Low serum bilirubin concentration is a novel risk factor for the development of albuminuria in patients with type 2 diabetes

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Available online at www.sciencedirect.com

Metabolism www.metabolismjournal.com

Low serum bilirubin concentration is a novel risk factor for the development of albuminuria in patients with type 2 diabetes Hiroshi Okada a , Michiaki Fukui a,⁎, Muhei Tanaka a , Shinobu Matsumoto a , Kanae Kobayashi a , Hiroya Iwase a , Kiichiro Tomiyasu b , Koji Nakano c , Goji Hasegawa a , Naoto Nakamura a a

Department of Endocrinology and Metabolism, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan b Department of Cardiology, Kyoto Yamashiro General Medical Center, Japan c Department of Endocrinology and Metabolism, Kyoto Yamashiro General Medical Center, Japan

A R T I C LE I N FO Article history:

AB S T R A C T Objective. Bilirubin has been recognized as an important endogeneous antioxidant.

Received 30 July 2013

Previous studies reported that bilirubin could prevent atherosclerosis. The aim of this study

Accepted 13 November 2013

was to investigate if serum bilirubin concentration could be a predictor for the development of albuminuria in patients with type 2 diabetes.

Keywords:

Materials and Methods. We measured serum bilirubin in 320 consecutive patients with

Endogeneous antioxidant

normoalbuminuria. We performed follow-up study to assess the development of

Diabetic nephropathy

albuminuria, mean interval of which was 3.2 ± 0.9 years. Cox proportional hazards

Diabetes

regression was used to examine the relationship between serum bilirubin concentration and the development of albuminuria. Results. During follow-up duration, 43 patients have developed albuminuria. In multivariate analysis, after adjusting for comprehensive risk factors, the risk of developing albuminuria was higher in the lowest quartile of serum bilirubin concentrations than that in the highest quartile of serum bilirubin concentrations (Hazard ratio, 5.76; 95% CI, 1.65 to 24.93). Conclusions. Low serum bilirubin concentration could be a novel risk factor for the development of albuminuria in patients with type 2 diabetes. © 2014 Elsevier Inc. All rights reserved.

1.

Introduction

Cardiovascular disease (CVD) has become the most common cause of mortality and morbidity in patients with type 2 diabetes, and several risk factors including smoking, hyper-

tension and dyslipidemia have been shown to accelerate the progression of CVD [1,2]. Albuminuria, the earliest manifestation of nephropathy, is a marker of increased cardiovascular mortality and the progression of CVD [3,4]. The finding of microalbuminuria is an indication for screening for possible

Abbreviations: CVD, cardio vascular disease; UAE, urinary albumin excretion; BMI, body mass index; ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blockers; ANOVA, analysis of variance; SBP, systolic blood pressure; CI, confidence interval; CCB, calcium-channel blockers; LDL, low-density lipoprotein; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intercellular adhesion molecule 1. ⁎ Corresponding author. Tel.: +81 75 251 5505; fax: + 81 75 252 3721. E-mail address: [email protected] (M. Fukui). 0026-0495/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.metabol.2013.11.011

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vascular disease and aggressive intervention to reduce all cardiovascular risk factors [5]. Bilirubin has been shown to be an effective endogenous antioxidant [6–9], to suppress the oxidation of lipids and lipoproteins, especially low-density lipoprotein cholesterol [10], and to be directly related to the total serum antioxidant capacity in humans [9]. Because of its antioxidant, bilirubin could act against plaque formation and subsequent atherosclerosis [11]. Previous studies reported that serum bilirubin has been consistently shown to be inversely related to CVD [12–14]. Inoguchi et al. [15] reported lower prevalence of vascular complications such as coronary artery disease, cerebrovascular disease, retinopathy, or macroalbuminuria in patients with diabetes and Gilbert syndrome, a congenital hyperbilirubinemia. Moreover, recent studies showed serum bilirubin to be associated with CVD-related factors such as hypertension [16,17], abnormal glucose tolerance [18], body mass index [16,19,20], metabolic syndrome [19,21], and coronary artery calcification scores [22]. Serum bilirubin has also been inversely related to the severity of nonalcoholic fatty liver disease, a condition closely related to type 2 diabetes and CVD [23]. And, we previously reported that the serum bilirubin concentration is associated with urinary albumin excretion (UAE) in patients with type 2 diabetes in a cross-sectional study [24]. However, a relationship between serum bilirubin concentration and the development of albuminuria in patients with type 2 diabetes has not been investigated. Therefore, we evaluated the relationship between serum bilirubin concentration and the development of albuminuria in patients with type 2 diabetes.

2.

Methods

2.1.

Patients and study design

conducted in accordance with Declaration of Helsinki, and informed consent was obtained from all participants.

2.2.

Data collection

All data had been retrieved from a database. Overnight fasting blood samples were taken in the morning at baseline. Total serum bilirubin concentrations were measured by an enzymatic method with bilirubin oxidase on an automatic analyzer (Hitachi 7600; Hitachi High-Tchnologies, Tokyo, Japan). Serum total cholesterol and triglyceride concentrations were assessed using standard enzymatic methods. Hemoglobin A1c was assayed using high-performance liquid chromatography and expressed with the unit defined by the National Glycohemoglobin Standardization Program. Urinary albumin and creatinine concentration was determined in an early morning spot urine. UAE was measured with an immunoturbidimetric assay. A mean value for UAE was determined from 3 urine collections. As a follow-up study, we collected urine samples for calculation of UAE at the end of the follow-up interval. The development of albuminuria was defined as UAE equal to or more than 30 mg/g Cr [5].

2.3.

Statistical analysis

Means and frequencies of potential confounding variables were calculated. The participants were categorized according to the quartiles of their serum bilirubin concentrations in order to examine the association between patient characteristics at baseline and serum bilirubin concentration: < 0.58, 0.58–0.72,

Table 1 – Characteristics of patients.

We performed a retrospective cohort study with 320 participants recruited from the outpatient clinic at the Kyoto Prefectural University of Medicine and Kyoto Yamashiro General Medical Center from April, 2006 to June, 2013. Type 2 diabetes was diagnosed according to the Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus [25]. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Patients were classified as never smokers, previous smokers, or current smokers according to a self-administered questionnaire. Patients with albuminuria, defined as UAE equal to or more than 30 mg/g creatinine, those with hematuria, bacteriuria, advanced renal dysfunction (serum creatinine more than 2.0 mg/dL), liver cirrhosis, malignancy or hematologic disease, and those with major cardiovascular event during a follow up were excluded from this study. Moreover, we excluded patients (n = 42) who were newly prescribed angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARB) for the first time during follow up period of this study because antihypertensive drugs such as ARB could delay the development of albuminuria in patients with diabetes [5]. We then calculated the hazard ratio for development of albuminuria in patients with normoalbuminuria. This study was approval by the local Research Ethics Committee and was

n Age (y) Sex (male/female) Duration of diabetes (y) Body mass index (kg/m2) Average systolic blood pressure (mmHg) Bilirubin (mg/dL) Hemoglobin A1c (%) Total cholesterol (mmol/L) Triglycerides (mmol/L) Aspartate aminotransferase (IU/L) Alkaline phosphatase (IU/L) Uric acid (μmol/L) Creatinine (μmol/L) Smoking (never/previous/current) Retinopathy (NDR/SDR/PDR) Urinary albumin excretion (mg/g creatinine) History of cardiovascular disease (−/+) Antidiabetic treatment (insulin/OHA/diet only) Antihypertensive drug (calcium channel blockers/ ACE inhibitor/ARB/diuretic drug/alpha blockers/ beta blockers) Statin (−/+)

320 64.0 (10.1) 197/123 12.6 (10.4) 23.4 (4.2) 128.8 (12.9) 0.77 (0.28) 7.4 (1.3) 5.0 (0.8) 1.4 (0.8) 23.6 (15.7) 23.9 (13.7) 302.5 (86.8) 65.2 (21.1) 210/69/41 245/36/39 12.8 (7.5) 269/51 92/197/31 84/14/118/23/ 14/15 179/141

Data are expressed as mean (SD) or absolute number. NDR, no diabetic retinopathy; SDR, simple diabetic retinopathy; PDR, proliferative diabetic retinopathy; OHA, oral hypoglycemic agent; ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blockers.

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0.73–0.91, and > 0.91 mg/dL. The means or percentages were reported for each quartile, and associations were assessed by analysis of variance (ANOVA) or chi-square test, respectively. The differences of general characteristics at baseline according to the development of albuminuria at follow up were assessed by t-test or chi-square test. The relationships between serum bilirubin concentration and other variables were examined by Pearson’s correlation analyses. The association between serum bilirubin concentration and the development of albuminuria was analyzed in Cox proportional hazards regression models. Because triglycerides showed skewed distributions, log transformation was carried out before performing Cox proportional hazards regression models. An unadjusted model and a multivariate model were used for the estimation of association between serum bilirubin concentration and the development of albuminuria. Covariates included in the multivariate model were statistically significant variables in univariate analysis and those known to be related factors for the development of albuminuria. Model 1 is adjusted for hemoglobin A1c and logarithm of triglycerides. Model 2 was adjusted for all variables

in Model 1 plus age, duration of diabetes, average systolic blood pressure (SBP) and baseline UAE. Model 3 was adjusted for all variables in Model 2 plus sex, BMI, total cholesterol, uric acid, smoking status, the use of calcium-channel blockers (CCB), ACE inhibitors, ARB, diuretic drug, alpha blockers, beta blockers and statin. All continuous variables are presented as the mean ± SD, absolute number or percentages. A P value < 0.05 was considered statistically significant. The size, direction, and statistical significance of relationships were estimated by the hazard ratio with 95% confidence interval (CI). The statistical analyses were performed using the JMP version 10 software (SAS Institute, Cary, North Carolina).

3.

Results

Characteristics of the 320 patients with type 2 diabetes enrolled in this study are shown in Table 1. Mean serum bilirubin was 0.77 ± 0.28 mg/dL. The average duration of follow up was 3.2 ± 0.9 years. During the study period, 50 patients have developed

Table 2 – Characteristics at baseline according to quartiles of serum bilirubin. Bilirubin (mg/dL)

n Age (y) Male Duration of diabetes (y) BMI (kg/m2) Average SBP (mmHg) Hemoglobin A1c (%) T-CHO (mmol/L) Triglycerides (mmol/L) AST (IU/L) ALT (IU/L) Uric acid (μmol/L) Creatinine (μmol/L) Smoking Never Previous Current Retinopathy UAE History of CVD Antidiabetic treatment Insulin OHA Diet only Antihypertensive drug CCB ACE inhibitor ARB Diuretic drug Alpha blockers Beta blockers Statin Bilirubin (mg/dL)

Quartile 1

Quartile 2

Quartile 3

Quartile 4

< 0.58

0.58–0.72

0.73–0.91

> 0.91

80 64.0 52.5 13.6 24.3 129.6 7.5 4.9 1.7 21.7 22.6 300.4 65.3

(11.8) (9.8) (5.1) (13.8) (1.5) (0.9) (1.0) (13.1) (12.2) (80.8) (18.4)

80 63.5 62.5 11.6 23.3 128.5 7.4 5.1 1.5 24.3 23.7 314.0 69.8

(8.9) (9.9) (3.7) (13.7) (1.2) (0.8) (0.7) (12.6) (10.9) (101.1) (28.9)

80 64.0 60.0 13.2 22.9 128.3 7.4 5.1 1.4 22.7 23.9 295.8 62.6

(9.6) (12.0) (3.8) (12.0) (1.2) (0.8) (0.7) (11.5) (12.3) (87.7) (17.3)

80 64.4 71.3 12.1 23.2 129.0 7.1 5.0 1.2 25.8 25.4 300.3 63.4

P-value

(10.1) (9.8) (4.3) (12.2) (1.2) (0.8) (0.7) (22.7) (18.1) (78.6) (17.5)

63.8 20 16.2 31.2 12.4 (8.0) 22.5

61.3 22.5 16.2 31.2 14.1 (7.5) 12.5

65.0 22.5 12.5 16.2 12.8 (7.9) 15.0

72.4 21.3 6.3 15.0 11.8 (6.6) 13.8

36.3 59.9 3.8

31.3 59.9 8.8

27.5 62.5 10.0

20.0 63.7 16.3

38.8 2.5 41.3 13.8 5 3.8 52.5 0.46 (0.09)

25.0 7.5 38.8 7.5 6.3 7.5 47.5 0.64 (0.04)

20.0 3.8 32.5 1.3 1.3 3.8 38.8 0.80 (0.05)

21.3 3.8 35.0 6.25 5 3.8 37.5 1.15 (0.21)

0.97 0.33 0.67 0.25 0.94 0.33 0.42 0.03 0.46 0.70 0.71 0.20 0.20

0.08 0.33 0.31 0.03

0.03 0.29 0.61 0.02 0.29 0.46 0.17 < 0.0001

Data are expressed as mean (SD) or percent. BMI, body mass index; SBP, systolic blood pressure; T-CHO, total cholesterol; AST, Aspartate aminotransferase; ALT, Alkaline phosphatase; UAE, urinary albumin excretion; CVD; cardiovascular disease; OHA, oral hypoglycemic agent; CCB, calcium channel blockers; ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blockers. P values are from chi-square tests (for categorical data) or from ANOVA (for continuous data).

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Table 3 – Unadjusted hazard ratios and multivariate adjusted hazard ratios for development of albuminuria. Quartile of serum bilirubin concentration Quartile 1 Crude Model 1 Model 2 Model 3

3.95 3.59 4.00 5.76

(1.47–13.70) (1.29–12.71) (1.37–14.62) (1.65–24.93)

Quartile 2 2.38 2.19 2.53 2.29

(0.81–8.61) (0.74–8.03) (0.84–9.35) (0.67–9.44)

Quartile 3 1.18 1.14 1.19 1.35

(0.34–4.64) (0.32–4.47) (0.32–4.87) (0.33–6.23)

P Quartile 4 1 1 1 1

(reference) (reference) (reference) (reference)

0.005 0.01 0.01 0.005

Model 1 is adjusted for hemoglobin A1c and logarithm of triglycerides. Model 2 includes all variables in Model 1 plus age, duration of diabetes, systolic blood pressure and baseline urinary albumin excretion. Model 3 includes all variables in Model 2 plus sex, body mass index, total cholesterol, uric acid, smoking status, the use of calcium-channel blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, diuretic drug, alpha blockers, beta blockers and statin.

albuminuria. Age (P = 0.01), average SBP (P = 0.0002) and baseline UAE (P < 0.0001) were higher at baseline in patients with the development of albuminuria than those without. Duration of diabetes was longer at baseline in patients with the development of albuminuria than those without (P = 0.03). Serum bilirubin concentration was lower at baseline in patients with the development of albuminuria than those without (P = 0.0004). Patients with the development of albuminuria more frequently used ARB (P = 0.02) or diuretic drugs (P = 0.004) at baseline. Serum bilirubin concentration correlated negatively with hemoglobin A1c (r = -0.13 P = 0.03) and logarithm of triglycerides (r = -0.19, P = 0.002). No significant correlations were found between serum bilirubin concentration and age, duration of diabetes, BMI, average SBP, total cholesterol, uric acid, creatinine and baseline UAE. Table 2 shows characteristics of the study participants at the baseline according to quartiles of serum bilirubin concentrations. Serum triglyceride concentration, antidiabetic treatment and the use of CCB or diuretic drug differ among bilirubin quartiles. Participants in the lowest quartile of serum bilirubin concentrations had higher triglycerides than those in the highest quartile. Participants in the lowest quartile of serum bilirubin concentrations more frequency used insulin, CCB or diuretic drugs. The results of the unadjusted and adjusted Cox regression analyses are shown in Table 3. The unadjusted Cox regression analysis revealed that the risk for the development of albuminuria was higher in the lowest quartile of serum bilirubin concentrations than that in the highest quartile of serum bilirubin concentrations (Hazard ratio, 3.95; 95% CI, 1.47 to 13.70). Additionally, the unadjusted Cox regression analyses demonstrated that age (Hazard ratio, 1.06; 95% CI, 1.02 to 1.11), duration of diabetes (Hazard ratio, 1.03; 95% CI, 1.00 to 1.05), average SBP (Hazard ratio, 1.03; 95% CI, 1.01 to 1.06), logarithm of triglycerides (Hazard ratio, 4.44; 95% CI, 1.10 to 18.31) or baseline UAE (Hazard ratio, 1.07; 95% CI, 1.03 to 1.11) were associated with an increased hazard of the development of albuminuria. The adjusted Cox regression analysis revealed that the risk for the development of albuminuria was higher in the lowest quartile of serum bilirubin concentrations than that in the highest quartile of serum bilirubin concentrations (Model 1, 2 and 3).

4.

Discussion

The major finding of our study is that low serum bilirubin concentration is a risk factor for the development of albu-

minuria after adjustment for the other risk factors. Previous studies reported that low serum bilirubin concentration has been associated with a risk of CVD [12–14], CVD-related factors [16–20,22] and diabetic complications including nephropathy in a cross-sectional study [24] and retinopathy in a cohort study [26]. Then, to the best of our knowledge, this is the first study to investigate the association between the development of albuminuria and serum bilirubin concentration in patients with type 2 diabetes. The postulated pathogenesis in the development of diabetic nephropathy included increased polyol pathway flux, over expression of transforming groth factor-β [27], increased advanced glycation end-product formation [28], activation of protein kinase C [29], increased oxidative stress [30–32] and microinflammation [33–35]. To support the mechanisms of bilirubin actions in preventing atherosclerosis and diabetic nephropathy, several mechanisms have been reported. First, bilirubin inhibits oxidation of low-density lipoprotein (LDL) cholesterol [6,8]. LDL cholesterol is highly susceptible to oxidation, and it is known that the atherogenic process involves uptake of oxidized LDL by intimal macrophages leading to the accumulation of lipid-rich foam cells. Yesiloza et al. [36] showed that the lipophilic bilirubin is more effective at protecting lipids from oxidation than the watersoluble antioxidants such as glutathione and that oxidized LDL concentrations were significant lower in Gilbert’s syndrome compared to healthy control. Second, bilirubin inhibits oxidative stress through an inhibitory effect on the activity of NAD(P)H oxidase as an antioxidant [37]. The oxidative stress in the kidney is increased in diabetic condition compared to control [38]. Sasaki et al. [39] reported that the marker of oxidative stress such as urinary 8-hydroxy-2’-deoxyguanosine was reduced in the diabetic patients with Gilbert’s syndrome. Fujii et al. [40] showed that bilirubin and biliverdin, the precursor of bilirubin, protect against diabetic nephropathy through inhibition of NADPH-dependent superoxide production. Lastly, bilirubin has anti-inflammatory property. Mazzone et al. [41] showed that bilirubin inhibits the tumor necrosis factor-α induced gene upregulation of endothelial adhesion molecules including E-selectin, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). E-selectin may play a key role in leukocyte infiltration into the renal interstitium [42]. ICAM-1 and VCAM-1 are critically involved in the pathogenesis of diabetic nephropathy [43]. Moreover, Tapan et al. [44] reported that the soluble forms of CD40 ligand and high sensitive C reactive

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protein concentrations were significantly lower in Gilbert’s syndrome compared to the controls and both CD40 ligand and high sensitive C reactive protein were negatively correlated with serum bilirubin. The properties of bilirubin described above might be some of the important mechanisms which explain the association between serum bilirubin concentration and the development of albuminuria. There are several limitations in this study. Duration of hypertension could be also clinically significant variable in terms of the development of albuminuria and physical exercise may affect UAE. Unfortunately, however, we have no data about duration of hypertension and physical exercise. The study population consisted of Japanese men and women, therefore, it is uncertain whether these findings are generalized in other ethnic groups. In addition, the number of patients available for analysis was not large and the follow-up was rather short and the number of patients who developed albuminuria was small in this study. Because this study is retrospective study, data had been retrieved from database. Large prospective trials are needed to better assess the relationship between serum bilirubin concentration and the development of albuminuria in patients with type 2 diabetes. Bilirubin could have an important role for the prevention of the development of albuminuria, however, interventional methods to increase serum bilirubin concentration have not been established in human. Then, patients with low serum bilirubin concentration might require aggressive lifestyle modifications and medication to lower blood glucose and blood pressure for the prevention of the development of albuminuria. To the best of our knowledge, this is the first study to investigate if serum bilirubin concentration could be a predictor for the development of albuminuria in patients with type 2 diabetes. In conclusion, low serum bilirubin concentration could be a novel risk factor for the development of albuminuria.

Author contributions H.O. researched data and wrote manuscript. M.F., M.T. and G.H. researched data and contributed to discussion. S.M., K.K., H.I. K.T. and K.N. researched data. N.N. researched data and reviewed and edited the manuscript. M.F. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Conflict of interest No potential conflicts of interest relevant to this article were reported.

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