NOx concentration (combined nitrite, nitrate and nitroso species) was 2.5 times higher in DDAH1−/− mice compared to control mice. Yet, there were no significant differences in plasma concentration or urinary excretion of ADMA and NOx. Rodionov et al. (2014) studied in mice the effects of bilateral nephrectomy on plasma ADMA levels and on enzyme activities of DDAH and AGXT2, as measured by conversion of isotope-labeled ADMA to its enzyme-specific products (28). Although liver DDAH and AGXT2 activities were not significantly different between nephrectomized and sham rats, they found that plasma ADMA levels were not significantly higher in nephrectomized rats compared to sham-operated rats. Plasma SDMA levels, however, increased by a factor of 4. The
findings of Tomlinson et al. and Rodionov et al. suggest that decreased renal DDAH activity is well compensated by other organs’ DDAH potential.
One of the reasons why plasma ADMA concentrations increase only marginally compared to the considerable change in urinary ADMA excretion in our study may lie in the biochemical properties of ADMA. Specifically, ADMA is known as a biochemically active metabolite, namely an NO synthase inhibitor, that is potentially harmful in the renal and cardiovascular systems. Thus, it can be hypothesized that it would be beneficial to keep stable plasma ADMA levels at levels as they are measured in healthy adults. Consequently, we can hypothesize that the dynamic changes that may occur with decreased renal function may be increased enzymatic metabolism of ADMA, presumably in liver (32). Accordingly, given the relatively narrow range of plasma ADMA concentration due to tight regulation and the hypothesis that circulating ADMA exerts biological activity, urinary ADMA excretion may, in this context, be a better marker of true ADMA homeostasis.
The hypothesized response of ADMA metabolism rate to renal function is supported by our observations from the linear regression analyses of the association of plasma citrulline concentration with mGFR. Although this association was only significant in RTR and not in donors post-donation, this discrepancy is more likely due to difference in renal function impairment between RTR and donors post-donation, rather than due to difference in the hypothesized ADMA metabolism: plasma citrulline concentration remains associated with mGFR despite adjustment for being an RTR and there is no interaction of being an RTR with plasma citrulline concentrations. The association of plasma citrulline concentration with mGFR drastically decreased after adjustment for urinary ADMA excretion, suggesting that urinary ADMA excretion explains a large part of the association between plasma citrulline concentration and renal function.
Other interesting hypotheses on the discrepant change in plasma and urine ADMA levels with renal function impairment are decreased breakdown of arginine-methylated proteins, decreased dietary ADMA intake, or possibly decreased protein methylation of arginine residues. Urea is a product of amino acid catabolism and urinary urea excretion can, therefore, be viewed as a marker of amino acid and thereby protein turnover. In the present study, we observed only slightly and non-significantly lower urea excretion in donors post-donation compared to pre-donation. Therefore, decreased arginine-methylated protein turnover would unlikely be the main cause of decreased urinary ADMA excretion with renal
function impairment. It is interesting to study the role of dietary protein intake on circulating and urinary ADMA levels. In lean and obese healthy men, both low-fat and high-fat protein intake for several weeks were found not to increase long-term plasma ADMA, yet there was a temporary small post-prandial increase in plasma ADMA concentration (25,33). This is likely to have resulted from intake of ADMA-containing protein. It is notable that both animal and plant proteins have been reported to contain methylated proteins (34). Whether protein arginine-methylation changes with renal function impairment cannot be confirmed nor denied by our study. Yet, this is an interesting topic for future studies.
Strengths of our study are the availability of 24h urinary data on ADMA and other urinary markers, the possibility to compare eGFR to mGFR, and the use healthy kidney donors as a model of isolated renal function impairment. Although it is yet to be demonstrated whether renal ADMA excretion has a circadian rhythm, we believe that properly performed collection of 24h urine is a better measure of average renal excretion compared to spot urine samples. Limitations of our study are its cross-sectional cohort nature and the absence of data on plasma DDAH levels or renal DDAH activity. It is known that the remaining kidney after living kidney donation undergoes hypertrophic changes, which at least partially compensate for the loss of renal function (35,36). It is unknown whether the hypertrophic changes also result in increase in renal DDAH capacity. It would be interesting if future studies would include assessment of plasma DDAH levels and potentially also renal DDAH activity. Another limitation is the absence of symmetric dimethylarginine (SDMA) data, which is a structurally similar product to ADMA and eliminated primarily by renal excretion in unchanged form because it is not hydrolyzed by DDAH. It would be interesting to investigate whether there are changes in protein arginine-methylation or arginine-methylated protein breakdown with renal function impairment. Finally, our study is limited by the absence of data on AGXT2 activity or AGXT2 metabolites, such as asymmetric dimethylguanidinovaleric acid (ADGV) and symmetric dimethylguanovaleric acid (SDGV) (26,37). Since AGXT2 is expressed mainly in the kidneys (27,28), it would be interesting when future studies could incorporate data on AGXT2 activity or AGXT2 metabolites to get an even more complete picture of the effect of renal function on ADMA metabolism and homeostasis.
A potential implication of the current study is that renal function decline poses an increased renal and cardiovascular risk for subjects who are highly dependent
on renal excretion of ADMA in order to not accumulate ADMA in plasma. In rats, it has been found that oxidative stress induced by high glucose impairs DDAH activity, leading to an increase in plasma ADMA (38). Consequently, renal function decline may pose an additional risk for patients with a concomitant increase in oxidative stress, such as patients with diabetes mellitus, obesity, or hypertension (39).
To conclude, we found that renal function impairment has a disproportional effect on plasma levels and urinary excretion of ADMA, where plasma ADMA levels remain relatively stable and urinary ADMA excretion decreases drastically. This may be due to increased metabolism of ADMA by DDAH. Given the apparent tight regulation of plasma ADMA concentrations, urinary ADMA excretion may be a better marker of ADMA metabolism and homeostasis than plasma ADMA concentrations.
Compliance with Ethical Standards
Conflict of interest
R.M. Douwes is supported by the applied science division of the Dutch Technology Foundation (Stichting voor Technische Wetenschappen-Nederlandse Organisatie voor Wetenschappelijk Onderzoek; STW-NWO) in a partnership program with DSM Animal Nutrition and Health, a manufacturer of animal nutrition and nutritional products; project number: 14939.
Informed consent and ethical approval
All procedures performed in this study were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.
Acknowledgements
N. de Ruiter, E. Jonkers, P de Blaauw, and J. van der Krogt, technicians of the laboratory of metabolic diseases, are gratefully acknowledged for citrulline and arginine analyses. Funding was provided by Stichting voor de Technische Wetenschappen (NL) & DSM Animal Nutrition and Health (14939).
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