Interleukin-4 (IL4) -590C/T (rs2243250) gene polymorphism is not associated with diabetic nephropathy (DN) in Caucasians with type 2 diabetes mellitus (T2DM)

Authors

  • Matej Završnik Department for Diabetes and Metabolic Diseases, Clinic for Internal Medicine, University Medical Centre Maribor, Maribor, Slovenia
  • Jernej Letonja Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
  • Jana Makuc Department of Internal Medicine, General Hospital Slovenj Gradec, Slovenj Gradec, Slovenia
  • Maja Šeruga Department of Internal Medicine, General Hospital Murska Sobota, Murska Sobota, Slovenia
  • Ines Cilenšek Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
  • Daniel Petrovič Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

DOI:

https://doi.org/10.17305/bjbms.2018.2688

Keywords:

Interleukin 4, rs2243250, diabetic nephropathy, association study

Abstract

Diabetic nephropathy (DN) is a microvascular complication that affects up to 40% of diabetic patients and can lead to end-stage kidney disease. Inflammatory cytokines such as interleukin 1 (IL-1), IL-6, IL-18 and tumor necrosis factor-α (TNFα) have been linked to the development and progression of DN. The aim of our study was to examine the relationship between interleukin-4 (IL4) -590C/T (rs2243250) gene polymorphism and DN in patients with type 2 diabetes mellitus (T2DM). This study is a continuation of our previous research on the association between angiotensinogen (AGT) gene polymorphisms and DN in patients with T2DM. We included 651 unrelated Slovenian (Caucasian) patients who had had T2DM for at least 10 years. The participants were classified into a group of T2DM patients with DN (276 cases) and a group without DN (375 controls). IL4 rs2243250 polymorphism was analyzed using a TaqMan SNP genotyping assay and StepOne Real-Time PCR System. The frequencies of rs2243250 TT, CT and CC (wild type) genotypes were 3.2%, 29.4% and 67.4%, respectively in patients with DN, and 2.7%, 34.4% and 62.9%, respectively in controls. Our logistic regression analysis adjusted for gender, age, diabetes duration, and glycated hemoglobin showed no association between rs2243250 and the risk for DN (OR 1.06; CI 0.37-3.05; p = 0.9). IL4 rs2243250 is not associated with DN in our subset of Slovenian patients with T2DM.

Downloads

Download data is not yet available.

Author Biographies

Matej Završnik, Department for Diabetes and Metabolic Diseases, Clinic for Internal Medicine, University Medical Centre Maribor, Maribor, Slovenia

Department for Diabetes and Metabolic Diseases

Jana Makuc, Department of Internal Medicine, General Hospital Slovenj Gradec, Slovenj Gradec, Slovenia

Department of Internal Medicine

Maja Šeruga, Department of Internal Medicine, General Hospital Murska Sobota, Murska Sobota, Slovenia

Department of Internal Medicine

References

Ogurtsova K, da Rocha Fernandes JD, Huang Y, Linnenkamp U, Guariguata L, Cho NH, et al. IDF diabetes atlas: Global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract 2017;128:40-50. https://doi.org/10.1016/j.diabres.2017.03.024.

Boyle JP, Honeycutt AA, Narayan KM, Hoerger TJ, Geiss LS, Chen H, et al. Projection of diabetes burden through 2050: Impact of changing demography and disease prevalence in the U.S. Diabetes Care 2001;24(11):1936-40. https://doi.org/10.2337/diacare.24.11.1936.

Ayodele OE, Alebiosu CO, Salako BL. Diabetic nephropathy - A review of the natural history, burden, risk factors and treatment. J Natl Med Assoc 2004;96(11):1445-54.

Afkarian M, Sachs MC, Kestenbaum B, Hirsch IB, Tuttle KR, Himmelfarb J, et al. Kidney disease and increased mortality risk in type 2 diabetes. J Am Soc Nephrol 2013;24(2):302-8. https://doi.org/10.1681/ASN.2012070718.

Freedman BI, Bostrom M, Daeihagh P, Bowden DW. Genetic factors in diabetic nephropathy. Clin J Am Soc Nephrol 2007;2(6):1306-16. https://doi.org/10.2215/CJN.02560607.

Navarro-González JF, Mora-Fernández C. The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol 2008;19(3):433-42. https://doi.org/10.1681/ASN.2007091048.

Paul WE. Interleukin-4: A prototypic immunoregulatory lymphokine. Blood 1991;77(9):1859-70.

Vitetta ES, Ohara J, Myers CD, Layton JE, Krammer PH, Paul WE. Serological, biochemical, and functional identity of B cell-stimulatory factor 1 and B cell differentiation factor for IgG1. J Exp Med 1985;162(5):1726-31. https://doi.org/10.1084/jem.162.5.1726.

Mosmann TR, Coffman RL. Th1 and Th2 cells: Different pattern of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1898;7:145-73. https://doi.org/10.1146/annurev.iy.07.040189.001045.

Seder RA, Paul WE, Davis MM, Fazekas de St Groth B. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J Exp Med 1992;176(4):1091-8. https://doi.org/10.1084/jem.176.4.1091.

Zamorano J, Rivas MD, Pérez-GM. Interleukin-4: A multifunctional cytokine. Inmunología 2003;22(2):215-24.

Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996;14:397-440. https://doi.org/10.1146/annurev.immunol.14.1.397.

Steinke JW, Borish L. Th2 cytokines and asthma-interleukin-4: Its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin-4 receptor antagonists. Respir Res 2001;2(2):66-70. https://doi.org/10.1186/rr40.

Mi QS, Ly D, Zucker P, McGarry M, Delovitch TL. Interleukin-4 but not interleukin-10 protects against spontaneous and recurrent Type 1 diabetes by activated CD1d-restricted invariant natural killer T-cells. Diabetes 2004;53(5):1303-10. https://doi.org/10.2337/diabetes.53.5.1303.

Mire-Sluis AR. Interleukin-4. In: Cytokines. London: Academic Press; 1998. p. 53-68. https://doi.org/10.1016/B978-012498340-3/50005-1.

Liang J, Liu Y, Xue R, Chen L, Chen H, Shao L, et al. Interleukin 4 -590C/T (rs2243250) polymorphism is associated with increased risk of atopic dermatitis: Meta-analysis of case-control studies. Dermatitis 2017;28(2):144-51. https://doi.org/10.1097/DER.0000000000000265.

Qiu LJ, Ni J, Cen H, Wen PF, Zhang M, Liang Y, et al. Relationship between the IL-4 gene promoter -590C/T (rs2243250) polymorphism and susceptibility to autoimmune diseases: A meta-analysis. J Eur Acad Dermatol Venereol 2015;29(1):48-55. https://doi.org/10.1111/jdv.12435.

Liu S, Li T, Liu J. Interleukin-4 rs2243250 polymorphism is associated with asthma among Caucasians and related to atopic asthma. Cytokine 2012;59(2):364-9. https://doi.org/10.1016/j.cyto.2012.05.006.

Makuc J, Šeruga M, Završnik M, Cilenšek I, Petrovič D. Angiotensinogen (AGT) gene missense polymorphisms (rs699 and rs4762) and diabetic nephropathy in caucasians with Type 2 diabetes mellitus. Bosn J Basic Med Sci 2017;17(3):262-7. https://doi.org/10.17305/bjbms.2017.1823.

Zheng Z, Zheng F. Immune cells and inflammation in diabetic nephropathy. J Diabetes Res 2016;2016:1841690. https://doi.org/10.1155/2016/1841690.

Kazemi Arababadi M. Interleukin-4 gene polymorphisms in Type 2 diabetic patients with nephropathy. Iran J Kidney Dis 2010;4(4):302-6.

Neelofar K, Ahmad J, Ahmad A, Alam K. Study of IL4-590C/T and IL6-174G/C gene polymorphisms in type 2 diabetic patients with chronic kidney disease in North Indian population. J Cell Biochem 2017;118(7):1803-9. https://doi.org/10.1002/jcb.25853.

Alsaid A, El-Missiry M, Hatata ES, Tarabay M, Settin A. Association of IL-4-590 C>T and IL-13-1112 C>T gene polymorphisms with the susceptibility to Type 2 diabetes mellitus. Dis Markers 2013;35(4):243-7. https://doi.org/10.1155/2013/107470.

Cilenšek I, Hercegovac A, Nikolajević Starčević J, Vukojević K, Saraga Babić M, Milutinović Živin A. Polymorphisms of interleukin-4, -10 and 12B genes and diabetic retinopathy. Central Eur J Biol 2011;6(4):558-64. https://doi.org/10.2478/s11535-011-0036-6.

Interleukin-4 (IL4) -590C/T (rs2243250) gene polymorphism is not associated with diabetic nephropathy (DN) in Caucasians with type 2 diabetes mellitus (T2DM)

Downloads

Additional Files

Published

2018-11-07

How to Cite

1.
Završnik M, Letonja J, Makuc J, Šeruga M, Cilenšek I, Petrovič D. Interleukin-4 (IL4) -590C/T (rs2243250) gene polymorphism is not associated with diabetic nephropathy (DN) in Caucasians with type 2 diabetes mellitus (T2DM). Biomol Biomed [Internet]. 2018Nov.7 [cited 2023Dec.4];18(4):347-51. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/2688

Issue

Vol. 18 No. 4 (2018)

Section

Translational and Clinical Research

INTRODUCTION

Diabetes mellitus (DM) is a group of metabolic disorders characterized by chronic hyperglycemia which develops as the result of reduced insulin secretion and/or ineffectiveness of produced insulin. Global prevalence of diabetes mellitus (DM) in adults aged 20 to 79 years is estimated to rise from 8.8% in 2015 to 10.4% by the year 2040 [1]. Moreover, in the United States alone, a 165% increase in the prevalence of DM is expected between 2000 and 2050 [2].

Complications of diabetes can be classified as macrovascular and microvascular. A common microvascular complication is diabetic nephropathy (DN), a kidney disease defined by the presence of abnormal levels of urinary albumin and/or decreased glomerular filtration rate (GFR) that affects up to 40% of all patients with diabetes [3]. Patients with DN have a higher mortality rate and are more prone to cardiovascular diseases (CVDs) compared to diabetic patients without DN [4].

Familial aggregation of albuminuria, end-stage kidney disease and chronic kidney disease, as well as differences in the prevalence of DN between populations, suggest a strong genetic component of DN [5]. Thus, identification of genes that might be involved in the onset and progression of DN is important for the development of new preventive and therapeutic strategies.

Immune-mediated inflammatory processes have been implicated in the pathophysiology of diabetes and its complications, and a number of studies investigated the specific roles of inflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-18, and tumor necrosis factor-α (TNFα) in the development of DN [6].

IL-4 is an anti-inflammatory cytokine involved in the regulation of the immune system at different levels. For example, IL-4 stimulates the proliferation of activated T and B cells, regulates the differentiation of B cells, promotes type 2 T helper (Th2) and inhibits type 1 T helper (Th1) cell differentiation [7-10]. In addition to lymphoid cells, IL-4 is able to modulate the differentiation, proliferation and apoptosis of other hematopoietic as well as non-hematopoietic cell populations [11].

IL-4 and the related signaling pathways have been linked to the development of autoimmune [12] and allergic diseases [13]. For instance, an association of allergic asthma with IL-4 was clinically demonstrated by a significant increase in airway hyperresponsiveness in patients with mild asthma after administration of IL-4 with a nebulizer [13]. Generally, it is suggested that allergic diseases are dependent on Th2 cells and related cytokines, whereas autoimmune diseases depend on Th1 cells and cytokines produced by monocytes [12]. The anti-inflammatory role of IL-4 in autoimmune diseases has been indicated based on its protective effects in murine models of diabetes and rheumatoid arthritis [11,14].

The IL4 gene, located at the long arm of chromosome 5 (5q31), is 0.9 kb long and contains 4 exons [15]. Several studies have shown that a polymorphism in the promoter region of IL4 (IL4 -590C/T, rs2243250) might be associated with genetic susceptibility to atopic dermatitis [16], multiple sclerosis, rheumatoid arthritis [17], and atopic asthma [18].

In this study, we investigated the relationship between IL4 -590C/T (rs2243250) polymorphism and DN in patients with type 2 diabetes mellitus (T2DM).

MATERIALS AND METHODS

Patients

This study is a continuation of our previous research on the association between angiotensinogen (AGT) gene polymorphisms and DN in patients with T2DM [19]. We included 651 unrelated Slovenian (Caucasian) patients who had had T2DM for at least 10 years. The participants were classified into a group of T2DM patients with DN (276 cases) and a group without DN (375 controls) [19]. The diagnosis of T2DM and DN was made according to the World Health Organization diagnostic criteria. To avoid the confounding effect of impaired kidney function, patients with overt nephropathy were not enrolled in the study. Additional exclusion criteria were poor glycemic control, significant heart failure [New York Heart Association (NYHA) Classification II-IV], alcoholism, infection, and the presence of other causes of renal disease.

The study was approved by the national medical ethics committee and performed in compliance with the Helsinki declaration. All participants provided informed consent. Information on age, sex, blood pressure, duration of T2DM and hypertension, body mass index (BMI), smoking status, incidence of microvascular complications of T2DM (diabetic retinopathy [DR], DN, diabetic foot [DF]), duration of DR, estimated GFR (eGFR), hemoglobin (Hb), total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol levels was obtained by questionnaire [19].

Biochemical analyses

Plasma glucose, Hb, glycated hemoglobin (HbA1c), urea, creatinine, cystatin C, total cholesterol, LDLs, HDLs, and triglycerides (TGs) were determined by standard biochemical methods. Albumin/creatinine ratio (ACR) was determined in three urine samples for each patient [19].

Genotyping

Genomic DNA was extracted from 100 µl of peripheral blood using a DNeasy Blood and Tissue Kit (Qiagen, Germany). IL4 rs2243250 polymorphism was analyzed using a TaqMan single nucleotide polymorphism (SNP) genotyping assay and 48-well StepOne Real-Time PCR System, according to the manufacturer’s instructions (Applied Biosystems, USA). The volume of PCR reaction (5 µl) was calculated with StepOne real-time PCR Systems software, and the following components were included in the reaction mix: TaqMan Universal Master Mix, mix of fluorescent dye (VIC/FAM)-labeled oligonucleotide primers, RNase-free H2O, and 0.5 µl of DNA. The amplification was carried out under the following conditions: step 1, pre-PCR plate read for 30 seconds at 60°C; step 2, pre-denaturation for 10 minutes at 95°C, repeating 35 cycles with denaturation for 15 seconds at 95°C and annealing/extension for 60 seconds at 60°C; step 3, post-PCR plate read for 30 seconds at 60°C. Three control samples with known genotypes and negative control were applied in each series.

Statistical analysis

Statistical analysis was conducted using IBM SPSS Statistics for Windows, Version 21.0. (IBM Corp., Armonk, NY). Continuous variables were compared by either unpaired Student’s t-test or Mann–Whitney U test. Chi-square test was used to compare discrete variables. The relationship between IL4 rs2243250 polymorphism and DN was assessed by logistic regression analysis, adjusted for gender, age, diabetes duration, and HbA1c concentration. The value of p < 0.05 was considered statistically significant. The deviation from Hardy-Weinberg equilibrium (HWE) was assessed by the Fisher’s exact test (http://ihg.gsf.de/).

RESULTS

Demographic and clinical characteristics of T2DM patients with and without DN are summarized in Table 1 and 2. Higher systolic blood pressure, higher ACR, presence of CVD, and increased levels of serum fasting glucose, HbA1c, urea, creatinine, and triglycerides were significantly more frequent in patients with DN. In addition, the prevalence of DR and DF was significantly higher in patients with DN compared to those without DN (Table 1). Similarly, serum cystatin C and creatinine levels were significantly higher in patients with DN (Table 2).

TABLE 1: Clinical characteristics of T2DM patients with and without DN
TABLE 2: Laboratory characteristics of T2DM patients with and without DN

The distribution of IL4 rs2243250 genotypes and alleles is shown in Table 3. rs2243250 was in HWE in both groups and there was no statistically significant difference in the genotype distribution between T2DM patients with and without DN (Table 3).

TABLE 3: Distribution of IL4-590C/T (rs2243250) genotypes and alleles in T2DM patients with DN (cases) and without DN (controls)

A logistic regression analysis was used to evaluate whether rs2243250 polymorphism was independently associated with DN after adjusting for gender, age, diabetes duration, and HbA1c concentration. The results indicated no relationship between rs2243250 polymorphism and DN in our cohort of T2DM patients (Table 4).

TABLE 4: Association of IL4-590C/T (rs2243250) polymorphism with DN in T2DM patients

DISCUSSION

Chronic inflammation is considered to be the underlying cause of DN. In tissue and organs, different types of immune cells function as internal sensors of damage and thus have a primary role in the process of inflammation [20]. IL-4 is involved in the differentiation of T cells and a change in the gene sequence might affect the anti-inflammatory function of protein, and consequently influence the pathogenesis of DN [9]. This assumption was the basis of our study, however, in our group of patients with T2DM we did not observe an association between IL4 -590C/T (rs2243250) polymorphism and DN.

Up until now, only two other studies have investigated the association between IL4 -590C/T polymorphism and DN. Arababadi [21] showed a significant difference in the genotype and allele frequencies of -590C/T SNP between T2DM patients with DN and healthy controls in a Rafsanjan population from the southeast of Iran [21]. Similarly, Neelofar et al. [22] found an association between IL4 -590C/T and IL6 -174G/C polymorphisms and chronic kidney disease in North Indian patients with T2DM [22]. In addition, Alsaid et al. [23] indicated that, in their group of Egyptian patients with diabetes, the heterozygous genotypes of IL4 -590 (CT) and IL13 -1112 (CT) could be considered as risk factors while the homozygous wild-type genotypes of both genes (CC) might be considered protective for T2DM [23].

Comparable to the results of the current study, Cilenšek et al. showed no association between IL4 -590C/T SNP and another microvascular complication of T2DM, proliferative DR, in Caucasians with T2DM [24].

We assume that the differences in the frequency of -590C/T alleles between diverse and geographically distant populations (in this case Iranian and Indian versus Slovenian population) might, at least partially, explain the discrepant results and the lack of association between IL4 rs2243250 and DN in our study.

Overall, we did not find an association between IL4 -590C/T gene polymorphism and DN in Slovenian (Caucasian) patients with T2DM. This implies that IL4 rs2243250 may not be a marker for DN in Caucasians with T2DM.

DECLARATION OF INTERESTS

The authors declare no conflict of interests.

REFERENCES

  1. , , , , , (). IDF diabetes atlas: Global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract. https://doi.org/10.1016/j.diabres.2017.03.024
  2. , , , , , (). Projection of diabetes burden through 2050: Impact of changing demography and disease prevalence in the U.S. Diabetes Care. https://doi.org/10.2337/diacare.24.11.1936
  3. , , (). Diabetic nephropathy - a review of the natural history, burden, risk factors and treatment. J Natl Med Assoc.
  4. , , , , , (). Kidney disease and increased mortality risk in type 2 diabetes. J Am Soc Nephrol. https://doi.org/10.1681/ASN.2012070718
  5. , , , (). Genetic factors in diabetic nephropathy. Clin J Am Soc Nephrol. https://doi.org/10.2215/CJN.02560607
  6. , (). The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol. https://doi.org/10.1681/ASN.2007091048
  7. (). Interleukin-4: A prototypic immunoregulatory lymphokine. Blood.
  8. , , , , , (). Serological, biochemical, and functional identity of B cell-stimulatory factor 1 and B cell differentiation factor for IgG1. J Exp Med. https://doi.org/10.1084/jem.162.5.1726
  9. , (). Th1 and Th2 cells: Different pattern of lymphokine secretion lead to different functional properties. Annu Rev Immunol. https://doi.org/10.1146/annurev.iy.07.040189.001045
  10. , , , (). The presence of interleukin 4 duringin vitropriming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J Exp Med. https://doi.org/10.1084/jem.176.4.1091
  11. , , (). Interleukin-4: A multifunctional cytokine. Inmunología.
  12. , , (). Role of cytokines in rheumatoid arthritis. Annu Rev Immunol. https://doi.org/10.1146/annurev.immunol.14.1.397
  13. , (). Th2 cytokines and asthma-interleukin-4: Its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin-4 receptor antagonists. Respir Res. https://doi.org/10.1186/rr40
  14. , , , , (). Interleukin-4 but not interleukin-10 protects against spontaneous and recurrent Type 1 diabetes by activated CD1d-restricted invariant natural killer T-cells. Diabetes. https://doi.org/10.2337/diabetes.53.5.1303
  15. (). Interleukin-4. Cytokines. https://doi.org/10.1016/B978-012498340-3/50005-1
  16. , , , , , (). Interleukin 4 -590C/T (rs2243250) polymorphism is associated with increased risk of atopic dermatitis: Meta-analysis of case-control studies. Dermatitis. https://doi.org/10.1097/DER.0000000000000265
  17. , , , , , (). Relationship between the IL-4 gene promoter -590C/T (rs2243250) polymorphism and susceptibility to autoimmune diseases: A meta-analysis. J Eur Acad Dermatol Venereol. https://doi.org/10.1111/jdv.12435
  18. , , (). Interleukin-4 rs2243250 polymorphism is associated with asthma among Caucasians and related to atopic asthma. Cytokine. https://doi.org/10.1016/j.cyto.2012.05.006
  19. , , , , (). Angiotensinogen (AGT) gene missense polymorphisms (rs699 and rs4762) and diabetic nephropathy in caucasians with Type 2 diabetes mellitus. Bosn J Basic Med Sci. https://doi.org/10.17305/bjbms.2017.1823
  20. , (). Immune cells and inflammation in diabetic nephropathy. J Diabetes Res. https://doi.org/10.1155/2016/1841690
  21. (). Interleukin-4 gene polymorphisms in Type 2 diabetic patients with nephropathy. Iran J Kidney Dis.
  22. , , , (). Study of IL4-590C/T and IL6-174G/C gene polymorphisms in type 2 diabetic patients with chronic kidney disease in North Indian population. J Cell Biochem. https://doi.org/10.1002/jcb.25853
  23. , , , , (). Association of IL-4-590 C>T and IL-13-1112 C>T gene polymorphisms with the susceptibility to Type 2 diabetes mellitus. Dis Markers. https://doi.org/10.1155/2013/107470
  24. , , , , , (). Polymorphisms of interleukin-4, -10 and 12B genes and diabetic retinopathy. Central Eur J Biol. https://doi.org/10.2478/s11535-011-0036-6