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Published ahead of print on July 12, 2006
J Am Soc Nephrol 17: 2333-2340, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2006010066
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Clinical Transplantation

IL-6 Promoter Polymorphism –174 Is Associated with New-Onset Diabetes after Transplantation

Jamal Bamoulid*,{dagger}, Cécile Courivaud*,{dagger}, Marina Deschamps*,{ddagger}, Patricia Mercier*,{ddagger}, Christophe Ferrand*,{ddagger}, Alfred Penfornis§, Pierre Tiberghien*,{ddagger},||, Jean-Marc Chalopin*,{dagger},||, Philippe Saas*,{ddagger} and Didier Ducloux*,{dagger},||

* INSERM, U645, University Besançon, and IFR133, {dagger} CHU Saint Jacques, Department of Nephrology, Dialysis, and Renal Transplantation, {ddagger} EFS Bourgogne Franche-Comté, Plateforme de Biomonitoring, § CHU Jean Minjoz, Department of Endocrinology, and || CHU Saint Jacques, CIC Biothérapie, Besançon, France

Address correspondence to: Dr. Didier Ducloux, CHU St. Jacques, 2 Place Saint Jacques, Besançon 25000, France. Phone: +33-38-121-8782; Fax: +33-38-121-8781; E-mail: dducloux{at}chu-besancon.fr

Received for publication January 29, 2006. Accepted for publication May 29, 2006.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
New-onset diabetes after transplantation (NODAT) is a serious complication of transplantation. This study tested whether IL-6 production capacity may influence the development of NODAT in two different groups of patients. The occurrence of NODAT was analyzed with respect to IL-6 gene promoter polymorphism at position –174 (G->C) and other relevant risk factors retrospectively in 217 renal transplant recipients and prospectively in 132. A linear increase in both circulating IL-6 (P = 0.09) and C-reactive protein (an indicator of basal IL-6 secretion; P = 0.03) concentrations from the CC genotype to the GG genotype was observed. In the multivariate model, the CC genotype was associated with a decreased risk for NODAT compared with the GG genotype in the two cohorts. Homeostasis Model Assessment for Insulin Resistance also revealed lesser insulin sensitivity in the GG carriers than in the CC carriers (2.15 ± 2 versus 1.32 ± 1.03; P = 0.03). Subgroup analysis showed that the influence of IL-6 gene promoter polymorphism on the development of NODAT was restricted mostly to overweight patients. These results highly suggest that IL-6 production capacity influences the development of NODAT and that diabetes-inducing drug administration should be limited in overweight patients who carry the GG genotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
New-onset diabetes after transplantation (NODAT) is a known serious long-term complication of transplantation. A number of risk factors for development of NODAT have been established (1); age, body mass index (BMI), ethnicity, hepatitis C infection, and immunosuppressive therapy are among the most important (1). Chronic inflammation now is supposed to play a pivotal role in the pathogenesis of type 2 diabetes (2). Indeed, the results of both cross-sectional and prospective studies suggest a contributory role for low-grade activation of the immune system in the development of type 2 diabetes, and both C-reactive protein (CRP) and IL-6 levels have been identified as risk factors for the future development of type 2 diabetes (3–6). Moreover, despite some controversial data (7,8), the IL-6 gene promoter polymorphism at position –174 (G->C), which is associated with different transcription rates, seems to predict type 2 diabetes (9). Nevertheless, whether chronic inflammation may promote NODAT is unknown. Babel et al. (10) previously reported no association between diabetes and the IL-6 gene promoter polymorphism at position –174 (G->C) in long-term renal transplant recipients (RTR). Nevertheless, the relevance of this study is seriously hampered by selection bias, lack of definition of diabetes, and confusion between type 2 diabetes and NODAT.

In this study, we tested whether the IL-6 gene promoter polymorphism at position –174 may influence the later development of NODAT in two separate cohorts of RTR. This hypothesis first was generated by the exploratory analysis of a retrospective cohort and then validated in a second, prospectively established, verification cohort.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Design and Populations
We analyzed two groups of patients to assess whether the IL-6 gene promoter polymorphism at position –174 (G->C) may be a risk factor for the later development of NODAT independent of the effects of age, obesity, and maintenance immunosuppressive regimens. First, we conducted a nested genetic study in a retrospective cohort of patients who were participants in a previously reported study that assessed the association between lymphopenia and atherosclerotic outcomes after renal transplantation (11). A total of 302 consecutive RTR had been enrolled consecutively in this trial. We included participants in this study when their pretransplantation baseline examination showed no previous diagnosis of diabetes and recorded a fasting plasma glucose (FPG) of <6.1 mmol/L. DNA was available from 224 (74%) of the 302 patients. These patients received a transplant between January 1990 and June 2001. Reasons for DNA unavailability were death (12%) and loss to follow-up, primarily as a result of return to dialysis (14%). Seven patients had pretransplantation diabetes and were excluded. The remaining 217 individuals did not differ significantly from the 85 patients who were not evaluated in any relevant baseline parameter (age, gender, pretransplantation BMI [weight (kg)/height2 (m2)]), and primary renal disease), but they were marginally less prone to atherosclerotic events, graft loss, and death after transplantation (data not shown).

Second, we studied a prospective cohort of 144 consecutive patients who received a transplant between July 2001 and December 2004 in our center. All of these patients received tacrolimus-based immunosuppression. Twelve patients had pretransplantation diabetes and were excluded.

Age, gender, BMI, history of cardiovascular disease, hypertension, previous renal transplantation, cytomegalovirus donor/recipient status, hepatitis C virus (HCV) status, HLA compatibility, and panel-reactive antibodies were analyzed as covariates. Dialysis mode (none, hemodialysis, or peritoneal dialysis) and its duration before transplantation were recorded. All parameters were collected at the transplant time. Immunosuppressive regimens were assessed. The cumulative dose of steroids in the first year after transplantation, the use of calcineurin inhibitors, and the use of tacrolimus versus cyclosporin A were considered as a potential covariate.

Approval was obtained from the Besançon ethical committee for these studies. Informed consent was provided according to the Declaration of Helsinki.

NODAT
NODAT was defined according to the 2003 International Consensus Guidelines on Diabetes Mellitus in Transplantation (1), and its definition was applied to our database that existed from 1996. This definition is based on the currently accepted criteria proposed by the Canadian Diabetes Association. In summary, diabetes is defined by a casual glucose value of 11.1 mmol/L, or an FPG value of ≥7 mmol/L, or a plasma glucose value of ≥11.1 mmol/L 2 h after a 75-g glucose load. In our cohort, all of the patients with NODAT were identified using FPG. Patients with FPG ≥7 mmol/L or antidiabetic treatment (oral antidiabetic drugs or insulin) were considered to have NODAT.

DNA Extraction and Analysis of the IL-6 Gene Promoter Polymorphism at Position –174 (G->C)
Genomic DNA (gDNA) was extracted from white blood cells using standard salting out procedure (12). Analysis of the IL-6 gene promoter locus then was studied using a PCR-based genotyping assay (13). Briefly, in this assay, primers were designed to amplify a fragment that contained an additional enzyme recognition site in the mutant C allele, allowing the discrimination of mutant and wild-type alleles by 2% standard agarose gel electrophoresis. Primer sequences were the following (sense primer is indicated first): 5'-TTGTCAAGACATGCCAAAGTG-3' and 5'-TCAGACATCTCCAGTCCTATA-3' (13). After PCR amplification of the polymorphic regions of interest, PCR products were digested overnight with Nla III enzyme (New England Biolabs, Beverly, MA), according to the supplier’s recommendations. For each PCR, negative (PCR amplification without gDNA) and positive (plasmid DNA carrying mutated allele) controls were included. Sequencing of undigested PCR products confirmed restriction fragment length polymorphism–PCR electrophoresis profiles for representative samples. Researchers and laboratory personnel had no access to identifiable information and could identify samples by number only.

Functional Validation of the IL-6 Gene Promoter Polymorphism
The functional effects of IL-6 gene promoter polymorphism were analyzed by measuring serum levels of IL-6 6 mo after transplantation. IL-6 was measured with high-sensitivity ELISA kits (Quantikine HS IL-6 Immunoassay, #HS600B; R&D Systems, Lille, France) according to the manufacturer’s instructions. Briefly, the profile of IL-6 secretion was determined according to the genotype of the promoter. Three different possible genotypes at the –174 position of the IL-6 gene promoter then could be defined and associated with a high (G/G), medium (G/C), or low (CC) secretion profile (14). CRP (high-sensitivity determination) serum levels were measured by nephelometry (Beckman Coulter, Villepinte, France), because IL-6 is known as the main inducer for hepatic CRP synthesis (15).

Insulin Resistance and Secretion Status of Patients
Insulin resistance and secretion were evaluated in 45 RTR who did not have diabetes and had normal FPG and different genotypes (GG; n = 15), GC; n = 15), and CC; n = 15) for IL-6 gene promoter polymorphism by appropriate Homeostasis Model Assessment (HOMA) index. Serum fasting insulin (normal range 6 to 20 µU/ml) was measured with an enzyme immunoassay (AIA-PACK IRI; Tosoh Corp., Minato-Ku, Japan). The HOMA for insulin resistance (HOMA-IR), used as a marker of insulin resistance, was calculated as described previously (16): [fasting insulin (µU/ml) x fasting glucose (mmol/L)/22.5]. The HOMA for insulin secretion (HOMA-beta) was calculated as described previously (16) to study the possible role of beta cell dysfunction: [20 x fasting insulin (µU/ml)/fasting glucose (mmol/L) – 3.5].

Statistical Analyses
Arithmetic means were calculated and expressed ± SD. Circulating IL-6 concentrations were not normally distributed, and their values in the three different genotypes were compared using the Kruskal-Wallis test. Differences in circulating IL-6 values between CC and G carriers were evaluated by the Mann-Whitney test. For normally distributed variables, G carriers and CC carriers were compared using the {chi}2 test for dichotomous variables and the t test for continuous variables. Hardy-Weinberg equilibrium was assessed for the genotype distribution. A one-way ANOVA was performed to exclude potential differences among the three genotype groups. Relationships between numerical variables were evaluated with the Spearman/Pearson rank test. Using log-rank tests on Kaplan-Meier nonparametric estimates of the survival without NODAT distribution, we selected variables with a P ≤ 0.20. The selected variables were included into a Cox proportional hazard model, and a backward stepwise selection process was performed, this time at a classical {alpha} = 0.05. Patient baseline characteristics were assessed on the transplant day. Age was split into two classes separated by its median (45 yr); BMI was separated into two classes (<25 or ≥25 kg/m2). Results are expressed as hazard ratio (HR) and 95% confidence interval (CI), with a P value testing the null hypothesis: HR = 1. Therefore at P < 0.05, HR is significantly different from 1. Assumptions of Cox models (log linearity and proportionality of risk in time) were met in this analysis. Analysis was performed on Statview 5 (SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Study Population
Retrospective Cohort.
The cohort of 217 patients was followed for a mean duration of 98 ± 21 mo after transplantation. Mean follow-up was similar in patients with the different genotypes. There were no differences in transplant timing between the different genotypes (data not shown). Mean age was 43 ± 13 yr, and 139 RTR (64%) were men. Hypertension was present in 156 (72%) RTR. Mean dialysis duration was 20 ± 17 mo. Three patients were HCV positive. All patients but five were white.

Prospective Cohort.
A total of 132 patients were studied with a mean follow-up of 33 ± 11 mo. Five patients died during follow-up, and four lost their graft. There were no differences in transplant timing between the different genotypes (data not shown). Mean age was 47 ± 14 yr, and 86 (65%) RTR were men. Hypertension was present in 99 (75%) RTR. Mean dialysis duration was 19 ± 13 mo. Two patients were HCV positive. All patients but four were white.

Analysis of the IL-6 Gene Promoter Polymorphism
Reverse transcription–PCR reactions were performed, and a PCR product of 300 bp was obtained. Digestion with NlaIII enzyme and agarose gel electrophoresis allowed identification of two bands (a 233- and a 54-bp band) for the wild-type G allele and three bands (with 122, 111, and 54 bp length, respectively) for the C allele (Figure 1A). This contrasts with data reported by Depboylu et al. (13), who used the same primers and the same restriction enzyme. To confirm our results, we first sequenced undigested PCR products from wild-type, heterozygote, and homozygote RTR. Sequence analysis confirmed restriction fragment length polymorphism–PCR analysis (Figure 1B). Then, we analyzed the sequence of the IL-6 gene promoter using online software provided by New England Biolabs (www.neb.com). Again, we confirmed our results and found, in addition to position –174, two other NlaIII restriction sites in the PCR product. The first one, located within the sense primer, generated a 13-bp product that was not discriminated by electrophoresis. The second one, at position –63, generated a 54-bp product (Figure 1A) in all 361 RTR, excluding the presence of a polymorphism at this position.


Figure 1
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  		Figure 1. Analysis of the IL-6 gene promoter polymorphism –174 (G/C) in four representative samples: Undigested (UD), wild-type (WT), heterozygote (H/h), and homozygote mute (H). (A) Agarose gel electrophoresis of restriction fragment length polymorphism–PCR (RFLP-PCR) after NlaIII overnight restriction digest. MW represents the molecular weight marker. (B) Sequencing analysis of undigested PCR products, confirming RFLP-PCR analysis. N in the sequence determined by the sequence analysis software (Applied Biosystems, Foster City, CA), and arrows in H/h samples depict overlay of two different allele-specific nucleotides.

 
IL-6 Gene Promoter –174 Genotype Frequencies in RTR
Retrospective Study.
Ninety-five (43.8%) patients were GG homozygotes (wild type), 92 (42.4%) were GC heterozygotes, and 30 (13.8%) were CC homozygotes (mutant; Table 1). There were no differences in pretransplantation characteristics, acute graft rejection episodes, and cumulative steroid dose in the first year after transplant between patients with the different genotypes (Table 2). The observed allele frequencies were in Hardy-Weinberg equilibrium.


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  		Table 1. Genotype frequencies of the IL-6 gene promoter –174 (G/C) polymorphism in the two cohorts

 

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  		Table 2. Characteristics of patients with the different IL-6 gene promoter –174 genotypesa

 
Prospective Study.
Genotype frequencies were similar in the prospective cohort (GG 42.7%; GC 45.9%; CC 11.4%; Table 1). Nevertheless, in this cohort, CC patients were older than patients with the G allele (53 ± 13 versus 46 ± 14; P = 0.04; Table 2). Gender ratio also was different, with more women being CC carriers than G allele carriers (60 versus 33%; P = 0.03; Table 2). The observed allele frequencies were in Hardy-Weinberg equilibrium.

Relation between the IL-6 Promoter –174 Genotype and Both Serum IL-6 and CRP Levels
Serum IL-6 concentrations were measured in 36 RTR (CC genotype 12; GC genotype 12; and GG genotype 12). Serum samples were selected as follows: Frozen serum samples from 12 patients with the CC genotype and absence of acute rejection or infection at 6 mo after transplant were available in our serum library. We selected an equal number of patients with the GC and GG genotypes after matching for age and BMI. None of these patients experienced either acute rejection or infection at the date of serum collection. Circulating IL-6 concentrations were closely related to BMI (r = 0.55, P = 0.0005). There was a trend toward a linear increase in serum IL-6 concentrations from the CC genotype to the GG genotype (median values and ranges were, respectively, 2.77 lsqb]1.02 to 9.26], 3.48 [1.11 to 76.85], and 4.19 pg/ml [2.02 to 100] in CC, GC, and GG carriers; P = 0.09; Figure 2A). When analysis was restricted to patients who were overweight, serum IL-6 concentration was found to be significantly lower in CC patients than in G carriers (4.2 [1.02 to 5.1] versus 7.3 pg/ml [4.4 to 100]; P = 0.025). By contrast, in patients who were not overweight, IL-6 levels were similar in CC and G carriers (2.2 [1.4 to 9.3] versus 2.7 pg/ml [1.1 to 17.8]; P = 0.27).


Figure 2
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  		Figure 2. Functional validation of the IL-6 gene promoter polymorphism –174 (G/C) in renal transplant recipients. (A) Circulating IL-6 concentrations were determined by ELISA in the sera of 36 renal transplant recipients (RTR; 12 with the CC genotype, 12 with the GC genotype, and 14 with the GG genotype). Results are given in pg/ml on a logarithm scale. Solid black bars represent median values. (B) C-reactive protein concentrations were determined as described in Materials and Methods in the sera of 217 RTR (30 with the CC genotype, 92 with the GC genotype, and 95 with the GG genotype). Results are given in mg/ml. Solid black bars represent mean values.

 
CRP levels were assessed in the 217 patients of the retrospective cohort. Posttransplantation CRP concentrations increased gradually from the CC genotype to the GG genotype (mean values and SD: 3.2 ± 1.6, 4.1 ± 2.2, and 4.4 ± 2.1 mg/L in 30 CC, 92 GC, and 95 GG patients, respectively; P = 0.03). Similar results were observed in the prospective cohort (3.0 ± 1.5, 4.2 ± 2.3, and 4.7 ± 2.4 mg/L in CC, GC, and GG patients, respectively; P = 0.02; Figure 2B).

Relation between the IL-6 Promoter Polymorphism –174 and NODAT
Retrospective Cohort.
Thirty-three (15.2%) patients experienced NODAT. The distribution of IL-6 genotypes was significantly different between patients with and without diabetes. The CC genotype was less common in patients with (3%) than without (16.4%) diabetes (P = 0.025). The incidence of NODAT was significantly higher in patients with the GG genotype than in patients with the CC genotype (19 versus 3%; P = 0.02). We observed a linear increase in the incidence of NODAT from the CC genotype to the GG genotype (3, 14, and 19% in CC, GC, and GG carriers, respectively; P = 0.001).

CC carriers developed NODAT less frequently than G carriers (3 versus 17.1%; P = 0.032). In the subgroup analysis, we observed that in overweight patients (n = 58), CC carriers had a reduced risk for NODAT compared with G carriers (0 versus 47%; P = 0.015), whereas an effect of the C allele could not be demonstrated in other than overweight patients (4.3 versus 5.9%; P = 0.31).

In the monovariate analysis, age, BMI, autosomal dominant polycystic kidney disease (ADPKD), cumulative steroid dose, and CC genotype (mutant type) were associated with NODAT. In the multivariate model (Table 3), age (HR 4.54; 95% CI 1.53 to 13.45; P = 0.006) and BMI (HR 8.79; 95% CI 3.58 to 21.61; P < 0.0001) were independent risk factors for the later development of NODAT. The CC genotype also conferred a reduced risk for NODAT compared with the GG genotype (HR 0.08; 95% CI 0.01 to 0.71; P = 0.023; Table 3). The risk for NODAT was marginally lower in GC carriers than in GG carriers (HR 0.15; 95% CI 0.02 to 1.17; P = 0.074; Table 3). There was a clear interaction between the IL-6 genotype and BMI. Indeed, individual adjustment for this variable was shown to modify significantly the HR of the IL-6 genotype for NODAT. When the BMI variable was entered in the model, we observed an increased effect of the IL-6 genotype on the risk for NODAT. This suggests a higher influence of high IL-6 production capacity on NODAT in overweight patients. A marginally higher risk for NODAT was observed in patients with ADPKD (HR 3.37; 95% CI 0.92 to 12.27; P = 0.07).


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  		Table 3. Cox model: HR of NODAT and 95% CI (retrospective cohort)a

 
Prospective Cohort.
Twenty-six (19.7%) patients experienced NODAT. The incidence of NODAT was marginally higher in patients with the GG genotype than in patients with the CC genotype (28.3 versus 13.3%; P = 0.12).

In the monovariate analysis, as previously shown for the retrospective cohort, age, BMI, ADPKD, cumulative steroid dose, and GG (wild type) genotype were associated with NODAT. In the multivariate model (Table 4), age (HR 5.49; 95% CI 1.70 to 17.75; P = 0.005), BMI (HR 6.70; 95% CI 1.65 to 27.25; P = 0.008), and ADPKD (HR 3.89; 95% CI 1.08 to 13.08; P = 0.037) were independent risk factors for the later development of NODAT. The CC genotype conferred an independent lower risk for NODAT than did the GG genotype (HR 0.15; 95% CI 0.03 to 0.99; P = 0.049; Table 4). Most of the data that were obtained from the prospective cohort confirmed the data that were obtained from the retrospective cohort.


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  		Table 4. Cox model: HR of NODAT and 95% CI (prospective cohort)

 
HOMA Indexes in Patients without Diabetes According to Their IL-6 Promoter –174 Genotype
Insulin resistance and secretion were evaluated in 45 patients in the retrospective cohort. The anthropometric characteristics of these 45 patients are shown in Table 5. Patient age, gender, and BMI were similar in the three genotype groups. FPG levels increased gradually from the CC to the GG genotype and were significantly higher in GG carriers than in CC carriers (4.8 ± 0.4 versus 5.5 ± 0.7 mmol/L; P = 0.02). We also observed a trend toward an increase in fasting insulin levels from the CC to the GG genotype. HOMA-IR index revealed reduced insulin sensitivity in the GG genotype carriers than in the CC genotype carriers (2.15 ± 2 versus 1.32 ± 0.91; P = 0.03). By contrast, HOMA-beta index was similar in all genotypes (Table 5). This suggests that IL-6 gene promoter genotype at position –174 is associated with insulin resistance rather than with beta cell function.


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  		Table 5. Insulin resistance and secretion according to the different IL-6 gene promoter –174 genotypesa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IL-6 gene promoter polymorphism at position –174 (G->C) has been associated previously with type 2 diabetes (7) and insulin resistance (17,18). In this study, we demonstrate that such polymorphism in the IL-6 promoter region is associated with the later development of NODAT. More precise, we found the risk for NODAT to be significantly higher in homozygous (GG) wild-type patients than in homozygous (CC) mutant patients independent of age, BMI, and other confounding factors. The incidence of NODAT increased linearly from low to intermediate and high IL-6 production capacity. Moreover, insulin sensitivity as assessed by HOMA-IR index was lower in homozygous wild-type GG RTR without diabetes than in homozygous CC mutant RTR without diabetes, suggesting a role for IL-6 in insulin resistance.

It is interesting that the IL-6 G/C promoter polymorphism at position –174 has functional significance (14,19). A previous study using in vitro reporter gene assay reported that transcription of IL-6 gene is more strongly activated by the G allele in both unstimulated and LPS/IL-1–stimulated HeLa cells (14). The functional effect of the IL-6 gene –174G/C promoter polymorphism on IL-6 levels also was examined in vivo after coronary artery bypass graft surgery, a widely known inflammatory stimulus for IL-6 production (19). We also tested the functionality of the IL-6 gene promoter polymorphism at position –174 in our transplant population and showed that the G allele is associated with higher IL-6 serum levels. Whereas some studies previously reported the significance of this IL-6 gene promoter polymorphism in transplant patients, none of them evaluated its functionality in the settings of immunosuppression (20,21). Our study is the first to confirm the influence of the IL-6 promoter polymorphism at position –174 on the IL-6 production capacity in patients who receive immunosuppressive drugs. This result is in accordance with recent findings indicating that only high steroid doses influence IL-6 production in RTR (22). Our results also suggest that the impact of such IL-6 gene promoter polymorphism on circulating IL-6 levels is higher in overweight patients. This effect probably is because white adipose tissue, which contains adipocytes, is an important source of basal IL-6 secretion (23). It is interesting that we also showed an interaction between the IL-6 promoter polymorphism at position –174 and overweight in predicting NODAT. Indeed, the increased risk for NODAT in G carriers seems to be restricted to overweight patients. Finally, we also found higher posttransplantation CRP concentrations in patients with the IL-6 promoter GG genotype. Collectively, these data are consistent with a role for genetic determinants of inflammation in the development of NODAT, especially in overweight patients.

NODAT has become a major challenge facing transplant clinicians. A number of clinical studies have shown that NODAT is associated with lower overall graft and patient survival (1,24,25).

Our group also recently demonstrated that NODAT was associated with an increased risk for atherosclerotic events (26). In this setting, a better definition of patients who are at risk for NODAT is relevant and may serve to reduce the rate of this serious complication. Nevertheless, for a genetic test to be relevant in clinical management, it obviously is critical that the test have additional predictive power over and above the currently available and easily assessable risk factors. Our study takes into account the major risk factors for NODAT and demonstrates that the IL-6 gene promoter polymorphism at position –174 independently contributes to this serious complication. The CC genotype is associated with a reduced risk for NODAT, and its negative predictive value reaches 85%, suggesting that it may serve to characterize independently a low-risk population. By contrast, the G allele increases the risk for NODAT, but its predictive positive value remains low (20%) in the overall population. Therefore, the presence of the G allele should be interpreted with respect to other risk factors to eventually guide preventive measures. For instance, the predictive positive value of the G allele for the development of NODAT reaches 50% in overweight patients. In such patients who carry the G allele, prevention of NODAT can be achieved by reducing diabetes-inducing drugs (e.g., tacrolimus, high steroid doses).

Although several in vitro and in vivo studies have examined the effects of IL-6 on insulin resistance, the underlying mechanism that links IL-6 and insulin resistance still is unclear. Data from animal models indicate a complex involvement of IL-6 in body composition and glucose metabolism (27–29). Some studies have shown that IL-6 alters hepatic glucose metabolism (30). In addition, Senn et al. (31) found that IL-6 pretreatment inhibited insulin receptor signal transduction in primary mouse hepatocytes as well as in a human hepatoma cell line. More recently, Kim et al. (32) reported that IL-6 induced defects in hepatic insulin action and signaling activity both in liver and in skeletal muscles. Taken together, these findings suggest that IL-6 interacts with insulin metabolism and contributes to insulin resistance. In our study, reduced insulin sensitivity, assessed by the HOMA-IR index in GG carriers without diabetes compared with RTR who carry the CC genotype and do not have diabetes, also suggests that IL-6 interferes with insulin signaling.

The retrospective study might suffer from a selection bias. All of the results indicate that we included low-risk patients regarding the risks for cardiovascular disease, graft failure, and death. As a consequence, a survival bias cannot be totally excluded. A differential rate of graft and patient survival or DNA availability between patients with the C or the G allele also may have influenced our results. Nevertheless, all of these limitations should favor the null hypothesis and not contribute to a false-positive result. Moreover, NODAT is an early posttransplantation event, and a differential rate of later events should not influence our results. The incidence of NODAT was similar in untested patients, and these patients did not differ in age and BMI, the major predictive factors of NODAT, from those who were included in the study. We also observed that the prevalence and impact of the IL-6 gene promoter polymorphism at position –174 was similar in patients with available pretransplantation DNA and in those who were investigated after transplantation. Because a bias related to DNA availability is improbable, our results are likely to be applicable to the whole transplant population. By the nature of the recruitment process, clinical information that is obtained prospectively usually is of higher quality than retrospective data, and our retrospective study can be used only to generate hypotheses that have to be validated in a prospective cohort. Analysis of our prospective cohort confirmed the results that were obtained in the retrospective cohort. The IL-6 gene promoter polymorphism at position –174 (G->C) was not significantly associated with NODAT in the monovariate analysis because of age distribution discrepancies in the different genotypes. Nevertheless, the correlation became significant after adjustment for age. Allele frequencies were comparable between the two cohorts and with previous studies in white individuals (9,33). Alternatively, although this seems improbable, the association of the IL-6 genotype with NODAT could be explained by linkage disequilibrium of the G allele with a nearby causative polymorphism or by confounding as a result of population stratification.

Finally, our results highly suggest that IL-6 production capacity modifies sensitivity and influences the development of NODAT, especially in overweight patients. The IL-6 gene promoter polymorphism at position –174 may serve as a genetic marker to help physicians in determining recipient risk profiles and in optimizing pre- and posttransplantation treatment strategies.


   Acknowledgments
 
This study was supported by grants from the Fondation Transplantation and the Etablissement Français du Sang (Appel d’offres 2003). J.B. and C.C. received financial support from the Fondation transplantation (ET-031211 and ET-050320, respectively).

We thank Prof. Claude Férec (INSERM U613, EFS Bretagne, Brest, France) for helpful discussion and Jackie Kerveillant for help in preparing this manuscript.


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

J.B. and C.C. contributed equally to this work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Davidson J, Wilknson A, Dantal J, Dotta F, Haller H, Hernandez D, Kasiske BL, Kiberd B, Krentz A, Legendre C, Marchetti P, Markell M, van der Woude FJ, Wheeler DC; International Expert Panel: New-onset diabetes after transplantation: 2003 International consensus guidelines. Proceeding of an international expert panel meeting. Barcelona, Spain, 19 February 2003. Transplantation 75[Suppl]: SS3–SS24, 2003[CrossRef][Medline]
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