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Mini-Review: GSDME-Mediated Pyroptosis in Diabetic Nephropathy

Pyroptosis is a recently identified type of lytic programmed cell death, in which pores form in the plasma membrane, and cells swell, rupture, and then release their contents, including inflammatory cytokines. Molecular studies indicated that pyroptosis may occur via a gasdermin D (GSDMD) and caspase-1 (Casp1) -dependent classical pathway, a GSDMD and Casp11/4/5-dependent non-classical pathway, or a gasdermin E (GSDME) and Casp3-dependent pathway. Studies of animal models and humans indicated that pyroptosis can exacerbate several complications of diabetes, including diabetic nephropathy (DN), a serious microvascular complication of diabetes. Many studies investigated the mechanism mediating the renoprotective effect of GSDMD regulation in the kidneys of patients and animal models with diabetes. As a newly discovered regulatory mechanism, GSDME and Casp3-dependent pyroptotic pathway in the progression of DN has also attracted people’s attention. Z-DEVD-FMK, an inhibitor of Casp3, ameliorates albuminuria, improves renal function, and reduces tubulointerstitial fibrosis in diabetic mice, and these effects are associated with the inhibition of GSDME. Studies of HK-2 cells indicated that the molecular and histological features of secondary necrosis were present following glucose stimulation due to GSDME cleavage, such as cell swelling, and release of cellular contents. Therefore, therapies targeting Casp3/GSDME-dependent pyroptosis have potential for treatment of DN. A novel nephroprotective strategy that employs GSDME-derived peptides which are directed against Casp3-induced cell death may be a key breakthrough. This mini-review describes the discovery and history of research in this pyroptosis pathway and reviews the function of proteins in the gasdermin family, with a focus on the role of GSDME-mediated pyroptosis in DN. Many studies have investigated the impact of GSDME-mediated pyroptosis in kidney diseases, and these studies used multiple interventions, in vitro models, and in vivo models. We expect that further research on the function of GDSME in DN may provide valuable insights that may help to improve treatments for this disease.


INTRODUCTION
There are several programmed cell death (PCD) pathways (Abbreviations in the manuscript are listed in Supplementary Table 1), and each pathway depends on different genes, signals, and cellular activities. Each of the PCD pathways removes functionally dispensable, infected, or potentially neoplastic cells. PCD is therefore an important component of homeostasis and also provides host defense against pathogens, cancer, and other pathologies (Bedoui et al., 2020). The major types of PCD are pyroptosis, apoptosis, and necroptosis (Wang and Kanneganti, 2021). Disturbances of extracellular or intracellular homeostasis that destroy innate immunity can lead to pyroptosis, also referred to as gasdermin (GSDM)dependent programmed necrosis, the most recently defined type of PCD (Jorgensen and Miao, 2015). Pyroptosis is a form of PCD characterized by greatly increased inflammation, and it most commonly occurs after a serious infection, but can also occur in chronic diseases Galluzzi et al., 2018).
Histological studies indicated that pyroptosis has some features of apoptosis and some features of necrosis. More specifically, cells undergoing pyroptosis develop pores in the cytoplasmic membrane, become swollen, and then rupture, leading to the release pro-inflammatory mediators, such as interleukin-1β (IL-1β) and interleukin-18 (IL-18) (Galluzzi et al., 2018). At the molecular level, pyroptosis is characterized by increased activity of the canonical pathway of caspase-1 (Casp1) and/or the non-canonical pathway of Casp4/5 (humans) and Casp11 (mice), as well as their downstream execution protein gasdermin D (GSDMD) (Kayagaki et al., 2015;Shi et al., 2015). A recent study by Rogers et al. found that Casp3 successfully induced apoptosis and then cleaved GSDME, leading to generation of an N-terminal fragment (N-GSDME) and induction of pyroptosis (Rogers et al., 2017). Wang et al. confirmed that Casp3 cleavage led to formation of N-GSDME, and this interacted with 4,5-diphosphate phosphatidylinositol (PI[4,5]P2), and that this was followed by the perforation of liposomes and release of their contents (Wang et al., 2017). Therefore, Casp3, which has a well-established role in apoptosis (Cohen, 1997), also functions in pyroptosis by cleavage of GSDME. The cleavage of GSDME after treatment of cancer with chemotherapy agents provides a cross-talk between the pro-inflammatory process of pyroptosis and the anti-inflammatory process of apoptosis (Zheng and Li, 2020). Thus, GSDME may be regarded as a "molecular switch", in that its cleavage status determines whether cells undergo pyroptosis or apoptosis. There is also evidence that the effects of GSDME are downstream and upstream of Casp3, in that it connects the endogenous and exogenous apoptosis pathways and it also increases activation of Casp3, thereby functioning in a positive feedback loop . The expression of GSDME therefore controls whether cell death occurs via apoptosis or pyroptosis. In the presence of GSDME, some chemotherapy drugs induce Casp3 activation and lead to cell death via pyroptosis (Rogers et al., 2017;Wang et al., 2017) because pyroptosis is more rapid than apoptosis .
Pyroptosis functions in several different clinical pathologies, including type-2 diabetes. The ninth Edition of the International Diabetes Federation Diabetes Atlas reported in a worldwide survey that there will be 1.25 billion people with diabetes by 2030 and 1.50 billion by 2045 (Saeedi et al., 2019). Diabetic kidney disease (DKD) is a very serious microvascular complication of diabetes, and the incidence rate of DKD is also increasing worldwide (Harjutsalo and Groop, 2014;Huang et al., 2019). There is therefore a critical need for novel treatments of diabetic nephropathy (DN), because it is a primary cause of endstage renal disease throughout the world (Podgórski et al., 2019). Increased renal inflammation and renal fibrosis play critical roles in the onset and progression of DN (Sabapathy et al., 2019;Liu et al., 2020).
Recent research confirmed that Z-DEVD-FMK, a tetrapeptide that inhibits Casp3, reduced albuminuria, renal dysfunction, and tubulointerstitial fibrosis in diabetic mice (Wen et al., 2020). The nephroprotective effects of Z-DEVD-FMK were presumably due to its inhibition of GSDME activation. Other in vitro studies reported the presence of molecular and morphological features of secondary necrosis in HK-2 cells that were stimulated by glucose, and this was accompanied by GSDME cleavage, swelling of cells, and release of cellular contents (Wen et al., 2020). It is therefore believed that disruption of Casp3/GSDME-dependent pyroptosis has potential as a treatment for DN. We examined therapies that focus on pyroptosis and the Casp3/GSDME signaling pathway in DN in this mini-review. We first compare pyroptosis with other forms of PCD, introduce the history of scientific studies of pyroptosis, and then describe the functions of proteins in the GSDM family, especially GSDME and describe the role of GSDME-mediated pyroptosis during DN.

COMPARISON OF PYROPTOSIS, NECROPTOSIS, FERROPTOSIS, AND APOPTOSIS
The development and homeostasis of tissues and organisms requires regulation of cell proliferation and the removal of cells that are unnecessary or harmful. This includes the removal of damaged cells that may undergo neoplastic transformation and of cells whose functions are controlled by pathogenic microbes to promote pathogen replication (Bedoui et al., 2020). Cell death can be broadly classified as PCD or non-PCD (Durand et al., 2011). The different types of PCD depend on the regulation of specific genes that encode signals or activities that remove functionally dispensable, infected, or potentially neoplastic cells (Bedoui et al., 2020). The major types of PCD are pyroptosis, necroptosis, ferroptosis, and apoptosis (Green, 2019;Bedoui et al., 2020); the first three types are considered lytic cell death, and apoptosis is considered non-lytic. These different forms of PCD can be distinguished by different initiators, effectors, and executioners (Wang and Kanneganti, 2021).
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 780790 characterized by activation of inflammasomes by pathogenassociated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). This leads to recruitment of Casp1, which promotes the dimerization and activation of these compounds. Activated Casp1 then leads to synthesis and secretion of multiple inflammatory factors, including IL-1β and IL-18 (Wang et al., 2019b). In contrast, the non-canonical pathway of pyroptosis depends on the function of Casp4/5 in mice (corresponding to Casp11 in humans), which recognize lipopolysaccharide (LPS) via the caspase activation and recruitment domains (CARDs) (Kayagaki et al., 2011;Shi et al., 2014;Kovacs and Miao, 2017). The canonical and noncanonical pathways of pyroptosis are both characterized by inflammatory caspases that activate the pore-formation protein GSDMD . Recent studies confirmed that Casp3 also functions in pyroptosis Shen et al., 2021).
In particular, Casp3 can be activated by the intrinsic apoptosis initiator (Casp9) or by the extrinsic apoptosis initiator (Casp8). Upon activation, Casp3 induces pyroptosis via cleavage of GSDME.
Necroptosis is another form of lytic PCD that is defined by the destruction of the cytoplasmic membrane and cellular swelling (Tonnus et al., 2019). Numerous signaling molecules can induce necroptosis via death receptors or pathogen recognition receptors (PRRs) (Galluzzi et al., 2014;Choi et al., 2019). Three proteins have important functions in necroptosis: receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) (Belavgeni et al., 2020). Notably, necroptosis is considered independent of capsases (Conrad et al., 2016). Instead, upon initiation by receptors, necroptosis is mediated by RIPK1 or RIPK3-induced MLKL phosphorylation, followed by the formation of MLKL pores (Wang and Kanneganti, 2021). Interestingly, necroptosis usually occurs when the extrinsic pathway of apoptosis is non-functional (Frank and Vince, 2019). Recent research found that the occurrence of necroptosis was inhibited by Casp8 (Nailwal and Chan, 2019), which can inactivate RIPK1 via proteolytic cleavage (Newton et al., 2019). However, viral infections can inhibit Casp8, thus driving necroptosis (Belavgeni et al., 2020). Ferroptosis, a newly described form of PCD, is driven by lipid peroxidation and depends on the availability of iron and the generation of reactive oxygen species (ROS) (Galluzzi et al., 2018). A key feature of ferroptosis is intracellular overload iron and catalysis of lipid peroxidation (Belavgeni et al., 2020). Ferroptosis differs from the other forms of PCD in that it is characterized by small dysmorphic mitochondria with fewer cristae, a ruptured outer membrane, and a condensed inner membrane (Wang et al., 2020a;Sun et al., 2020), Ferroptosis functions in a variety of biological processes, including oxidative stress, iron metabolism, and lipid metabolism (Qiu et al., 2020). In return, the levels of iron, ROS, glutathione (GSH), and related regulatory molecules can regulate ferroptosis (Qiu et al., 2020). Ferroptosis occurs mainly due to the disruption of endogenous cellular antioxidant defense systems. Glutathione peroxidase 4 (GPX4), is the only glutathione peroxidase that affects ferroptosis, in that it functions as an endogenous inhibitor of this form of PCD (Ursini et al., 1982). In particular, GPX4 catalyzes the reduction of GSH, thereby limiting lipid peroxidation and ferroptosis (Imai et al., 2017).
Apoptosis is the best known form of PCD and was first described in 1972, although many earlier studies described several of the key feature of this form of PCD (Kerr et al., 1972). Unlike the other types of PCD, apoptosis is an active series of steps that occurs during normal physiological processes, such as development and aging, to maintain homeostasis; Apoptosis also occurs during pathological states, such as when cells are injured by a disease or toxic agents (Norbury and Hickson, 2001). Histologically, apoptosis is characterized by shrinkage and pyknosis of cells, condensation of chromatin, fragmentation of nuclei, and the formation of apoptotic bodies and cytoskeletal disintegration, but no obvious histological changes in the mitochondria (Elmore, 2007;Li et al., 2020c).
Apoptosis may occur via an intrinsic pathway (initiation by intracellular signals) or an extrinsic pathway (initiation by extracellular signals), and each pathway requires distinct caspases (Elmore, 2007). The intrinsic (mitochondrial) pathway is driven by numerous intracellular events, such as DNA damage, ROS formation, endoplasmic reticulum stress, and others. These stimuli then produce a change in the transmembrane potential of the mitochondria, leading to the release of pro-apoptotic proteins, especially cytochrome c (Cyto C), into the cytoplasm (Kiraz et al., 2016). Subsequently, Cyto C interacts with Apf-1 to activate Casp9, which then cleaves pro-Casp3 into Casp3, the active form which functions in the execution phase (Li et al., 1997).
The extrinsic pathway of apoptosis begins upon activation of the plasma membrane-bound death receptor and the dependence receptor. The death receptor is activated by binding to ligand(s), and the dependence receptor is activated when the levels of associated ligands decline below a certain threshold (Galluzzi et al., 2018). Once triggered, pro-Casp8 is recruited and processed to Casp8 (Muzio et al., 1996), which then activates the downstream executioners, Casp3 and Casp7 (Stennicke et al., 1998;Twiddy et al., 2006), leading to a series of histological and molecular changes.
It is important to note that pyroptosis, necroptosis, ferroptosis, and apoptosis have different initiators, effectors, and executioners (Wang and Kanneganti, 2021), and this is associated with differences in their histological and biochemical features (Belavgeni et al., 2020;Li et al., 2020c). All types of PCD except apoptosis are passive processes (Norbury and Hickson, 2001). In addition, the different modes of PCD may play different roles in immune defense. For example, pyroptosis is primarily a cellular reaction to potential damage or insult, such as pathogen invasion (Frank and Vince, 2019). Necroptosis is critical in the host response to infections, especially by viruses (Brault and Oberst, 2017), and it functions as a backup mechanism of cell death and defense when apoptosis is blocked, such as during pathogen infection (Frank and Vince, 2019). Ferroptosis is a form of cellular antioxidant defense against oxidative stress and it Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 780790 functions in a variety of diseases, not just infectious diseases (Kuang et al., 2020). Apoptosis is regarded as a mechanism of selfprotection that eliminates dead or damaged cells that form during the physiological process of aging (Tomei and Umansky, 1998;Argüelles et al., 2019). Apoptosis also has an important function in the suppression of tumor growth, and some anticancer drugs function by stimulation of apoptosis (Kaczanowski, 2016). Although these distinct types of PCD have different functions in different physiological states and in a host's response to external damage, there is clear evidence of crosstalk or interactions (Wang and Kanneganti, 2021), and some of the same proteins function in multiple types of PCD. For instance, Casp3 functions in apoptosis and GSDME-mediated pyroptosis . Casp8 initiates the extrinsic apoptosis pathway, and can also inhibit necroptosis (Newton et al., 2019). In addition, the same stimulant may induce different types of PCD, depending on its intensity or action time (Bedoui et al., 2020).

HISTORICAL PERSPECTIVE OF RESEARCH ON PYROPTOSIS
The term pyroptosis derives from the Greek words 'pyro' (fever) and 'ptosis' (falling) (Cookson and Brennan, 2001). This newly identified form of PCD is characterized by the formation of large pores in the plasma membrane, followed by cell swelling and rupture (Galluzzi et al., 2018). Before this word was adopted, this form of PCD was described by Friedlander in 1986, who treated primary mouse macrophages with anthrax toxin and reported the rapid release of the cell contents (Friedlander, 1986). In 1992, researchers first described this inflammatory PCD as a type of apoptosis in their studies of infection by Shigella flexneri (Zychlinsky et al., 1992). Five years later, Hilbi et al. discovered that Shigella dysenteriae activated Casp1 in host cells (Hilbi et al., 1997). In 1999, Hersh et al. confirmed that knocking out Casp1 blocked Salmonella-induced PCD (Hersh et al., 1999).
The year 2001 was an important turning point in our understanding of pyroptosis. Boise et al. observed macrophage death induced by bacterial infection, and identified a type of PCD that different from apoptosis, and named this pathway "caspase-1-dependent programmed necrosis" (Boise and Collins, 2001;Cookson and Brennan, 2001). Cookson et al. then proposed using the word "pyroptosis" for this proinflammatory mode of PCD (Cookson and Brennan, 2001). In 2015, researchers identified the GSDMD protein and identified it as the executioner of pyroptosis; they also reported that pyroptosis is auto-inhibited in normal and healthy cells, but that cleavage of GSDMD by Casp1 and Casp4/5 (Casp11 in humans) activated pyroptosis (Kayagaki et al., 2015;Shi et al., 2015). Subsequent research identified other proteins in the GSDM family (GSDMA, GSDMB, GSDMC, GSDME, and DFNB59) and confirmed their roles in membrane perforation and induction of pyroptosis (Martirosyan and Gorvel, 2013;Ding et al., 2016). In 2017, Rogers et al. reported that activated Casp3 induced apoptosis and then cleaved GSDME into N-GSDME, and this induced pyroptosis (Rogers et al., 2017). Wang et al. confirmed that N-GSDME interacted with PI(4,5)P2, and this led to the perforation of liposomes and loss of their phospholipids in a Casp3-mediated process (Wang et al., 2017). Therefore, to emphasize the role of multiple proteins in the GSDM family, Feng Shao and others characterized pyroptosis as "GSDM familymediated programmed necrosis" .
In 2018, Kambara et al. discovered that neutrophil elastase (NE) promoted the cleaveage of GSDMD, and this led to a process they called "neutrophil pyroptosis" (Kambara et al., 2018). Also in 2018, the Nomenclature Committee on Cell Death (NCCD) defined pyroptosis as a form of regulated cell death (RCD) that was characterized by pore formation in the plasma membrane due to the actions of proteins in the GSDM family (Galluzzi et al., 2018). Many recent studies have described the function of pyroptosis during the onset and progression of multiple diseases or pathological states, such as cancer, inflammation, and fibrosis (Aglietti and Dueber, 2017;Kovacs and Miao, 2017;Shi et al., 2017;Xia et al., 2020). Figure 1A summarizes the functions of different signals, molecules, and pathways in pyroptosis.
Pyroptosis is now accepted as a pro-inflammatory form of PCD that functions as a host defense mechanism against invading pathogens and in response to other types of cellular injury (Aglietti and Dueber, 2017). Initial studies suggested that pyroptosis only occurred in immune cells (macrophages, monocytes, dendritic cells, and T cells) (Schroder and Tschopp, 2010;Jorgensen et al., 2017), and that stimulation of these cells by pathogens or their products led to pyroptosis due to the increased activity of pro-inflammatory caspases (Casp1 in humans and mice; Casp4/5 in humans, and Casp11 in mice) Yuan et al., 2016). The original descriptions of pyroptosis thus characterized the Casp1-dependent classical pathway and the Casp4/5/11-dependent non-classical pathway.
The activation of pyroptosis begins when the initiator directly or indirectly detects microbial ligands, followed by Casp1 activation (Orning et al., 2019). This induces proteolysis of substrate proteins, including GSDMD, and activates several pro-inflammatory cytokines, includingIL-1β and IL-18. However, Casp11 can also mediate non-canonical activation of inflammasomes. This non-canonical pathway acts on GSDMD without the NLR sensor and adaptor proteins that function in the canonical pathway. LPS or host-derived oxidized phospholipids activate Casp11 in mice (corresponding to Casp4/5 in humans) (Kayagaki et al., 2011;Hagar et al., 2013;Kayagaki et al., 2013;Shi et al., 2014;Zanoni et al., 2016). GSDM functions as the effector protein in the canonical and non-canonical pathways, and its cleavage by pro-inflammatory caspases at Asp275 in humans (Asp276 in mice) leads to formation of the active form, N-GSDMD Kayagaki et al., 2015;Shi et al., 2015).
Interestingly, there is evidence that pyroptosis can also occur in the absence of Casp1, and even in the absence of GSDMD (Schneider et al., 2017). In fact, some studies found that Casp3, a pro-apoptotic caspase can induce pyroptosis (Rogers et al., 2017;Wang et al., 2017). More typically, the death receptor-mediated pathway of apoptosis activates Casp3 via activation of Casp8 or the mitochondrial apoptotic pathway activates Casp3 via Casp9 FIGURE 1 | (A) Pyroptosis may occur via a GSDMD/Casp1-dependent canonical pathway, a caspase-11 (4/5) -dependent non-canonical pathway, and a Casp3/ GSDME pathway. Left: In the canonical pathway, viruses, toxins (including DAMPs and PAMPs), or bacteria bind to inflammasome sensors, which then binds to an initiator protein (NLRP1, NLRP3, AIM2, . . . ). This leads to activation of an adaptor protein (ASC), an executioner caspase (typically a Casp1 initiator protein such as NLRP3 or AIM2), and then an executioner caspase (typically Casp1). Once activated, Casp1 induces the proteolysis of substrate proteins, including GSDMD and the pro-inflammatory cytokines IL-1β and IL-18, followed by membrane perforation and pyrolysis. Middle: In the noncanonical pathway, intracellular LPS or toxins activate Casp11 in mice (Casp4/5 in humans). This leads to the same cascade of reactions as in the canonical pathway. Right: In the Casp3/GSDME pathway, a death receptor or the mitochondrial pathway activates Casp3. This leads to cleavage of GSDME to N-GSDME, membrane perforation, and pyroptosis. The generation of N-GSDME also permeabilizes the mitochondrial membrane, leading to release of cytochrome C and induction of Casp3-mediated apoptosis, a positive feedback loop; (B) Structure of the wildtype and mutant GSDME gene. The wildtype GSDME has 10 exons and three domains: globular domain A (exon 2-6), a hinge region (exon 6-7), and globular domain B (exon 7-10). The hinge region differs in length and sequence among different GSDME variants. In wildtype, the N-terminal domain (exons 2-6) is responsible for production of plasma membrane pores and subsequent pyroptosis, and is only active upon cleavage from the C-terminal domain. However, individuals with GSDME-mediated hearing loss have a deletion of exon eight and protein truncation, leading to spontaneous formation of plasma membrane pores and pyroptosis. Abbreviations: GSDMD, gasdermin D; Casp3, caspase-3; GSDME, gasdermin E; DAMPs, danger-associated molecular patterns; PAMPs, pathogen-associated molecular patterns; NLRP1, NACHT, LRR, and PYD domains-containing protein 1; NLRP3, NACHT, LRR, and PYD domains-containing protein 3; AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; IL-1β/IL-18, interleukin-1β/interleukin-18; LPS, lipopolysaccharide; N-GSDME, N-terminal fragment of gasdermin E.
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 780790 6 (Li and Yuan, 2008). During the mediation of pyroptosis by the extrinsic or intrinsic pathways of apoptosis, GSDME functions as the effector. Several anti-cancer agents promote the cleavage of GSDME into N-GSDME, thereby promoting the death of cancer cells (Rogers et al., 2017;Wang et al., 2017;Zhou et al., 2018). N-GSDMD and N-GSDME function as executioners because they promote the formation of pores on the plasma membrane.
IL-1β and IL-18 (pro-inflammatory cytokines) are processed from precursors into biologically active forms. In contrast to other cytokines, cytokines in the IL-1 family are not released via typical protein secretion pathways, and the mechanisms underlying the release of IL-1β and IL-18 were only recently determined. In particular, recent studies found that several GSDMs (GSDMD, GSDMA3, and GSDME) undergo proteolytic activation of their N-terminal domains and then form large transmembrane pores (Aglietti et al., 2016;Chen et al., 2016;Ding et al., 2016;Liu et al., 2016;Wang et al., 2017). Cryo-electron microscopic analysis of the structure of the mouse GSDMA3 pore provided important insights regarding the mechanism of this pore formation (Ruan et al., 2018). In particular, the pore consists of a 27-fold symmetric, ∼0.8-MDa oligomeric assembly and has an inner diameter of 18 nm (Ruan et al., 2018). This pore allows the transfer of many soluble cytosolic contents to the extracellular space including proinflammatory cytokines IL-1β and IL-18 (Heilig et al., 2018;Xia et al., 2021a). However, lactate dehydrogenase (LDH), a ∼140 kDa protein whose release is often used as a marker of complete membrane rupture and lytic cell death, is too large to pass through this pore (Heilig et al., 2018) , (Tinton et al., 1993).

GSDMS AND GSDME
The GSDM family is so named because these proteins have high expression along the gastrointestinal tract and dermis (Saeki et al., 2000;Tamura et al., 2007). This protein family includes GSDMA, GSDMB, GSDMC, GSDMD, GSDME (DFNA5), and DFNB59 (Pejvakin) in humans (corresponding to GSDMA1-3, GSDMC1-4, GSDMD, GSDME, and DFNB59 in mice) (Nagarajan et al., 2019). These proteins have 45% sequence homology, and most of them have similar functional domains (Feng et al., 2018). Except for DFNB59, each protein has a N-terminal domain that is responsible for pore formation domain, a C-terminal domain that functions as a regulator, and a flexible "hinge" region that joints these two domains (Op de Beeck et al., 2011). A recent study reported that the N-GSDM domain of GSDMA, GSDMD, and GSDME executed cell death by pore formation (Ding et al., 2016), and that the C-terminal domain (C-GSDM) of the full-length protein inhibited this action. Although GSDMD was the first identified and most studied member of this family, recent studies of the pyroptosis of tumors have focused on GSDME (Feng et al., 2018;Nagarajan et al., 2019).
A 1998 study first identified the GSDME gene (also called deafness autosomal dominant 5, DFNA5) on chromosome 7p15.3. Patients with specific genetic variants have autosomal dominant, progressive, sensorineural, and non-syndromic hearing loss (Van Laer et al., 1998). The wildtype GSDME has 10 exons, encodes a protein with 496 amino acids, and has a molecular weight of ∼55 kDa. Most GSDME mutations affect the inhibitory C-terminal domain, thus leading to spontaneous formation of pores and pyroptosis (Gregan et al., 2003). Although families with GSDME-mediated hearing loss have different mutations at the DNA level, they all have a deletion of exon eight and protein truncation (Yu et al., 2003;BISCHOFF et al., 2004;Cheng et al., 2007;Park et al., 2010;De Beeck et al., 2012;Chai et al., 2014;Nishio et al., 2014;Li-Yang et al., 2015;Nadol et al., 2015;Wang et al., 2018a;Booth et al., 2018) ( Figure 1B). GSDME expression varies among cells and tissues. In humans, GSDME is normally expressed in the placenta, heart, brain, and kidneys; however, in mice GSDME is expressed in the cochlea, thymus, colon, lung, brain, spleen, and small intestine (Van Laer et al., 1998;Wu et al., 2009), but not in the breasts, gastric system, or colorectal cancer due to hypermethylation of its promotor (Akino et al., 2007;Kim et al., 2008;Stoll et al., 2017). Recent studies found that chemotherapy drugs activated the Casp3/ GSDME-dependent pyroptosis pathway via cleavage of GSDME to N-GSDME and C-GSDME, and that this led to pore formation in the membranes and pyroptosis (Rogers et al., 2017;Wang et al., 2017). Rogers et al. demonstrated that the human and mouse DFNA5 protein contained a putative Casp3 recognition motif at residues 267-270 (267DMPD270) (Rogers et al., 2017), whereas Wang et al. identified a Casp3-cleavable site at 267DMPD270 in the human protein and at 267DMLD270 in the mouse protein (Wang et al., 2017). The potential cause of these different findings may be the different methods used for identification of this motif. For example, Rogers et al. identified the Casp3 recognition site in GSDME by searching for a consensus Casp3 recognition motif in the linker region between the N-GSDME and C-GSDME domains, and then they confirmed the Casp3cleavable site by substituting Asp270 with Glu270 in the human protein using site-directed mutagenesis (Rogers et al., 2017). In contrast, Wang et al. identified the Casp3 recognition site by using self-made Casp3 protein to cleave GSDME that was used for liposome-binding and leakage assays (Wang et al., 2017).
Interestingly, GSDME can act downstream and upstream of Casp3. In particular, generation of the N-GSDME by Casp3 leads to permeabilization of the mitochondrial membrane, the release of Cyto C, and the inducion of Casp3-mediated apoptosis (Tait and Green, 2010;Rogers et al., 2019). This cross-talk of the endogenous and exogenous pathways of apoptosis also promotes the activation of Casp3, thus forming a positive feedback loop . GSDME cleavage into N-GSDME leads to the formation of large pores in the plasma membrane and pyroptosis, and N-GSDME also permeabilizes the mitochondrial membrane and this activates apoptosis via the mitochondrial pathway. Although Casp3 has pro-apoptotic effects, GSDME may be considered a "switch" that determines whether a cell undergoes pyroptosis or apoptosis; in that its level determines the type of PCD that occurs in cells with activated Casp3. In particular, Wang et al. reported that the expression of GSDME determined whether cells underwent apoptosis or pyroptosis, with a high level of GSDME leading to pyroptosis and a low level leading to apoptosis (as in patients receiving chemotherapy). In the presence of GSDME, chemotherapy drug-induced Casp3 activation often leads to pyroptosis (Rogers et al., 2017;Wang et al., 2017), because pyroptosis is a more rapid process than apoptosis . However, several studies have challenged the interpretation that GSDME drives pyroptosis upon stimulation of apoptosis. For example, GSDME-deficient macrophages still underwent plasma membrane damage in response to Casp3 activators (Lee et al., 2018). Another study found that during mitochondrial apoptosis, GSDME was not required for channel formation, and that it had a redundant role in the lysis of macrophage that was downstream of the ripoptosome (Chen et al., 2019). In agreement, Tixeira et al. reported that GSDME was unnecessary for the pyroptosismediated regulation of human T cells and monocytes (Tixeira et al., 2018) and Lee et al. demonstrated that GSDME was not required for pyroptosis in caspase-1 −/− caspase-11 −/− bone morrow-derived macrophages treated with flagellin, cytochrome, or the Fas ligand (Lee et al., 2018). Taking together, these findings suggest that GSDME-mediated pyroptosis occurs only in cells that have specific internal conditions or only in specific types of cells.

Pyroptosis Mediates Different Types of Renal Cell Damage in DN
The increasing prevalence of diabetes, and DN indicates the need for an improved understanding of the mechanisms underlying these diseases. DN is a complex process involving multiple factors. Excessive accumulation of glucose and lipids are the initial inducers of DN. The consequent induction of inflammation, fibrosis, hemodynamic alterations, oxidative stress, and apoptosis are the main pathogenic characteristics of DN (Barrera-Chimal and Jaisser, 2020; Kushwaha et al., 2020;Opazo-Ríos et al., 2020;Thongnak et al., 2020), but there is emerging evidence that additional signaling molecules function in the pathogenesis of DN. These include renal endothelial-related molecules (endothelial sirtuin 3 [SIRT3] (Wang et al., 2019c), endothelial glucocorticoid receptors [E-GRs] (Srivastava et al., 2021a), and endothelial fibroblast growth factor receptor 1 [FGFR1]) (Typiak et al., 2021), and the podocyteglucocorticoid receptor (P-GR) (Srivastava et al., 2021b). E-GR and P-GR have anti-fibrotic effects, and their loss or knock-outpromotes fibrosis and accelerates DN (Srivastava et al., 2021a;Srivastava et al., 2021b).

Pyroptosis Mediates Damage of Endothelial Cells in DN
Previous studies (Shahzad et al., 2015) identified the activation of inflammasomes in endothelial cells and podocytes in vivo (humans and mice with diabetes), and in vitro (glucose-stressed glomerular endothelial cells and podocytes). Blocking the expression of NLRP3 or Casp1 in bone marrow-derived cells did not prevent the development of DN in mice; however, mice that were deficient in NLRP3 were protected from DN even when receiving transplantation of wild-type bone marrow. An in vitro study of glomerular endothelial cells (GECs) reported that addition of a high dose of glucose (HG) promoted pyroptosis, but this was inhibited by silencing of GSDMD, thus indicating the participation of pyroptosis in the injury of these cells (Gu et al., 2019).

Pyroptosis Induces Podocyte Injury in DN
Podocytes are important for the proper function of the glomerular filtration barrier, and their injury or loss can lead to proteinuria. Cheng et al. (2021) studied mice with DN and showed that the increased expression of Casp11 and the increased cleavage of GSDMD in podocytes, correlated with reduced expression of two podocyte markers (nephrin and podocin), and with the loss and fusion of podocyte foot processes. Knockout of Casp11 or GSDMD mitigated these changes in DN mice model. Their studies of human podocytes (Cheng et al., 2021) treated with HG led to similar results. Specifically, knockdown of Casp4 (or GSDMD) with siRNA significantly reduced the levels of Casp4 (or N-GSDMD), and also reduced the levels of NF-κB, IL-1β, and IL-18, nephrin, and podocin. Zhang et al. Gao et al. (2014Gao et al. ( , 2015) found that HG-induced activation of NADPH oxidase was driven by thioredoxin interacting protein (TXNIP), and that this increased NLRP3 inflammasome activation in podocytes and ultimately led to podocyte injury. Thus, blockage of this signaling may have promise as a treatment for DN. Yang et al. Liu et al. (2018)) demonstrated that TLR4 knockdown attenuated HG-induced podocyte injury by targeting the NALP3/ASC/Casp1 signaling pathway.

Pyroptosis Causes Renal Tubular Epithelial Cell Injury in DN
The renal damage induced by pyroptosis in DN is not limited to GECs and podocytes. In fact, GSDMD is a critical protein in pyroptosis, its role in regulating renal tubular injury in DN was Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 780790 8 widely discussed. As the executioner of pyroptosis, GSDMD functions in various pathways that contributes to this injury. TLR-4/NF-kappaβ signaling is a common pathway in inflammation. Wang et al. (2019a), reported that inhibition of NF-κB decreased the levels of Cas1, N-GSDMD, and secretion of IL-18 and IL-1β. Ke et al. (2020) studied a rat model of DN and reported that activation of the TXNIP/NLRP3 axis led to induction of inflammation and pyroptosis, and silencing of TXNIP inhibited inflammation and pyroptosis, as well as renal damage. They performed in vitro to examine pyroptosismediated damage of NRK-52E cells (Ke et al., 2020). microRNA miR-200a, which is degraded by an endoplasmic reticulum stress (ERS)-related factor (IRE1α), binds to TXNIP. Thus, inhibiting IRE1α increased the level of miR-200a, and decreased the level of TXNIP. Conversely, inhibiting miR-200a in HG-induced NRK-52E cells partially blocked pyroptosis that was mediated by STF-083010, an IRE1α RNase specific inhibito. Li et al. (2017), investigated the role of long noncoding RNA MALAT1 and its downstream molecules in the pyroptosisinduced injury of HK-2 cells. Their data demonstrated that HG-treated HK-2 cells had an increased level of MALAT1 and a decreased level of miR-23c, and this led to NLRP3 activation and initiation of pyroptosis.
Sun and his team  found an increased level of mtROS-TXNIP-NLRP3 inflammasomes in the kidneys of patients with DN and in diabetic (db/db) mice. Addition of MitoQ to HK-2 cells prevented the dissociation of TRX from TXNIP and blocked the interaction of TXNIP and NLRP3, and this inhibited the activity of NLRP3 inflammasomes and the maturation of IL-1β. Pretreatment of these cells with TXNIP siRNA increased the effect of MitoQ, and pretreatment with monosodium urate (MSU) and TRX siRNA blocked this effect. This suggests that activation of mitochondrial ROS-TXNIP/NLRP3/IL-1β signaling leads to tubular oxidative injury, and that this can be blocked by MitoQ due to its inhibition of mtROS production .
In addition, long noncoding RNAs (lncRNAs) also reduced the severity of DN via their regulation of miRNAs in HK-2 cells. In particular, Xie et al. (2019) found that HG-treated HK-2 cells had decreased expression of growth arrest specific 5 (lncRNA GAS5), and that GAS5 overexpression downregulated several proteins related to pyroptosis Casp1,NLRP3,. Notably, interference by miR-452-5p led to the same changes as overexpression of GAS5 in these same cells, and inhibition of GAS5 reversed the effect of miR-452-5p interference. This led to the conclusion that the lncRNA GAS5/miR-452-5p axis regulated HGinduced pyroptosis in renal tubular cells. In addition, studies of the lncRNA KCNQ1OT1/miR-506-3p pathway have provided additional insights into the pathogenesis and possible treatment of DN. In particular, downregulation of KCNQ1OT1 in HG-treated HK-2 cells led to upregulation of miR-506-3p, and this was followed by reduced pyroptosis, inflammation, and oxidative stress .

Pyroptosis Leads to Glomerular Mesangial Cell Injury in DN
Other studies examined the role of pyrolysis of glomerular mesangial cells in DN. For example, Zhan et al. (2020) established a rat model of DN and found that mesangial cell pyroptosis occurred during the pathogenesis of DN, and that upregulation of the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) increased the levels of pyroptosisassociated proteins. They also examined the interactions of NEAT1 with pyroptosis using an in vitro DN model (HGtreated HBZY1 cells). Their in vitro results indicated that NEAT1 altered the miR-34c/NLRP3 axis and thereby regulated pyroptosis in these cells.

GSDME Mediated Pyroptosis Presents Great Potential Targeting DN
A 2017 study first reported that chemotherapy drugs specifically cleaved GSDME, and that this generated N-GSDME, pore formation in plasma membranes, and pyroptosis (Rogers et al., 2017). Casp3 is thus a pro-apoptotic caspase and is also responsible for GSDME cleavage, and GSDME status determines whether a cell undergoes pyroptosis or apoptosis. In agreement with this interpretation, pyroptosis occurs in normal human primary cells that express GSDME (epidermal keratinocytes, placental epithelial cells, and umbilical artery smooth muscle cells) and in cancer cells (neuroblastoma, skin melanoma, and gastric cancer cells) (Wang et al., 2017;Wang et al., 2018b;Zhang et al., 2019). However, cells that do not express GSDME undergo apoptosis. For example, mice that lack GSDME (GSDME −/-) were protected from tissue injury due to chemotherapy (Wang et al., 2017). Indeed, traditional chemotherapeutic drugs, such as cisplatin, are commonly used to treat various cancers, including lung, bladder, and ovarian cancer (Pabla and Dong, 2008;BROXTERMAN et al., 2009;Ozkok and Edelstein, 2014;Gabizon et al., 2016). Chemotherapeutic drug-induced nephrotoxicity reportedly occurs in one-third of cancer patients, which limits the clinical application of these drugs (Izzedine, 2018;Volarevic et al., 2019). Chen and his team demonstrated that renal tubular epithelial cell pyroptosis induced by chemotherapy drugs (cisplatin or doxorubicin) was mediated by ROS-JNK-Casp3-GSDME signaling (Shen et al., 2021). Therefore treatments that target GSDME signaling may reduce this specific cause of drug-induced nephrotoxicity. Their research also examined the role of GSDME in renal cellular pyroptosis and in the pathogenesis of cisplatininduced acute kidney injury (AKI) in mice that received cisplatin. The results indicated that GSDME −/− mice had less severe renal injury AKI and reduced inflammation. This role of GSDME in promotion of pyroptosis also occurred in human tubular epithelial cells (TECs) in vitro. Interestingly, inhibition of Casp3 blocked the cleavage of GSDME to N-GSDME, and this ameliorated cisplatin-induced pyroptosis and kidney dysfunction (Xia et al., 2021b).
Caspase/GSDME-mediated pyroptosis also occurs in other inflammatory diseases. Tan et al. studied bone marrow-derived macrophages (BMDMs) that were treated with the MCC950 (a specific inhibitor of the NLRP3) and RAW264.7 cells that lcaked in ASC. Their results showed that cell lysis induced by extracellular ATP had histological similarities to the canonical pyroptosis in these cells, but Casp1 was not activated, and there Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 780790 was no cleavage of GSDMD. Instead, this activated Casp8/9 (apoptotic initiator) and Casp3/7 (apoptotic executioner), and this led to formation of N-GSDME and pyroptosis. Notably, a Casp3 inhibitor reduced the N-GSDME production and cell lysis that were induced by extracellular ATP were ameliorated by. When BMDMs did not receive MCC950 treatment, the addition of ATP led to the formation of ASC that co-localized with Casp8, but there was no such effect in BMDMs that received MCC950 treatment. Their sdudies of the RAW264.7 cells indicated that knockdown of GSDME by an siRNA attenuated ATP-induced cell lysis and the release of high mobility group box-1 protein (HMGB1). Collectively, the results of these studies indicate that ATP induces pyroptosis of macrophages via the Casp3/GSDME axis when there is blockage of the canonical NLRP3 pathway. This suggests there is an alternative mechanism for prevention of pathogen invasion . In addition to inflammatory diseases, pyroptosis also functions in fibrotic diseases. For example, research on obstructive nephropathy by Xu et al. showed that Casp3/ GSDME-mediated pyroptosis occurred in renal parenchyma but not in hematopoietic cells. The studies of renal parenchyma indicated pyroptosis was related to the onset of renal tubule injury due to ureteral obstruction, and subsequently exacerbated hydronephrosis, inflammation, and fibrosis. Deletion of Casp3 or GSDME reduced renal tubule damage, inflammation, hydronephrosis, and kidney fibrosis (Li et al., 2021b).
In a study of microelement molybdenum and environmental heavy metal contaminant cadmium-induced nephrotoxicity, GSDME was confirmed to be involved in this injury . In addition, it was reported that GSDMEb-mediated pyroptosis promoted LPS-induced AKI in zebrafish and renal tubule cell injury from zebrafish larvae. Gene knockout of GSDMEb can block these damages induced by LPS (Wang et al., 2020b). Many studies have examined the effect of GSDMEmediated pyroptosis in kidney diseases, and these studies used multiple interventions, in vitro models, and in vivo models. Table  1 summarized present studies exploring GSDME-mediated pyroptosis in kidney diseases.
Although there are a few reports about the role of GSDMEdependent pyroptosis in DN, its specific roles are uncertain. A recent study confirmed the underlying mechanism of renoprotection provided by GSDME regulation in human tubular cells. In particular, Wen et al. showed that the Casp3 inhibitor Z-DEVD-FMK reduced albuminuria, improved renal function, and blocked tubulointerstitial fibrosis in mice with diabetes. It is likely that these nephroprotective effects were due to its inhibition of GSDME. Studies of HG-treated HK-2 cells reported molecular and histological features of secondary necrosis, with evidence of GSDME cleavage, swelling of the cells, and release of cellular contents (Wen et al., 2020). In addition to fibrosis, inflammation also contributes to the progression of DN (Wada and Makino, 2016). Renal inflammation and fibrosis are crucial in promoting the onset and progression of DN, and the findings presented above demonstrate that Casp3/GSDME-triggered pyroptosis contributed to the inflammation and fibrosis that are characteristic of AKI and CKD. Figure 2 shows the potential mechanism of pyroptosis in the pathogenesis of DN. FIGURE 2 | Potential mechanism of pyroptosis in the pathogenesis of DN. Hyperglycemia-induced production of ROS plays a key role in the initiation and progression of DN. Left: One ROS signaling pathway leads to dissociation of TXNIP from Trx and subsequent activation of NLRP3 and the Casp1/GSDMD canonical pyroptosis pathway. Right: Another ROS signaling pathway leads to activation of Casp3 via Casp8 or via mitochondrial dysfunction; in both cases, there is induction of Casp3/GSDME-mediated pyroptosi, leading to inflammation and fibrosis. Abbreviation: DN, diabetic nephropathy; ROS, reactive oxygen species; TXNIP, thioredoxin interacting protein; Trx, thioredoxin; NLRP3, NOD-like receptor family pyrin domain containing three; Casp1, caspase-1; Casp3, caspase-3; GSDME, gasdermin E.
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 780790 Therefore, we speculate that experimental interventions that target Casp3/GSDME dependent pyroptosis will provide novel insights for the development of new treatments for DN. There are currently several GSDME-derived Casp3 inhibitors that differ from previously described classical Casp3 inhibitors (e.g., Z-DEVD-FMK). For example, the human/mouse GSDME cleavage site peptide DMPD/DMLD binds to the catalytic domain of Casp3. In addition, the derived inhibitor Ac-DMPD/DMLD-CMK significantly suppressed Casp3 activation and reduced the levels of downstream effectors GSDME, thereby preventing apoptosis and pyroptosis. Initial studies of these inhibitors showed they helped to prevent in vivo acute hepatic failure that was induced ligation of bile duct due to their blockage of apoptosis and pyroptosis in hepatocytes and macrophages (Xu et al., 2021).

Pharmacological Inhibitors, Future Directions and Perspective in DN
The many studies discussed above clearly indicate that Casp3/ GSDME-dependent pyroptosis occurs during the onset and progression of DN. The presence of this type of pyroptosis was also confirmed in HG-treated HK-2 cells, and a selective Casp3 inhibitor (Z-DEVD-FMK) inhibited pyroptosis and fibrogenesis. However, several remaining questions must be addressed. First, because glomerular injury is responsible for albuminuria and is common during the early stages of DN (Setyaningsih et al., 2021), does Casp3/GSDME-mediated pyroptosis contribute to the injury of other renal cells in addition to damaging renal tubular epithelial cells? Second, does Casp3/GSDME-mediated pyroptosis interact with other signaling pathways, and if so, are these interactions upstream or downstream?
The selective Casp3 inhibitor Z-DEVD-FMK shows great potential as a treatment for DN. There is also evidence that the Ac-DMPD/DMLD-CMK (a GSDME-derived inhibitor) protects against acute hepatic failure by regulating apoptotic and pyroptotic events in hepatocytes and macrophages. Thus, does Ac-DMPD/DMLD-CMK also have a renoprotective effect in DN? If so, which is more effective, Z-DEVD-FMK or Ac-DMPD/ DMLD-CMK? Finally, can gene knockout or interference, especially at the organ or tissue level, be an effective treatment? Although there is still a long way to go, further research of Casp3/GSDME-mediated pyroptosis may provide answers to these and other important questions and help to identify new therapies for DN.

AUTHOR CONTRIBUTIONS
LM and WC decided to elaborate this subject and helped revise the manuscript. WL was responsible for collection of published paper and drove the manuscript. JS helped revise the manuscript. XZ and YL made a contribution of literature collection. All authors have read and approved the final manuscript.

FUNDING
This study was supported by the Natural Science Foundation of Jilin Province (No. 20200201352JC).

ACKNOWLEDGMENTS
Thanks for the Department of Nephropathy in the Second Hospital of Jilin University providing computers to search for the related materials.