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DKK3 promotes oxidative stress injury and fibrosis in HK-2 cells by activating NOX4 via β-catenin/TCF4 signaling

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Abstract

Oxidative stress and fibrosis may accelerate the progression of chronic kidney disease (CKD). DKK3 is related to regulating renal fibrosis and CKD. However, the molecular mechanism of DKK3 in regulating oxidative stress and fibrosis during CKD development has not been clarified, which deserves to be investigated. Human proximal tubule epithelial cells (HK-2 cells) were treated with H2O2 to establish a cell model of renal fibrosis. The mRNA and protein expressions were analyzed using qRT-PCR and western blot, respectively. Cell viability and apoptosis were evaluated using MTT assay and flow cytometry, respectively. ROS production was estimated using DCFH-DA. The interactions among TCF4, β-catenin and NOX4 were validated using luciferase activity assay, ChIP and Co-IP. Herein, our results revealed that DKK3 was highly expressed in HK-2 cells treated with H2O2. DKK3 depletion increased H2O2-treated HK-2 cell viability and reduced cell apoptosis, oxidative stress, and fibrosis. Mechanically, DKK3 promoted formation of the β-catenin/TCF4 complex, and activated NOX4 transcription. Upregulation of NOX4 or TCF4 weakened the inhibitory effect of DKK3 knockdown on oxidative stress and fibrosis in H2O2-stimulated HK-2 cells. All our results suggested that DKK3 accelerated oxidative stress and fibrosis through promoting β-catenin/TCF4 complex-mediated activation of NOX4 transcription, which could lead to novel molecules and therapeutic targets for CKD.

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Data availability

All data generated or analysed during this study are included in this published article.

Abbreviations

CKD:

Chronic kidney disease

ROS:

Reactive oxygen species

NADPH:

Nicotinamide adenine dinucleotide phosphate

NOX4:

NADPH oxidase 4

IPF:

Idiopathic pulmonary fibrosis

DKK3:

Dickkopf-3

UUO:

Unilateral ureteral obstruction

qRT-PCR:

Quantitative real-time polymerase chain reaction

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

ChIP:

Chromatin immunoprecipitation

Co-IP:

Co-immunoprecipitation

dn-TCF4:

Dominant-negative TCF-4

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by National Natural Science Foundation (82160143) and The Engineering Research Center of Kidney Disease in Jiangxi Province (20164BCD40095).

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Authors and Affiliations

  1. Department of Nephrology, The Second Affiliated Hospital of Nanchang University, No. 1, Minde Road, Nanchang, 330006, Jiangxi Province, People’s Republic of China

    Jianling Song, Yanxia Chen, Yan Chen, Minzi Qiu, Wenliu Xiang, Ben Ke & Xiangdong Fang

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  1. Jianling Song
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  2. Yanxia Chen
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  3. Yan Chen
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Contributions

JS: Conceptualization, Methodology, Writing—Original draft preparation, Investigation, Validation, Visualization. YC: Data curation, Software. YC: Data curation. MQ: Software. WX: Data curation. BK: Conceptualization, Writing—Original draft preparation, Supervision, Writing—Reviewing and Editing. XF: Conceptualization, Writing—Original draft preparation, Supervision, Writing—Reviewing and Editing

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Correspondence to Ben Ke or Xiangdong Fang.

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11010_2023_4789_MOESM1_ESM.tif

Supplementary file1 (TIF 3043 KB) Figure S1 (A) HK-2 cells were treated with different concentrations of H2O2 (25, 50, 100, 200, 500, 800 μM) for 24 h and treated with 500 μM H2O2 for 6, 12, 24 and 36 h, and cell viability was tested by MTT assay. (B-C) HK-2 cells were treated with 500 μM H2O2 for 6, 12, 24 and 36 h, and DKK3 and NOX4 expression levels in cells were determined using qRT-PCR and western blot. (D) HK-2 cells were subjected to sh-DKK3 transfection upon H2O2 treatment, and β-catenin location was analyzed using IF. The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001

11010_2023_4789_MOESM2_ESM.tif

Supplementary file2 (TIF 853 KB) Figure S2 LF3 treatment weakened the effect of H2O2 on HK-2 cells. (A-B) NOX4 expression level in cells was determined using qRT-PCR and western blot. (C) Cell viability was tested by MTT assay. (D) Cell apoptosis was analyzed by flow cytometry. The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001

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Song, J., Chen, Y., Chen, Y. et al. DKK3 promotes oxidative stress injury and fibrosis in HK-2 cells by activating NOX4 via β-catenin/TCF4 signaling. Mol Cell Biochem (2023). https://doi.org/10.1007/s11010-023-04789-x

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