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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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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This research was approved by the Medical Ethics Committee of the **’an Jiaotong University (No. XJTULAC2021-1461).
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Dong, B., Ding, C., **ang, 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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DOI: https://doi.org/10.1007/s00011-022-01648-1