Relationship between hepcidin and GDF15 in anemic patients with type 2 diabetes without overt renal impairment

diabetes research and clinical practice 109 (2015) 64–70

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Relationship between hepcidin and GDF15 in anemic patients with type 2 diabetes without overt renal impairment Jun Hwa Hong a, Yeon-Kyung Choi a, Byong-Keol Min b,c, Kang Seo Park d, Kayeon Seong e, In Kyu Lee a,c, Jung Guk Kim a,* a

Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, South Korea Department of Biomedical Science, Graduate School, Kyungpook National University, Daegu, South Korea c BK21 Plus KNU Biomedical Convergence Program (Brain Korea 21 Plus Project for Bio-Medical Convergence Program for Creative Talent), Kyungpook National University, Daegu 702-701, South Korea d Department of Internal Medicine, Eulji University School of Medicine, Daejeon, South Korea e College of Nursing, Taegu Science University, Daegu, South Korea b

article info

abstract

Article history:

Aims: Despite the absence of overt renal impairment and decreased erythropoietin (EPO)

Received 17 February 2015

levels, patients are usually anemic. Hepcidin, which is induced by inflammatory stimuli,

Received in revised form

plays an important role in anemia in chronic disease. Growth differentiation factor 15

20 April 2015

(GDF15) is a putative anti-inflammatory cytokine that is elevated in type 2 diabetes (T2DM).

Accepted 1 May 2015

Hence, we investigated the relationship between hepcidin and GDF15 in anemic T2DM

Available online 11 May 2015

patients without overt renal impairment. Methods: Among 1150 patients who visited Kyungpook National University Hospital for

Keywords:

T2DM between June 2006 and June 2014, we selected 55 anemic patients without overt renal

Anemia

impairment (serum creatinine <1.5 mg/dL or estimated glomerular filtration rate >60 mL/

Type 2 diabetes

min/1.73 m2) and other co-morbid diseases, including malignancy, thyroid disease, rheu-

Hepcidin

matic arthritis, liver disease, iron-deficiency anemia and other endocrine disease. We

GDF15

measured anthropometric and metabolic parameters, as well as measured the serum iron, ferritin, interleukin-6 (IL-6), erythropoietin, hepcidin-25 and GDF15 levels. Results: Anemic T2DM patients without overt renal impairment presented a greater inflammatory state, with increased serum hsCRP, ESR and IL-6 levels compared with non-anemic T2DM patients. Both hepcidin and GDF15 levels were increased and showed a positive correlation in anemic T2DM patients. Conclusion: In the absence of overt renal impairment, anemia in T2DM is associated with chronic inflammation, inducing elevation of hepcidin and GDF15 levels independently of the erythropoietin level. # 2015 Elsevier Ireland Ltd. All rights reserved.

* Corresponding author. Tel.: +82 53 420 5566; fax: +82 53 426 2046. E-mail address: [email protected] (J.G. Kim). http://dx.doi.org/10.1016/j.diabres.2015.05.001 0168-8227/# 2015 Elsevier Ireland Ltd. All rights reserved.

diabetes research and clinical practice 109 (2015) 64–70

1.

Introduction

In Korea, 10.9% of females and 2.3% of males have anemia, and the prevalence of anemia increases with age [1]. Type 2 diabetes (T2DM) patients are more frequently anemic. The major contributing factor of anemia is renal impairment accompanying T2DM [2,3]. The deficient production of erythropoietin (EPO) is regarded as a mechanism underlying the development of anemia in T2DM [4]. However, according to Craig et al., anemia is also present in T2DM, despite an elevated level of EPO in the absence of nephropathy [5]. Thus, we sought to identify the factors underlying the development of anemia in T2DM patients without overt renal impairment. Hepcidin, a cysteine-rich peptide, was discovered in 2001. Hepcidin is a urinary antimicrobial peptide that is produced in the liver [6]. Further studies have elucidated the role of hepcidin as a regulator of cellular iron efflux by binding and internalization of ferroportin, an iron efflux channel. Inhibition of ferroportin by hepcidin negatively regulates intestinal iron absorption and iron efflux from macrophages, thus balancing the level of circulating iron [7]. Hepcidin is closely correlated with anemia of chronic disease (ACD). Inflammation during chronic disease increases the expression and production of hepcidin in the liver, and the elevated level of hepcidin inhibits the function of ferroportin. Thus, decreased efflux of iron to serum aggravates ACD [8]. Anemia of chronic inflammation with elevated hepcidin has been reported in rheumatic arthritis, sepsis, and malignancy, which are chronic inflammatory diseases [9–11]. Several pathways are involved in the regulation of hepcidin expression. Among them, interleukin-6 (IL-6) induced by chronic inflammation is suggested to be a transcriptional regulator of hepcidin in ACD. Growth differentiation factor-15 (GDF15), a putative anti-inflammatory cytokine, is also suggested to be a regulator of hepcidin. In T2DM, GDF15 was associated with insulin resistance, and GDF15 levels were upregulated in patients with compared to those without T2DM [12–14]. We investigated the expression of hepcidin in anemic T2DM patients without overt renal insufficiency and analyzed the relationship between hepcidin and GDF15.

2.

Materials and methods

2.1.

Recruitment and eligibility

Between June 2006 and June 2014, we collected overnightfasting blood samples from outpatients and hospitalized patients with T2DM who visited Kyungpook National University Hospital, Daegu, Korea and who provided informed consent to participate in this research. A total of 1150 T2DM patients were recruited in this study. Of them, we selected anemic patients using the World Health Organization (WHO) criteria of anemia, a hemoglobin level <13 g/dL in males and <12 g/dL in females [15]. We excluded patients with renal impairment of serum creatinine levels over 1.5 mg/dL, or estimated glomerular filtration rate (eGFR) levels below 60 mL/min/1.73 m2. According to a review of

65

electronic medical records (EMRs), we excluded additional patients who were diagnosed with a malignancy, thyroid disease, rheumatic arthritis, liver disease, iron-deficiency anemia and other endocrine diseases; we also excluded those who were taking steroid medication. Finally, a total of 55 anemic patients with T2DM and a total of 63 age-matched non-anemic T2DM patients as controls were enrolled.

2.2.

Anthropometric and biochemical measurements

Weight and height were measured in light outdoor clothing without shoes. The body mass index (BMI) (kg/m2) was calculated as the weight (kg) divided by the square of the height (m2). The waist-to-hip ratio was calculated using the waist (cm) and hip (cm) girths at the end of expiration. All blood samples were taken in the morning after an overnight fast of >8 h and were immediately centrifuged and stored at 80 8C until thawing for analysis. The fasting plasma glucose was measured using a Beckman Glucose Analyzer (Beckman Instruments, Irvine, CA, USA). Homeostasis model assessment of insulin resistance (HOMA-IR) values, representing the insulin resistance, were calculated as follows: fasting plasma glucose (mg/dL)  fasting insulin level (mU/mL)/405. The glycosylated hemoglobin (HbA1c) level was analyzed using a Cobas1 Integra 800 automated biochemistry analyzer (Roche, Basel, Switzerland). The serum lipid profiles, including the total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-cholesterol) and low-density lipoprotein cholesterol (LDL-cholesterol) levels, were measured by enzymatic methods (Dimension AR; Dade Behring Inc., Deerfield, IL, USA). The estimated glomerular filtration rate (eGFR) was also calculated using the Cockcroft-Gault equation as follows: eGFR = (140-age)  body weight (kg)/72/serum creatinine level (mg/dL)  (0.85, if female).

2.3.

Iron, ferritin, IL-6, EPO measurement

Serum iron was measured using a colorimetric assay, and serum ferritin was measured using a electrochemiluminescence immunoassay (ECLIA). Both iron and ferritin were measured using the E170 for Modular Analytics (Roche Diagnostics, Mannheim, Germany). Erythropoietin measurements using a chemiluminescent immunoassay (CLIA) with IMMUNOLITE 2000 EPO (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) were analyzed by the Immulite 2000 XPI system (Siemens). The IL-6 level was measured by enzyme-linked immunosorbent assay (ELISA) using the Quantikine1 HS Human IL-6 immunoassay kit (R&D Systems, Minneapolis, MN, USA). All ELISA data were analyzed using a microplate reader (VersaMaxTM; Molecular Devices, Sunnyvale, CA, USA).

2.4.

Hepcidin-25 and GDF15 measurement

We measured the levels of hepcidin-25, the bioactive form (hormone form with 25 amino acids) of hepcidin. We used DRG1 hepcidin-25 (bioactive) ELISA (catalog number: EIA5258), which is a solid-phase ELISA based on the competitive principle, for the quantitative measurement of serum hepcidin-25. We applied the quantitative sandwich enzyme

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diabetes research and clinical practice 109 (2015) 64–70

immunoassay technique to measure fasting serum GDF15 levels using ELISA (R&D systems, Minneapolis, MN, USA; Quantikine1 ELISA, Human GDF15, catalog number: DGD150) according to the manufacturer’s instructions.

Table 1 – Baseline characteristics of T2DM patients with or without anemia.

2.5.

Age (years) Sex (M/F) Weight (kg) Height (cm) BMI (kg/m2) WHR WBC (103/mL) Hb (g/dL) MCV (fL) MCHC (g/dL) Creatinine (mg/dL) eGFR (mL/min/1.73 m2) ACR (mg/g creatinine) HbA1c (%) (mmol/mol) DM duration (years) T Chol (mg/dL) TG (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) AST (IU/L) ALT (IU/L) FPG (mg/dL) Insulin (mIU/mL) C-peptide (ng/mL) HOMA-IR

Statistical analysis

All of the parameter values are presented as means  stanstandard deviation (SD). Independent t-test was used for twogroup analysis of the anemic and non-anemic T2DM patient groups. To analyze the relationships between hepcidin and GDF15 with anemic T2DM, Pearson’s correlation coefficient was used. Logistic regression analysis was applied to evaluate the contribution of hepcidin to the risk of anemia in T2DM without overt renal impairment. Statistical significance was considered at a p value less than 0.05 (two-tailed analysis). Statistical analysis was performed using SPSS 20 for Windows (IBM Statistical Package for Social Sciences, Chicago, IL, USA).

2.6.

Ethics

The study protocol was reviewed and approved by the Institutional Review Board of Kyungpook National University Hospital. Oral and written informed consents were obtained from all participants in this study.

3.

Results

3.1.

Anthropometric analysis of the study population

The clinical characteristics of the study population are shown in Table 1. The mean hemoglobin levels were 14.4  0.8 mg/dL in the non-anemic T2DM group and 11.3  0.9 mg/dL in the anemic T2DM group. All of the hemoglobin levels of the enrolled patients were in the mild-to-moderate anemic range without any severe anemic level by the WHO criteria. A female with the lowest level of hemoglobin at 8.8 mg/dL showed negative results in stool occult blood test and endoscopy. All of the participants with anemic T2DM had normocytic normochromic anemia. To rule out the effect of aging on anemia, we selected non-anemic T2DM patients randomly in an agematched manner. The mean age of the study participants was approximately 57 years, and there was no significant difference between the two groups. The prevalence of anemia without renal impairment in T2DM was 4.7% (25/537) in males, 4.9% (30/613) in females and 4.8% (55/1150) in total. The sex ratio showed no significant difference between the two groups. Anemic T2DM patients presented a lower weight (58.2  11.9 vs. 64.9  9.6 kg, respectively; p = 0.001) and lower BMI (22.3  3.7 vs. 24.2  2.3 kg/m2, respectively; p = 0.001) than non-anemic T2DM patients. However, there was no difference in the waist-to-hip ratio (0.92  0.04 vs. 0.93  0.04, respectively; p = 0.501) between the two groups.

3.2.

Metabolic parameters

All of the participants were T2DM patients, and the median duration of diabetes was 10.3  6.4 years in the non-anemic

Non-anemic T2DM (N = 63)

Anemic T2DM (N = 55)

p-Value

57.8  8.7 32/31 64.9  9.6 163.4  8.7 24.2  2.3 0.93  0.04 6.6  1.3 14.4  0.8 89.84  3.90 34.47  0.97 0.76  0.15 91.5  16.2

54.1  11.1 25/30 58.2  11.9 161.5  10.0 22.3  3.7 0.92  0.04 6.2  1.7 11.3  0.9 89.40  5.00 33.82  1.04 0.72  0.22 91.6  37.7

0.714 0.411 0.001 0.264 0.001 0.501 0.155 <0.001 0.603 0.001 0.266 0.985

18.5  21.1

53.2  74.0

0.001

8.4  1.5 (68.3  16.4) 10.3  6.4

9.2  2.6 (77.0  28.4) 12.5  8.8

0.051 0.117

173.8  44.0 137.0  64.3 110.5  40.9 46.9  12.2 22.4  9.4 27.9  13.4 155.9  53.7 11.7  41.3 2.32  1.37 4.36  15.36

158.0  43.3 125.1  77.4 97.9  33.0 46.4  16.6 23.5  11.1 21.3  12.3 172.0  85.1 10.2  13.2 2.07  2.00 4.32  13.71

0.055 0.367 0.076 0.847 0.594 0.007 0.261 0.747 0.459 0.990

Values are presented as means  standard deviation. T2DM, type 2 diabetes mellitus; BMI, body mass index; GDF15, growth differentiation factor-15; WHR, waist-to-hip ratio; WBC, white blood cell; Hb, hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; eGFR, estimated glomerular filtration rate; ACR, albumin creatinine ratio; T Chol, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; AST, aspartate transaminase; ALT, alanine transaminase; FPG, fasting plasma glucose; HOMA-IR, homeostatic model assessment-insulin resistance.

T2DM group and 12.5  8.8 years in the anemic T2DM group; that was not significant difference. Few patients had drugnaı¨ve diabetes. In the glycemic control state, anemic T2DM patients presented with a higher HbA1c level (9.2  2.6%) than non-anemic T2DM patients (8.4  1.5%). Although the anemia may affect the elevated HbA1c level, there was no significant difference between the two groups. Furthermore, there was no significant difference in the fasting plasma glucose, insulin, and c-peptide levels. HOMA-IR also did not show any difference between the two groups. Comparing the inflammatory parameters, anemic T2DM patients showed a higher hsCRP (2.78  2.68 mg/L vs. 1.03  0.81 mg/L, respectively; p < 0.001) and ESR (23.2  19.5 vs. 10.1  9.3, respectively; p < 0.001) than non-anemic T2DM patients. The level of IL-6 was also elevated in anemic T2DM patients (4.54  2.34 ng/mL vs. 1.69  1.05 ng/mL, respectively; p < 0.001). Although we excluded T2DM patients with overt renal impairment using

67

diabetes research and clinical practice 109 (2015) 64–70

serum creatinine and eGFR, anemic patients presented with a higher level of ACR (53.2  74.0 mg/g vs. 18.5  21.1 mg/g creatinine, respectively; p = 0.001). In the analysis of lipid profiles, total cholesterol, LDL-cholesterol, triglyceride and HDL-cholesterol levels showed no significant difference.

3.3. Elevation of hepcidin-25 and GDF15 were in anemic T2DM patients The mean serum hepcidin-25 level of anemic T2DM patients was more than twofold that of non-anemic patients (13.52  5.59 ng/mL vs. 6.29  2.86 mg/mL, respectively; p < 0.001). The mean serum GDF15 level of anemic patients was also more elevated than that of non-anemic patients (1609.26  691.70 pg/mL vs. 713.63  168.96 pg/mL, respectively; p < 0.001). However, the erythropoietin level showed no significant difference between anemic and non-anemic T2DM patients in the absence of renal impairment (10.41  7.20 mIU/ mL, 12.95  14.67 mIU/mL, p = 0.119). The serum iron level was lower in the anemic T2DM group than in the non-anemic T2DM group (71.60  29.56 mg/dL vs. 88.12  46.90 mg/dL, respectively; p = 0.038). The mean serum ferritin level was lower in anemic patients, albeit not significantly so (Table 2).

3.4. Correlations of hepcidin with GDF15 and metabolic parameters The correlation of hepcidin with metabolic parameters by Pearson’s coefficient correlation analysis is shown in Table 3. Serum hepcidin-25 presented a negative correlation with serum hemoglobin (r = 0.526, p < 0.001) and positive correlation with IL-6 and hsCRP levels (r = 0.483, p < 0.001 and r = 0.341, p < 0.001, respectively). Serum GDF15 had a strongly positive correlation with serum hepcidin-25 (r = 0.643, p < 0.001) (Fig. 1).

3.5. Contribution of hepcidin to the risk of anemia in T2DM without overt renal impairment In logistic regression analysis, hepcidin contributed to the risk of anemia in T2DM without overt renal impairment (odds ratio: 1.847; 95% confidence interval: 1.434–2.379, p < 0.001)

Table 2 – Iron status and inflammatory parameters of T2DM patients with or without anemia.

hsCRP (mg/L) ESR (mm/h) IL-6 (pg/mL) Iron (mg/dL) Ferritin (ng/mL) EPO (mIU/mL) Hepcidin-25 (ng/mL) GDF15 (pg/mL)

Non-anemic T2DM (N = 63)

Anemic T2DM (N = 55)

p-Value

1.03  0.81 10.1  9.3 1.69  1.05 88.12  46.90 212.02  301.10 10.41  7.20 6.29  2.86

2.78  2.68 23.2  19.5 4.54  2.34 71.60  29.56 140.02  107.97 12.95  14.67 13.52  5.59

<0.001 <0.001 <0.001 0.038 0.119 0.246 <0.001

713.63  168.96

1609.26  691.70

<0.001

hsCRP, high-sensitivity C-reactive protein; ESR, erythrocyte sedimentation rate; IL-6, interleukin-6; EPO, erythropoietin; GDF15, growth differentiation factor-15.

Table 3 – Correlation analysis of hepcidin with metabolic parameters. Pearson’s coefficient (r) Age DM duration BMI Hemoglobin Iron Ferritin eGFR Erythropoietin HbA1c HOMA-IR ACR IL-6 hsCRP GDF15

0.000 0.223 0.180 0.526 0.142 0.023 0.062 0.104 0.013 0.004 0.138 0.483 0.341 0.643

p-Value 0.999 0.015 0.051 <0.001 0.165 0.822 0.504 0.264 0.891 0.968 0.135 <0.001 <0.001 <0.001

BMI, body mass index; eGFR, estimated glomerular filtration rate; HOMA-IR, homeostatic model assessment-insulin resistance; ACR, albumin creatinine ratio; IL-6, interleukin-6; hsCRP, high-sensitivity C-reactive protein; GDF15, growth differentiation factor-15.

when adjusted for age, sex, eGFR, HbA1c, EPO and DM duration (Table 4).

4.

Discussion

The present cross-sectional study showed that T2DM patients without overt renal impairment also presented with anemia with an incidence of 5.2% (5.0% in males, 5.4% in females). The elevated hepcidin level might be associated with the presence of anemia in normal renal function. Regarding T2DM as a chronic inflammatory disease, the level of GDF15, an antiinflammatory cytokine, was also increased and showed a positive correlation with hepcidin. The incidence of anemia in T2DM with normal renal function is unknown. Additionally, no large population-based study of this issue has been preformed. Some studies have reported the incidence of anemic T2DM patients as approximately 15%. Adejumo et al. reported the incidence of anemia in T2DM without renal impairment as 15.3% and that poorly controlled diabetes (elevated HbA1c) and old age were correlated with the anemia [16]. Many studies have validated the risk factors for anemia in T2DM without renal impairment. Poorly controlled glucose in T2DM was suggested to be a risk factor for anemia [17]. In that study, autonomic neuropathy, which is provoked by poor glycemic control, decreased the production of erythropoietin prior to renal impairment. However, the mechanism underlying the correlation between autonomic neuropathy and the attenuated production of erythropoietin remains unknown. Other studies have suggested risk factors for anemia, including damage to the renal glomerulus by advanced glycemic end products [18] and depressed androgen levels [19]. Oxidative stress and systemic inflammation were reported to increase the risk of anemia [20]. That study showed an inverse correlation of oxidative stress and inflammatory markers with eGFR, thus affecting the presence of anemia. In the progressed renal disease state,

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diabetes research and clinical practice 109 (2015) 64–70

Fig. 1 – Correlation of hepcidin with GDF15 in T2DM without overt renal impairment.

highly activated or acute inflammation might be a major contributing factor to the development of anemia. T2DM is a chronic inflammatory disease, and the degree of inflammation is occasionally mild in the absence of complications, including nephropathy [21]. From a pathophysiological view of the development of anemia in T2DM, ACD is an appropriate model. Patients with ACD often show low plasma iron and transferrin saturation, despite normal or elevated body iron stores or levels of ferritin [22]. The major factor contributing to

Table 4 – Determination of the risk of anemia in T2DM without overt renal impairment by logistic regression analysis. Variables Age Sex eGFR HbA1c Erythropoietin DM duration Hepcidin

Odds ratio

95% CI

p-Value

1.001 2.379 0.987 1.289 1.074 1.046 1.847

0.934–1.073 0.711–7.960 0.950–1.025 0.954–1.742 1.000–1.153 0.958–1.142 1.434–2.379

0.978 0.160 0.492 0.098 0.049 0.313 <0.001

eGFR, estimated glomerular filtration rate; DM, diabetes mellitus; CI, confidence interval. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/ certificate/R1ow0A.

disrupted iron balance is hepcidin. Acute or chronic inflammatory stimuli increase the level of the inflammatory cytokine IL-6 and then stimulate hepcidin expression in the liver [23,24]. In our study, serum IL-6 levels were increased in anemic T2DM patients, despite the absence of renal dysfunction. Consequently, the serum level of hepcidin also increased, suggesting that patients with T2DM without overt renal impairment were also exposed to inflammation, and thus could develop anemia. A prospective study is required to confirm that anemia with elevated hepcidin levels without overt renal impairment could be used to identify candidates for renal replacement therapy. GDF15 is a member of the transforming growth factor-beta (TGF-b) superfamily [25]. The expression of GDF15 is elevated in many types of malignancies, cardiovascular diseases, inflammatory diseases and type 2 diabetes [26–29]. Although the exact role and mechanism are as-yet-unknown, the putative role of GDF15 is as an anti-inflammatory cytokine. Several studies have investigated the role of GDF15 in the regulation of hepcidin. According to Tanno et al., higher levels of GDF15 suppressed the expression of hepcidin in thalassemia patients [30]. In contrast to high levels of GDF15, the expression of hepcidin and GDF15 levels showed a positive and increasing correlation until GDF levels were <1000 pg/ml. The suppressive effect of GDF15 was present at GDF15 levels >10,000 pg/ml. Another study concerning the relationship between GDF15 and hepcidin showed a bidirectional correlation in cancer patients. At GDF15 levels <2000 pg/ml, there was a positive correlation between GDF15 and the expression

diabetes research and clinical practice 109 (2015) 64–70

of hepcidin. However, GDF15 levels >6000 pg/ml showed a negative correlation between GDF15 and hepcidin [31]. GDF15 levels are occasionally elevated in cancer patients [32]. Higher GDF15 levels were associated with more advanced tumor stages and a high prevalence of metastasis, and thus a positive correlation with risk of death from cancer [33,34]. Elevated GDF15 levels in advanced cancers suppress the expression of hepcidin and induce the iron overload that is frequently observed in cancer-related anemic patients. In T2DM, the GDF15 level was in the range 700 to 1100 pg/ml [14]. In our data, the GDF15 level in T2DM was in the range 700 to 1600 pg/ml, suggesting that mild inflammation in T2DM resulted in secretion of a lower level of GDF15 than in thalassemia and malignancies. In that GDF15 concentration range, the two prior studies and our findings showed a positive correlation of GDF15 with hepcidin. Interestingly, our data showed that anemic T2DM patients without renal impairment presented a lower BMI than nonanemic patients with T2DM without renal impairment. Obesity is an inflammatory disease associated with ACD [35,36]. Similarly, obesity associated with chronic inflammation increased the expression of hepcidin, thus showing an ACD phenotype. However, obese participants with ACD in the previous study displayed severe obesity and a BMI over 30 kg/ m2. The correlation of severe obesity with chronic inflammation is well known and is associated with metabolic syndrome and metabolic diseases [37]. Contrary to the aforementioned theory, Peter et al. reported a high prevalence of anemia in unmarried females with a lower BMI [38]. The average BMI of participants was 19 kg/m2. The causes of anemia in that study included a low socioeconomic status, low food intake, and a weight loss tendency. In our study, the BMI range of the participants was 22 to 24 kg/m2. These values are included in the normal to overweight criteria according to the Korean Society for the Study of Obesity (KSSO). Although we did not investigate the nutritional and socio-economic statuses of all of the participants, our data might exclude the effect of inflammation derived from severe obesity and associated inflammatory signals. To elucidate the low BMI of anemic T2DM patients without overt renal impairment in this study, further analysis of lifestyle will be required. Additionally, although the anemic and non-anemic T2DM groups were in the normal eGFR range and did not differ significantly, our data showed that anemic T2DM patients excreted more urinary albumin (ACR: 53.2  74.0 mg/g creatinine vs. 18.5  21.1 mg/g creatinine, respectively; p < 0.001). The pathophysiology of microalbuminuria suggests that endothelial dysfunction and podocyte dysfunction might alter the charge-selective permeability barrier, and thus reduce the reabsorption of filtered albumin in the kidney [39,40]. In a large epidemiologic study, microalbuminuria was associated with worsening renal function to more advanced stages and endstage renal disease, and is also a risk factor for cardiovascular mortality [41,42]. In a large, collaborative meta-analysis, a urinary ACR above 10 mg/g creatinine increased the risk of allcause mortality and cardiovascular mortality. Even in subjects with normal renal function (eGFR >90 mL/min/1.73 m2), the hazard ratio for cardiovascular mortality was 1.63 (ACR range 10 to 20 mg/g creatinine) and 1.82 (ACR range 30 to 299 mg/g creatinine), and the decreasing eGFR over time accentuated

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the increase in ACR [43]. According to our data, isolated microalbuminuria without renal dysfunction was associated with anemia in T2DM. Although ACR didn’t show the significant correlation with hepcidin in Table 3, the level of GDF15 presented positive correlation with urinary ACR (r = 0.217, p = 0.018, data are not shown). The research for the investigation of GDF15 and urinary albuminuria is required to predict the progression of renal dysfunction in T2DM.

5.

Conclusions

Anemia in T2DM without overt renal impairment is associated with elevated levels of hepcidin and GDF15, a marker of chronic inflammation, independently of the erythropoietin level.

Conflict of interest statement The authors declare that they have no conflicts of interest.

Acknowledgment This work was supported by Biomedical Research Institute grant, Kyungpook National University Hospital (2013).

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