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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Published on: January 7, 2013
Inhibition of GRK2 mitigates cisplatin-induced acute and chronic nephrotoxicity by targeting the NADPH oxidase
Fei Duan1, Lei Ni1, Qianlei Wang1,2
1Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Anhui Medical University, Key Laboratory of Anti-Inflammatory and Immune Medicine, Ministry of Education, Anhui Collaborative Innovation Centre of Anti-Inflammatory and Immune Medicine, Center of Rheumatoid Arthritis of Anhui Medical University, Hefei, China.
Background And Purpose:
Cisplatin nephrotoxicity involves oxidative stress, with NADPH oxidase 4 (NOX4) in renal tubular epithelial cells (RTECs) as a potential source of key reactive oxygen species (ROS). Although G protein-coupled receptor kinase 2 (GRK2) contributes to tissue injury, its regulatory role in cisplatin nephrotoxicity via NOX4 remains undefined. We have investigated the underlying mechanisms of GRK2 in cisplatin-induced acute and chronic nephrotoxicity.
Experimental Approach:
Hemizygous Grk2 knockout mice and homozygous Grk2 knockout RTECs were utilised. The GRK2-NOX4 interaction was investigated by co-immunoprecipitation or glutathione S-transferase pull-down. Kinase assays and ubiquitination experiments were performed to investigate the regulatory effect of GRK2 on the phosphorylation and ubiquitination of NOX4. Acute, sub-chronic and chronic cisplatin models, as well as syngeneic tumour models, evaluated the renoprotection afforded by the GRK2 inhibitor CP-25 and its impact on chemotherapy efficacy.
Key Results:
Grk2 deficiency attenuated cisplatin-induced nephrotoxicity in vivo and reduced RTEC death and epithelial-mesenchymal transition (EMT) in vitro. Grk2 knockout or knockdown downregulated NOX4 expression, which repressed NOX4-mediated ROS release and oxidative stress, preventing cisplatin-induced RTEC death and EMT. GRK2 promoted NOX4 phosphorylation and suppressed ubiquitin-mediated degradation, thereby increasing NOX4 stability and ROS production. CP-25 effectively ameliorated cisplatin-induced nephrotoxicity, suppressed oxidative stress, and reduced renal apoptosis and EMT without compromising its anticancer efficacy. These protective effects also were observed in vitro.
Conclusion And Implications:
GRK2 plays a crucial role in cisplatin-induced nephrotoxicity by modulating NOX4, suggesting that targeting GRK2 is an effective renoprotective strategy for cisplatin-based cancer therapy.
Insights
Targeting G protein-coupled receptor kinase 2 (GRK2) protects against cisplatin nephrotoxicity by inhibiting NADPH oxidase 4 (NOX4)-mediated oxidative stress. This discovery offers a renoprotective strategy for cancer therapy.
Area of Science:
- Nephrology
- Oncology
- Molecular Biology
Background:
- Cisplatin nephrotoxicity is linked to oxidative stress, with NADPH oxidase 4 (NOX4) in renal tubular epithelial cells (RTECs) as a key reactive oxygen species (ROS) source.
- G protein-coupled receptor kinase 2 (GRK2) is implicated in tissue injury, but its role in cisplatin nephrotoxicity via NOX4 is unclear.
Purpose of the Study:
- To investigate the mechanisms by which GRK2 contributes to cisplatin-induced acute and chronic kidney injury.
- To explore the regulatory role of GRK2 on NOX4 activity in the context of cisplatin nephrotoxicity.
Main Methods:
- Utilized hemizygous Grk2 knockout mice and homozygous Grk2 knockout RTECs.
- Investigated GRK2-NOX4 interaction using co-immunoprecipitation and glutathione S-transferase pull-down assays.
- Assessed the effect of GRK2 inhibition (CP-25) on cisplatin-induced nephrotoxicity in vitro and in vivo models, including tumor models.
Main Results:
- Grk2 deficiency attenuated cisplatin nephrotoxicity, RTEC death, and epithelial-mesenchymal transition (EMT).
- Grk2 deficiency downregulated NOX4 expression, reducing ROS production and oxidative stress.
- GRK2 inhibition with CP-25 ameliorated cisplatin nephrotoxicity and EMT without compromising anticancer efficacy.
Conclusions:
- GRK2 plays a critical role in cisplatin-induced nephrotoxicity by modulating NOX4.
- Targeting GRK2 represents a promising renoprotective strategy for patients undergoing cisplatin-based cancer therapy.
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