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N-acetylcysteine regulates oxalate induced injury of renal tubular epithelial cells through CDKN2B/TGF-β/SMAD axis

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Abstract

This study was aimed to investigate the preventive effects of N-acetyl-l-cysteine (NAC) against renal tubular cell injury induced by oxalate and stone formation and further explore the related mechanism. Transcriptome sequencing combined with bioinformatics analysis were performed to identify differentially expressed gene (DEG) and related pathways. HK-2 cells were pretreated with or without antioxidant NAC/with or silencing DEG before exposed to sodium oxalate. Then, the cell viability, oxidative biomarkers of superoxidase dismutase (SOD) and malondialdehyde (MDA), apoptosis and cell cycle were measured through CCK8, ELISA and flow cytometry assay, respectively. Male SD rats were separated into control group, hyperoxaluria (HOx) group, NAC intervention group, and TGF-β/SMAD pathway inhibitor group. After treatment, the structure changes and oxidative stress and CaOx crystals deposition were evaluated in renal tissues by H&E staining, immunohistochemical and Pizzolato method. The expression of TGF-β/SMAD pathway related proteins (TGF-β1, SMAD3 and SMAD7) were determined by Western blot in vivo and in vitro. CDKN2B is a DEG screened by transcriptome sequencing combined with bioinformatics analysis, and verified by qRT-PCR. Sodium oxalate induced declined HK-2 cell viability, in parallel with inhibited cellular oxidative stress and apoptosis. The changes induced by oxalate in HK-2 cells were significantly reversed by NAC treatment or the silencing of CDKN2B. The cell structure damage and CaOx crystals deposition were observed in kidney tissues of HOx group. Meanwhile, the expression levels of SOD and 8-OHdG were detected in kidney tissues of HOx group. The changes induced by oxalate in kidney tissues were significantly reversed by NAC treatment. Besides, expression of SMAD7 was significantly down-regulated, while TGF-β1 and SMAD3 were accumulated induced by oxalate in vitro and in vivo. The expression levels of TGF-β/SMAD pathway related proteins induced by oxalate were reversed by NAC. In conclusion, we found that NAC could play an anti-calculus role by mediating CDKN2B/TGF-β/SMAD axis.

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The data used to support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (NSFC, No.81370803).

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Authors and Affiliations

  1. Department of Urology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150081, China

    Wei Cao & Jingbo Zhang

  2. Department of Urology, The First Affiliated Hospital of Harbin Medical University, 23 YouZheng Street, HarbinHarbin, Heilongjiang, 150001, China

    Shiliang Yu, Xiuguo Gan & Ruihua An

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Contributions

Conception and design: Ruihua An, Wei Cao, Jingbo Zhang. Data analysis and interpretation: Wei Cao, Shiliang Yu, Xiuguo Gan. Manuscript writing: Wei Cao, Jingbo Zhang, Ruihua An. Final approval of manuscript: all authors.

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Correspondence to Ruihua An.

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Approval was obtained from the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University and the animal study procedures were conformed to the Animal Care and Use guidelines provided by the National Institutes of Health (NIH).

The experimental protocol was performed in accordance with the relevant guidelines and regulations of the Basel Declaration. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

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Cao, W., Zhang, J., Yu, S. et al. N-acetylcysteine regulates oxalate induced injury of renal tubular epithelial cells through CDKN2B/TGF-β/SMAD axis. Urolithiasis 52, 46 (2024). https://doi.org/10.1007/s00240-023-01527-2

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