Pyroptosis in Kidney Disease

Contact: emily.li@wecistanche.com

Yujia Wang, Yinshuang Li and Yanfang Xu1

Abstract

In the last several decades, apoptosis interference has been considered clinically irrelevant in the context of renal injury. The recent discovery of programmed necrotic cell death, including necroptosis, ferroptosis, and pyroptosis refreshed our understanding of the role of cell death in kidney disease. Pyroptosis is characterized by a lytic pro- inflflammatory type of cell death resulting from gasdermin-induced membrane permeabilization via activation of inflflammatory caspases and inflammasomes. The danger-associated molecular patterns (DAMPs), alarmins, and pro-inflflammatory cytokines are released from pyroptotic cells in an uncontrolled manner, which provokes inflflammation, resulting in secondary organ or tissue injuries. The caspases and inflammasome activation-related proteins and pore-forming effector proteins known as GSDMD and GSDME have been implicated in a variety of acute and chronic microbial and nonmicrobial kidney diseases. Here, we review the recent advances in pathological mechanisms of pyroptosis in kidney disease and highlight the potential therapeutic strategies in the future.

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Introduction

Programmed cell death (PCD) plays a critical role in both the development of multicellular organisms and the maintenance of adult tissues. In sharp contrast to accidental cell death (e.g., necrosis) which is instantaneous, catastrophic, and uncontrollable, PCD is tightly regulated, which allows pharmacological and genetic modulations.1 Over the last few decades, distinct forms of PCDs have been identifified, including noninflammatory processes (e.g., apoptosis) and highly inflflammatory processes (e.g., pyroptosis and necroptosis). In addition to the presence or absence of inflflammation, apoptosis and pyroptosis can be distinguished by morphology. The characteristic morphological changes of apoptosis include cell shrinkage, membrane blebbing, apoptotic body formation, and DNA fragmentation, but no inflflammatory content release.2 Conversely, pyroptosis features cell swelling, membrane rupture, and uncontrolled release of inflflammatory contents. Necroptosis shares common characteristics with pyroptosis, including obvious inflflammation and loss of membrane integrity.3

Although pyroptosis and apoptosis are both caspase-dependent, the specific caspase family members involved are completely different. Apoptotic caspases include caspases-8/9/10 as initiator and caspases-3/6/7 as executors,4 while pyroptosis relies on caspases-1/4/5/11 in gasdermin D (GSDMD)-mediated pyroptosis and caspases-3 in gasdermin E (GSDME)-mediated pyroptosis.5,3 Necroptosis proceeds via the step-wise activation of receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL), which could be inhibited by caspase-8. That is different from GSDMs in pyroptosis, whose activation is caspase-dependent. The relationship between gender mins and MLKL under physiological conditions has become a topic of interest for research.3 Ferroptosis is a peroxidation-controlled system that does not involve caspases, usually accompanied by a large amount of iron accumulation and lipid peroxidation.6 Quite little is known about the interaction between ferroptosis and pyroptosis.

Growing experimental evidence supports the hypothesis that pyroptosis is a signifificant factor in kidney diseases. In this review, we discuss the molecular mechanisms of pyroptosis, the existing data on pyroptosis in kidney diseases, and potential therapies.

Overview of Pyroptosis

Executors

Pyroptosis is defined as gasdermin-dependent for its key step of pore formation in the plasma membrane is executed by masterminds, which makes it quite distinct from other types of PCDs. GSDMD is the first member of the gasdermin family that was found to be involved in pyroptosis.7–9 Full-length GSDMD remains in an autoinhibited state through the binding of C-terminal domain to the N-terminal domain. Upon cleavage, the N-terminal fragment of GSDMD (GSDMDN) can oligomerize to form pores (with an inner diameter of 12–14 nm) on the plasma membrane,10 which impairs the permeability barrier and causes cell lysis. GSDMD cleavage is mediated through canonical or noncanonical pathways involving caspase-1 or caspases-4/5/11 respectively.5,11 GSDMDmediated pyroptosis is defined as inflammasome dependent given that caspases-1/4/5/11 are activated by different inflflammasomes. Recent studies revealed that GSDMD cleavage can also be induced by neutrophil elastase in aging neutrophils,12 and by caspase-8 in macrophages during Yersinia infection.13,14

Genetic mutation of GSDME, another member of the gasdermin family, was firstly identifified as the cause of autosomal dominant nonsyndromic hearing loss in humans.15 Recent studies have revealed the role of GSDME in pyroptosis.16,17 GSDME is highly expressed in various kinds of normal tissues; however, it is silenced in most tumor cells due to promoter hypermethylation, and modulation of its expression by DNA methyltransferase inhibitor would improve chemotherapy sensitivity.17 Different from GSDMD, GSDME is cleaved into GSDME-C and GSDME-N by activated caspase-3 and is independent of inflammasome formation. GSDME-mediated pyroptosis occurs downstream of the mitochondrial apoptotic pathway, and high expression of GSDME and persistent mitochondrial permeability would switch the manner of cell death from apoptosis to pyroptosis.18

Other members of the gasdermin family, including GSDMA, GSDMB, and GSDMC, share highly conserved gasdermin-N and gasdermin-C domains with GSDMD and GSDME.19 GSDMB can be cleaved and activated by granzyme A from cytotoxic lymphocytes.20 A recent study showed that programmed death-ligand 1 switched tumor necrosis factor (TNF)-an-induced apoptosis to pyroptosis in cancer cells, and this and mediated by enhancing GSDMC gene transcription. GMC activation was induced by caspase-8 following TNF-a treatment, under the hypoxia condition.21 However, the mechanism by which GSDMA would be activated remains unexplored.

Signaling pathways

GSDMD-mediated pyroptosis. GSDMDmediated pyroptosis is initiated by the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular pattern molecules (DAMPs) with subsequent inflammasome activation.22 The first and most extensively studied form of GSDMD-mediated pyroptosis is the canonical pyroptotic pathway, also known as the caspase-1 dependent pathway. Caspase-1 has long been implicated in cell death, which was previously mistaken for apoptosis23,24 and necroptosis.25 In 2001, the form of PCD involving caspase-1 was distinguished from apoptosis and necroptosis by its inflflammatory response and characteristic morphology including the early membrane integrity loss.26,27

In response to pathogen invasion or host immunological challenge, pattern-recognition receptors (PRRs) perceive both PAMPs and/or DAMPs. In the canonical pyroptotic pathway, several PRRs such as absent in melanoma 2 (AIM2)-like receptors (ALRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) or pyrin proteins, and bridge adaptors such as apoptosis-associated speck-like proteins (ASCs), would assemble a multi-protein platform (i.e. inflammasome) which recruits pro-caspase-1 and then generates active caspase-1.28 As an exception, NLRC4 can bind to members of the NLR family of apoptosis inhibitory proteins (NAIPs) rather than ASC to form inflammasome, and subsequently activate procaspase-1.29 Activated caspase-1 has the activity of processing pro-IL-1b and pro-IL-18 into mature forms,30 which endows pyroptosis with proinflammatory functions, distinguishing it from apoptosis. In parallel with the process of inflflammatory cytokines, activated caspase-1 cleavages GSDMD to form membrane pores22,31,32 (Figure 1). The formation of membrane pores leads to the disruption of cell membrane integrity, the release of inflflammatory intracellular contents, and eventually cell lysis. NLRP3 is the most widely characterized inflammasome.33 NLRP3 responds to a wide variety of stress, including intracellular pathogens and extra-cytoplasmic inflammatory stimuli.34 The topic of NLRP3 activation has attracted a great deal of attention, and researchers have proposed different mechanisms including oxidized mitochondrial DNA, the generation of mitochondrial reactive oxygen species (ROS), potassium efflflux out of the cell, and cathepsin release after lysosomal destabilization.35 Despite years of intensive efforts, the exact mechanisms of NLRP3 activation are still under debate. It is interesting that NLRP3 can be triggered by necroptosis signaling,36–38 suggesting potential crosstalk between necroptosis and pyroptosis. The AIM2 inflammasome behaves differently from NLR inflflammasomes as it can recognize and bind cytosolic dsDNA, which can be released during pathogen infection39,40 or generated in tumor cells.41,42

In the non-canonical pyroptotic pathway, caspase-11 (in mice) and caspases-4/5 (in humans) are responsible for GSDMD cleavage (Figure 1). In 2011, Kayagaki et al.43 reported that loss of caspase-11 but not caspase-1 could block IL-1b maturation and secretion induced by gram-negative bacteria, thereby protecting mice from endotoxic shock. Further studies discovered that lipopolysaccharide (LPS), the major toxin of the Gram-negative bacteria, is the trigger of the noncanonical inflammasome.44,45 Independent of PRRs, caspase-11 itself could directly recognize LPS by binding to the lipid A component of LPS.46 Activated caspase-11 ultimately cleaved GSDMD to induce pyroptosis, similar to the canonical pathway. Lately, human caspase-4/5 were demonstrated to have the same function as mouse caspase-11. Poelzl et al.47 reported that tyrosine kinase 2, a cytokine receptor-associated kinase, could function as a critical upstream regulator of caspase-11 and permitted non-canonical inflammasome activation during endotoxemia.

In addition to these canonical and non-canonical pyroptotic pathways, recent studies have shed new light on GSDMD-mediated pyroptosis. Kambara et al. found that z-YVAD-fmk, a caspase inhibitor, failed to block neutrophil lysate-elicited GSDMD cleavage, suggesting that GSDMD cleavage by neutrophil lysate might be caspase-independent.12 They further identifified that the neutrophil serine protease ELANE was the GSDMD cleaver in neutrophils. Orning et al.13 and Sarhan et al.14 made a major discovery on a new form of GSDMD cleavage induced by caspase-8 in Yersinia-infected macrophages. These two groups demonstrated that inhibition of transforming growth factor (TGF)b-associated kinase (TAK1) during Yersinia infection would promote the formation of a cell-death complex composed of RIPK1 and caspase-8, and drive the cleavage of GSDMD in macrophage. Furthermore, Demarco et al.48 revealed the details of caspase-8 involvement in GSDMD cleavage and provided compelling evidence that caspase-8–dependent GSDMD activation would promote TNF-a-induced lethality independent of inflammasome activation.

GSDME-mediated pyroptosis. Unlike other caspase family members, caspase-3, the cleaver of GSDME, has long been defined as the main apoptotic executor for both intrinsic and extrinsic pathways. GSDME-mediated pyroptosis shares the same upstream effectors with apoptosis, in which mitochondrial damage plays a crucial role (Figure 1). GSDME overexpression in tumor cells switches apoptosis induced by chemotherapy to pyroptosis.17 In contrast, when the expression of the GSDME gene is abrogated, macrophages remain in the apoptotic phase characterized by membrane blebbing and apoptotic body formation, instead of entering into the pyroptotic phase characterized by membrane swelling and ballooning.16 Besides the influences of GSDME expression, Xu et al.18 found that persistent mitochondrial permeability transition could drive the assembly of an Apaf-1-caspase-4/11 pyroptosome that triggers GSDME-dependent pyroptosis, while mitochondrial outer membrane permeabilization prompts the formation of apoptosome to induce apoptosis. Controversially, other researchers reported that mitochondrial outer membrane permeabilization caused by BAX/BAK activation49 or Tom20/BAX assembly50 also induces GSDME-dependent pyroptosis in tumor cells. Moreover, GSDME-N could permeabilize the mitochondria, release cytochrome c from mitochondria, and enhance caspase-3 activation, forming a self-amplifying positive feed-forward loop.51

Pyroptosis in Kidney Disease

In recent years, the discovery of pyroptotic signaling pathways has broadened our understanding of the pathogenesis of renal diseases beyond apoptosis. Emerging as an effector mechanism in innate immune responses, pyroptosis was firstly observed in macrophages and other phagocytes.52 A lot of efforts were made to determine the distribution and expression of masterminds, especially GSDMD and GSDME, in various tissues and cell types, which have demonstrated that pyroptosis is not limited to phagocytes.5 A variety of kidney diseases induced by microbial and non-microbial stimuli are usually characterized by the death of renal cells and activated immune responses. Some experiments showed that pyroptosis occurs in resident renal cells and makes a great contribution to the development of various renal diseases. Further studies have elucidated that pyroptosis in bone marrow-derived infiltrating immune cells, which has been triggered by renal cell death, could aggravate and maintain the injury of the kidney. Recent research has further pinpointed the role of pyroptosis as well as related inflflammatory signaling pathways in several types of kidney diseases including acute kidney injury, diabetic kidney disease, crystal-induced kidney disease, and allograft injury in kidney transplantation. (Table 1) (Figure 2)

Acute Kidney Injury (AKI)

Cell death is the pivotal pathological event during the development of AKI. Apoptosis was previously considered responsible for AKI pathogenesis.53 However, pharmacological inhibition of apoptotic signaling pathways failed to effectively prevent or alleviate AKI. Recent advances have revealed the role of pyroptosis in AKI. In 2018, Zhang et al.54 reported that caspases-4/5/11 were required for contrast-induced tubular epithelial cell death. They further demonstrated that these inflflammatory caspases were responsible for GSDMD and IL-1b cleavage in damaged tubular epithelial cells, which uncovered a requisite role of epithelial pyroptosis in contrast-induced AKI. Miao et al.55 detected a cleaved fragment of GSDMD by western blot analysis of whole kidney lysates in both cisplatin-induced and ischemia–reperfusion-induced AKI models. And knockout of caspase-11 or GSDMD signifificantly protected the mice from cisplatin-induced AKI but this analysis was not performed in the ischemia-reperfusion (I/R) model. The authors also observed GSDMD-N translocation onto the plasma membrane promoted by caspase-11. A constructive comment on this work made by Tonnus et al.56 suggested that the possible target of GSDMD-N on intracellular membranes and more work on the subcellular distribution of the cleaved fragment of GSDMD was required to understand a potentially important formation of pores in intracellular organelles. Additional studies supported the contribution of GSDMD-mediated pyroptosis to AKI induced by cisplatin.57 Our previous study revealed that GSDMD-mediated pyroptosis synergized with RIPK3/MLKL-mediated necroptosis to amplify inflflammatory signaling and enhance tissue injury in the process of sepsis.58 We found cumulative protection against sepsis induced by cecal ligation and puncture in mice after combined deletion of Gsdmd and Ripk3 (or Mlkl). These two forms of necrosis collaborated to contribute to acute tissue injury including kidney, lung, liver, and intestine, and systemic coagulation in the sepsis animal model. Our studies on bone marrow transplantation suggested that necroptosis and pyroptosis in both myeloid and non-myeloid cells greatly contributed to the progression of sepsis. There is a growing interest in the combined effects of different cell death pathways in a single pathological condition.59–61 Another study on coagulation cascades in sepsis also emphasized the central role of caspase-11/ GSDMD-mediated pyroptosis for the initiation of disseminated intravascular coagulation (DIC), providing strong supporting evidence for our fifindings.62 Inhibition of TNF-a, as well as high-mobility group box protein 1 (HMGB1), could exert negative control of GSDMD-mediated pyroptosis, and provide protective effects on AKI and acute liver injury caused by sepsis, delineating this pyroptotic pathway from a regulatory view.63

A recent study from our research group illuminated how GSDME-mediated pyroptosis boosted the development of ureteral obstruction-induced renal tubule injury and subsequent renal fifibrosis.64 We firstly found the upregulated expression and activation of Casp3 and GSDME in the obstructed kidney of the unilateral ureteral obstruction (UUO) model, suggesting the involvement of GSDME-mediated pyroptosis in this kind of kidney injury. We next confirmed that GSDME-mediated pyroptosis was indeed responsible for AKI after UUO and the subsequent nephropathy with the use of Gsdme
/
and Casp3-/- mice. Studies of bone marrow transplantation pointed out that GSDMEmediated pyroptosis in bone marrow-derived infiltrating immune cells was not responsible for the pathogenesis of obstructive nephropathy. Renal tubular cells (RTCs)-specific and hematopoietic cells-specifific caspase3-defificient mice were generated to further verify that GSDME-mediated pyroptosis occurs in RTCs, but not in infiltrating immune cells, predominantly contributes to the progression of kidney injury and fifibrosis induced by ureteral obstruction. To investigate the inflflammatory molecular mechanisms in the process, we monitored the infiltration of inflflammatory cells and the production of pro-inflflammatory cytokines in the kidney. The results showed that tubular cell pyroptosis could promote HMGB1 secretion and macrophages and neutrophils recruitment post-UUO. Notably, other studies showed that GSDME-mediated pyroptosis might contribute to chemotherapy drug-induced nephrotoxicity.65–66

It is worth noting that other lytic cell deaths besides pyroptosis have also been reported to contribute to AKI. In addition to the collaborative effects of pyroptosis and necroptosis in sepsis-induced AKI mentioned above, a growing body of evidence supports the pivotal role of necroptosis in AKI induced by certain stimuli. Linkermann et al.67 identifified the signifificant role of necroptosis in ischemia–reperfusion-induced and cisplatin-induced AKI as RIPK3-knockout mice and the application of RIPK1 kinase inhibitor necrostatin (Nec)-1 could both attenuate kidney injury. Our research group has demonstrated that necroptosis is a major mechanism of proximal tubular cell death in cisplatin-induced nephrotoxic AKI.68 Our data showed that the inhibition of any of the core components of the necroptotic pathway (RIPK1, RIPK3 or MLKL) either by gene knockout or a chemical inhibitor, could diminish cisplatin-induced proximal tubule damage in mice. In contrast, overexpression of RIPK1 or RIPK3 enhanced cisplatin-induced necroptosis in vitro. We also identifified the essential effects of necroptosis in the pathophysiology of IR injury and subsequent progression to chronic kidney disease (CKD), during which NLRP3 inflammasome activation was triggered.69 Whether the activation of NLRP3 by necroptosis can act upstream of the pyroptotic pathway in AKI is unclear yet. In addition, several studies demonstrated RIPK1 as a signifificant driver of NF-jB activation, an effective trigger for NLRP3. In Yersinia-infected macrophages, RIPK1 exerted positive control for caspase-8-induced GSDMD cleavage, during which RIPK3/MLKL-mediated necroptosis was inhibited.13 However, the RIPK1-caspase-8-GSDMD axis has not been discovered in renal cells. Ferroptosis has been reported to dominate in certain forms of AKI including cisplatin-induced renal injury70 and rhabdomyolysis.71–72 Tonnus et al.73 found that dysfunction of the key ferroptosis-surveilling systems would hypersensitize mice to tubular necrosis during AKI. Martin-Sanchez, D. et al.74 studied the timing of cell death pathways during AKI development and found that ferroptosis and necroptosis mediated the first and second waves, respectively, of cell death in folic acid (FA)-induced AKI. It was shown that ferroptosis was responsible for initiating FA-induced AKI and necroptosis allowed AKI to persist. ROS, produced in mitochondria during oxidative phosphorylation or upon endoplasmic reticulum (ER) stress, drives lipid peroxidation— the core process of ferroptosis—and interacts with the glutathione (GSH)-glutathione peroxidase 4 (GPX4) system, which potently prevents ferroptosis. Although there is a lack of solid evidence for a detailed connection between ferroptosis and other PCDs, mitochondria damage and ROS generation in ferroptosis give potential clues for its crosstalk with necroptosis and pyroptosis (Figure 3).

Diabetic Kidney Disease (DKD) DKD is a glucose-driven pathogenetic process that occurs in the kidneys of diabetic patients. The key factors regulating DKD, including excessive glucose flux, advanced glycosylation end products (AGEs), and ROS, act as critical roles in the process of pyroptosis either as the inducers of inflammasome or the regulators of the signaling pathway.75 Among various inflflammasomes that can induce pyroptosis, NLRP3 has been under the most intensive investigation in DKD. Toll-like receptor (TLR)2 and TLR4, PRRs upstream of NLRP3 activation, can be upregulated by high glucose, and diabetic patients with renal failure exhibit signifificant increases in TLR2 and TLR4 mRNA expression in peripheral blood.76 Stimulation of TLR2 and TLR4 is essential for NLRP3 activation.77–78 Thioredoxin interacting protein (TXNIP) belongs to the thioredoxin system and can bind to thioredoxin (TRX), decreasing TRX’s inhibitory effects on oxidative stress.79 It has been well established that TXNIP action is critical for NLRP3 activation and cell destructive responses to diverse DAMPs including pyroptosis.80–82 Diabetic kidneys show elevated expression of TXNIP and inhibition of TXNIP attenuates kidney injury induced by high glucose, positioning TXNIP as a potential target for DKD treatment.83,84 ROS has been proposed as a common signal for NLRP3 activation and can activate NLRP3 through different pathways, among which the NF-jB pathway is the most extensively studied.85 NLRP3 inflammasome can be effectively oligomerized into their active form through ROStriggered NF-jB signaling pathway.86–88 In DKD, suppressing the ROS/NF-jB /NLRP3 pathway appears to reduce inflflammatory responses and control the development of progressive kidney injury.89,90 ER stress has been identifified as another trigger of NLRP3 inflammasome.91,92 Hyperglycemia can induce ER stress in podocytes, tubular epithelial cells, and mesangial cells.93–95 After activation, NLRP3 drives inflflammatory responses through the processing of caspase-1, IL-1b, and IL-18, which contribute to the DKD progression. Recent research has provided evidence that inhibiting NLRP3/caspase-1-mediated pyroptosis alleviates pathological changes in the diabetic kidney, suggesting its therapeutic potential in DKD.96–98

Pyroptosis in different renal resident cells contributes to the development of DKD. Glomerular endothelium disruption alters glomerular permselectivity and occurs in the early stage of DKD. Partial colocalization of NLRP3 or cleaved caspase-1 with glomerular endothelial cells in histological sections of diabetic human or mice kidneys has been observed by confocal microscopy.98 Further study confirmed high glucose-induced glomerular endothelial cell injury via GSDMD-mediated pyroptosis.99 Podocytes have been considered the “weakest link” in DKD development,100, and their cell number is highly correlated with albuminuria in diabetic patients.101 The loss of podocytes is a concomitant phenomenon in the progression of DKD, and pyroptosis has been reported to be one of the most important mechanisms of podocyte loss. Genetic deletion of TXNIP or inhibiting NADPH oxidase to reduce ROS, both of which negatively control the NLRP3 activation, signifificantly attenuates high-glucose-induced podocyte injury, giving a clue for the role of pyroptosis in diabetic podocyte loss.102 Liu et al.103 found increased caspase-1 activity and elevated IL-1b and IL-18 levels in podocytes exposed to high glucose and Li et al.104 detected cleaved GSDMD-N in high-glucose injured podocytes, providing more confident evidence for pyroptosis in the diabetic podocyte. It is worth mentioning that the non-canonical pyroptotic pathway also contributes to podocyte injury in DKD with caspase-11/4 involved in the process.105 Although it was once believed that tubular dysfunction occurred in the later stage of DKD and developed as a result of glomerular injury, more and more researches appear to challenge this hypothesis, which discovered that tubule injury can be observed in the early stage of DKD and even play a driving role.106,107 In the kidneys of db/db mice and in HK-2 cells exposed to high glucose conditions, the ROS/TXNIP/NLRP3/IL-1b axis was observed to be signifificantly upregulated, and blocking this axis would alleviate tubular injury.108 Wang et al.109 discovered that TLR4/ NF-jB and GSDMD-N expressions were increased in HK-2 cells under high glucose ambiance and inhibition of TLR4/NF-jB signaling alleviated caspase-1/ GSDMD-mediated pyroptosis of tubular cells, indicating that pyroptosis induced by TLR4/NF-jB took part in the diabetic tubular injury. It is noteworthy that pyroptosis may not be the only important cell death pathway in DKD. Ferroptosis,110 necroptosis111, and apoptosis112 have been investigated in many instances. However, clear distinction and/or linkage among different cell death pathways in DKD development have not been fully delineated.

Crystal-induced kidney disease

Crystals can trigger kidney injuries ranging from mild/transient to severe/irreparable depending on the localization and dynamics of crystal deposition. The molecular mechanisms of crystal-induced renal diseases remain largely unknown despite substantial research efforts. A breakthrough study revealed that crystals could stimulate the activation of NLRP3 and demonstrated the pivotal role of the inflammasome in crystal-induced dysfunction or diseases.113 Later, NLRP3/caspase-1/IL-1b axis has been identifified as the main cause of the development of oxalate nephropathy114 and calcium oxalate nephropathy,115 shifting the hotspot in the research of crystal-induced renal disease from crystal deposits to inflflammatory responses. Crystals depositing in renal arteries and tubules can both activate the NLRP3 inflammasome and induce IL- 1b-dependent inflflammation. Cholesterol crystal is the most common culprit for renovascular damage in renovascular crystallopathies. Dowell et al.116 discovered that cholesterol crystals could activate NLRP3 inflammasome and promote IL-1b production in macrophages, which is central to the pathogenesis of crystal-induced atherosclerosis. Tubular crystallopathies, which result from the precipitation of crystals inside the tubular lumen, can promote both AKI and chronic kidney lesions.117 NLRP3 inflflammasomes are highly correlated with AKI and CKD caused by tubular crystal deposition. Liu et al.118 detected elevated expression of GSDMD and IL-18 expression in tubular cells exposed to calcium oxalate crystals through liquid chromatography-mass spectrometry-based tandem mass tag labeling quantitative proteomic analyses and identifified inflflammatory pyroptosis-related biological behaviors through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Further results confirmed NLRP3 activation and GSDMD-dependent pyroptosis in tubular epithelial cells in calcium oxalate crystal-induced models. Ding et al.119 also detected increased protein levels of NLRP3 in kidney homogenates of mice with glyoxylate-induced nephrolithiasis and in HK-2 cells exposed to calcium oxalate monohydrate crystals. Simultaneously increased protein expression levels of IL-1b and GSDMD were observed in tubular epithelial cells isolated from kidneys of glyoxylate-treated mice, supporting the notion that crystal-exposed tubular cells underwent GSDMDdependent pyroptosis. A large number of experiments provided evidence for NLRP3 activation by crystals in immune cells including macrophage and dendritic cells in different ways,120–122 which was confirmed in crystal-induced nephropathy.115,119 Crystals precipitating within tubules also cause persistent stimuli which foster the development and progression of CKD. Knauf et al.114 showed that the NLRP3-IL-1b axis was upregulated in mice with oxalate nephropathy while NLRP3-/- mice were completely protected from the progressive renal failure induced by feeding with a diet high in soluble oxalate. Another groundbreaking study performed by Mulay et al.115 depicted a more profound scene in which renal failure due to oxalate nephropathy was strongly dependent on the NLRP3/ASC/caspase-1/IL-1b axis. Yet there has been no direct detection of GSDMD cleavage in the process of crystal-associated renal fifibrosis. Notably, other forms of PCD including the RIPK3-MLKL necroptotic pathway123,124 and ferroptosis125 have been reported to contribute to renal injury induced by crystals. Whether these different cell deaths are collaborative or independent is still under investigation. The effect of crystal type and dynamics on the cellular fate of renal cells is also of outstanding interest.

Allograft injury in kidney transplantation

Kidney transplantation remains to be the optimal option for patients with end-stage renal disease (ESRD). Given the growing population requiring kidney transplantation over recent decades, increasing attention has been paid to allograft injury in both the donors and the recipients. HMGB1 could be released from cells undergoing pyroptosis.3 Kru¨ger et al.126 found that tubules from deceased- but not living-donor kidneys stained positively for HMGB1. They further demonstrated that HMGB1 could stimulate proinflammatory responses through TLR4, which contributed to graft inflflammation and sterile injury following cold preservation and transplantation. Thierry et al.127 detected elevated urinary HMGB1 as soon as 30 min after reperfusion following human kidney transplantation and addressed its possible role as innate-immune mediators in such injury. Inflflammatory cytokines IL-1b was reported to increase in brain-dead organ donors,128,129 possibly indicating the involvement of pyroptosis in the process of organ injury. Intensive efforts have been made by researchers in order to study the inflammasome activation in allograft dysfunction. Design et al.130 found that genetic variants in the NLRP3 gene affect the acute rejection risk following kidney transplantation. Wu et al.131 demonstrated that mice defificient in the TLR adaptor protein MyD88 were protected from both acute and chronic allograft rejection and increased their survival after transplantation compared with wild-type controls. Another study reported that donor MyD88 defificiency inhibited inflflammatory cytokine expression and ER stress, and was associated with improved graft function and prolonged allograft survival.132 Furthermore, a recent study revealed that the NLRP3 inhibitor Mcc950 could ameliorate tubular damage and improve allograft function in a renal transplantation model. Decreased caspase-1 activation and GSDMD-N levels were observed after Mcc950 treatment.133 These results provide clues for the involvement of pyroptosis in allograft injury in kidney transplantation.

Potential Therapies TargetingPyroptosis for Kidney Disease

Inhibition of pyroptosisConsidering the important role of pyroptosis in internal diseases, modulating this process is of great concern. Potential targets in the pyroptotic

pathway include inflflammasomes, caspases, masterminds, and IL-1b (Figure 4). Necroinflflammation triggered by immune responses to necrosis including necroptosis, ferroptosis, and pyroptosis leads to tissue injury. Linkermann et al.134,135 highlighted the clinical significance of targeting necroinflammation in kidney injury. Ascertain stimuli may trigger more than one single cell death pathway in injury cells and tissues, the potential of combined targeted strategies on different cell death pathways has attracted increased interest.

Targeting NLRP3 inflammasome. NLRP3 inflflammasomes run upstream of caspase-1/ GSDMD-mediated pyroptosis and several medications targeting NLRP3 have been developed with different inhibiting mechanisms.136 Mcc950 is a potent and selective small-molecular inhibitor for NLRP3 whose therapeutic potential has been assessed in several NLRP3-associated syndromes.137 In a murine model of crystal nephropathy induced by diets rich in oxalate or adenine, Mcc950 markedly reduced inflammasome activation in renal dendritic cells and subsequent IL-1b and IL-18 production. This NLRP3 inhibitor also effectively prevented crystal-induced renal fifibrosis.138 BAY 11-7082, which was initially reported to inhibit IjBa phosphorylation and NFjB activation,139 has been identifified as an inhibitor of NLRP3 ATPase. Juliana et al.140 elucidated that BAY 11-7082 inhibited NLRP3 inflammasome activation in LPS-primed macrophages independent of its effect on NF-jB and prevented NLRP3- induced ASC pyroptosome formation by inhibiting NLRP3 ATPase activity. In mice fed a high-fat, high-fructose diet, BAY 11-7082 attenuated the diet-induced increase in NLRP3 inflammasome expression, resulting in inhibition of caspase-1 activation and interleukin (IL)-1b and IL-18 production in liver and kidney. Chronic BAY 11-7082 administration signifificantly limited the diet-induced renal damage by improving the albumin-to-creatinine ratio.141 In a rat model of diabetic nephropathy, BAY 11-7082 treatment was found to signifificantly recover kidney histological architecture and normalized levels of inflflammatory cytokines including IL- 1b, supporting the renoprotective effect of BAY 11-7082 for DKD.142 b-Hydroxybutyrate (BHB), produced during energy deprivation or a lowcarbohydrate diet, inhibited NLRP3 activation and prevented renal fifibrosis induced by nephrocalcinosis.143 However, the protective effects were not necessarily mediated by intrarenal IL-1 release, and whether BHB affects pyroptosis remains to be clarified. Other NLRP3 inhibitors including fluoro fenidone144 and tranilast145 have been reported to play renoprotective roles in the unilateral ureteral obstruction (UUO) model while the molecular mechanism underlying pyroptosis is still unclear.

Targeting pyroptotic caspases. Inhibitors of pyroptotic caspases have been under intensive investigation. Ac-YVAD-CMK, a caspase-1 inhibitor, can attenuate the renal function impairment in AKI rat model146 and reduce pyroptosis of glomerular endothelial cells in diabetic nephropathy mouse model.99 VX-765, another caspase-1 inhibitor, was sufficient to decrease the proinflammatory macrophage population in the later stage of UUO.147 Chu et al.148 demonstrated that the oxidized phospholipid 1-palmitoyl-2-arachidonic noyl-sn-glycerol-3-phosphorylcholine (oxPAPC) could bind directly to caspase-4 and caspase-11, compete with LPS binding, and consequently inhibit LPS-induced pyroptosis, providing novel evidence for the precise role of oxPAPC in inflflammatory response. oxPAPC and its derivatives could provide new insight for therapies targeting non-canonical pyroptosis. Choi et al.149 reported that serpin family B member 1 (SERPINB1) limited the activity of caspase-1/4/5/11 by suppressing their caspaserecruitment domain (CARD) oligomerization and enzymatic activation, revealing a new checkpoint mechanism for inflflammatory caspases. SERPINB1 collaborated with Forkhead box protein O1 (FoxO1) to ameliorate ROS production-induced oxidative stress in DKD.150 However, the therapeutic potential of targeting this checkpoint protein is still under exploration.

Targeting mastermind. Identification of masterminds as the executors of pyroptosis raises new hope for targeted therapy of pyroptosis-associated diseases. Hu et al.151 performed high throughput biochemical screening and identifified disulfiram as an effective inhibitor of GSDMD pore formation. Another study showed that disulfiram can ameliorate GSDMD-driven mortality in murine sepsis.152 In addition, disulfifiram also negatively controls necroptosis by binding MLKL.153 The safety profile of disulfiram in humans has been well established over six decades and repurposing this old drug for renal diseases is promising. A GSDMD-derived inhibitor, Ac-FLTD-CMK, can inhibit GSDMD cleavage by caspases-1/4/5/11 by directly binding to the catalytic region of these caspases, therefore suppressing pyroptosis downstream of both canonical and noncanonical pathways.154 A 2020 study demonstrated that calcium (Ca)2+ influx was a prerequisite for the function of GSDMD-N and magnesium (Mg)2+ blocked Ca2+ influx by inhibiting the ATP-gated Ca2+ channel P2X7, thereby hampering the function of GSDMD-N and inhibiting LPS-induced noncanonical pyroptosis. These results suggest potential clinic applications of magnesium supplementation for sepsis treatment.155

Targeting IL-1. IL-1 has become a research hot spot for immunomodulation strategies targeting inflammasome-associated diseases. In particular, the human IL-1b monoclonal antibody canakinumab has been under intensive investigation. The CANTOS study (Canakinumab Anti-Inflflammatory Thrombosis Outcomes Study) evaluated the effificacy of canakinumab in 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of  2 mg/ L.156 A sub-analysis of the CANTOS data showed that IL-1b inhibition with canakinumab reduced major adverse cardiovascular event rates among high-risk atherosclerosis patients with CKD, particularly among those with a robust anti-inflflammatory response to initial treatment, indicating the utility of the anti-inflflammatory agent development in CKD.157

Synchronized regulation of different PCDs

Recently, more attention has been paid to the crosstalk and coordination of different PCDs in certain diseases. Collaborative effects of GSDMDmediated pyroptosis and MLKL-mediated necroptosis have been revealed in AKI58 and inflflammatory bowel diseases.59 Furthermore, a newly emerging concept termed PANoptosis was proposed to describe the co-regulation between pyroptosis, necroptosis, and apoptosis.158 Z-DNA binding protein 1(ZBP1), transforming growth factor beta-activated kinase 1 (TAK1), FS7-associated cell surface antigen (Fas)-associated death domain (FADD), and Caspase-8 may cooperate to act as master regulators for PANoptosis in orchestrating multiple cell death pathways.159,59–61 Tonnus et al.73 generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets RIPK1 and ferroptosis and proved its protective effects in AKI. These fifindings lay the foundation for effective therapeutic approaches to targeting multifaceted cell death pathways in kidney diseases.

Conclusions

A body of evidence has suggested the crucial roles of pyroptosis in kidney disease and revealed a number of factors involved in pyroptosis such as NLRP3, caspase-1, GSDMDand IL-1, which might be modulated genetically and/or pharmacologically for therapeutic use. Canonical GSDMD-mediated pyroptosis has been proven to play a central role and possess the potential as a therapeutic target in various renal diseases, including AKI, DKD, crystal-induced kidney disease, and allograft injury in kidney transplantation. Findings on noncanonicalGSDMD-mediated pyroptosis, which is stimulated by LPS, have opened up a new avenue for treating sepsis-induced organ damage. And the recent discovery of GSDME-mediated pyroptosis as a key regulator of AKI and subsequent AKI-CKDprogression provides novel insights into the management of AKI and the prevention of CKD. In addition, synchronized regulation of multiple cell death pathways may exhibit more beneficial effects on kidneys, since certain stimuli usually induce more than one death pathway.

Nevertheless, our understanding of pyroptosis in the context of kidney disease is still limited. The published studies about pyroptosis in kidney disease were based on a limited number of animal and cell experiments. Reproduction of the current data as well as the translation of experimental data to clinical settings is still required. The insight mechanisms of different pyroptotic pathways under various stress circumstances and/or in distinct types of renal cells, as well as the crosstalk and interplay between pyroptosis and other forms of cell death, are still largely unknown. It is of considerable importance to further investigate the pyroptotic mechanisms as well as the orchestration of various PCDs in renal pathophysiology, which would help to tap the potential of targeted therapies for solving the clinical dilemmas and breaking the clinical bottleneck in kidney diseases.

From: 'Pyroptosis in Kidney Disease' by Yujia Wang, Yinshuang Li and Yanfang Xu1

---Journal of Molecular Biology