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Our reference: NUMECD 1373 P-authorquery-v9
AUTHOR QUERY FORM
Journal: NUMECD
Article Number: 1373
Please e-mail or fax your responses and any corrections to:
� The GLUT9 polymorphism, powerful marker of hyperuricemia, predicts faster CKD progression.� ADMA is a strong predictors of CKD progression.� ADMA and uric acid levels are directly related each other in several conditions.� We found that ADMA amplifies the risk for CKD progression due to the risk allele of GLUT9 gene.
Please cite this article in press as: Testa A, et al., Synergism between asymmetric dimethylarginine (ADMA) and a genetic marker of uricacid in CKD progression, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/j.numecd.2014.10.016
http://dx.doi.org/10.1016/j.numecd.2014.10.0160939-4753/ª 2014 Published by Elsevier B.V.
Abstract Background & aims: We have recently reported that a polymorphism (rs734553) in amajor urate transporter gene (GLUT9) is a strong predictor of incident renal events in stage 2e5CKD patients implying that life-time exposure to high uric acid levels may be causally implicatedin CKD progression. Since disturbed NO bioavailability is a major pathway whereby high uric maycause renal damage, we tested the interaction between the major endogenous inhibitor of NOsynthase, asymmetric-dimethylargine (ADMA), and the rs734553 polymorphism for CKD pro-gression in the same cohort.Methods & results: Over a 29 � 11 months follow-up the risk for incident renal events was higherin patients harboring the risk allele of the polymorphism (T) as compared to those without therisk allele (HR: 2.35, 95% CI: 1.25e4.42, P Z 0.008) (p Z 0.01). Similarly, patients withADMA > median value had an increased risk for the same outcome (HR: 1.37, 95% CI: 1.06e1.76, P Z 0.016). Interaction analysis showed a strong amplification by ADMA of the risk forrenal events associated to the T allele because in adjusted (P Z 0.016) and bootstrapping vali-dated (P Z 0.020) analyses the risk excess associated to this allele was progressively higheracross increasing ADMA levels.Conclusions: The rs734553 polymorphism, the strongest genetic marker of uric acid levelsdiscovered so far, interacts with ADMA in determining the risk for CKD progression in CKD pa-tients. This synergic interaction conforms to biological knowledge indicating that disturbed NObio-availability is a critical pathway whereby life time exposure to high uric acid may engenderrenal damage.ª 2014 Published by Elsevier B.V.
Introduction
In a recent study based on the Mendelian randomizationapproach [1], we showed that the strongest (P Z 10�206)genetic marker of uric acid levels identified so far [2], i.e. apolymorphism (rs734553) in a gene which regulates thesynthesis of amajor tubular transporter of urate (GLUT9), is apowerful predictor of progression toward kidney failure inpatientswith chronic kidney disease (CKD). This observationis of relevance because genetic polymorphisms are randomlydistributed at gamete formation and therefore the risk allelein this polymorphism (T allele) represents an unbiased,
* Corresponding author. c/o EUROLINE di Barillà Francesca, Via Val-lone Petrara 55-57, 89100 Reggio Calabria, ItalyQ1 . Tel.: þ39 (0)965397010; fax: þ39 0965 26879.
E-mail address: [email protected] (C. Zoccali).1 MAURO Study Investigators: Audino A, Bruzzese V, Caglioti A, Campo
S, Caridi G, Caruso A, Chiarella S, Cicchetti T, Curti C, D’Anello, EniaGiuseppe, Fabiano F, Fatuzzo P, Fersini S, Garozzo M, Grandinetti F,Guido A, Gullo M, Maimone I, Mancuso F, Mannino M, Marino F, NataleG, Palma L, Papalia T, Parisi T, Parlongo G, Pinciaroli A, Pinna M, PlutinoD, Postorino Maurizio, Pugliese M, Rapisarda F, Reina A, Santoro O,Tramontana D.Collaborators: Parlongo RM.
Please cite this article in press as: Testa A, et al., Synergism between asymmetric dimethylarginine (ADMA) and a genetic marker of uricacid in CKD progression, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/j.numecd.2014.10.016
http://dx.doi.org/10.1016/j.numecd.2014.10.0160939-4753/ª 2014 Published by Elsevier B.V.
environment independent, marker of life-time exposure tohyperuricemia. The endogenous inhibitor of nitric oxidesynthesis, asymmetric dimethylarginine (ADMA), is anotherenvironmental risk factor of paramount importance in CKD[3]. The plasma concentration of this substance has beenassociated to uric acid levels in various populations includingCKD patients [4], patients with chronic heart failure [5] andwomen with coronary artery disease [6], oxidative stressbeing held as a likely mechanism underlying such an asso-ciation [5]. The uric acid-ADMA link may be of particularrelevance inCKDbecause bothhighADMA[7,8] andhighuricacid levels [9] predict a high risk of kidney failure. Avariety ofexperimental [10,11] and clinical studies [12,13] show thathigh levels of these two compounds associate with reducedNO bio-availability and endothelial dysfunction, i.e. apathway which may engender and/or aggravate renal dam-age in experimental models [14]. Furthermore, the majordrug used to treat hyperuricemia and gout, allopurinol, is apotent anti-oxidant and also an ADMA lowering agent [5].For the foregoing, testing whether there is a synergic inter-action between the genetic polymorphism which regulatesserum uric acid levels and ADMA in the risk of CKD pro-gression is a relevant pathophysiological question.
With this background in mind, we have now measuredADMA in the same cohort and tested the interaction be-tween ADMA and the GLUT9 polymorphism (rs734553) inrenal disease progression in a sizable series of CKD patients.
Methods
The study protocol was in conformity with the ethicalguidelines of our institution and informed consent was ob-tained fromeachparticipant. Theexperimentalprotocols andthe process for obtaining informed consent were approvedby the appropriate institutional review committee. Awritteninformed consent was obtained from each participant.
CKD patients
All patients were consecutively recruited from 22Nephrology Units in Southern Italy. Patient enrollment wasperformed between October 2005 and October 2008. Thesource CKD cohort of this study was composed by 826 CKDpatients. Thirty-four patients were lost to observation afterthe baseline visit, thirty-three were erroneously enrolledand in 4 patients no DNA sample was available. Thus 755stage 2e5 CKD patients (91% of the whole cohort) wereincluded in the present analysis. All patients were in stableclinical condition and none had intercurrent infections oracute inflammatory processes. Inclusion criteria were: nonacute or rapidly evolving renal diseases; age ranging from18 to 75 years; non-transplanted; non-pregnant, notaffected by cancer or diseases in the terminal phase. Thiscohort has been accurately described elsewhere [1].
Healthy population
To confirm the link between uric acid levels and thers734553 polymorphism in the GLUT9 gene, we studied a
sample of 211 consecutive healthy volunteers enrolledbetween January 1st, 2001eJuly 12th, 2011 of the samegeographic area of CKD patients, without renal disease(GFRCKD-EPI > 60 ml/min 1.73 m2 and no proteinuria),invited to provide a control group for the study.
Study outcome
Patients were followed up for an average time of 29 � 11months (range: 1.4e48 months). The study end-point wasa pre-specified composite renal end point, i.e. >30%decrease in the GFR (estimated by MDRD186 study equa-tion formula), dialysis or transplantation [15].
Laboratory measurements
Serum creatinine, lipids, albumin, calcium, and hemoglo-bin were measured by standard methods in the routineclinical laboratory. Serum C-Reactive Protein (CRP) wasmeasured by a high sensitivity commercially available RIAkit [intra-assay coefficient of variation (CV): 3.5%; inter-assay CV: 3.4% (Dade Behring, Marburg, Germany)]. ADMAwas measured in plasma by a validated ELISA (DLD-Diag-nostika, Hamburg, Germany) [16]. All CKD patients alsounderwent a 24 h urinary collection for the measurementof proteinuria.
Genotyping of GLUT9 gene polymorphism
Genomic DNA was extracted by salting-out technique. Theparticipants were genotyped for the rs734553 GLUT9polymorphism, studied by a validated TaqMan SNP Geno-typing Assay, as previously described [1]. A random 5% ofsamples were independently repeated to confirm geno-typing results. The genotype results for these sampleswere completely consistent.
Statistical analysis
Data are expressed as mean � SD, median and inter-quartile range or as percent frequency and comparisonsbetween two groups were made by T Test, ManneWhitneyTest or Chi Square test, as appropriate. Comparisonsamong more than 2 groups were made by P for trend.
The relationship between GLUT9 SNP and the incidencerate of renal events was investigated by KaplaneMeiersurvival curves and univariate and multivariate Coxregression analyses. The effect modification by ADMA onthe relationship between GLUT9 polymorphism and renaloutcomes was investigated by introducing ADMA, thepolymorphism and their interaction term [ADMA*GLUT9polymorphism (0 Z GT þ TT; 1 Z GG)] into the same Coxmodel. The same analysis was performed for testing theinteraction between ADMA and uric acid levels. The hazardratios of GLUT9 polymorphism for renal events throughoutplasma ADMA levels were calculated by the linear com-bination method. The internal validation of study resultswas carried out by a bootstrapping resampling technique[17]. Data analysis was performed by commercially
Please cite this article in press as: Testa A, et al., Synergism between asymmetric dimethylarginine (ADMA) and a genetic marker of uricacid in CKD progression, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/j.numecd.2014.10.016
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available statistical software (SPSS for WindowseVersion9, Chicago, Illinois, USA and STATA 9 by Stata Corp LP,College Station, USA).
Results
The main demographic, clinical and biochemical charac-teristics of the CKD population categorized according tothe rs734553 polymorphism in the GLUT9 gene are sum-marized in Table 1.
Correlates of GLUT9 SNP in CKD patients and in controls
In CKD patients, the distribution of alleles in this poly-morphism (rs734553, GG: 8.1%; GT: 35.4%; TT: 56.6%)slightly deviated from HardyeWeinberg equilibrium(c2 Z 4.30, P Z 0.04), while in healthy normotensivesubjects (rs734553, GG: 7.3%; GT: 45.2%; TT: 47.5%,c2 Z 1.91, P Z 0.17) no such deviation was noted. In CKDpatients, 24 h proteinuria was substantially higher(P Z 0.006) in homozygotes and heterozygotes (GT þ TT)(0.70 g/24 h, 0.20e0.14) for the T allele in comparison withpatients without such an allele (GG) (0.30 g/24 h,0.11e1.10) (Table 1). As expected from Mendelianrandomization, no difference for demographic, clinical andbiochemical rick factors was found between GG and GT/TT
CKD patients and plasma ADMA levels did not differ in thetwo genetic groupings. However, there was a higher pro-portion of smokers in patients harboring the T allele (GT orTT patients, Table 1).
In normal subjects (Table 2), an analysis according to adominant model showed that uric acid was higher(P < 0.05) in individuals with at least one T allele (GT þ TT:4.7 � 1.4 mg/dL) than in those without such an allele (GG:4.1 � 1.5 mg/dL).
Rs734553 polymorphism and ADMA synergism in theprogression of CKD
Two hundred and forty-four patients developed incidentrenal events during the 29 � 11 months follow-up period(range 1.4e47.0 months). The cumulative risk for theseevents was significantly higher in GT and TT as comparedto GG patients (Fig. 1, left panel) and the risk for renalevents was more than twice higher in patients with atleast one T allele (GT þ TT) higher (HR: 2.35, 95% CI:1.25e4.42, P Z 0.008) than in those without this allele(HR: 1, reference group). By the same token patients withADMA > median value (>0.82 mMol/L) had an increasedrisk for CKD progression (HR: 1.37, 95% CI: 1.06e1.76,PZ 0.016) (Fig. 1, right panel). Of note, on crude analysis aswell as in fully adjusted (P Z 0.016) and bootstrappingvalidated (P Z 0.02) analyses (Table 3) there was a syn-ergic interaction between the rs734553 polymorphismand ADMA (Table 3) for explaining the incidence of renaloutcomes. Indeed, the hazard ratio for renal events asso-ciated to the risk allele (T) was closely dependent onADMA values (Fig. 2) being progressively higher acrossincreasing ADMA values. At ADMA levels of 1.2 mMol/Lsuch risk was about 12 times higher than at 0.8 mMol/L(the upper limit of the normal range). The inclusion ofeGFR into this model did not affect these results becausethe ADMA-rs734553 interaction maintained an almostunchanged statistical significance (p Z 0.02).
Discussion
This study shows that a polymorphism in the GLUT9 gene,a major genetic regulator of serum uric acid levels,
Table 1 Main clinical and biochemical data of the study population(n Z 755, stage 2e5 CKD).
Please cite this article in press as: Testa A, et al., Synergism between asymmetric dimethylarginine (ADMA) and a genetic marker of uricacid in CKD progression, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/j.numecd.2014.10.016
interacts with ADMA levels in predicting renal diseaseprogression in CKD patients.
Hyperuricemia has been associated with the risk forCKD in observational studies in the general population[18e22], as well as with faster progression to kidney fail-ure in patients with CKD [23,24] and potential mecha-nisms whereby uric acid may engender/or amplify renaldamage have been lucidly reviewed by Johnson et al. [9].On the other hand, evidence that lowering uric acid levelsmay retard CKD progression has been investigated just in asmall number of clinical studies [25,26] and in a recentlypublished, very large observational study in patients withhyperuricemia [27], but we still lack well powered ran-domized trials with uric acid lowering agents proving thatreducing asymptomatic hyperuricemia may impact uponkidney outcomes in CKD patients. In the lack of such a trialwe further explored the issue by adopting the T allele of
the rs734553 polymorphism, the genetic variant mostconsistently associated (p < 10�206) with hyperuricemia[2], as an unbiased and unconfounded marker of long termexposure to high uric acid levels. According to the workinghypothesis that hyperuricemia may accelerate CKD pro-gression, we found that the risk allele for hyperuricemiapredicts faster progression toward kidney failure [1]. Likein the previously mentioned meta-analysis [2], in thepresent study we found that the T allele of GLUT9 gene isstrongly associated with hyperuricemia in healthy sub-jects. However, in the previous cohort study [1] whichoriginated the present study we found no such an asso-ciation in patients with CKD. This observation is in keepingwith remarks by Jalal et al. [28] that the link betweenactual uric acid levels and CKD progression is difficult toinvestigate in observational studies because such a link isconfounded by environmental factors including treatment
Figure 1 Cumulative incidence of combined renal endpoint according to the presence/absence of T risk allele (left panel) and low/high ADMAvalues (right panel). The comparison between curves was done by log rank test.
Table 3 Univariate and multiple Cox regression models of GLUT9 polymorphism for explaining the incidence rate of renal events.
Variables (units of increase) Crude Fully adjusted analysis Bootstrapping validatedmodel
GLUT9* ADMA 1.49 (1.03e2.16), P Z 0.03 1.56 (1.09e2.24), P Z 0.016 0.06 (0.001e1.28), P Z 0.02GLUT9 polymorphism
(0 Z GG; 1 Z GT þ TT)0.10 (0.006e1.58), P Z 0.10 0.06 (0.004e0.93), P Z 0.04 0.71 (0.39e1.02), P Z 0.07
ADMA (0.1 mMol/L) 0.78 (0.54e1.12), P Z 0.17 0.03 (0.0009e1.19), P Z 0.06 1.56 (1.08e2.80), P Z 0.08Age (years) 0.99 (0.98e1.01), P Z 0.45 1.00 (0.98e1.01), P Z 0.53Male sex 0.98 (0.69e1.40), P Z 0.92 1.02 (0.72e1.49), P Z 0.92Smoking (0 Z no; 1 Z yes) 1.24 (0.91e1.71), P Z 0.17 1.25 (0.90e1.74), P Z 0.20Diabetes (0 Z no; 1 Z yes) 0.85 (0.62e1.16), P Z 0.31 0.85 (0.95e1.17), P Z 0.39Cholesterol (1 g/dL) 1.00 (0.99e1.01), P Z 0.62 1.00 (0.99e1.01), P Z 0.63Systolic BP (5 mmHg) 1.01 (1.00e1.01), P Z 0.002 1.06 (1.02e1.11), P Z 0.08Anti-hypertensive treatment
(0 Z no; 1 Z yes)1.18(0.44e3.19), P Z 0.74 1.18 (0.50e18.54), P Z 0.76
BMI (1 kg/m2) 0.98 (0.95e1.01), P Z 0.20 0.98 (0.95e1.01), P Z 0.23Hemoglobin (1 g/dL) 0.85 (0.78e0.93), P < 0.001 0.85 (0.78e0.92), P Z 0.002Albumin (1 g/dL) 0.66 (0.48e0.90), P Z 0.009 0.66 (0.45e0.90), P Z 0.02Phosphate (1 mg/dL) 1.21 (1.02e1.44), P Z 0.028 1.22 (0.96e1.54), P Z 0.08CRP (1 mg/L) 0.99 (0.98e1.00), P Z 0.32 0.99 (0.97e1.00), P Z 0.20Urinary protein (1 g/24 h) 1.27 (1.18e1.37), P < 0.001 1.27 (1.19e1.45), P Z 0.001On treatment with allopurinol
(0 Z no; 1 Z yes)1.16 (0.89e1.51), P Z 0.27 1.16 (0.84e1.55), P Z 0.31
Data are expressed as hazard ratio, 95% confidence intervals and P values.
Please cite this article in press as: Testa A, et al., Synergism between asymmetric dimethylarginine (ADMA) and a genetic marker of uricacid in CKD progression, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/j.numecd.2014.10.016
with allopurinol, diuretics and acidebase status in thesepatients. Again, along with Jalal’s remark, uric acid levelsper se (data not shown) failed to interact with ADMA forthe prediction of renal events in the present study.
Although theprecisemechanismwherebyserumuric acidis associated with renal outcomes in CKD is still undefined,oxidative stress, a common pathway of several nephrotoxins[29], and interaction with factors impinging upon endothe-lial function [30] appear of relevance in the pathogenesis ofuric acid-dependent renal damage. ADMA is an establishedrisk marker of oxidative stress, endothelial dysfunction andother cogent alterations in CKD patients [3], including renaldisease progression [7,8]. Uric acid levels have been associ-ated with plasma ADMA concentration in at least threestudies [4e6]. Thus, it appears biologically plausible thatADMA may amplify the effect of hyperuricemia on renaldamage. In line with this hypothesis, we found a synergisticinteraction between ADMA and the risk of CKD progressionportended by the T allele of the rs734553 polymorphism.Indeed, this allele signaled a progressively higher risk acrossincreasing ADMA levels. The synergic rs734553-ADMAinteraction is hypothesis generating and provides a ratio-nale for detailed biological studies exploring the uric acid-ADMA link. Findings in the present study suggest that uricacid lowering interventions may be particularly beneficial inpatientswith highADMA, a hypothesis that couldbe retestedin future experimental studies investigating the effect ofurate lowering therapy on CKD progression. Furthermore,future studies should also test the effect modification byanother relevant endogenous inhibitor of NO, i.e. the ADMAenantiomer symmetric dimethylarginine (SDMA). Thismethylarginine competes with the parent aminoacid L-argi-nine for a cell transporter and inhibits NO synthesis by thispathway. Of note, there is increasing evidence that, inde-pendently of ADMA, SDMA may per se impinge upon car-diovascular and renal outcomes [31].
Our study has limitations. Although large, our samplesize was limited. However, the rs734553 polymorphism isan established, strong marker of uric acid levels [2] and we
confirmed this association in healthy subjects of ourgeographical area. Furthermore, we confirmed the internalvalidity of the rs734553-ADMA interaction by boot-strapping analysis. Our findings remain to be confirmed inother ethnicities and, more in general, in other CKD co-horts. Appropriate clinical trials are needed to conclusivelydemonstrate that reducing uric acid levels may improvethe risk for adverse renal outcomes in CKD patients withhigh ADMA levels.
In summary, the rs734553 genetic variant interactswith ADMA in the risk of CKD progression. This interactionsuggests that the synergism between ADMA and this ge-netic marker of hyperuricemia may contribute to the riskof renal disease progression. This hypothesis can beformally tested in clinical trials investigating the effect ofurate lowering drugs on the progression of CKD.
Disclosure
The authors have nothing to disclose.
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Figure 2 Effect modification by ADMA levels on the relationship be-tween presence/absence of T risk allele and the incidence rate of thecombined end point. The hazard ratio (GT þ TT versus TT patients) isreported on the left scale. The continuous line represents the shape ofthe hazard ratios throughout ADMA levels and the dotted lines thecorresponding 95% CI. In the background the distribution of ADMA isplotted and the number of patients corresponding to each column ofthe histogram is reported on the right scale.
Please cite this article in press as: Testa A, et al., Synergism between asymmetric dimethylarginine (ADMA) and a genetic marker of uricacid in CKD progression, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/j.numecd.2014.10.016
Original text:
Inserted Text
As Refs. [5,15] and [9,26] were identical, the latter have been removed from the reference list and subsequent references have been renumbered.
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