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USP7 accelerates FMR1-mediated ferroptosis by facilitating TBK1 ubiquitination and DNMT1 deubiquitination after renal ischemia–reperfusion injury

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Abstract

Background

Renal ischemia/reperfusion (I/R) leads to acute kidney injury and is associated with cell ferroptosis, an oxidative programmed cell death. This study aims to explore whether USP7 regulates ferroptosis in rat kidneys suffered I/R and the underlying mechanisms.

Methods

Human renal tubular epithelial cells HK-2 were treated with hypoxia/reoxygenation (H/R) to establish a cell model. The expression of ubiquitin specific peptidase 7 (USP7) in H/R-treated cells was determined. USP7 siRNA was transfected into H/R-treated cells, followed by the detection of cell proliferation, iron ion concentration, oxidative stress levels and glutathione peroxidase 4 (GPX4) and solute carrier family 7-member 11 (SLC7A11) protein levels. Western blotting and immunoprecipitation analyses were performed to detect the effects of USP7 on the ubiquitination of TANK-binding kinase 1 (TBK1) and DNA methyltransferase 1 (DNMT1). Then, H/R-treated cells were transfected with USP7 siRNA alone or together with TBK1 siRNA. Co-immunoprecipitation was used to detect binding relationship between TBK1 and FMRP translational regulator 1 (FMR1). The level of DNMT1 and methylation ratio of the FMR1 promoter region were determined with chromatin immunoprecipitation and methylation specific PCR assays, respectively. Furthermore, USP7 siRNA and FMR1 siRNA were transfected alone or together into H/R-treated cells, followed by the detection of cell functions. An I/R rat model was constructed to analyze the effects of USP7 on renal function in rats.

Results

USP7 was significantly upregulated in H/R-treated cells. USP7 interference markedly increased HK-2 cell proliferation and the protein levels of GPX4 and SLC7A11, restrained the iron ion concentration, and ameliorated oxidative stress. USP7 promoted TRIM27-mediated TBK1 ubiquitination and degradation. USP7 inhibition resulted in increased ubiquitination and decreased stability of DNMT1. USP7 was able to recruit DNMT1 to the FMR1 promoter region, which increased promoter methylation rates and suppressed FMR1 expression. TBK1 or FMR1 overexpression could reverse the effects of USP7 on cell functions. Inhibition of USP7 alleviated renal ischemia–reperfusion injury in rats.

Conclusions

USP7 inhibition attenuated I/R-induced renal injury by inhibiting ferroptosis through decreasing ubiquitination of TBK1 and promoting DNMT1-mediated methylation of FMR1.

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Data availability

The datasets used during the present study are available from the corresponding author on reasonable request.

Abbreviations

I/R:

Renal ischemia/reperfusion

H/R:

Hypoxia/reoxygenation

GPX4:

Ubiquitin specific peptidase 7, glutathione peroxidases 4

SLC7A11:

Solute carrier family 7 member 11

TBK1:

TANK-binding kinase 1

DNMT1:

DNA methyltransferase 1

Co-IP:

Co-immunoprecipitation

FMR1:

FMRP translational regulator 1

ChIP:

Chromatin immunoprecipitation

AKI:

Acute kidney injury

RCD:

Regulated cell death

HO-1:

Heme oxygenase-1

NCOA4:

Nuclear receptor coactivator 4

FTH1:

Ferritin light chain 1

SAP:

Severe acute pancreatitis

DUBs:

Deubiquitinating enzymes

KD:

Kinase domain

SDD:

Scaffold dimerization domain

CCD2:

Coiled coil domain 2

ULD:

Ubiquitin like domain

PCNA:

Proliferating cell nuclear antigen

PTMs:

Post-translational modifications

HDACs:

Histone deacetylases

USP2B:

Ubiquitin specific protease

SD:

Sprague–Dawley

Cr:

Serum creatinine

BUN:

Blood urea nitrogen

DMEM:

Dulbecco’s modified eagle medium

FBS:

Fetal bovine serum

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene difluoride

Ub:

Ubiquitin

HRP:

Horseradish peroxidase

CCK-8:

Cell Counting Kit-8

ChIP:

Chromatin immunoprecipitation assay

ELISA:

Enzyme-linked immunosorbent assay

SOD:

Superoxide dismutase

MDA:

Malondialdehyde

MSP:

Methylation-specific PCR

CpG:

Cytosine-phosphate-guanine

ICP-MS:

Inductively coupled plasma mass spectrometry

SEM:

Standard error of mean

rAAV:

Recombinant adeno-associated virus

CKD:

Chronic kidney disease

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Authors and Affiliations

  1. Department of Renal Transplantation, The First Affiliated Hospital of Xi’ an Jiaotong University, 277 West Yanta Street, Xi’ an, 710061, Shaanxi, China

    Boqing Dong, Chenguang Ding, Heli Xiang, Jin Zheng, Xiao Li, Wujun Xue & Yang Li

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  1. Boqing Dong
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  2. Chenguang Ding
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  3. Heli Xiang
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  4. Jin Zheng
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  5. Xiao Li
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  6. Wujun Xue
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  7. Yang Li
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Contributions

YL: designed this study and wrote the manuscript. BD: performed the experimental work. XL: provided the majority of statistical analysis as well as provided the figures and tables for the manuscript. JZ, CD, HX and WX: collected a large amount of data for the data set. All authors read and approved the final manuscript.

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Correspondence to Yang Li.

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This research was approved by the Medical Ethics Committee of the Xi’an Jiaotong University (No. XJTULAC2021-1461).

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Dong, B., Ding, C., Xiang, H. et al. USP7 accelerates FMR1-mediated ferroptosis by facilitating TBK1 ubiquitination and DNMT1 deubiquitination after renal ischemia–reperfusion injury. Inflamm. Res. 71, 1519–1533 (2022). https://doi.org/10.1007/s00011-022-01648-1

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