Relationship between serum bilirubin concentrations and diabetic nephropathy in Shanghai Han’s patients with type 1 diabetes mellitus
- Xu Li†1,
- Lei Zhang†1,
- Haibing Chen†1,
- Kaifeng Guo1,
- Haoyong Yu1,
- Jian Zhou1,
- Ming Li1,
- Qing Li1,
- Lianxi Li1,
- Jun Yin1,
- Fang Liu1,
- Yuqian Bao1,
- Junfeng Han1Email author and
- Weiping Jia1Email author
© The Author(s). 2017
Received: 27 October 2015
Accepted: 24 March 2017
Published: 31 March 2017
Recent studies highlight a negative association between total bilirubin concentrations and albuminuria in patients with type 2 diabetes mellitus. Our study evaluated the relationship between bilirubin concentrations and the prevalence of diabetic nephropathy (DN) in Chinese patients with type 1 diabetes mellitus (T1DM).
A total of 258 patients with T1DM were recruited and bilirubin concentrations were compared between patients with or without diabetic nephropathy. Multiple stepwise regression analysis was used to examine the relationship between bilirubin concentrations and 24 h urinary microalbumin. Binary logistic regression analysis was performed to assess independent risk factors for diabetic nephropathy. Participants were divided into four groups according to the quartile of total bilirubin concentrations (Q1, 0.20–0.60; Q2, 0.60–0.80; Q3, 0.80–1.00; Q4, 1.00–1.90 mg/dL) and the chi-square test was used to compare the prevalence of DN in patients with T1DM.
The median bilirubin level was 0.56 (interquartile: 0.43–0.68 mg/dL) in the DN group, significantly lower than in the non-DN group (0.70 [interquartile: 0.58–0.89 mg/dL], P < 0.001). Spearman’s correlational analysis showed bilirubin concentrations were inversely correlated with 24 h urinary microalbumin (r = -0.13, P < 0.05) and multiple stepwise regression analysis showed bilirubin concentrations were independently associated with 24 h urinary microalbumin. In logistic regression analysis, bilirubin concentrations were significantly inversely associated with nephropathy. In addition, in stratified analysis, from the first to the fourth quartile group, increased bilirubin concentrations were associated with decreased prevalence of DN from 21.90% to 2.00%.
High bilirubin concentrations are independently and negatively associated with albuminuria and the prevalence of DN in patients with T1DM.
KeywordsType 1 diabetes mellitus Diabetic nephropathy Bilirubin concentrations
Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide, which remains a major cause of morbidity and mortality in patients with T1DM . Oxidative stress may be a common pathway linking diverse, seemingly distinct, potential mechanisms underlying the pathogenesis of complications in diabetes, including nephropathy . Bilirubin is the end product of haem catabolism and it acts as a powerful biological antioxidant [3, 4]. A study on Gilbert syndrome (GS) reported that the prevalence of ischemic heart disease (IHD) was 2% in GS patients (who are characterised by high bilirubin concentrations) compared to 12.1% in the general population, indicating that chronic hyperbilirubinemia prevents the development of IHD by increasing the antioxidant capacity of serum . In a recent study that used the deoxycorticosterone acetate (DOCA)-salt model of hypertension in Heine oxygenase (HO)-1-/- and HO-1+/+ mice, systolic arterial pressure was significantly elevated in HO-1-/- mice treated with DOCA salt but not in HO-1+/+ mice; in addition, DOCA-salt impaired vasorelaxation was noted in wild-type rats but not in hyperbilirubinemic rats . These results suggest that the HO-1 isozyme and the product bilirubin may have protective effects on vascular disease. This finding has also been confirmed in a model of balloon injury . In addition, diabetic hyperbilirubinemic Gunn j/j rats excrete significantly less urinary albumin than diabetic non-hyperbilirubinemic Gunn j/+ rats and administration of biliverdin (5 mg/kg) protects against both albuminuria and renal mesangial expansion in db/db mice . These findings suggest that bilirubin and biliverdin may protect against DN. A population-based study showed that high bilirubin concentrations in serum are associated with reduced risk of DN . Furthermore, several longitudinal studies on healthy subjects and type 2 diabetes mellitus (T2DM) also demonstrated that low serum bilirubin concentration could be a novel risk factor for the development of albuminuria in patients with type 2 diabetes [10, 11]. However, renal disease remains a major cause of morbidity and mortality in patients with T1DM and the association between bilirubin concentrations in serum and the prevalence of diabetic nephropathy in patients with T1DM has not yet been studied. Therefore, in the present study, we evaluated the association between bilirubin concentrations in serum and the prevalence of DN, and we hypothesized that bilirubin concentrations may inversely associate with the prevalence of DN in Chinese patients with T1DM.
This was a cross-sectional, population-based study involving 258 patients with T1DM. Participants were hospitalised patients who presented at the Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People’s Hospital between January 2008 and January 2013. T1DM was defined as anti-glutamate decarboxylase (GAD) antibody level ≥ 1.5 U/mL and injecting insulin at least three times daily or using an insulin pump, and DN were diagnosed according to KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for Diabetes and Chronic Kidney Disease for 2007 . Hypertension was diagnosed if the patient had a blood pressure greater than 140/90 mmHg or used anti-hypertensive drugs. Subjects were excluded for the following reasons: absence of data (bilirubin, haemoglobin A1c [HbA1c]), abnormal thyroid function (hyperthyroidism or hypothyroidism), elevated serum levels of creatinine (>124 μmol/L), serum bilirubin concentrations > 2.0 mg/dL, and T2DM or specific diabetes. In addition, patients were excluded if they had confounding hepatobiliary or haemolytic disease, hepatitis B or C, alcoholic liver disease, gallstones, cirrhosis, IgA nephropathy, or urinary tract infections.
Clinical and laboratory measurements
The clinical parameters investigated included age, height, weight, duration of diabetes, systolic blood pressure (SBP), diastolic blood pressure (DBP), waist circumference (WC), hip circumference, and waist-to-hip ratio (WHR). Biochemical variables were analysed after an overnight fast of at least 10 h and included fasting plasma glucose (FPG), 2 h postprandial glucose (2hPG), glycated haemoglobin A1c (HbA1c), glycated albumin (GA), C-reactive protein (CRP), 30-min postprandial venous C peptide, 120-min postprandial venous C peptide, albumin, aspartate aminotransferase (AST), alamine aminstransferase (ALT), gamma glutamytransferase (γ-GT), total cholesterol (TC), triglyceride level (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), haemoglobin (Hb), 24 h urinary microalbumin, total GFR, uric acid, creatinine (Cr), and total bilirubin.
Subject height was measured to the nearest 0.1 cm with subjects not wearing shoes and weight was measured to the nearest 0.1 kg while the subjects wore light clothing; BMI was calculated as weight (kg) divided by the square of the height (m). SBP and DBP were measured after participants had rested for at least 5 min. WC (cm) was measured midway between the costal margin and the iliac crest at the end of a normal expiration and the hip circumference was measured as the circumference around both greater femoral trochanters. The WHR was calculated by dividing waist by hip circumference (cm). All biochemical determinations were performed using the same standard laboratory methods. After overnight fasting, blood was drawn early in the morning from the antecubital vein into vacuum tubes to determine the concentrations of fasting plasma glucose (FPG), C peptide, CRP, and concentrations of lipid components and liver enzymes. Bilirubin concentrations from serum samples were determined using the vanadate oxidation method.
Measurement of GFR using the 99mTc-diethylene triamine pentaacetic acid (99mTc-DTPA) renal dynamic imaging method, and 99mTc-DTPA renal dynamic imaging (modified Gate’s method) was measured by Millennium TMMPR SPECT from General Electric Medical System . Urine albumin excretions were evaluated by calculating total amounts of 24 h urinary microalbumin. The stage of albuminuria was defined as normal if no more than 30 mg/24 h, microalbuminuria if 24 h urinary microalbumin was 30–299 mg/24 h, and macroalbuminuria if 24 h urinary microalbumin was equal to or higher than 300 mg/24 h .
Data are expressed as the means + standard deviation (SD) for a normal distribution of variables or as the median (interquartile range) for a skewed distribution of variables. For continuous data with a normal distribution and a skewed distribution between DN and non-DN patients, unpaired Student’s t-tests and nonparametric tests, respectively, were used for statistical analyses. Categorical variables were compared using a chi-square test. Spearman’s correlational analysis between 24 h urinary microalbumin, and other variables was performed. Multiple stepwise regression analysis was performed to assess the relationship between bilirubin concentrations and 24 h urinary microalbumin. Binary logistic regression analysis was performed to assess independent risk factors for diabetic nephropathy. Participants were divided into four groups according to the quartile of total bilirubin concentrations and the chi-square test was used to compare the prevalence of DN in patients with T1DM. The first quartile was 0.20–0.60 mg/dL, second quartile was 0.60–0.80 mg/dL, third quartile was 0.80–1.00 mg/dL, and fourth quartile was 1.00–1.90 mg/dL. All statistical analyses were performed using SPSS version 17.0 for Windows (SPSS, Chicago, IL, USA). A P-value less than 0.05 was considered to indicate statistical significance.
Patient demographics and laboratory data
Patient demographics and laboratory data
DN (n = 33)
Non-DN (n = 225)
Duration of diabetes (years)
12.74 + 6.73
14.03 + 5.57
Uric acid (mg/dL)
Fasting C peptide (ng/mL)
30-min postprandial venous C peptide (ng/mL)
120-min postprandial venous C peptide (ng/mL)
Total Bilirubin (mg/dL)
4.75 + 1.34
4.41 + 0.99
24 h urinary microalbumin (mg/24 h)
Total GFR (mL/min)
Bilirubin concentrations in serum are independently and negatively associated with 24 h urinary microalbumin
Correlations of 24 h urinary microalbumin with bilirubin concentrations and other characteristics in subjects with T1DM
24 h urinary microalbumin (unadjusted)
Duration of diabetes
Fasting C peptide
30-min postprandial venous C peptide
120-min postprandial venous C peptide
Multiple stepwise regression analysis showing the variables independently associated with 24 h urinary microalbumin
Bilirubin concentrations in serum are independently associated with diabetic nephropathy
Independent risk factors associated with the diabetic nephropathy in patients with type 1 diabetes
Duration of diabetes (years)
Total bilirubin (mg/d)
Bilirubin concentrations in serum are negatively associated with the prevalence of DN in patients with T1DM
Prevalence of DN in patients with T1DM according to quartiles of serum bilirubin concentrations
OR (95% CI)
OR (95% CI)
Q1 (n = 64)
Q2 (n = 76)
Q3 (n = 68)
Q4 (n = 50)
P trend for ORs
Several previous studies have reported that high bilirubin concentrations in serum are negatively associated with the incidence of hypertension  and T2DM . In addition, a recent study demonstrated that bilirubin concentrations were significantly negatively correlated with log (UAE) in patients with type 1 diabetes . We performed a cross-sectional study to examine whether bilirubin concentrations in serum are associated with the prevalence of DN in patients with T1DM. We found that bilirubin concentrations were independently and negatively associated with albuminuria and the prevalence of DN in patients with T1DM. We deduced a range of bilirubin concentrations (0.80–1.90 mg/dL) that may serve as protective factors for the development of DN in Chinese patients with T1DM and likely represent a pharmacologically attractive target for slowing the development of DN.
We found that bilirubin concentrations and HbA1c level were not relevant (r = 0.06, P = 0.334) in 258 patients with type 1 diabetes mellitus (33 patients with diabetic nephropathy, 225 patients without diabetic nephropathy). However, a study from Choi SW et al. evaluated the relationship between HbA1c and bilirubin in 690 patients with type 2 diabetes mellitus and found that bilirubin concentrations were negatively associated with HbA1c, independent of gender, age, and other confounding factors . Because several studies have confirmed that high bilirubin concentrations are inversely associated with insulin resistance [20–22], T1DM is possibly due to β-cell destruction and leads to absolute insulin deficiency rather than insulin resistance. Furthermore, results from Mianowska B et al. showed that serum total bilirubin concentration is an independent factor inversely associated with HbA1c level in young patients with type 1 diabetes (type 1 diabetes duration of more than 12 months, age from 2 to 18 years) , which means that in T1DM, the results are controversial. Thus, we infer that the unsynchronized results of relationship between serum bilirubin concentration and HbA1c in patients with diabetes mellitus were attributed to the age, duration of diabetes mellitus and glycemic control. Therefore, we admitted that further study should be designed to investigate the relationship between serum bilirubin concentration and HbA1c in type 1 diabetes mellitus.
Although several studies have been performed, the exact mechanism underlying the negative association between high bilirubin concentrations and the progression of T1DM remains unknown. A previous study demonstrated that diabetic hyperbilirubinemic Gunn j/j rats with high concentrations of unconjugated bilirubin in plasma excreted significantly less urinary albumin than diabetic non-hyperbilirubinemic Gunn j/+ rats, and that administration of biliverdin (5 mg/kg) protected against both albuminuria and renal mesangial expansion in db/db mice. The same authors subsequently discovered that treatment with bilirubin and biliverdin completely inhibited oxidative stress-induced increased expression of NOX4 mRNA and protein levels of cultured vascular endothelial and mesangial cells . These findings suggest that bilirubin and biliverdin significantly inhibit NADPH-dependent superoxide production. A study using an isolated, perfused rat kidney model demonstrated that micromolar dose administration of exogenous bilirubin resulted in significantly improved renal vascular resistance, urine output, glomerular filtration rate, tubular function, and mitochondrial integrity after ischemia-reperfusion injury (IRI) and showed that bilirubin pretreatment may have future clinical applications, particularly in IRI after organ transplantation . Furthermore, studies on diabetic rats have shown that exogenously administered CO or bilirubin can protect endothelial cells from oxidative stress-mediated injury . A study on hyperbilirubinemia caused by atazanavir treatment in 16 subjects with T2DM indicated that hyperbilirubinemia is associated with a significant improvement in endothelial function . Considering that oxidative stress, described as increased levels of reactive oxygen species, may be a common pathway linking diverse mechanisms underlying the pathogenesis of complications in diabetes  and that hyperglycemia-induced increases in blood pressure and changes in endothelial cells are reversed by antioxidants [27, 28], high concentrations of bilirubin in serum may potentially serve as protective factors against the development of DN through their antioxidative properties.
This study had several limitations. First, only total bilirubin concentrations in serum were measured, without distinguishing between conjugated versus unconjugated bilirubin. Second, this was a single-center, cross-sectional study, which prevented us from drawing conclusions regarding the temporal nature of the observed association between bilirubin concentrations and the prevalence of DN in Chinese patients with T1DM. Third, the study population consisted of Chinese males and females and whether our findings apply to other ethnic groups remains unclear.
In conclusion, our study evaluated the association between total bilirubin and albuminuria, the prevalence of DN in Chinese patients with T1DM. Our data indicate that high bilirubin concentrations in serum may be protective factors for the development of DN.
Glomerular filtration rate
Ischemic heart disease
Type 1 diabetes mellitus
The authors are grateful for support from the Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People’s Hospital. We are also extremely appreciative of all of the participants for their dedication in data collection and laboratory measurements.
This work was supported by a grant the National Natural Sciences Foundation of China (81200564) to Junfeng Han.
Availability of data and materials
The dataset supporting the conclusions of this article is available from the corresponding author on reasonable request.
JFH and WPJ participated in the design of the study. LZ and HBC performed the statistical analysis. XL is involved in drafting the manuscript. KFG, HYY, JZ, ML, QL, LXL, JY, FL, YQB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was performed according to the principles of the Declaration of Helsinki and was pre-approved by the Ethics Committee of Shanghai Jiaotong University Affiliated Sixth People’s Hospital, and all subjects provided written informed consent prior to participating.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Raine AE. The rising tide of diabetic nephropathy--the warning before the flood? Nephrol Dial Transplant. 1995;10:460–1.View ArticlePubMedGoogle Scholar
- Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40:405–12.View ArticlePubMedGoogle Scholar
- Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science. 1987;235:1043–6.View ArticlePubMedGoogle Scholar
- Balla J, Vercellotti GM, Jeney V, Yachie A, Varga Z, Eaton JW, Balla G. Heme, heme oxygenase and ferritin in vascular endothelial cell injury. Mol Nutr Food Res. 2005;49:1030–43.View ArticlePubMedGoogle Scholar
- Vítek L, Jirsa M, Brodanová M, Kalab M, Marecek Z, Danzig V, Novotný L, Kotal P. Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels. Atherosclerosis. 2002;160:449–56.View ArticlePubMedGoogle Scholar
- Nath KA, d’Uscio LV, Juncos JP, Croatt AJ, Manriquez MC, Pittock ST, Katusic ZS. An analysis of the DOCA-salt model of hypertension in HO-1-/- mice and the Gunn rat. Am J Physiol Heart Circ Physiol. 2007;293:H333–42.View ArticlePubMedGoogle Scholar
- Peyton KJ, Shebib AR, Azam MA, Liu XM, Tulis DA, Durante W. Bilirubin inhibits neointima formation and vascular smooth muscle cell proliferation and migration. Front Pharmacol. 2012;3:48.View ArticlePubMedPubMed CentralGoogle Scholar
- Han SS, Na KY, Chae DW, Kim YS, Kim S, Chin HJ. High Serum Bilirubin Is Associated with the Reduced Risk of Diabetes Mellitus and Diabetic Nephropathy. Tohoku J Exp Med. 2010;221:133–40.View ArticlePubMedGoogle Scholar
- Toya K, Babazono T, Hanai K, Uchigata Y. Association of serum bilirubin levels with development and progression of albuminuria, and decline in estimated glomerular filtration rate in patients with type 2 diabetes mellitus. J Diabetes Investig. 2014;5:228–35.View ArticlePubMedGoogle Scholar
- Jung CH, Lee MJ, Kang YM, Hwang JY, Jang JE, Leem J, Park JY, Kim HK, Lee WJ. Higher serum bilirubin level as a protective factor for the development of diabetes in healthy Korean men: a 4 year retrospective longitudinal study. Metabolism. 2014;63:87–93.View ArticlePubMedGoogle Scholar
- Okada H, Fukui M, Tanaka M, Matsumoto S, Kobayashi K, Iwase H, Tomiyasu K, Nakano K, Hasegawa G, Nakamura N. Low serum bilirubin concentration is a novel risk factor for the development of albuminuria in patients with type 2 diabetes. Metabolism. 2014;63:409–14.View ArticlePubMedGoogle Scholar
- KDOQI. KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for Diabetes and Chronic Kidney Disease. Am J Kidney Dis. 2007;49(2 Suppl 2):S12–154.Google Scholar
- Gates GF. Split renal function testing using Tc-99 m DTPA. A rapid technique for determining differential glomerular filtration. Clin Nucl Med. 1983;8:400–7.View ArticlePubMedGoogle Scholar
- Viberti GC, Hill RD, Jarrett RJ, Argyropoulos A, Mahmud U, Keen H. Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet. 1982;1:1430–2.View ArticlePubMedGoogle Scholar
- Mogensen CE, Christensen CK. Predicting diabetic nephropathy in insulin-dependent patients. N Engl J Med. 1984;311:89–93.View ArticlePubMedGoogle Scholar
- Chin HJ, Song YR, Kim HS, Park M, Yoon HJ, Na KY, Kim Y, Chae DW, Kim S. The bilirubin level is negatively correlated with the incidence of hypertension in normotensive Korean population. J Korean Med Sci. 2009;24(Suppl):S50–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Ohnaka K, Kono S, Inoguchi T, Yin G, Morita M, Adachi M, Kawate H, Takayanagi R. Inverse associations of serum bilirubin with high sensitivity C-reactive protein, glycated hemoglobin, and prevalence of type 2 diabetes in middle-aged and elderly Japanese men and women. Diabetes Res Clin Pract. 2010;88:103–10.View ArticlePubMedGoogle Scholar
- Nishimura T, Tanaka M, Sekioka R, Itoh H. Serum bilirubin concentration is associated with eGFR and urinary albumin excretion in patients with type 1 diabetes mellitus. J Diabetes Complications. 2015;29:1223–7.View ArticlePubMedGoogle Scholar
- Choi SW, Lee YH, Kweon SS, Song HR, Ahn HR, Rhee JA, Choi JS, Shin MH. Association between Total Bilirubin and Hemoglobin A1c in Korean Type 2 Diabetic Patients. J Korean Med Sci. 2012;27:1196.View ArticlePubMedPubMed CentralGoogle Scholar
- Song YS, Koo BK, Cho NH, Moon MK. Effect of Low Serum Total Bilirubin levels in serum (≤0.32 mg/dL) on Risk of Coronary Artery Disease in Patients With Metabolic Syndrome. Am J Cardiol. 2014;114:1695–700.View ArticlePubMedGoogle Scholar
- Kwak MS, Kim D, Chung GE, Kang SJ, Park MJ, Kim YJ, Yoon JH, Lee HS. Serum bilirubin levels are inversely associated with nonalcoholic fatty liver disease. Clin Mol Hepatol. 2012;18:383.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin LY, Kuo HK, Hwang JJ, Lai LP, Chiang FT, Tseng CD, Lin JL. Serum bilirubin is inversely associated with insulin resistance and metabolic syndrome among children and adolescents. Atherosclerosis. 2009;203:563–8.View ArticlePubMedGoogle Scholar
- Mianowska B, Kamińska A, Fendler W, Szadkowska A, Młynarski W. Bilirubin is an independent factor inversely associated with glycated hemoglobin level in pediatric patients with type 1 diabetes. Pediatr Diabetes. 2014;15:389–93.View ArticlePubMedGoogle Scholar
- Adin CA, Croker BP, Agarwal A. Protective effects of exogenous bilirubin on ischemia-reperfusion injury in the isolated, perfused rat kidney. Am J Physiol Renal Physiol. 2005;288:F778–84.View ArticlePubMedGoogle Scholar
- Rodella L, Lamon BD, Rezzani R, Sangras B, Goodman AI, Falck JR, Abraham NG. Carbon monoxide and biliverdin prevent endothelial cell sloughing in rats with type I diabetes. Free Radic Biol Med. 2006;40:2198–205.View ArticlePubMedGoogle Scholar
- Dekker D, Dorresteijn MJ, Pijnenburg M, Heemskerk S, Rasing-Hoogveld A, Burger DM, Wagener FA, Smits P. The Bilirubin-Increasing Drug Atazanavir Improves Endothelial Function in Patients With Type 2 Diabetes Mellitus. Arterioscler Thromb Vasc Biol. 2011;31:458–63.View ArticlePubMedGoogle Scholar
- Marfella R, Verrazzo G, Acampora R, La Marca C, Giunta R, Lucarelli C, Paolisso G, Ceriello A, Giugliano D. Glutathione reverses systemic hemodynamic changes induced by acute hyperglycemia in healthy subjects. Am J Physiol. 1995;268:E1167–73.PubMedGoogle Scholar
- Curcio F, Ceriello A. Decreased cultured endothelial cell proliferation in high glucose medium is reversed by antioxidants: new insights on the pathophysiological mechanisms of diabetic vascular complications. In Vitro Cell Dev Biol. 1992;28A:787–90.View ArticlePubMedGoogle Scholar