Background: Pyroptosis is a critical form of cell death during the
development of chronic kidney disease (CKD). Tripartite motif 6 (TRIM6) is an
E3-ubiquitin ligase that participates in the progression renal fibrosis (RF). The
aim of this study was to investigate the roles of TRIM6 and Glutathione
peroxidase 3 (GPX3) in oxidative stress-induced inflammasome activation and
pyroptosis in Ang-II treated renal tubular epithelial cells. Methods: To
study its role in RF, TRIM6 expression was either reduced or increased in human
kidney-2 (HK2) cells using lentivirus, and Ang-II, NAC and BMS-986299 were served
as reactive oxygen species (ROS) inducer, ROS scavenger and NLRP3 agonist
respectively. Pyroptosis and mitochondrial ROS were measured by flow cytometry.
The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase
(SOD) were determined using commercial kits, while the levels of IL-1,
IL-18, IL-6, and tumor necrosis factor- (TNF-) were
determined by Enzyme-Linked Immunosorbent Assay (ELISA). Co-immunoprecipitation
(Co-IP) assay was used to evaluate the interaction between TRIM6 and GPX3.
Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were
used to measure mRNA and protein expression, respectively. Results:
Treatment with Angiotensin II (Ang II) increased the protein and mRNA levels of
TRIM6 in HK2 cells. Ang II also increased mitochondrial ROS production and the
malondialdehyde (MDA) level, but decreased the levels of GSH and SOD. In
addition, Ang II enhanced HK2 cell pyroptosis, increased the levels of
IL-1, IL-18, IL-6, and TNF-, and promoted the expression of
active IL-1, NLRP3, caspase-1, and GSDMD-N proteins. These effects were
reversed by knockdown of TRIM6 and by treatment with N-acetyl-L-cysteine (NAC), a
ROS scavenger. BMS-986299, an NLRP3 agonist treatment, did not affect ROS
production in HK2 cells exposed to Ang II combined with NAC, but cell pyroptosis
and inflammation were aggravated. Moreover, the overexpression of TRIM6 in HK2
cells resulted in similar effects to Ang II. NAC and GPX3 overexpression in HK2
cells could reverse ROS production, inflammation, and pyroptosis induced by TRIM6
overexpression. TRIM6 overexpression decreased the GPX3 protein level by
promoting its ubiquitination, without affecting the GPX3 mRNA level. Thus, TRIM6
facilitates GPX3 ubiquitination, contributing to increased ROS levels and
pyroptosis in HK2 cells. Conclusions: TRIM6 increases oxidative stress
and promotes the pyroptosis of HK2 cells by regulating GPX3 ubiquitination. These
findings could contribute to the development of novel drugs for the treatment of
RF.