J Korean Med Sci.  2014 Sep;29(Suppl 2):S139-S145. 10.3346/jkms.2014.29.S2.S139.

Cobalt Chloride Attenuates Oxidative Stress and Inflammation through NF-kappaB Inhibition in Human Renal Proximal Tubular Epithelial Cells

• Oh SW
• Lee YM
• Kim S
• Chin HJ
• Chae DW
• Na KY

• KMID: 2069805
• DOI: http://doi.org/10.3346/jkms.2014.29.S2.S139

Abstract

We evaluated the effect of cobalt chloride (CoCl2) on TNF-alpha and IFN-gamma-induced-inflammation and reactive oxygen species (ROS) in renal tubular epithelial cells (HK-2 cells). We treated HK-2 cells with CoCl2 before the administration of TNF-alpha/IFN-gamma. To regulate hemeoxygenase-1 (HO-1) expression, the cells were treated CoCl2 or HO-1 siRNA. CoCl2 reduced the generation of ROS induced by TNF-alpha/IFN-gamma. TNF-alpha/IFN-gamma-treated-cells showed an increase in the nuclear translocation of phosphorylated NF-kappaBp65 protein, the DNA-binding activity of NF-kappaBp50 and NF-kappaB transcriptional activity and a decrease in IkappaBalpha protein expression. These changes were restored by CoCl2. We noted an intense increase in monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) production in TNF-alpha/IFN-gamma-treated cells. We demonstrated that this effect was mediated through NF-kappaB signaling because an NF-kappaB inhibitor significantly reduced MCP-1 and RANTES production. CoCl2 effectively reduced MCP-1 and RANTES production. The expression of HO-1 was increased by CoCl2 and decreased by HO-1 siRNA. However, knockdown of HO-1 by RNA interference did not affect MCP-1 or RANTES production. We suggest that CoCl2 has a protective effect on TNF-alpha/IFN-gamma-induced inflammation through the inhibition of NF-kappaB and ROS in HK-2 cells. However, CoCl2 appears to act in an HO-1-independent manner.

Keyword

Cobalt Chloride; Hemeoxygenase-1; Inflammation; Nuclear Factor-kappa B; Renal Tubular Epithelial Cells

MeSH Terms

Cell Line
Chemokine CCL2/metabolism
Chemokine CCL5/metabolism
Cobalt/*pharmacology
Epithelial Cells/cytology/metabolism
Heme Oxygenase-1/antagonists & inhibitors/genetics/metabolism
Humans
*Inflammation
Interferon-gamma/pharmacology
Kidney Tubules, Proximal/cytology
NF-kappa B/antagonists & inhibitors/genetics/*metabolism
NF-kappa B p50 Subunit/genetics/metabolism
Oxidative Stress/*drug effects
Phosphorylation
Protein Binding
RNA Interference
RNA, Small Interfering/metabolism
Transcription Factor RelA/metabolism
Tumor Necrosis Factor-alpha/pharmacology
Chemokine CCL2
Chemokine CCL5
Cobalt
Heme Oxygenase-1
Interferon-gamma
NF-kappa B
NF-kappa B p50 Subunit
RNA, Small Interfering
Transcription Factor RelA
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Effects of CoCl2 pre-treatment on TNF-α/IFN-γ-induced ROS generation. TNF-α/IFN-γ-induced increases in intracellular ROS were revealed by the fluorescence intensity of 2',7'-dichlorofluorescein. The fluorescence intensities of the cells are shown as the relative intensities of the experimental groups compared with control cells. A representative set of three independent experiments is shown. The results are expressed as the mean±s.d. *P<0.05, compared with control cells. †P<0.05, compared with TNF-α-+INF-γ-treated cells. C, control cells; TI, TNF-α/IFN-γ-treated cells; TIC, TNF-α/IFN-γ-treated cells with CoCl2 pre-treatment; Co, CoCl2-pre-treated cells.

• 

  Fig. 2 Effects of CoCl2 pre-treatment on TNF-α/IFN-γ-induced NF-κB activation. (A) Western blotting of NF-κB, phosphorylated NF-κB, and IκBα in nuclear and cytoplasmic extracts of HK-2 cell lysates. The intensities of the bands corresponding to phosphorylated NF-κB in nuclear extracts and IκBα in whole cell lysates are shown as the relative intensities of experimental groups compared with control cells. (B) The DNA-binding activity of NF-κBp50 was analyzed in the nuclear extracts of HK-2 cells using ELISA. (C) NF-κB-driven transcription is expressed as relative luciferase activity in HK-2 cells. A representative set of three independent experiments is shown. The results are expressed as the mean±s.d. *P<0.05, compared with control cells. †P<0.05, compared with TNF-α-+IFN-γ-treated cells. C, control cells; TI, TNF-α/INF-γ-treated cells; TIC, TNF-α/IFN-γ-treated cells with CoCl2 pre-treatment; Co, CoCl2-pre-treated cells.

• 

  Fig. 3 The expression of hemeoxygenase-1 (HO-1). (A) Western blotting of HO-1 in HK-2 cells. HK-2 cells were pre-treated with 150 µM CoCl2 for 24 hr and then treated with 5 ng/mL TNF-α and 50 ng/mL IFN-γ for an additional 24 hr. Beta-actin was used as a loading control. A representative set of three independent experiments is shown. The densitometric ratios of HO-1 and β-actin in cells are shown as the relative ratios of experimental groups compared with control cells. The results are expressed as the mean ± s.d. *P<0.01, compared with control cells. C, control cells; TI, TNF-α/IFN-γ-treated cells; TIC, TNF-α/IFN-γ-treated cells with CoCl2 pre-treatment; Co, CoCl2-pre-treated cells. (B) Western blotting of HO-1 in HK-2 cells. GAPDH was used as a loading control. A representative set of three independent experiments is shown. The densitometric ratios of HO-1 and GAPDH in cells are shown as the relative ratios of experimental groups compared with control cells. The results are expressed as the mean±s.d. *P<0.001, compared with control cells. †P<0.005, compared with CoCl2-pre-treated cells.

• 

  Fig. 4 Effects of CoCl2, an NF-κB inhibitor, and HO-1 RNA interference on TNF-α/IFN-γ-induced cytokine production. (A) The production of RANTES and MCP-1 was analyzed in the supernatants of HK-2 cells using ELISA. A representative set of three independent experiments is shown. The results are expressed as the mean ± s.d. *P<0.05, compared with control cells. †P<0.05, compared with TNF-α-+IFN-γ-treated cells. C, control cells; TI, TNF-α/IFN-γ-treated cells; TIC, TNF-α/IFN-γ-treated cells with CoCl2 pre-treatment; TICN, TNF-α/IFN-γ-treated cells with CoCl2 pre-treatment and negative control siRNA transfection; TIC-HO1, TNF-α/IFN-γ-treated cells with CoCl2 pre-treatment and HO-1 siRNA transfection; Co, CoCl2-pre-treated cells. (B) HK-2 cells were pre-treated with different doses of the NF-κB inhibitor PDTC for 2 hr before exposure to TNF-α and IFN-γ. TI, TNF-α/IFN-γ-treated cells; PDTC, pyrrolidinedithiocarbamate.

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