Association of Interleukin-6 −174 G>C Promoter Polymorphism with Increased Risk of Type 2 Diabetes Mellitus Patients with Diabetic Nephropathy in Turkey

Abstract

Objective: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus. We aimed to evaluate the interleukin (IL)-6 gene polymorphisms in type 2 DN and control subjects. Materials and Methods: The patients selected from the Department of Endocrinology and Metabolism Diseases included 43 type 2 diabetes mellitus patients without DN and 43 type 2 diabetes mellitus patients with DN and 340 healthy normal controls. All subjects underwent venous blood drawing for complete hormonal assays, lipid profile, glucose, and insulin and Il-6 gene polymorphism genetic analysis. Results: IL-6 −174 G>C genotype distribution was different between the control group and the type 2 diabetes mellitus patients (p=0.004). The higher frequency of the polymorphic G allele was also similar for the group with type 2 diabetes mellitus as for the control group. The frequency of the polymorphic G allele was 83.9% in diabetic patients with nephropathy versus 70.9% in those without nephropathy (p=0.039). Conclusion: We suggest that the −174 G>C polymorphism of the IL-6 gene is an independent risk factor for DN in Turkish type 2 diabetes mellitus patients.

Introduction

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (Shpichinetsky et al., 2000). It is a common cause of end stage renal disease. The etiology of DN is multifactorial and includes both environmental and genetic factors (Sun et al., 2004). There is a large body of evidence that genetic abnormalities are involved in the development of DN (Ksiazek et al., 2004).

Interleukin-6 (IL-6) is a multifunctional cytokine produced by many different cell types, including monocytes, lymphocytes, fibroblasts, endothelial cells, and mesangial cells (Fried et al., 1998; Mohamed-Ali et al., 1998). IL-6 stimulates mesangium cell proliferation and matrix production in vitro (Fernandez-Real et al., 2000a; Vozarova et al., 2003). IL-6 levels and polymorphisms have been associated with the risk of DN (Kitamura et al., 2002). Effects of the IL-6 gene may vary with ethnicity (Elbein et al., 1999, 2004; Das et al., 2004).

There are several polymorphisms in and close to IL6 (Fishman et al., 1998; Terry et al., 2000; Kristiansen et al., 2003). Studies investigating the genetic association between IL6 polymorphisms and disease—including type 1 diabetes, type 2 diabetes, insulin resistance, and other features of the metabolic syndrome—have mainly focused on the three common single-nucleotide polymorphisms (SNPs) in the IL6 promoter: the IL6 −174G>C, IL6 −572A>G, and IL6 −597A>G. The IL6 −174G>C promoter SNP, which has been suggested to functionally affect the IL6 promoter activity (Fishman et al., 1998), is a suitable haplotype marker for the common IL6 promoter polymorphisms (Terry et al., 2000).

The purpose of our study was to investigate whether the IL-6 −174 G>C polymorphism is associated with type 2 diabetes mellitus in Turkish patients with and without nephropathy and to evaluate the role of this polymorphism in the susceptibility to DN.

Materials and Methods

The patients selected from the Department of Endocrinology and Metabolism Diseases included 43 type 2 diabetes mellitus patients without DN and 43 type 2 diabetes mellitus patients with DN and 340 healthy normal controls. A detailed medical history of each patient was obtained. The phenotypic characteristics were determined. Age, sex, and blood pressure were recorded. The total cholesterol, triglycerides, HbA1c (%), plasma creatinine, plasma urea, and microalbuminuria were measured by the automated Olympus AU2700.

Genetic study

Restriction fragment length polymorphism-IL-6 gene analysis

DNA was extracted from cellular blood components by the salting-out method. The PCR was used to detect the IL-6 SfaNI restriction fragment length polymorphism (RFLP) by the method of Fernandez-Real et al. (2000b).

The SfaNI polymorphism is due to a replacement of G by C at position 174, and primers were designed to amplify the promoter region of the IL-6 gene. The primers used in the PCR were forward −5′-TGACTTCAGCTTTACTCTTTGT-3′ and reverse −5′-CTGATTGGAAACCTTATTAAG-3′ (TIB MOLBIOL Syntheselabor, Berlin, Germany). The reaction was carried out in a final volume of 25 mkL containing 1.5 mM of MgCl₂, 0.2 mM each dNTP (Promega, Madison, WI), 0.2 mM each primer, and 1.0 U Ampli Taq Polymerase (PE Applied Biosystems, Foster City, CA). DNA was amplified during 35 cycles with an initial denaturation of 10 min at 94°C and a final extension of 10 min at 72°C. The cycle program consisted of a 1-min denaturation at 94°C, a 1-min 35-s annealing at 55°C, and a 1-min extension at 72°C. PCR products were digested with SfaNI restriction enzyme (New England BioLabs, Beverly, MA) at 37°C overnight and electrophoresed on a 2% agarose gel. SfaNI RFLP was detected by ethidium bromide staining. The identified genotypes were named according to the presence or absence of the enzyme restriction sites, so SfaNI (G/G), (G/C), and (C/C) are homozygotes for the presence of the site (140/58 bp), heterozygotes for the presence and absence of the site (198/140/58 bp), and homozygotes for the absence of the site (198 bp), respectively.

Statistical analysis

All statistical analyses were conducted by using the SPSS statistical package, version 11.0. Distributions of continuous variables in groups are expressed as mean±SD. Categorical variables were compared between groups by use of the χ² test. A value of p<0.05 was considered significant.

Results

A total of 434 individuals were screened for the presence of the IL-6 gene polymorphism: 47 individuals who had type 2 diabetes mellitus with DN and 47 individuals who just had type 2 diabetes mellitus without DN, and the remaining 340 individuals who constituted the healthy control group. Nephropathy was defined as the presence of microalbuminurea (30-300 mg/day).

After obtaining the medical history of each patient, some physiological and biochemical tests were performed. Diastolic and systolic blood pressure, fasting serum glucose, postprandial serum glucose, A1c, total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, plasma creatinine, plasma urea, and microalbuminurea levels were significantly higher in type 2 diabetes mellitus patients either with or without DN than in the control group (Table 1).

Table 1.

Physiological and Biochemical Test Results in Diabetic Patients and Healthy Controls

	With DN (n=43)	Without DN (n=43)	Control group (n=340)
	Mean±SD	Mean±SD	Mean±SD	p
Age (years)	58.31±10.44	52.23±9.39	54.23±2.72	0.320
Duration of diabetes (years)	8.73±7.12	7.15±5.18		0.260
Systolic BP (mmHg)	129.62±21.00	120.70±11.10	114.36±11.22	0.0011^a
Diastolic BP (mmHg)	80.78±9.23	74.89±7.31	76.32±5.22	0.0122^a
Fasting serum glucose (mg/dL)	154.77±60.53	140.53±43.15	85.77±15.26	0.000^a
Postprandial serum glucose (mg/dL)	218.5±95.21	193.9±71.70	121.3±8.4	0.000^a
HbA1c (%)	8.02±2.39	6.68±1.39	5.13±0.13	0.000^a
Total cholesterol (mg/dL)	207.05±48.31	207.35±36.57	172.25±41.2	0.000^a
Triglyceride (mg/dL)	201.1±19.31	167.56±82.15	135.21±97.35	0.0042^a
HDL (mg/dL)	47.32±10.6	47.8±10.6	52.79±13.6	0.027^a
LDL (mg/dL)	131.17±38.39	135.8±31.64	108.72±43.6	0.001^a
Plasma creatinine (mg/dL)	1.00±0.0	0.87±0.00	0.7±1.3	0.012^a
Plasma urea (mg/dL)	37.49±14.01	31.35±8.8	27.36±6.82	0.001^a
Microalbuminuria (mg/day)	201.00±22.89	11.79±4.36	10.03±3.35	0.013^a

p<0.05.

n, Number of individuals; DN, diabetic nephropathy; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol.

The distributions of genotype and allele frequencies were compared between diabetic patients and controls (Table 2) as well as between patients with nephropathy and those without nephropathy (Table 3). We analyzed the frequency of IL-6 −174 G>C polymorphism in the type 2 diabetes mellitus and control groups. The genotype distribution was different between the control group and the type 2 diabetes mellitus patients (p=0.004). The higher frequency of the polymorphic G allele was also similar for the group with type 2 diabetes mellitus as for the control group. Genotype frequencies in diabetic patients with nephropathy were C/C 0%, C/G 32.6%, G/G 67.4% versus C/C 14%, C/G 30.2%; G/G 55.8% in those without nephropathy. The frequency of the polymorphic G allele was 83.9% in diabetic patients with nephropathy versus 70.9% in those without nephropathy (p=0.039). Thus, we suggest that the −174 G>C polymorphism of the IL-6 gene is an independent risk factor for DN in Turkish type 2 diabetes mellitus patients.

Table 2.

Genotype and Allele Frequencies of the Interleukin-6−174 G>C Polymorphism in Diabetic Patients and Healthy Controls

IL-6 gene	Diabetic patients (n=86)	Control groups (n=340)
Genotypes
CC	6 (7%)	26 (7.6%)
CG	27 (31.4%)
GG	53 (61.6%)	143 (42.1%)
Alleles
C	39 (22.7%)	223 (32.8%)
G	133 (77.3%)	457 (67.2%)

n, Number of patients who could be genotyped successfully; IL-6, interleukin 6.

Table 3.

Genotype and Allele Frequencies of the Interleukin-6−174 G>C Polymorphism With Diabetic Nephropathy and Without Diabetic Nephropathy

IL-6 gene polymorphism	Diabetic nephropathy (n=43)	Without diabetic nephropathy (n=43)
Genotypes
CC	0 (0%)	6 (14%)
CG	14 (32.6%)	13 (30.2%)
GG	29 (67.4%)	24 (55.8%)
Alleles
C	14 (16.5%)	25 (29.1%)
G	72 (83.7%)	61 (70.9%)

n, Number of patients who could be genotyped successfully.

Discussion

Renal glomerular basal membrane thickening and increased accumulation of extracellular matrix are the principal characteristics of the expanded mesangium in diabetic glomerulosclerosis. The mechanisms of mesangial cell proliferation and production of the extracellular matrix are incompletely understood, but it is possible that cytokines play a major role in the beginning and progression of extracellular matrices by mesangial cells. IL-6 is a candidate cytokine for autocrine and paracrine regulation of mesangial cell behavior (Ruef et al., 1990; Abbott et al., 1991; Coleman and Ruef 1992; Sekizuka et al., 1994). Suzuki et al. (1995) have reported that IL-6 mRNA was expressed in mesangial cells collectively with other resident and infiltrating cells in renal tissue of patients with DN, a finding that may be related to mesangial cell proliferation. Whereas the precise mechanism of IL-6 expression and production in diabetes is uncertain, several in vitro cell culture studies have confirmed that high glucose concentrations and AGE stimulate IL-6 production (Iida et al., 1994; Morohoshi et al., 1995). Although further research is needed to clarify the functional impact of other polymorphisms within the IL-6 promoter region, our results suggest that the presence of the G allele and more so the GG genotype would result in a higher IL-6 production in vivo after exposure to various inflammatory stimuli compared with the C allele. The decrease in the frequency of the CC genotype in patients with microalbuminuria consequently suggests that the CC genotype confers a protective influence against the progression of DN.

There are several studies demonstrating an association of polymorphisms within cytokine genes and DN (Freedman et al., 1997; Tarnow et al., 1997). To our knowledge, the present study is the first attempt to examine whether the −174 C/G polymorphism in the promoter region of the IL-6 gene is involved in the development and progression of DN. A G/C polymorphism at position −174 in the IL-6 promoter region, which is associated with both the increased expressions of IL-6 and elevated plasma levels of IL-6, has previously been reported by Fishman et al. (1998). However, in our preliminary study, we were observing the IL-6 −174G/C polymorphism in the Turkish population, which is in contrast to the findings of Nakajima et al. (1999). In this study, we found a significant positive association of the IL-6 −634G/G homozygote with macroalbuminuria in Japanese patients with type 2 diabetes. However, we found a significantly higher frequency of the occurrence of the heterozygous form of the −174C/G polymorphism and homozygous GG form. Thus, these observations suggest that the IL-6 −174 C/G polymorphism, at least when in the GG homozygous form in type 2 diabetic patients, contributes to the development of nephropathy. We were able to find an association between the −174 C/G polymorphism and microalbuminuria. In this regard, although microalbuminuria is regarded as a risk factor for the development of DN (Mogensen 1984; Mattock et al., 1992), it has been reported that microalbuminuria is not a good predictor of subsequent progression to overt nephropathy in patients with long-standing diabetes (Forsblom et al., 1992). Therefore, we assume that IL-6 −174C/G polymorphism could be an aggravating and initiating factor for DN in type 2 diabetic patients.

In conclusion, the results in this study suggest that IL-6 −174 C/G polymorphism was linked with progression of DN in Turkish patients with type 2 diabetes.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Abbott

, Ryan

, Ceska

, et al. (1991) Interleukin-1β stimulates human mesangial cells to synthesize and release interleukins-6 and -8. Kidney Int, 40:597-605.

Coleman

, Ruef

(1992) Interleukin-6: an autocrine regulator of mesangial cell growth. Kidney Int, 41:604-606.

Das

, Hasstedt

, Zhang

, Elbein

(2004) Linkage and association mapping of a chromosome 1q21-q24 type 2 diabetes susceptibility locus in Northern European Caucasians. Diabetes, 53:492-499.

Elbein

, Hoffman

, Teng

, et al. (1999) A genome-wide search for type 2 diabetes susceptibility genes in Utah Caucasians. Diabetes, 48:1175-1182.

Elbein

, Zheng

, Wang

, et al. (2004) Novel T2DM locus on chromosome 4 and confirmation of chromosome 1q21 T2DM linkage in multiplex African American families. Diabetes, 53(Suppl 2):A270.

Fernandez-Real

, Broch

, Vendrell

, et al. (2000a) Interleukin-6 gene polymorphism and insulin sensitivity. Diabetes, 49:517-520.

Fernandez-Real

, Broch

, Vendrell

, et al. (2000b) Interleukin-6 gene polymorphism and lipid abnormalities in healthy subjects. J Clin Endocrinol Metab, 85:1334-1339.

Fishman

, Faulds

, Jeffery

, et al. (1998) The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest, 102:1369-1376.

Forsblom

, Groop

, Ekstrand

, Groop

(1992) Predictive value of microalbuminuria in patients with insulin-dependent diabetes of long duration. Br Med J, 305:1051-1053.

10.

Freedman

, Yu

, Spray

, et al. (1997) Genetic linkage analysis of growth factor loci and end-stage renal disease in African Americans. Kidney Int, 51:819-825.

11.

Fried

, Bunkin

, Greenberg

(1998) Omental and subcutaneous adipose tissues of obese subjects release interleukin-6: depot difference and regulation by glucocorticoid. J Clin Endocrinol Metab, 83:847-850.

12.

Iida

, Miyata

, Inagi

, et al. (1994) β2-microglobulin modified with advanced glycation end products induces interleukin-6 from human macrophages: role in the pathogenesis of hemodialysis-associated amyloidosis. Biochem Biophys Res Commun, 30:1235-1241.

13.

Kitamura

, Hasegawa

, Obayashi

, et al. (2002) Interleukin-6 polymorphism (-634C/G) in the promotor region and the progression of diabetic nephropathy in type 2 diabetes. Diabet Med, 19:1000-1005.

14.

Kristiansen

, Nolsøe

, Larsen

, et al. (2003) Association of a functional 17beta-estradiol sensitive IL6-174G/C promoter polymorphism with early onset type 1 diabetes in females. Hum Mol Genet, 12:1101-1110.

15.

Ksiazek

, Bednarek-Skublewsk

, Buraczynska

(2004) The C677T methylenetetrahydrofolate reductase gene mutation and nephropathy in type 2 diabetes mellitus. Med Sci Monit, 10:BR47-BR51.

16.

Mattock

, Morrish

, Viberti

, et al. (1992) Prospective study of microalbuminuria as predictor of mortality in NIDDM. Diabetes, 41:736-741.

17.

Mogensen

(1984) Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabetes. N Engl J Med, 310:356-360.

18.

Mohamed-Ali

, Pinkney

, Coppack

(1998) Adipose tissue as an endocrine and paracrine organ. Int J Obes Relat Metab Disord, 22:1145-1158.

19.

Morohoshi

, Fujisawa

, Uchimura

, Numano

(1995) The effect of glucose and advanced glycosylation end products on IL-6 production by human monocytes. Ann N Y Acad Sci, 748:562-570.

20.

Nakajima

, Ota

, Yoshida

, et al. (1999) Allelic variants in the interleukin-6 gene and essential hypertension in Japanese women. Genes Immun, 1:115-119.

21.

Ruef

, Budde

, Lacy

, et al. (1990) Interleukin 6 is an autocrine growth factor for mesangial cells. Kidney Int, 38:249-257.

22.

Sekizuka

, Tomino

, Sei

, et al. (1994) Detection of serum IL-6 in patients with diabetic nephropathy. Nephron, 68:284-285.

23.

Shpichinetsky

, Raz

, Friedlander

, et al. (2000) The association between two common mutations C677T and A1298C in human methylenetetrahydrofolate reductase gene and the risk for diabetic nephropathy in type 2 diabetic patients. J Nutr, 130:2493-2497.

24.

Sun

, Xu

, Zhu

, Lu

(2004) Genetic polymorphism of methylenetetrahydrofolate reductase as a risk factor for diabetic nephropathy in Chinese type 2 diabetic patients. Diabetes Res Clin Pract, 64:185-190.

25.

Suzuki

, Miyazaki

, Naka

, et al. (1995) In situ hybridization of interleukin 6 in diabetic nephropathy. Diabetes, 44:1233-1238.

26.

Tarnow

, Pociot

, Hansen

, et al. (1997) Polymorphisms in the interleukin-1 gene cluster do not contribute to the genetic susceptibility of diabetic nephropathy in Caucasian patients with IDDM. Diabetes, 46:1075-1076.

27.

Terry

, Loukaci

, Green

(2000) Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation. J Biol Chem, 275:18138-18144.

28.

Vozarova

, Fernandez-Real

, Knowler

, et al. (2003) The interleukin-6 (-174) G/C promoter polymorphism is associated with type-2 diabetes mellitus in Native Americans and Caucasians. Hum Genet, 112:409-413.