Article

Association of IL-6 and a Functional Polymorphismin the IL-6 Gene with Cardiovascular Events inPatients with CKD

Belinda Spoto,* Francesco Mattace-Raso,† Eric Sijbrands,† Daniela Leonardis,* Alessandra Testa,* Anna Pisano,*Patrizia Pizzini,* Sebastiano Cutrupi,* Rosa M. Parlongo,* Graziella D’Arrigo,* Giovanni Tripepi,*Francesca Mallamaci,* and Carmine Zoccali*

AbstractBackground and objectivesHigh serum IL-6 is a major risk factor for cardiovascular disease (CVD) in the generalpopulation. This cytokine is substantially increased in patients with CKD, but it is still unknownwhether the linkbetween IL-6 and CVD in CKD is causal in nature.

Design, setting, participants, & measurements In a cohort of 755 patients with stages 2–5 CKD, consecutivelyrecruited from 22 nephrology units in southern Italy, this study assessed the relationship of serum IL-6 withhistory of CVD, as well as with incident cardiovascular (CV) events (mean follow up6SD, 31610 months) andused the functional polymorphism (2174 G/C) in the promoter of the IL-6 gene to investigate whether the linkbetween IL-6 and CV events is causal.

Results In adjusted analyses, serum IL-6 above the median value was associated with history of CVD (P,0.001)and predicted the incidence rate of CV events (hazard ratio, 1.66; 95% confidence interval [95% CI], 1.11 to 2.49;P=0.01). Patients homozygous for the risk allele (C) of the2174G/Cpolymorphismhad higher levels of IL-6 thandid those with other genotypes (P=0.04). Homozygous CC patients more frequently had a history of CVD (oddsratio, 2.15; 95% CI, 1.15 to 4.00; P=0.02) as well as a 87% higher rate of incident CV events (hazard ratio, 1.87; 95%CI, 1.02 to 3.44; P=0.04) compared with other genotypes.

Conclusions In patients with stages 2–5 CKD, high serum IL-6 is associated with history of CVD and predictsincident CV events. The parallel relationship with history of CVD and incident CV events of the 2174 G/Cpolymorphism in the IL-6 gene suggests that IL-6may be causally involved in the high CV risk in this population.

Clin J Am Soc Nephrol 10: ccc–ccc, 2014. doi: 10.2215/CJN.07000714

IntroductionClassic experimental studies by Russel Ross et al. inthe 1990s solidly established inflammation as a criticalcomponent of the atherosclerosis process (1). Duringthe last two decades, large cohort studies in the gen-eral population have shown strong links between bio-markers of inflammation and cardiovascular (CV)outcomes in the general population (2–5) and in patientswith CV disease (CVD) (6,7). Observational studies aremethodologically vulnerable for testing causality be-cause these studies are open to various sources ofbias and confounding. Mendelian randomization—i.e.,the random assortment of alleles at conception—offersan intriguing opportunity to limit problems inherent toobservational studies because categorization of patientsaccording to pertinent alleles is a sort of genetic ran-domization. Genetic variants may thus be used as indi-cators of environmental exposures in the observationalcontext (8). Large-scale Mendelian randomization stud-ies applying genetic polymorphisms of inflammatorycytokines (9) strongly support the hypothesis that, as

seen in experimental animals (10,11), the link betweeninflammation and atherosclerosis complication iscausal in nature.CV risk is a multifactorial problem in patients with

CKD (12). Systemic inflammation is common in thesepatients, particularly those with stage 5 CKD under-going dialysis (13,14). In line with studies in the gen-eral population (5,15), IL-6, a major proinflammatorycytokine, is an established strong predictor of adverseclinical outcomes in patients with stage 5D CKD (16–20).However, the relationship between IL-6 and CVD atearlier CKD stages was investigated in just one rela-tively small study by Barreto et al. (21), which was basedon a limited number of CV events (just 22 events). Inaddition, to date we lack specific proof that this relation-ship in the CKD population is causal.Circulating levels of IL-6 are genetically regulated.

Because transmission of genes is a random phenom-enon, gene polymorphisms modulating IL-6 synthesismay represent an unbiased means for testing whetherthe link between IL-6 and CV outcomes in patients

*National ResearchCouncil of Italy-Institute of ClinicalPhysiology & Instituteof Biomedicineand MolecularImmunology &Nephrology, Dialysisand TransplantationUnit of Reggio,Calabria, Italy; and†Department ofInternal Medicine,Erasmus UniversityMedical Center,Rotterdam, TheNetherlands.

Correspondence:Professor CarmineZoccali, CNR-IFC andNephrology, Dialysisand TransplantationUnit, Ospedali Riuniti,89124 ReggioCalabria, Italy, c/oEUROLINE, ViaVallone Petrara 55-57,89100 ReggioCalabria, Italy. Emailcarmine.zoccali@tin.it

www.cjasn.org Vol 10 February, 2014 Copyright © 2014 by the American Society of Nephrology 1

. Published on December 9, 2014 as doi: 10.2215/CJN.07000714CJASN ePress

with CKD is causal (Mendelian randomization). The 2174G/C single-nucleotide polymorphism is a functional vari-ant located in the promoter region of the IL-6 gene thatregulates the rate of IL-6 gene transcription (22–28) andtherefore represents a reliable research tool for testingthe nature (causal versus noncausal) of the link betweenIL-6 and CV outcomes in CKD. With this background inmind, we set out to confirm findings by Barreto et al. (21)in a large observational study with a carefully character-ized cohort of 755 patients with stages 2–5 CKD and to testwhether this relationship may underlie a causal link byapplying the Mendelian randomization approach (i.e., bystratifying the study population according to the func-tional 2174 G/C polymorphism in the IL-6 gene).

Materials and MethodsStudy ProtocolThe study protocol conformedwith the ethical guidelines

of our institution and was approved by ethical committeesof all participating units. Each participant provided writteninformed consent.

Patients with CKDOur study population included a genetically homogenous

series of 755 white patients from the same geographic area(southern Italy) (29), consecutively recruited from 22 ne-phrology units between October 2005 and September 2008.Eligible patients were age 18–75 years and in stable clinicalcondition. Exclusion criteria included acute or rapidly evolv-ing renal disease, kidney transplant, acute intercurrent infec-tions or acute inflammatory processes, pregnancy, cancer, ordiseases in the terminal phase. This cohort was described indetail elsewhere (30).

Control PopulationTo compare the allelic frequencies of the 2174 G/C

polymorphism observed in patients with CKD, westudied a sample of 463 consecutive blood donors of thegeneral population from the same geographical area ofpatients with CKD.

Follow-up and Study OutcomeAfter the initial assessment, patients were monitored for

a mean6SD of 31610 months (range, 0.3–48 months). Thestudy endpoint was fatal and nonfatal CV events as de-scribed elsewhere (30). These events included myocardialinfarction, documented by electrocardiography and bio-markers of myocardial injury; heart failure, defined asdyspnea in addition to two of the following conditions:raised jugular pressure, bi-basilar crackles, pulmonaryvenous hypertension, or interstitial edema on chest radi-ography requiring hospitalization; electrocardiography-documented arrhythmia; stroke; peripheral vascular disease;and major arterial or venous thrombotic episodes. Theseevents were accurately recorded during the follow-up period.The history of CVD was defined as the presence of at least

one of the following comorbidities at enrollment: myocar-dial infarction, heart failure, peripheral vascular disease,stroke, transient ischemic attack, or coronary surgery/angioplasty.

Laboratory MeasurementsIn the whole study population, blood sampling was

performed in the early morning after an overnight fast andplasma was stored at 280°C until analysis. Serum glucose,lipids, hemoglobin, albumin, creatinine, and C-reactiveprotein (CRP) were measured by standard methods in theroutine clinical laboratory. Serum IL-6 was measured byELISA (R&D Systems, Inc., Minneapolis, MN). The eGFRwas calculated by using the four-variables Modification ofDiet in Renal Disease study equation (31) and not by theCKD-Epidemiology Collaboration formula because the cre-atinine data were not traceable by isotope dilution massspectrophotometry during the study period. All patientswith CKD underwent a 24-hour urinary collection for themeasurement of proteinuria.

Genotyping of 2174G/C PolymorphismAllelic discrimination of 2174 G/C polymorphism was

performed using a custom TaqMan SNP Genotyping As-say provided by Applied Biosystems (Foster City, CA). Inthis assay, primers were designed to amplify a region thatincluded the mutation site specifically recognized by a fewprobes able to discriminate wild-type and mutated alleles.The sequences of primers and probes were: 59-CGACC-TAAGCTGCACTTTTCC-39 (forward primer) and 59-GGGCTGATTGGAAACCTTATTAAGATTG-39 (reverseprimer); 59-CCTTTAGCAT[G]GCAAGAC-39 (C allele-specificprobe) and 59- CCTTTAGCAT[C]GCAAGAC-39 (G allele-specific probe). Allelic discrimination was performed on a7900HT Fast Real-Time PCR platform and its accompany-ing Sequence Detection System software, version 2.4 (Ap-plied Biosystems). Briefly, genomic DNA was extractedfrom peripheral blood mononuclear cells using standardsalting out procedure (32). The reaction system contained20 ng of genomic DNA, 12.5 ml of 2X TaqMan UniversalPCR Master Mix No AmpErase UNG, 1,25 ml of 40X AssayMix (including unlabeled PCR primers, FAM and VIC dye-labeled TaqMan MGB probes), and H2O for a total volumeof 25 ml. A random 10% of samples were independentlyrepeated to confirm genotyping results. The genotype re-sults for these samples were completely consistent. Allanalyses were done blinded to clinical information.

Statistical AnalysesData were expressed as mean6SD, median and interquar-

tile range, or percentage frequency. Comparisons betweentwo groups were made by t-test, Mann–Whitney test, orchi-square test, as appropriate. The comparison amongmore than two groups was performed by ANOVA forlog-transformed variables, when appropriate. The devia-tion from Hardy–Weinberg equilibrium was assessed bythe chi-square test comparing observed and expected geno-type frequencies. The 95% confidence interval (95% CI) ofthe risk allele frequency was calculated as suggested by thestandard method (33).The functional form of serum IL-6 (as continuous, binary,

quartile, or quintile data) was formally investigated by theanalysis of Martingale residuals, and the binary form(below/above the median) provided the best data fitting.The relationships between serum levels of IL-6 and CVD

was evaluated by two approaches. First, we analyzed thebaseline association between serum IL-6 and history of

2 Clinical Journal of the American Society of Nephrology

CVD. Second, we investigated the predictive power ofserum IL-6 in the prospective cohort study. In both logisticand survival analyses, we considered variables that metcriteria to be confounders (i.e., variables related [P#0.10] toboth the exposure under investigation [serum IL-6 levelsbelow/above the median value] and history of CVD orincident fatal and nonfatal CV events, which are not aneffect of the exposure and are not in the causal pathwaybetween the exposure and outcome) (34). Tested covariatesincluded traditional risk factors (age, sex, smoking, dia-betes and glucose, cholesterol, and BP), factors specific toCKD (hemoglobin, albumin, eGFR, and urinary protein),antihypertensive treatment, body mass index, and CRP. Inboth logistic and Cox regression models, eGFR was alwaysforced because of the strong and significant correlationbetween eGFR and serum IL-6 (Table 1). To further inves-tigate the causal role of IL-6 in the pathway leading to CVevents in patients with CKD, we applied a Mendelian ran-domization approach (i.e., we stratified the study popu-lation according to the 2174 G/C polymorphism). Theseanalyses were appropriately adjusted for variables thatdiffered between CC and GC or GG patients and that ap-peared to be potential confounders (i.e., age, sex, choles-terol) for the interpretation of the link between the riskgenotype (CC) and the study outcomes.In the prospective cohort study, the potential distortion

on the study results due to the competing risks of deathwas assessed by comparing the incidence rate of death inexposed patients (high IL-6 levels or CC risk genotype) andunexposed patients (low IL-6 levels or GC/GG genotype).If a difference was found, the competing risk of death wasaccounted for by carrying out a survival analysis consid-ering a combined outcome: death/CV events (35). Datawere expressed as odds ratios (ORs; logistic regressionanalysis), hazard ratios (HRs; Cox regression model), 95%CIs, and P values. To internally validate the independentrelationships of serum IL-6 and 2174 G/C polymorphismswith history of CVD and incident CV events, a bootstrapresampling technique of 1000 samples (randomly extractedfrom the original sample) was performed (36). All potentialeffect modifications exerted by covariates on the relation-ship between the key exposures (serum IL-6 and2174 G/Cpolymorphism) and study outcomes were formally testedby introducing a multiplicative term into the models; nosignificant interaction was found.All calculations were made by using a standard statis-

tical package (IBM SPSS Statistics for Windows, version21.0. 0.1, IBM, Armonk, NY).

ResultsWe studied 755 patients with stages 2–5 CKD (stage 2,

3%; stage 3a, 22%; stage 3b, 38%; stage 4, 34%; stage 5, 3%).Four hundred fifty-three patients were male (60%), 263had type 2 diabetes (35%), and 98 were current smokers(13%). Two-hundred twenty-one patients (29%) had a his-tory of CVD (Table 1). One hundred nine patients had onlyone previous CV event, and 112 of them had two or moreprevious CV events. The first CV event in this populationwas myocardial infarction in 19 patients, heart failure in 26patients, peripheral vascular disease in 29 patients, strokein 12 patients, transient ischemic attack in 12 patients, and

coronary surgery/angioplasty in 11 patients. Six hundredninety-one patients (92%) were receiving antihypertensivetreatment: one medication in 19%, two medications in 29%,three medications in 28%, and four or more medications in16%. The mean eGFR was 36613 ml/min per 1.73 m2, andthe median 24-hour urinary protein excretion was 0.6 mg/24hours (interquartile range, 0.2–1.5 mg/24 hours). The medianIL-6 level was 2.5 pg/ml (interquartile range, 1.6–4.0 pg/ml).

Analyses Based on Serum IL-6 LevelsPatients with IL-6 above the median value were signif-

icantly older, more frequently had diabetes, and had higherserum glucose and 24-hour urinary protein values thanthose with IL-6 below this threshold (Table 1). Values foreGFR, hemoglobin, and albumin were lower in patientswith higher IL-6 than in those with IL-6 below the medianvalue. Systolic BP was higher and diastolic BP was lowerin patients with IL-6 levels above the median (Table 1). Asexpected, serum CRP levels were directly associated withIL-6 levels (r=0.57; P,0.001). On bivariate, multivariate,and bootstrapping validation analyses, patients with anIL-6 level above the median were more likely to havehad a history of CVD (P#0.01) than those with an IL-6level below the median (Table 2).During the follow-up, 42 patients died. The incidence

rate of mortality was three times higher (P=0.01) in patientswith IL-6 above the median (3 deaths per 100 person-years; 95% CI, 2.0 to 4.1 deaths per 100 patient-years)than in those with IL-6 below this threshold (1 death per100 person-years; 95% CI, 0.6 to 2.1 deaths per 100 patient-years). Overall, 117 patients had fatal and nonfatal CVevents. As shown in Table 3, on bivariate, multivariate,and bootstrapping validation analyses, the incidence rateof fatal and nonfatal CV outcomes was significantly higher(P#0.02) in patients with IL-6 levels above the median (8.6CV events per 100 person-years; 95% CI, 2.5 to 5.0 CVevents per 100 patient-years) than in those with IL-6 levelsbelow this threshold (3.6 CV events per 100 person-years;95% CI, 6.9 to 10.8 CV events per 100 patient-years) (Fig-ure 1, left panel). CKD stages did not modify the relation-ship between IL-6 and CV risk (P for interaction=0.33).Further analyses investigating IL-6 as a continuous vari-able confirmed this biomarker as a strong and indepen-dent risk factor for study outcomes (history of CVD:bivariate analysis OR (1 pg/ml), 1.16 [95% CI, 1.10 to 1.26],P,0.001, and multivariate analysis OR, 1.10 [95% CI, 1.02 to1.20], P=0.02; incident CV events, bivariate analysis HR(1 pg/ml), 1.14 [95% CI, 1.07 to 1.22], P,0.001, and multi-variate analysis HR, 1.12 [95% CI, 1.03 to 1.22], P,0.001).To account for the potential effect of competing risks due

to death on the relationship between IL-6 and CV out-comes, an additional adjusted analysis that considered acombined endpoint of death/CV events was performed.This multivariate analysis showed that the HR of thecombined endpoint was about 1.5 times higher (HR, 1.54;95% CI, 1.05 to 2.26; P=0.03) in patients with IL-6 levelsabove the median as compared with the remaining ones.

Analyses Based on the 2174 G/C PolymorphismIn patients with CKD, the genotype distribution of 2174

G/C polymorphism (GG, n=333 [44%]; GC, n=370 [49%];

Clin J Am Soc Nephrol 10: ccc–ccc, February, 2014 IL-6 and CV Risk in CKD, Spoto et al. 3

Tab

le1.

Maindem

ograp

hic

andclinical

charac

teristicsofthestudypopulationac

cordingto

serum

IL-6

leve

lsan

d2174G/C

polymorphism

Cha

racteristic

Who

leGroup

(n=75

5)Se

rum

IL-6

Lev

elGen

otyp

e

,2.5pg

/ml(n=

380)

$2.5pg

/ml(n=

375)

PValue

CC(n=52

)GC/GG

(n=70

3)PValue

Age

(yr)

62611

59612

6469

,0.00

159

613

62610

0.09

Men

(%)

453(60)

224(59)

229(61)

0.55

39(75)

414(59)

0.02

Diabe

tes(%

)26

3(35)

105(28)

158(42)

,0.00

121

(40)

242(34)

0.38

Current

smok

ers(%

)98

(13)

55(14)

43(11)

0.22

9(17)

89(13)

0.34

CVcomorbidities(%

)22

1(29)

82(22)

139(37)

,0.00

123

(44)

198(28)

0.01

Bod

ymassindex

(kg/

m2 )

28.264.7

27.364.3

29.064.8

,0.00

127

.963.8

28.264.7

0.54

SystolicBP(m

mHg)

1346

1813

2617

1356

190.03

1326

1613

4618

0.41

DiastolicBP(m

mHg)

78611

79610

77611

0.01

76610

78610

0.15

Antihyp

ertens

ivetreatm

ent(%)

691(92)

341(90)

350(93)

0.89

44(85)

647(92)

0.69

Glucose

(mg/

dl)

1166

4910

9641

1226

56,0.00

198

(87–

127)

99(88–

120)

0.65

Total

cholesterol(mg/

dl)

1876

4518

9643

1846

460.12

1736

4318

8645

0.02

Hem

oglobin(g/dl)

12.861.8

13.161.8

12.661.8

,0.00

112

62.0

1361.8

0.29

Album

in(g/dl)

4.26

0.5

4.26

0.5

4.16

0.5

0.01

4.16

0.4

4.26

0.5

0.53

CRP(m

g/L)

2.4(1.0–5.5)

1.4(0.7–2.8)

4.3(1.9–9.0)

,0.00

13.2(1.2–10

)2.3(1.0–5.4)

0.06

Phosph

ate(m

g/dl)

3.76

0.8

3.66

0.7

3.8 6

0.8

0.02

3.71

60.68

3.72

60.78

0.92

eGFR

(ml/min

per1.73

m2 )

36613

38613

34613

,0.00

134

612

36613

0.32

Urina

ryprotein(m

g/24

hr)

0.6(0.2–1.5)

0.5(0.2–10.2)

0.6(0.2–1.7)

0.01

0.7(0.2–1.6)

0.6(0.2–1.5)

0.53

Dataareexpressedas

mean6

SD,m

edianan

dinterqua

rtile

rang

e,or

percen

tage

freq

uen

cy,a

sap

prop

riate.CV,cardiova

scular;C

RP,C

-reactiveprotein.

4 Clinical Journal of the American Society of Nephrology

Tab

le2.

Logistic

regressionmodelsofhistory

ofca

rdiova

sculardisea

se

Variable

Bivariate

Multiva

riate

Boo

tstrap

ping

Validation

OR(95%

CI)

PValue

OR(95%

CI)

PValue

OR(95%

CI)

PValue

Serum

IL-6

value(0

[,2.5pg

/ml];1

[$2.5pg

/ml])a

2.14

(1.55to

2.95

),0.00

11.58

(1.11to

2.24

)0.01

1.58

(1.12to

2.24

)0.01

Age

(inyr)

1.05

(1.03to

1.07

),0.00

11.05

(1.03to

1.07

),0.00

1Diabe

tes(0=no

;1=ye

s)2.83

(1.99to

4.03

),0.00

12.83

(1.97to

4.08

),0.00

1Sy

stolicBP(inmmHg)

1.00

(0.99to

1.01

)0.93

1.00

(0.99to

1.01

)0.94

Hem

oglobin(ing/

dl)

1.04

(0.94to

1.16

)0.47

1.04

(0.93to

1.16

)0.49

Album

in(ing/

dl)

0.74

(0.50to

1.11

)0.15

0.74

(0.50to

1.11

)0.14

Phosph

ate(inmg/

dl)

1.02

(0.80to

1.30

)0.86

1.02

(0.78to

1.34

)0.87

Urina

ryprotein(inmg/

24hr)

1.01

(0.89to

1.13

)0.93

1.01

(0.88to

1.14

)0.93

eGFR

(ml/min

per1.73

m2 )

1.00

(0.98to

1.01

)0.57

1.00

(0.98to

1.01

)0.57

OR,o

ddsratio;

95%

CI,95

%confi

den

ceinterval.

a Below

/ab

ovethemed

ianva

lue(inpa

renthe

ses).T

hedescription

ofthemod

elbu

ildingstrategy

isrepo

rted

intheMaterialsan

dMetho

dssection.

Tab

le3.

Coxregressionmodelsofinciden

tca

rdiova

scularev

ents

Variable

Bivariate

Multiva

riate

Boo

tstrap

pingValidation

HR(95%

CI)

PValue

HR(95%

CI)

PValue

HR(95%

CI)

PValue

Serum

IL-6

value(0

[,2.5pg

/ml];1

[$2.5pg

/ml])a

2.37

(1.61to

3.51

),0.00

11.66

(1.11to

2.49

)0.01

1.66

(1.10to

2.52

)0.02

Age

(inyr)

1.07

(1.04to

1.10

),0.00

11.07

(1.03to

1.10

),0.00

1Diabe

tes(0=no

;1=ye

s)1.59

(1.09to

2.32

)0.02

1.59

(1.06to

2.37

)0.02

SystolicBP(inmmHg)

1.00

(1.00to

1.02

)0.31

1.00

(1.00to

1.02

)0.31

Hem

oglobin(ing/

dl)

0.91

(0.80to

1.02

)0.10

0.91

(0.81to

1.02

)0.09

Album

in(ing/

dl)

0.82

(0.53to

1.27

)0.38

0.82

(0.52to

1.29

)0.39

Phosph

ate(inmg/

dl)

1.13

(0.88to

1.44

)0.34

1.13

(0.88to

1.45

)0.35

Urina

ryprotein(inmg/

24hr)

1.05

(0.94to

1.17

)0.38

1.05

(0.93to

1.18

)0.44

eGFR

(ml/min

per1.73

m2 )

1.01

(0.99to

1.02

)0.44

1.01

(0.99to

1.02

)0.49

HR,h

azardratio.

a Below

/ab

ovethemed

ianva

lue(inpa

renthe

ses).T

hedescription

ofthemod

elbu

ildingstrategy

isrepo

rted

intheMaterialsan

dMetho

dssection.

Clin J Am Soc Nephrol 10: ccc–ccc, February, 2014 IL-6 and CV Risk in CKD, Spoto et al. 5

CC, n=52 [7%]) significantly deviated from Hardy–Weinbergequilibrium (chi-square=14.3; P=0.001), whereas this wasnot true in healthy persons (GG, n=277 [60%]; GC, n=170[37%]; CC, n=16 [3%]) (chi-square=2.70; P=0.10). Of note,the frequency of the C risk allele was significantly higher(P,0.02) in patients with CKD (31%; 95% CI, 28% to 34%)than in healthy study participants (22%; 95% CI, 18% to26%).Patients with CKD and CC genotype had higher levels of

IL-6 (median, 2.9 pg/ml [interquartile range, 1.7–6.9 pg/ml])than in those with GC (2.4 pg/ml [interquartile range, 1.6–3.8 pg/ml]) and GG (2.5 pg/ml [interquartile range, 1.7–4.0pg/ml]) (P=0.04) (Figure 2, left panel). This was also true forCRP (CC, median: 3.2 mg/L; interquartile range, 1.2–10.4mg/L; GC, 2.2 mg/L [interquartile range, 1.0–5.6 mg/L];GG, 2.4 mg/L [interquartile range, 1.0–5.1 mg/L]) (P=0.03)(Figure 2, right panel). These findings show that the relation-ship between 2174 G/C polymorphism and inflammatorybiomarkers is well described by a recessive model of inher-itance (CC genotype versus GC/GG genotypes). For thisreason, further data analysis was carried out using the re-cessive model. As shown in Table 1 and as expected fromMendelian randomization, CC patients and GC/GG pa-tients did not differ for demographic and clinical character-istics except for a slight excess of men (75% versus 59%,respectively; P=0.02) in CC patients and slightly lower cho-lesterol levels (173643 mg/dl versus 188645 mg/dl, respec-tively; P=0.02) in patients with the same genotype.The prevalence of a CVD history was higher in CC

patients (44%) than in GC/GG patients (28%) (P=0.01). TheOR for this outcome was more than twice as high in pa-tients harboring the CC risk genotype (OR, 2.15; 95% CI,1.15 to 4.00; P=0.02) as in those with other genotypes in amodel adjusting for age, sex, and cholesterol (i.e., the co-variates that differed [P#0.10] between patients with CCand those with GC/GG genotypes; see Table 1). A boot-strapping validation model confirmed these results (OR,2.14; 95% CI, 1.18 to 3.89; P=0.01).

In the cohort study, the incidence rate of mortality didnot differ between patients with and those without the CCrisk genotype (P=0.27), indicating that the competing riskof death on the link between 2174 G/C polymorphismand CV events could be excluded. In close parallel withthe strong association between serum IL-6 levels and CVevents, the incidence rate of CV outcomes in patients withCC genotype was 87% higher (HR, 1.87; 95% CI, 1.02 to3.44; P=0.04) than in those with the GC or GG genotypes(Figure 1, right panel) and this was also true in a boot-strapping validation model (HR, 1.87; 95% CI, 1.01 to 3.51;P=0.05).

DiscussionIn this study, high serum IL-6 levels were associated with

history of CVD and predicted the risk for incident CVevents in patients with stages 2–5 CKD. Furthermore, thefunctional polymorphism 2174 G/C in the promoter ofthe IL-6 gene is associated with history of CVD and pre-dicts the risk for future CV events in this population. Theseresults are compatible with the hypothesis that this inflam-matory cytokine is causally implicated in the high CV riskin CKD.Atherosclerosis is an inflammatory disease (1). Chronic

inflammation is pervasive at all CKD stages, particularlystage 5D (13,14). IL-6 is considered an orchestrator of theinflammatory response (37) and a key player in atheroscle-rosis in humans (38). The IL-6 effect on the cardiovascularsystem might be mediated via downstream acute-phaseproteins (39) or by IL-6 per se. IL-6 stimulates endothelialactivation, vascular smooth muscle cell proliferation (40),and leukocyte recruitment (41), thereby contributing to theprocess of atherosclerotic plaque growth (42) and instabil-ity (43). IL-6 mRNA is overexpressed in atheromatous ar-teries, and IL-6 expression co-localizes with macrophagesin areas of plaque rupture (43). In apoE-deficient mice,systemic administration of IL-6 accelerates atherosclerosis

Figure 1. | IL-6 and risk of cardiovascular events. Left: Cumulative risk for incident fatal and nonfatal cardiovascular (CV) event–free survival inpatients stratified according to serum IL-6 level. Data were adjusted for age, diabetes, systolic BP, hemoglobin, albumin, phosphate, urinaryprotein, and eGFR (see Table 3). The table under the x-axis indicates the number of patients at risk in each group (IL-6 above/below the medianvalue) at relevant time points. Right: Cumulative risk for incident fatal and nonfatal CVevent–free survival in patients stratified according to2174 G/C polymorphism in the IL-6 gene. Data were adjusted for age, sex, and cholesterol (see text). The table under the x-axis indicates thenumber of patients at risk in each group (CC and GC/GG genotype) at relevant time points. Data are expressed as hazard ratios (HRs), 95%confidence intervals (95% CIs), and P values.

6 Clinical Journal of the American Society of Nephrology

(10). Elevated levels of IL-6 are predictive of future CVevents in healthy men (15) and women (44) and are mark-ers of poor prognosis in patients with chronic angina (45)and acute coronary syndrome (46). IL-6 levels are mark-edly elevated in CKD, a phenomenon only in part ex-plained by reduced renal clearance of this cytokine.Furthermore, the observation of a parallel increase in IL-6and CRP in our study is in keeping with the notion that IL-6drives the synthesis of CRP. High IL-6 has been solidly as-sociated with mortality in patients with stage 5 CKD whoare being maintained on long-term dialysis (16–20).To the best of our knowledge, only one study has tested

the relationship between IL-6 and CV mortality in CKD(21). This seminal study was carried out in a small cohortof 125 patients, was based on a limited number of CVevents (n=22), and also included dialysis patients (34% ofthe whole cohort) (21). Our study, based on a large CKDcohort composed exclusively of predialysis patients andincluding a large number of CV events, confirmed pilotdata by Barreto et al. (21) and showed that high IL-6 iscoherently associated both with history of CVD as wellas with incident CV events.Being observational in nature, findings in studies discussed

above, including our analysis based on circulating IL-6,remain hypothesis-generating and as such leave unresolvedthe critical question of whether this cytokine is causally im-plicated in CV complications in patients with predialysis CKD.This question can be resolved only by a full-fledged clinical trial.The Mendelian randomization approach is useful step in

the pathway to discovery in clinical research. Since geneticpolymorphisms are distributed randomly at gamete for-mation and since genotypes precede phenotypes and donot change over time, comparing individuals harboring agiven risk allele for the expression of a corresponding riskfactor with those without the risk allele in question mayallow unbiased assessment of the link between the attendantrisk factor and relevant clinical outcomes. In this perspective,we used the functional polymorphism2174 G/C in the pro-moter of the IL-6 gene as a marker to further investigate thelink between IL-6 and CV events in patients with CKD. The

2174 G/C polymorphism is a common variant that regu-lates the serum concentration of IL-6 (22–28). In keepingwith previous studies in patients with CVD (28), coronaryartery bypass grafting surgery (23,25), carotid atherosclero-sis (24), abdominal aortic aneurysm (22), and dialysis (26),we found that patients with CKD and CC genotype hadhigher circulating levels of IL-6 and CRP than those harbor-ing GC or GG genotypes, specifically legitimating the use ofthis genetic marker as an unbiased means for assessing thecausal nature of the link between the gene product (IL-6) ofthis polymorphism and CV complications in CKD. Interest-ingly, this analysis showed that this polymorphism is inde-pendently associated both with the history of CVD as wellas with incident CV events. Such associations, which wentalong with the previously described relationships of serumIL-6 with the same outcomes, further strengthen the hypoth-esis that IL-6 is a direct player in atherosclerotic complicationsin CKD. Moreover, the observation that the distribution ofgenotypes frequency of the 2174 G/C polymorphism in pa-tients with CKD was not in Hardy–Weinberg equilibriumand that the frequency of the C allele was significantly higherin patients CKD compared with general population of thesame geographic area, offers additional circumstantial evi-dence that IL-6 is a causal risk factor for CV events in thispopulation (47).Mendelian randomization studies support causal inter-

pretations but do not constitute definitive proof for cau-sality, which demands specific experimental evidence (i.e.,a formal randomized clinical trial). In this respect, meta-analytic data from two genetic consortia exploring the ef-fect of a polymorphism in the IL-6 receptor on the risk ofcoronary artery disease showed the allele that attenuatedIL-6 signaling was significantly associated with reducedrisk of coronary heart disease (48). Notably, the relevanceof this genetic association for the control of inflammation isshown by a meta-analysis of clinical trials testing a mono-clonal antibody against the IL-6 receptor (tocilizumab) inrheumatoid arthritis and documenting that lowering serumlevels of IL-6 is an effective strategy to induce the remissionof this chronic inflammatory disease (49).

Figure 2. | Inflammatory biomarkers and 2 174 G/C polymorphism. Serum levels of IL-6 (left panel) and C-reactive protein (right panel)according to2174 G/C polymorphism in IL-6 gene. Data are expressed as medians. The interquartile ranges are indicated in parentheses. Thecomparison among groups was made by ANOVA.

Clin J Am Soc Nephrol 10: ccc–ccc, February, 2014 IL-6 and CV Risk in CKD, Spoto et al. 7

Some limitations should be acknowledged. First, eventhough our cohort study registered a sizable number ofcardiovascular events, the number of deaths (n=42) waslimited, preventing adequately adjusted analyses focusingon this major outcome. However, the analysis of the pri-mary outcome in this study, incident CV events, whichwas robustly based on 117 events, showed parallel linksbetween serum IL-6 and the genetic marker of this cyto-kine with the same events.Second, although Mendelian randomization is a powerful

approach for inferring causality in observational studies, itsapplication has potential limitations. Genetic variants can beindicators of environmental exposures on condition thatthere is no genetic admixture, pleiotropy or linkage disequi-librium. However, it is reasonable to believe that in our studyall these assumptions are fulfilled. Our population is genet-ically homogeneous (29) and the 2174G/C polymorphismis a functional variant directly responsible for serum levelsof IL-6 (22–28). Furthermore, pleiotropy seems highly un-likely because of the location of 2174 G/C polymorphismin the promoter region of the gene.Third, although we demonstrated that our findings had

high internal validity by bootstrap modeling, replication ofthe results in a second cohort is a required proof for theexternal generalizability of findings in observational studies.In this respect, our observational study in a large southernEuropean cohort confirms findings in a small central Europeancohort (21). Furthermore, our study is the first applying agenetic marker of IL-6 to express the nature (causal versusnot causal) of the link between IL-6 and CV events in pa-tients with CKD.In conclusion, high serum IL-6 is associated with both a

history of CVD and future CV events in patients with CKD,and these associations are fully confirmed by the applica-tion of a functional polymorphism in the IL-6 gene. Overall,this study is compatible with the hypothesis that the IL-6plays a causal role in the high CV risk of patients with CKD.

AcknowledgmentsWe thank Cristina Politi andMaria Cristina Sanguedolce for their

technical collaboration in genetic study.This study is part of the Syskid project, which is supported through

European Union’s FP7, grant agreement number HEALTH-F2-2009-241544.

Contributors: members of the Southern Italy CKD CohortWorking Group (in alphabetical order): Audino A, Bruzzese V,Caglioti A, Campo S, Caridi G, Catalano F, Chiarella S, Cicchetti T,D’Anello E, Enia G, Fabiano F, Fatuzzo P, Ferini S, Garozzo M,Grandinetti F, GulloM,MafricaA,Maimone I,Mancuso F,ManninoM,Marino F, Natale G, Palma L, Papalia T, ParlongoG, Pinciaroli A,PinnaM, Plutino D, PostorinoM, Pugliese A, Rapisarda F, SantoroO,Tramontana D.

DisclosuresNone.

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Received: July 14, 2014 Accepted: October 24, 2014

Published online ahead of print. Publication date available at www.cjasn.org.

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