J Korean Med Sci.  2014 Sep;29(Suppl 2):S155-S163. 10.3346/jkms.2014.29.S2.S155.

Effect of Bilirubin on Triglyceride Synthesis in Streptozotocin-Induced Diabetic Nephropathy

• Xu J
• Lee ES
• Baek SH
• Ahn SY
• Kim S
• Na KY
• Chae DW
• Chin HJ

• KMID: 2069807
• DOI: http://doi.org/10.3346/jkms.2014.29.S2.S155

Abstract

We aimed to elucidate the effect of bilirubin on dyslipidemia and nephropathy in a diabetes mellitus (DM) type I animal model. Sprague-Dawley rats were separated into control, DM, and bilirubin-treated DM (Bil) groups. The Bil group was injected intraperitoneally with 60 mg/kg bilirubin 3 times per week and hepatoma cells were cultured with bilirubin at a concentration of 0.3 mg/dL. The Bil group showed lower serum creatinine levels 5 weeks after diabetes onset. Bilirubin treatment also decreased the amount of mesangial matrix, lowered the expression of renal collagen IV and transforming growth factor (TGF)-beta1, and reduced the level of apoptosis in the kidney, compared to the DM group. These changes were accompanied by decreased tissue levels of hydrogen superoxide and NADPH oxidase subunit proteins. Bilirubin decreased serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), free fatty acids, and triglycerides (TGs), as well as the TG content in the liver tissues. Bilirubin suppressed protein expression of LXRalpha, SREBP-1, SCD-1, and FAS, factors involved in TG synthesis that were elevated in the livers of DM rats and hepatoma cells under high-glucose conditions. In conclusion, bilirubin attenuates renal dysfunction and dyslipidemia in diabetes by suppressing LXRalpha and SREBP-1 expression and oxidative stress.

Keyword

Bilirubin; Diabetes-Related Complications; Triglyceride; Fibrosis; Transforming Growth Factor beta

MeSH Terms

Animals
Bilirubin/pharmacology/*therapeutic use
Cell Line, Tumor
Creatine/blood
Diabetes Mellitus, Experimental/chemically induced/complications/*pathology
Diabetic Nephropathies/*drug therapy/etiology
Disease Models, Animal
Kidney/pathology
Lipoproteins, HDL/blood
Liver/metabolism
Male
Mice
Mice, Inbred C57BL
NADPH Oxidase/metabolism
Orphan Nuclear Receptors/antagonists & inhibitors/genetics/metabolism
Oxidative Stress/drug effects
Rats
Rats, Sprague-Dawley
Reactive Oxygen Species/metabolism
Streptozocin/toxicity
Triglycerides/analysis/*biosynthesis/blood
Bilirubin
Creatine
Lipoproteins, HDL
Orphan Nuclear Receptors
Reactive Oxygen Species
Triglycerides
Streptozocin
NADPH Oxidase

Figure

• 

  Fig. 1 Changes in kidney tissue. (A) Representative image of PAS staining in kidney tissue (×400). (B) The ratios of mesangial area to total glomerular area were averaged from 30 glomeruli per rat. Ratios were increased in DM rats compared to controls, an effect that was decreased by bilirubin treatment (Bil group). *P = 0.002, †P = 0.248 compared to the control group and ‡P = 0.035 compared to DM group, as determined by analysis of variance (ANOVA and post-hoc Tukey test). (C) Western blot analysis of collagen IV in kidney tissue lysates. (D) The relative density ratios of collagen IV in each group compared to control group, normalized to actin. Collagen IV was increased in the DM group compared to control group and was decreased by bilirubin treatment. *P = 0.045, †P = 0.999 compared to the control and ‡P = 0.048 compared to DM group (ANOVA and Tukey post-hoc test). (E) Collagen IV expression in cultured mesangial cells in high glucose media (HG; 450 mg/dL of D-glucose). Bilirubin (0.3 mg/dL) was dissolved in DMSO prior to culturing cells in HG conditions. (F) The concentration of TGF-β1, measured in kidney tissue protein extracts by ELISA, was also increased in DM group. This effect was attenuated in the Bil group. *P = 0.019, †P = 0.991 compared to the control and ‡P = 0.014 compared to DM group (ANOVA and Tukey post-hoc test). (G) TUNEL staining of glomeruli. The red arrow indicates apoptotic cells with condensed nuclear DNA. (H) The ratio of TUNEL positive to total glomerular cells. *P < 0.001, †P = 0.020 compared to the control and ‡P = 0.001 compared to the DM group (ANOVA and Tukey post-hoc test). The bars on the graph indicate the 95% confidence interval of the mean values.

• 

  Fig. 2 NADPH oxidase subunits and hydrogen peroxide in kidney tissues. (A) The relative amount of hydrogen peroxide in kidney lysates (100 mg protein). Peroxide levels were lower in the Bil group than in the DM group. (B) Western blot analysis of NOX4 and p22phox levels of kidney tissue extracts. The levels of NOX4 (C) and p22phox (D) were significantly decreased by bilirubin treatment (0.3 mg/dL). The bars on the graph indicate the 95% confidence interval of the mean values. P values were estimated using the Student's t-test.

• 

  Fig. 3 Expression of factors that regulate TG synthesis. Western blot analysis of factors related to synthesis of TG in the kidney (A) and liver (B). Diabetic conditions increased the levels of LXRα, SREBP-1, FAS, and SCD-1 in kidney and liver tissues. These effects were attenuated by bilirubin treatment. The relative expression of LXRα and SREBP-1 in the kidney (C, D) and liver (E, F), normalized to actin are shown. *P < 0.05 in F compared to the DM group (ANOVA and Tukey post-hoc test). The bars on the graph indicate standard errors of the mean values.

• 

  Fig. 4 Expression of factors that regulate TG synthesis in hepatoma cells. (A) Western blot analysis of factors related to synthesis of TG in hepatoma cell lines. The relative levels of SREBP-1 and LXRα normalized to actin were increased in cells cultured in HG medium compared to those cultured in normal glucose medium. These effects were attenuated by bilirubin treatment (B). *P < 0.001 compared to the HG group (ANOVA and Tukey post-hoc test). (C) Western blot analysis of SREBP-1 in untransfected cells and those transfected with LXRα siRNA, cultured in high glucose media. HC; untreated control cells (no bilirubin or siRNA); HL: cells transfected with siRNA; BilC cells treated with bilirubin but not transfected. (D) The relative level of SREBP-1 normalized to actin. *P < 0.001, †P = 0.003 compared to the HG group and ‡P = 0.010 compared to the HL group (ANOVA and Tukey post-hoc test). The bars on the graph indicate the 95% confidence interval of the mean values.

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