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.