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PU.1/Spi1 exacerbates ischemia-reperfusion induced acute kidney injury via upregulating Gata2 and promoting fibroblast activation

Acta Pharmacologica Sinica volume 46, pages 2251–2266 (2025)Cite this article

Abstract

Previous studies on acute kidney injury (AKI) have predominantly focused on renal tubular cells, while the specific role of fibroblasts has been largely neglected. Recent evidence shows that PU.1/Spi1, a transcription factor, is an important modulator of fibroblast activation, whereas pharmacological and genetic silencing of PU.1/Spi1 disrupts the fibrotic network and reprograms activated fibroblasts into quiescent fibroblasts. In this study we investigated whether and how PU.1/Spi1 regulated renal fibroblast activation during AKI. An AKI model was established in male mice by clamping bilateral renal arteries for 30 min. Mice were sacrificed and blood and kidney samples were collected 48 h after the surgery. We showed that the expression level of PU.1/Spi1 was significantly upregulated in ischemia/reperfusion (I/R)-induced AKI and PU.1/Spi1 was specifically localized in fibroblasts. Meanwhile, we observed that a massive activation of fibroblasts occurred at the early stage of AKI. PU.1/Spi1 knockout significantly attenuated the activation of fibroblasts along with the decreased release of inflammatory factors and tubular injury. Bioinformatic analysis revealed that GATA binding protein 2 (Gata2), an evolutionarily conserved gene, might be a downstream target gene of PU.1/Spi1. In primary cultured mouse kidney fibroblasts subjected to hypoxia/reoxygenation (H/R), the expression levels of PU.1/Spi1, Gata2 and α-SMA were significantly upregulated. Activated fibroblasts exhibited elevated proliferative capacity, evidenced by upregulated proliferating cell nuclear antigen (PCNA) and cell cycle proteins such as cyclin B1 and cyclin D3. The secretion of inflammatory factors was increased in the activated fibroblasts. Conditioned medium from H/R-treated fibroblasts induced tubular cell injury and increased apoptosis. Using chromatin immunoprecipitation and promoter-luciferase assays, we demonstrated that PU.1/Spi1 was able to bind to the promoter region of Gata2 and enhanced its transcription. Our results show that interstitial fibroblasts are activated at the early stage of I/R-induced AKI and involved in renal injury. Upregulated PU.1/Spi1 stimulates fibroblast activation by upregulating its downstream gene Gata2. Inhibiting the activation of fibroblasts may have a beneficial effect on AKI.

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Fig. 1: PU.1/Spi1 was elevated in I/R-induced AKI and located in fibroblasts.
Fig. 2: Knockdown on PU.1/Spi1 inhibited H/R-induced fibroblast activation.
Fig. 3: PU.1/Spi1 enhanced Gata2 transcription in H/R-treated fibroblasts.
Fig. 4: Overexpression of PU.1/Spi1 upregulated Gata2 along with fibroblast activation.
Fig. 5: Knockdown on Gata2 blunted overexpression of PU.1/Spi1 induced fibroblast activation.
Fig. 6: Overexpression of Gata2 promoted fibroblast activation.
Fig. 7: Inflammatory factor secretion was enhanced in activated fibroblasts and stimulated tubular cell apoptosis.
Fig. 8: PU.1/Spi1 knockout alleviated the activation of fibroblasts in I/R induced AKI.
Fig. 9: PU.1/Spi1 knockout attenuated tubular cell apoptosis with decreased interstitial inflammatory factor levels and improved AKI.

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Acknowledgements

This research was financially supported by the Natural National Science Foundation of China (No. 82370718 to Jian-ying Niu, No. 82370695, 82070712 to Li-min Lu), and National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University (2024KF2004) to Li-min Lu, the Science and Technology Commission of Shanghai Municipality (14DZ2260200, the project of Shanghai Key Laboratory of Kidney and Blood Purification).

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Authors and Affiliations

  1. Department of Nephrology, Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, 200240, China

    Chen Zong, Guo-li Xu, Li-hong Zhang, Zhen-hao Yang & Jian-ying Niu

  2. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China

    Ming Ning, Heng-jiang Guo, Li Zhou, Chen Xu & Li-min Lu

  3. Department of Nephrology, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, 200011, China

    Jing-yao Li

  4. Department of Nephrology, Children’s Hospital of Fudan University, Shanghai, 201103, China

    Xin Wang

  5. National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, 200040, China

    Li-min Lu

  6. Center of Community-based Health Research, Fudan University, Shanghai, 200240, China

    Jian-ying Niu

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Contributions

CZ and LML conceived and designed the study. CZ performed the experiment and drafted the manuscript. GLX and MN contributed to the material preparation and data collection. JYL, XW, HJG, LHZ and ZHY performed formal analysis. LZ and CX contributed technical support. LML and JYN supervised the findings and reviewed the final manuscript. All the authors declared no competing interests.

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Correspondence to Li-min Lu or Jian-ying Niu.

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Zong, C., Xu, Gl., Ning, M. et al. PU.1/Spi1 exacerbates ischemia-reperfusion induced acute kidney injury via upregulating Gata2 and promoting fibroblast activation. Acta Pharmacol Sin 46, 2251–2266 (2025). https://doi.org/10.1038/s41401-025-01530-w

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