J Korean Med Sci. 2014;29 Suppl 2(Suppl 2):S139-45.

Cobalt chloride attenuates oxidative stress and inflammation through NF-κB inhibition in human renal proximal tubular epithelial cells.

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제1저자 오세원 · 교신저자 나기영

PMID 25317018 PMC4194284 DOI 10.3346/jkms.2014.29.S2.S139 IF 3.2 신장내과

소속 (3)

Department of Internal Medicine, Inje University College of Medicine, Ilsan Paik Hospital, Goyang, Korea.
Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea.
Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea. ; Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.

Abstract

We evaluated the effect of cobalt chloride (CoCl2) on TNF-α and IFN-γ-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-α/IFN-γ. 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-α/IFN-γ. TNF-α/IFN-γ-treated-cells showed an increase in the nuclear translocation of phosphorylated NF-κBp65 protein, the DNA-binding activity of NF-κBp50 and NF-κB transcriptional activity and a decrease in IκBα 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-α/IFN-γ-treated cells. We demonstrated that this effect was mediated through NF-κB signaling because an NF-κB 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-α/IFN-γ-induced inflammation through the inhibition of NF-κB and ROS in HK-2 cells. However, CoCl2 appears to act in an HO-1-independent manner.

Keywords

Cobalt ChlorideHemeoxygenase-1InflammationNuclear Factor-kappa BRenal Tubular Epithelial Cells

Figures

**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.

그림 출처: PubMed Central 공개 논문 (PMC4194284)

Conflict of interest statement

The authors have no potential conflicts of interest relevant to this article to disclose.

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