The patient group included 80 clinically stable patients receiving maintenance HD for > 3 months in Ewha Womans University Mokdong Hospital. The duration of HD per session was 4 to 6 hours; the frequency of HD was individualized to achieve a Kt/V > 1.2. We assessed the dialysis adequacy by calculating the Kt/V for each patient. Patients were treated with synthetic membranes (polysulfone or polyamide), and dialyzers were not re-used. Heparinization during the dialysis session was done by continuous infusion using unfractionated heparin at a dose of 500 to 1,000 U/hr according to patient weight. None of the patients had a history of acute diseases, such as recent myocardial infarction, unstable angina, acute pulmonary embolism, acute neurologic disorders, malignancies, and overt systemic infections within 3 months before blood sampling for IL-6. The access patent interval was defined as the time from the access formation to the time of first vascular stenosis in each patient. Acute obstructions due to mechanical factors related to surgery within a few days of the access creation, physical compression after the end of a dialysis session, or frequent hypotension during dialysis were excluded from the definition of vascular access patency. To analyze the relationship between IL-6 polymorphisms and vascular access patency, we categorized patients into two groups: the shorter access survival group (patients with access patency < 1 year at the time of enrollment), and longer access survival group (patients with vascular access patency > 1 year). We also performed the same analysis for patients with native arteriovenous fistula (AVF) excluding patients with an arteriovenous graft (AVG). We reviewed medical records from each patient and examined medical histories of patients with diabetes mellitus (DM), HTN, and drug histories for use of renin-angiotensin system blockers (angiotensin converting enzyme inhibitors or angiotensin receptor blockers), statins, and anti-platelet agents. We assessed the nutritional status of each patient by measuring the normalized protein catabolic rate.

An additional 89 healthy individuals without medical illnesses such as DM, HTN, or renal disease were enrolled as the control group. The study was conducted in accordance with the guidelines of the Declaration of Helsinki after receiving approval from Institutional Review Board of Ewha Womans University Mokdong Hospital. Written informed consent was obtained from all subjects.

Venous blood samples were taken from each patient just prior to the dialysis session and analyzed immediately. Samples were obtained from the HD needle puncture site 72 hours after the last dialysis. All patients were required to fast after midnight (> 6 hours). The standard laboratory tests included complete blood cell and platelet counts and blood chemistries, including serum albumin, protein, blood urea nitrogen, creatinine, cholesterol, and low density lipoprotein. High sensitive C-reactive protein (hsCRP) was measured via a nephelometric immunoassay (Handok Pharm Co., Seoul, Korea). IL-6 levels were determined using a commercial enzyme-linked immunosorbent assay kit (Pierce Biotechnology, Rockford, IL, USA).

DNA was isolated from peripheral blood leukocytes by a standard method. All subjects were genotyped for -634 C/G and -174 G/C polymorphisms using the polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Genotyping for the IL-6 -634 C/G and -174 G/C polymorphisms was performed by PCR followed by BsrB I and Hsp92 II digestion, respectively. The primers for each region were as follows: forward 5'-GAGACGCCTTGAAGTAACTG-3' and reverse 5'-AACCAAAGATGTTCTGAACTGA-3' for -634 C/G; and forward 5'-TGACTTCAGCTTTACTCTTGT-3' and reverse 5'-CTGATTGGAAACCTTATTAAG-3' for -174 G/C. The final reaction volume (30 µL) contained 200 ng of genomic DNA, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 200 mmol/L each dNTP, 1 µmol/L of each primer, and 2 U Taq polymerase (all reagents from Fermentas GmbH, St. Leon-Rot, Germany). Initial denaturation at 94℃ for 6 minutes was followed by 35 cycles of 94℃ for 1 minute, annealing at 60℃ for 1 minute, and extension at 72℃ for 1 minute. The final extension step was at 72℃ for 1 minute. The PCR product (-634 C/G, 180 bp; -174 G/C, 191 bp) was digested with the respective restriction enzyme (BsrB I for -643 C/G; Hsp92 II for -174 G/C) and 3% agarose gel. With respect to the -634 C/G polymorphism, CC homozygotes had a 180 bp fragment, CG heterozygotes had 180 bp and 120 + 60 bp fragments, and GG homozygotes had 120 + 60 bp fragments. With respect to the -174 G/C, the G allele had 2 fragments (161 + 30 bp) and the C allele had 3 fragments (116 + 45 + 30 bp).

All values are expressed as the mean ± SD or number (%). The Hardy-Weinberg equilibrium was assessed using the χ² test. Genotype and allele frequencies were compared between two groups using the χ² test or Fisher's exact test (values < 5) and the odds ratio (OR) was calculated. Differences in the continuous variables between groups were assessed using ANOVA and an unpaired Student's t test. Logistic regression analysis was used to assess risk factors for vascular access failure. All data were analyzed using SPSS for Windows version 9.0 (SPSS Inc., Chicago, IL, USA). A p < 0.05 was considered to be statistically significant.

Table 3 lists the distribution of IL-6 genotypes and each allelic frequency. The distribution of the IL-6 -634 genotypes in HD patients and healthy controls was in Hardy-Weinberg equilibrium. Interestingly, the genotype distribution of the IL-6 -634 C/G polymorphisms in the HD patient group differed significantly compared with that in the control group. The GG genotype appeared more frequently in the patient group. Furthermore, the -634 G allele frequency was significantly more common in the patient group than in the control group (OR, 1.77; 95% confidence interval [CI], 1.08 to 2.92; p = 0.009). This finding was also observed after excluding diabetic patients (OR, 2.13; 95% CI, 1.18 to 3.87; p = 0.005) (Table 3).

For the -174 G/C polymorphism, the -174 C allele was not detected in the patient or control group.

No significant differences appeared in clinical parameters based on genotypes (Table 4). Results were similar after diabetic patients were excluded (Table 5).

To assess the association between the IL-6 -634 polymorphism and vascular access patency, the -634 G allele frequency was examined with respect to the access patent interval (Table 6). The distribution of the -634 genotype and G allele frequency did not differ significantly based on the access patent interval. However, after excluding diabetic patients, the -634 genotype distribution differed significantly between the two groups and the G allele frequency exhibited a higher tendency in the access survival < 1 year sub-group (OR, 3.45; 95% CI, 0.88 to 13.57; p = 0.071). These findings were preserved in the analysis of patients with native AVF. However, the G allele did not reveal any statistical significance in logistic regression analysis for risk factors of early vascular access failure in either whole nondiabetic patients (Table 7) or patients with AVF (data not shown).