#999 PDK4 triggers ferroptosis in diabetic kidney disease by promoting NRF2 ubiquitination
Authors: Tian Shasha , Yang Xiaopeng , Li Xinran , Zhou Saijun , Yu Pei 
Published: May 2024

Abstract

Background and Aims
Diabetic kidney disease (DKD) is the principal cause of chronic kidney disease and end-stage renal disease. Despite this burden, the detailed understanding of the pathogenic mechanism that promote the initiation and progression of DKD remain to be fully elucidated. This study aims to investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in diabetic renal tubular cell death from the perspectives of mitochondrial injury, metabolic reprogramming and ferroptosis.

Method
Cellular transcriptomics was employed to screen the differential genes between normal glucose (as control) and 35 mM glucose combined with 120 uM palmitic acid (as HGL) groups. The vitro experimental groups include Ctl, HGL, HGL/DCA (5 mM PDK4 inhibitor, sodium dichloroacetate), HGL/DMSO, culturing for 48 h. C57BL/6J mice were used to perform the vivo experiments. A total of 40 C57BL/6J mice were randomly divided into 5 groups respectively labeled as control, DKD, DKD/DCA (25 mg/kg, daily), DKD/DMSO (1% DMSO, daily) and DKD/Fer-1 (1 mg/kg, daily) group. Western blot, qRT-PCR, Si-RNA transfection, transmission electron microscopy, ROS probe, FerroOrange/Green probes, liperfluo probe, immunofluorescence and Co-IP assay were used in this study to detect the indicators of metabolic reprogramming, mitochondrial function, oxidative stress and ferroptosis, as well as the NRF2 ubiquitination level.

Results
Based on the cellular transcriptomics, PDK4 mRNA was significantly elevated in the HK-2 cells treated with HGL. Likewise, mRNA and protein levels were also elevated in the kidneys of mice treated with streptozotocin and high fat diet.
In kidneys with PDK4 inhibitor DCA treatment or PDK4 genetic knockdown, glycolipid toxicity-induced metabolic reprogramming and destruction of mitochondrial function were markedly ameliorated. In addition, mitochondrial reactive oxygen species and the oxidative stress markers NADPH oxidase 2 and 4 (NOX2, NOX4) were diminished in DCA-treated or PDK4 knockdown HK-2 cells, while superoxide dismutase 1 and 2 (SOD1, SOD2) and catalase (CAT) expression and glutathione (GSH) levels were restored. DCA intervention or PDK4 knockdown in DKD mice or HK-2 cells alleviated the signs of ferroptosis caused by glycolipid toxicity, demonstrated as up-regulation of SLCA11, GPX4, FSP1 and GSH to maintain the balance of oxidative stress, increase of FTH1 and FPN1 to promote Fe2+ storage and discharge, decrease of TFR1 to reduce Fe2+ intake, as well as reduction of lipid peroxidation products including 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA).
PDK4 promoted NRF2 ubiquitination under HGL condition, since inhibition or knockdown of PDK4 in HK-2 cells increased the protein level of NRF2, while the mRNA level was unchanged.

Conclusion
Collectively, we identified a new ferroptosis inducer gene, PDK4, which is commonly up-regulated in diabetes. PDK4-induced metabolic reprogramming, mitochondrial damage and oxidative stress promote ROS accumulation and ultimately lead to ferroptosis in DKD, this effect can be achieved in part by promoting NRF2 ubiquitination.