J Korean Diabetes Assoc.  2007 May;31(3):261-273. 10.4093/jkda.2007.31.3.261.

Activation of NF-kappaB and AP-1 in Peripheral Blood Mononuclear Cells Isolated from Patients with Diabetic Nephropathy

• Nam J
• Cho MH
• Park JS
• Lee GT
• Kim HJ
• Kang ES
• Lee YM
• Ahn CW
• Cha BS
• Lee EJ
• Lim SK
• Kim KR
• Ha HJ
• Lee HC

• KMID: 1523015
• DOI: http://doi.org/10.4093/jkda.2007.31.3.261

Abstract

BACKGROUND: We evaluated the role of oxidative stress in diabetic nephropathy by measuring intracellular reactive oxygen species (ROS) and redox-sensitive transcription factors in isolated peripheral mononuclear cells (PBMC).
METHODS
From 66 diabetic patients with or without diabetic nephropathy (Group III and II, respectively) and 49 normal control subjects (Group I), spontaneous and stimulated ROS levels, activities of nuclear factor-kappa B (NF-kappaB), activator protein-1 (AP-1), and specificity protein1 (Sp1) in PBMC, urinary and PBMC TGF-beta1 (transforming growth factor-beta1), and 24-hour urinary albumin excretion (UAE) were measured.
RESULTS
Spontaneous ROS was significantly higher in group III and II than group I (60.7 +/- 3.3 vs. 60.0 +/- 3.0 vs. 41.1 +/- 2.4%, respectively), and stimulated ROS were significantly higher in Group III compared to Group II (Increment of H2O2-induced ROS production: 21.8 +/- 2.2 vs. 11.1 +/- 2.0%, respectively; increment of PMA-induced ROS production 23.5 +/- 4.5 vs. 21.6 +/- 2.2%, respectively). The activities of NF-kappaB and AP-1, but not of Sp1, were significantly higher in Group III than in Group II (2.53 vs. 2.0 vs. 1.43-fold, respectively). Both PBMC- and urinary TGF-beta1 levels were higher in Group III than Group II (3.23 +/- 0.39 vs. 1.99 +/- 0.68 ng/mg in PBMCs, 16.88 +/- 6.84 vs. 5.61 +/- 1.57 ng/mL in urine, both respectively), and they were significantly correlated with activities of NF-kappaB and AP-1 and 24-hour UAE.
CONCLUSIONS
Increased intracellular ROS generation in PBMCs of diabetic patients is involved in the pathogenesis of diabetic nephropathy through activation of NF-kappaB and AP-1, but not Sp1, and increased expression of TGF-beta1.

Keyword

AP-1; Diabetic nephropathy; NF-kappaB; Oxidative stress; Sp1; TGF-beta1

MeSH Terms

Diabetic Nephropathies*
Humans
NF-kappa B*
Oxidative Stress
Reactive Oxygen Species
Sensitivity and Specificity
Transcription Factor AP-1*
Transcription Factors
Transforming Growth Factor beta1
NF-kappa B
Reactive Oxygen Species
Transcription Factor AP-1
Transcription Factors
Transforming Growth Factor beta1

Figure

• 

  Fig. 1 Intracellular ROS in PBMC isolated from healthy control subject, diabetic patients without nephropathy, and patients with diabetic nephropathy. Fig. 1-A, spontaneous ROS level; 1-B, H2O2 stimulated ROS level; 1-C, PMA stimulated ROS level in Group I (normal healthy control group); 1-D, spontaneous ROS level; 1-E, H2O2 stimulated ROS level; 1-F, PMA stimulated ROS level in Group II (diabetic patients without nephropathy); 1-G, spontaneous ROS level; 1-H, H2O2 stimulated ROS level; 1-I, PMA stimulated ROS level in Group III (patients with diabetic nephropathy). Spontaneous, H2O2 and PMA-induced ROS production in ex vivo isolated peripheral blood mononuclear cells was measured spectrophotometrically using the fluorescent dye technique.

• 

  Fig. 2 The specificity of oligonucleotides for NF-κB, AP-1 and Sp1. In order to evaluate the reliability of this study, we analyzed the specificity of oligonucleotide for NF-κB, AP-1 and Sp1 by competetion EMSA with labeled and unlabeled ("cold") oligonucleotides, respectively. The band formed from interaction between nuclear extract and labeled NF-κB disappeared when "cold NF-κB" was added, and the same effect was seen with AP-1 and Sp1, thus showing the specificity of oligonucleotides for NF-κB, AP-1 and Sp1.

• 

  Fig. 3 The activities of NF-κB, AP1, and Sp1 in PBMC in different groups. Activities of transcription factors were quantified using EMSA and densitometry (Biorad). *P < 0.05, compared to group I, †P < 0.05, compared to group II. Group I, Normal healthy control; Group II, Diabetic patients without nephropathy; Group III, Diabetic patients with nephropathy; UAE, urine albumin excretion.

• 

  Fig. 4 The expressions of urinary and PBMC-TGF-β1 in each group. TGF-β1 was measured in PBMC lysate and in urine by quantitative sandwich enzyme immunoassay. *P < 0.05, compared to group I, †P < 0.05, compared to group II. Group I, Normal healthy control; Group II, Diabetic patients without nephropathy; Group III, Diabetic patients with nephropathy; PBMC-TGF-β1, TGF-β1 in PBMC; uTGF-β1, urinary TGF-β1. The expression of TGF-β1 in PBMC and urine was significantly higher in patients with diabetic nephropathy than in diabetic patients without nephropathy.

• 

  Fig. 5 Correlations between 24 h UAE and urinary TGF-β1 (A), activity of NF-κB and urinary TGF-1 β(B), activity of AP-1 and urinary TGF-β1 (C), and activity of Sp1 and urinary TGF-β1 (D). PBMC-TGF-β1: TGF-β1 in PBMC; uTGF-β1: urinary TGF-β1; ROD: Relative optical density. Urinary TGF-β1 protein was significantly correlated with 24-hour albumin excretion (r = 0.729, P = 0.001). Moreover, the expression of TGF-β1 was not found to be significantly correlated with the Sp1 activity, but was significantly correlated with NF-κB and AP-1 activities (r = 0.786, r = 0.826, respectively, all P < 0.001).

• 

  Fig. 6 Correlations between 24 h UAE and PBMC-TGF-β1 (A), activity of NF-κB and PBMC-TGF-β1 (B), activity of AP-1 and PBMC-TGF-β1 (C), and activity of Sp1 and PBMC-TGF-β1 (D). PBMC-TGF-β1: TGF-β1 in PBMC; uTGF-β1: urinary TGF-β1; ROD: Relative optical density. TGF-β1 protein in PBMC was found to be significantly correlated with 24-hour albumin excretion, NF-κB, and AP-1 activities (r = 0.694, r = 0.797, r = 0.086, respectively, all P = 0.001), but was not correlated with Sp1 activity (r = 0.549, P = 0.071).

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