4 | DISCUSSION
Despite recent advances in understanding the basis of acute renal injury
(Nakazawa et al., 2017; Tran et al., 2016), refractory nephrotic
syndrome in patients with AKI continues to be a challenge for clinical
practice. This study suggests that HDAC inhibitors can alleviate AKI by
regulating PSTPIP2 to inhibit the occurrence of AAI-induced PANoptosis.
Aristolochic acid nephropathy (AAN) is a rapidly progressing
tubulointerstitial disease caused by AA. The major nephrotoxic
constituent of aristolochic acid (AA) is
8-methoxy-6-nitrophenanthrene-(3,4-d)- 1,3-dioxo-5-carboxylic acid
(aristolochic acid I, AAI) (Shibutani, Dong, Suzuki, Ueda, Miller &
Grollman, 2007). There is evidence that oxidative stress injury is
induced in the kidneys of mice after AAI exposure, resulting in tubular
epithelial cell apoptosis(Wang et al., 2022) and histopathological
features of tubular necrosis in a short-term mouse model (Baudoux et
al., 2012). The assembled NLRP3 inflammasome can activate caspase-1 to
induce gasdermin D-dependent pyroptosis (Huang, Xu & Zhou, 2021; Shi et
al., 2015). NLRP3 or caspase-1 deficiency protects against renal injury
in a mouse model of acute AAN(Wang et al., 2019). Therefore, we
investigated whether programmed cell death occurred in AAI-induced AKI.
Programmed cell death (PCD) is an evolutionarily conserved process that
plays a central role in maintaining organismal homeostasis. Three key
PCD pathways have been studied in detail: pyroptosis
(inflammasome-dependent PCD executed by gasdermin family members),
apoptosis (PCD mediated by the apoptosome and executioner caspases), and
necroptosis (PCD mediated by RIPK3 and the downstream effector
MLKL)(Samir, Malireddi & Kanneganti, 2020). Apoptosis is a form of
programmed cell death that is executed by a family of proteases called
caspases that can be activated by cell surface death receptors
(extrinsic pathway) or perturbation of the mitochondrial membrane
(intrinsic pathway) (Lewis et al., 2005). The released apoptotic factors
can activate the caspase family, eventually resulting in apoptosis (Hsu
& Hsueh, 2000). Apoptosis played a pivotal role in the progression of
nephrotoxicity and AKI (Chen et al., 2010; Pozdzik et al., 2008;
Romanov, Whyard, Bonala, Johnson & Grollman, 2011). Necroptosis, a
necrotic type of cell death that follows a signaling pathway closely
related to apoptosis, is the most thoroughly examined form of regulated
necrosis, executed by RIPK3 and its substrate, pseudokinase mixed
lineage kinase domain-like protein (MLKL) (Belavgeni, Meyer, Stumpf,
Hugo & Linkermann, 2020; Muller et al., 2017). Recently, necroptosis
has been implicated in acute kidney injury (AKI), acute tubular necrosis
(ATN), generation of necrotic casts, and reduction of renal blood flow
(Huang et al., 2019; Liu et al., 2018; Martin-Sanchez et al., 2018;
Schreiber, Rousselle, Becker, von Massenhausen, Linkermann & Kettritz,
2017). Necroptosis is characterized by cellular swelling, rapid membrane
permeabilization, and the concomitant release of damage-associated
molecular patterns (DAMPs) into the extracellular space. Necroptosis has
been implicated in the development of autoimmune, neurodegenerative, and
inflammatory diseases, such as acute pancreatitis and ischemic injury
(Pasparakis & Vandenabeele, 2015). AKI is characterized by the damage
or death of tubular epithelial cells, in which pyroptosis has been
reported to play a role in the progression of AKI (Zhang et al., 2018).
Pyroptosis can resist intracellular infections by eliminating the
damaged cells. It is an important natural immune response that plays a
crucial role in antagonizing infection and endogenous danger signals
(Miao et al., 2010). Pyroptosis is activated by inflammatory caspases,
leading to cell swelling, pore formation, cell membrane disruption, and
consequent release of inflammatory cytokines (Xia et al., 2021).
Pyroptosis participates in various kidney diseases, as evidenced by the
activation of inflammatory caspases, massive release of IL-1β, and
increased cleavage of GSDMD (Li et al., 2020; Ye et al., 2019). We found
that in vivo, the kidneys had increased cleaved-caspase-3, GSDMD-N, and
pMLKL in response to AAI treatment compared with the control group. In
vitro, mTECs cells also showed increased cleaved-caspase-3, GSDMD-N, and
pMLKL following AAI treatment compared with mock treatment. Studies have
revealed that various cell death programs play alternating roles and
exhibit extensive crosstalk (Chen et al., 2016; Wang et al., 2018).
Cells carry out multiple regulated cell death programs via extensive
crosstalk, which can be activated simultaneously under specific
conditions. This is consistent with the recently proposed concept of
“PANoptosis” (Karki et al., 2021). Whether induced in response to
infection, during organismal homeostasis, or in the context of
autoinflammation, PANoptosis is executed by a molecular complex called
the PANoptosome that integrates apoptotic, necroptotic, and inflammasome
components (Samir, Malireddi & Kanneganti, 2020). Our results
demonstrate that AAI could co-use PANoptosis in the kidney.
However, in recent years, there has been significant growth in our
knowledge about the involvement of histone modifications in gene
regulation, which are known to play a role in normal cell physiology and
pathology. HDACs play essential roles in cellular physiology and gene
regulation (Iizuka & Smith, 2003). Histone acetylation plays a crucial
role in chromatin remodeling and regulation of gene transcription. The
presence of acetylated lysine in histone tails is associated with a more
relaxed chromatin state and activation of gene transcription, whereas
deacetylation of lysine residues is associated with a more condensed
chromatin state and transcriptional gene silencing(Iizuka & Smith,
2003; Ropero & Esteller, 2007). In our study, AAI induced a large
increase in HDAC1 and HDAC2 expression, whereas expression of HDAC 3, 4,
7, 8, and 11 was downregulated. To determine whether the AAI-induced
increase in HDAC expression mediated AAI-induced nephrotoxicity, HDAC1
and -2 specific inhibitors (romidepsin or FK-228), or vehicle were
administered with AAI. AAI administration significantly increased
tubular epithelial cell PANoptosis in the kidneys, which was largely
suppressed by FK-228 treatment. These results indicated that HDAC
inhibitors can inhibit AAI-induced PANoptosis.
Zhu et al. reported PSTPIP2 inhibits acute kidney injury (Zhu et al.,
2020). PSTPIP2 was silenced by the deacetylase activity. Our chromatin
immunoprecipitation assay supports the view that increased acetylation
of histones in the PSTPIP2 promoter region may enhance PSTPIP2
transcription. Thus, it appears that inhibition of deacetylase activity
promotes PSTPIP2 gene transcription. PSTPIP2 belongs to the F-BAR family
and contains a conserved Fes CIP4 homology (FCH) domain in the
N-terminal (Liu et al., 2014). PSTPIP2 participates in macrophage
activation, neutrophil migration, cytokine production, and osteoclast
differentiation (Xu et al., 2021). Most tissue macrophages and
osteoclasts are regulated by colony-stimulating factor-1 (CSF-1, also
known as macrophage CSF). PSTPIP2 regulates the morphology and motility
of macrophages downstream of the CSF-1R PSTPIP2 deficiency and causes
both an expansion of macrophage progenitors and increased responsiveness
of mature macrophages to stimulating stimuli, which together prime the
organism for exaggerated and sustained responses leading to
autoinflammatory disease (Chitu et al., 2009; Chitu et al., 2005; Pixley
& Stanley, 2004). Our results prove that the HDAC inhibitor FK-228 can
block apoptosis, pyroptosis, and necroptosis by regulating PSTPIP2 in
AAN, a complex disorder involving insulin resistance, lipid metabolism
dysfunction, oxidative stress, inflammation, and various types of cell
death. We demonstrated that AAI induces kidney injury and PANoptosis in
mice. However, AAI did not induce glomerulonephritis in PSTPIP2-KI mice.
These results are consistent with those of previous studies. However,
the regulation of AAI-mediated cell death by PSTPIP2 requires further
investigation.
Prajwal et al. found a significantly higher number of caspase-8 in
Pstpip2cmo neutrophils than in WT controls (Gurung, Burton &
Kanneganti, 2016). In “PANoptosis,” the traditional promoter of
apoptosis, caspase-8, plays a pivotal role. Researchers have found that
caspase-8 can switch the modes of cell death. Even when the
caspase-8-dependent apoptotic pathway and MLKL-dependent necroptotic
pathway are inhibited, the catalytically inactive caspase-8 (C362A) can
mediate pyroptosis (Newton et al., 2019). Thus, caspase-8 has attracted
significant attention as a molecular switch between these three types of
cell death. Our studies show that this suppression of PANoptosis is
mediated through the downregulation of caspase-8 expression in
epithelial cells.
In conclusion, our results showed that administration of the HDAC1 and
HDAC2 inhibitor FK-228 inhibited PANoptosis of renal tubular epithelial
cells. The protective activity of HDAC inhibitors is mediated by the
upregulation of a novel anti-inflammatory and anti-apoptotic protein,
PSTPIP2. This study highlights that PSTPIP2 alleviates kidney injury in
response to AAI treatment by suppressing PANoptosis, which may guide the
design of a better therapeutic strategy for treating patients with AKI.