Citation: Cell Death and Disease (2016) 7, 2125; doi:10.1038/cddis.2016.

37
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Review

Necroptosis in acute kidney injury: a shedding light

S Wang1,2, C Zhang1,2, L Hu*,3 and C Yang*,2,4

Acute kidney injury (AKI) is a common and severe clinical condition with a heavy healthy burden around the world. In spite of
supportive therapies, the mortality associated with AKI remains high. Our limited understanding of the complex cell death
mechanism in the process of AKI impedes the development of desirable therapeutics. Necroptosis is a recently identified novel
form of cell death contributing to numerable diseases and tissue damages. Increasing evidence has suggested that necroptosis
has an important role in the pathogenesis of various types of AKI. Therefore, we present here the signaling pathways and main
regulators of necroptosis that are potential candidate for therapeutic strategies. Moreover, we emphasize on the potential role and
corresponding mechanisms of necroptosis in AKI based on recent advances, and also discuss the possible therapeutic regimens
based on manipulating necroptosis. Taken together, the progress in this field sheds new light into the prevention and management
of AKI in clinical practice.
Cell Death and Disease (2016) 7, 2125; doi:10.1038/cddis.2016.37; published online 3 March 2016

Facts • What is the mechanism of cell-type-specific vulnerability to

necroptosis? And what are the clinical implications of this
• Necroptosis is a kind of regulated necrosis, a novel form of selective sensitivity in AKI?
cell death with morphologic features of necrosis but • How does necroptosis interplay with inflammation specifi-
molecularly controlled. cally during AKI?
• Necroptosis can be widely triggered by death receptors, toll- • What is the relative contribution of necroptosis and other
like receptors, interferons (IFNs), and intracellular protein forms of regulatory cell death in AKI?
DNA-dependent activator of IFN regulatory factors in
response to viruses. Various upstream signaling converge Cell death, serving as an essential process throughout life,
on mediator receptor-interacting protein kinase 3 and share has been extensively investigated for decades. Historically,
the common executor mixed lineage kinase domain-like cell death was classified as distinctive forms: apoptosis,
protein. autophagic cell death and necrosis.1 The term ‘apoptosis’,
• Necroptosis has been implicated in the pathogenesis of deriving from ancient Greek, has long been recognized
diverse types of AKI to different extents. synonymously as the programmed cell death, which is
• Blocking necroptotic pathways by pharmacological inhibi- developmentally programmed and molecularly controlled.
tors or genetic manipulation alleviates renal injuries in vitro Characteristic morphologic changes of apoptosis include cell
and in vivo, indicating a promising outlook in the manage- shrinkage, nuclear and cytoplasmic condensation, DNA
ment of AKI. fragmentation, and the formation of apoptosomes.2 Autopha-
gic cell death is a newly defined, highly regulated cell death,
which is characterized by the markers of autophagy
Open Questions pathway.3,4 In contrast, necrosis was thought to be accidental
cell death, and results in cellular swelling, breakdown of
• How to monitor necroptosis in vivo? plasma membrane integrity and release of intracellular
• Does necroptosis relate to different stages of AKI? If contents, all of which are absolutely distinct from programmed
yes, how? cell death.5 Cell death, no matter which specific form, has a

1
Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China; 2Shanghai Key Laboratory of Organ Transplantation, Shanghai, China; 3Department of
Urology, The First Affiliated Hospital of Soochow University, Suzhou, China and 4Department of Plastic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
*Corresponding author: L Hu, Department of Urology, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, China. Tel: +86 512 67780128;
Fax: +86 512 67780128; E-mail: [email protected]
or C Yang, Department of Plastic Surgery, Zhongshan Hospital, Fudan University; Shanghai Key Laboratory of Organ Transplantation, 180 Fenglin Road, Shanghai 200032,
China. Tel: +86 21 64031990; Fax: +86 21 64037269; E-mail: [email protected]
Abbreviations: AKI, acute kidney injury; CsA, cyclosporin A; CYLD, cylindromatosis; DAI, DNA-dependent activator of IFN regulatory factors; DAMP, damage-associated
molecular pattern; FADD, Fas-associated death domain; cFLIP, cellular FADD-like interleukin (IL)-1β- converting enzyme (FLICE)-inhibitory protein; cIAPs, cellular inhibitor
of apoptosis proteins; IDO, indolamin-2, 3-dioxygenase; IFN, interferon; IFNAR, IFN-α receptor; IRI, ischemia–reperfusion injury; LPS, lipopolysaccharide; LUBAC, linear
ubiquitin chain assembly complex; MLKL, mixed lineage kinase domain-like protein; Nec-1, necrostatin-1; poly(I:C), polyinosine–polycytidylic acid; PKR, protein kinase R;
RHIM, RIP homotypic interaction motif; RIP1, receptor-interacting protein kinase 1; RIP3, receptor-interacting protein kinase 3; TLR, toll-like receptor; TNF, tumor
necrosis factor; TNFR, TNF receptor; TWEAK, TNF-related weak inducer of apoptosis; TEC, tubular epithelia cell
Received 27.11.15; revised 01.2.16; accepted 02.2.16; Edited by L Galluzzi
 Necroptosis in AKI
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Figure 1 Schematic overview of necroptotic pathway. Necroptosis is triggered by various stimuli, including engagement of death receptors and Toll-like receptors, IFNs and
intracellular protein DAI in response to viruses. Diverse upstream signals converge on mediator RIP3, and consequently activate the executor MLKL. Especially, an autocrine loop
via de novo-transcribed IFNs/IFNAR1 signaling is believed to play a crucial role in sustaining the activation of necroptosis. Physiological regulators or pharmacological inhibitors
can regulate necroptotic pathway at different molecular levels. Necrosulfonamide only acts on human MLKL

key role in maintaining tissue homeostasis as well as other mechanism in the process of AKI. Recent progress suggested
crucial biological processes. that besides apoptosis, other forms of cell death, including
However, the full extent of cell death has not been explored. regulated necrosis (such as, necroptosis, necrosis by mito-
Recent breakthroughs in this field shed light on regulated chondrial permeability transition, pyroptosis, parthanatos and
necrosis, a new type of cell death with morphologic features of ferroptosis), autophagic cell death and mitotic catastrophe,
necrosis but genetically determined.6 It has been well- also significantly contribute to the pathogenesis of AKI.12,13 As
documented that necroptosis is highly orchestrated regulated the best-characterized form of regulated necrosis, necroptosis
necrosis dependent on receptor-interacting protein kinase has been intensively explored in the setting of AKI. Under-
3 (RIP3) and mixed lineage kinase domain-like protein standing the precise role of necroptosis in AKI might provide
(MLKL).7,8 It not only involves in the development of organism potential therapeutic regimens. Therefore, this review sum-
but also participates in various pathophysiological processes. marized the potential role and corresponding mechanisms of
An accumulating body of evidence has demonstrated that necroptosis in AKI based on recent advances, and also
necroptosis contributes to the pathogenesis of numerous discussed the possible therapeutic strategies based on
diseases and tissue damages, including ischemic brain injury, manipulating necroptotic pathways.
myocardial infarction, pancreatitis as well as liver and renal
injury.8
The signaling pathways of necroptosis
Acute kidney injury (AKI) is still a critical problem in clinical
practice, with a heavy health burden around the world.9 AKI As the identification of necrostatins as specific inhibitors
affects ~ 13.3 million individuals and contributes to ~ 1.7 targeting receptor-interacting protein kinase 1 (RIP1), the
million deaths globally per year.10 Apoptosis was recognized signaling pathways of necroptosis have been extensively
as the main form of cell death that is responsible for renal determined.14,15 Engagement of death receptors and Toll-like
dysfunction in AKI.11 Therefore, for a long time, strategies receptors (TLRs), interferon (IFN) signals, as well as intracel-
targeting the apoptosis pathway have been widely explored for lular stimuli from protein DNA-dependent activator of IFN
AKI treatment. Despite the substantial therapeutic effect in regulatory factors (DAI) in response to viruses are able to initiate
animal models, the efficient anti-apoptosis intervention stra- necroptosis8 (Figure 1). Among them, tumor necrosis factor
tegies are still absent in clinic. This could be partly ascribed (TNF)-α-induced necroptosis in the presence of caspase
to our limited understanding of the complex cell death inhibition is the best-characterized model. After TNF-α

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binds to TNF receptor (TNFR)1, the adaptor molecules signals converge on the RIP3 and share the same down-
Fas-associated death domain (FADD) and TNF-receptor- stream executing pathway.
associated death domain recruit RIP1 that subsequently
combines with RIP316–18 to form a complex termed ‘necro-
Key regulators of necroptosis
some’.19 It is believed that the oligomerization driven by the RIP
homotypic interaction motif (RHIM) domain on RIP3 and RIP1 RIP1. Why Rip1-deficient mice die perinatally remains an
leads to the autophosphorylation of RIP3, which results in the intriguing question for a long time. Recent exciting findings
activation of RIP3.17,18 In accordance, other initial participants showed that kinase-inactive Rip1 mutant D138N and K45A
like Toll/IL-1 receptor domain-containing adaptor inducing knockin mice are viable.30,31 Importantly, in addition, cells
IFN-β (TRIF) and DAI use the RHIM domain to activate RIP3, derived from these mice are resistant to necroptosis, but
indicating that RHIM domain has an important role in triggering mediate NF-κB pathway normally. These studies indicate
necroptosis. Activated RIP3 recruits and phosphorylates that the kinase activity of RIP1 is indispensable and might
the downstream MLKL, which serve as the executor serve as a ‘permission switch’ in the necroptotic pathways.
of necroptosis.20,21 It is demonstrated that phosphorylation of Ironically, on the other hand, RIP1 itself might act as an
MLKL induces a molecular switch that enable MLKL to inhibitor of necroptosis when its kinase activity is functionally
translocate to the membrane and consequently disrupt the absent. There probably exists an underlying physiological
plasma membrane. However, the exact mechanism needs to mechanism regulating the ‘permission switch’ of RIP1,
be further illustrated.7,22 Moreover, it seems that MLKL could thereby providing a negative feedback loop to restrict the
cause mitochondria fission via phosphoglycerate mutase family magnitude of necroptosis. According to this theory, loss of
member 5 and dynamin-related protein 1.23 Whereas, the RIP1 could result in the overreaction of necroptosis that might
depletion of mitochondria has been determined not to prevent account for the lethality of Rip1-knockout mice. Consistently,
necroptosis in another study, suggesting that the mitochondrial Rip1−/− cells have high sensitivity to necroptotic stimuli.32
fragmentation by MLKL may contribute little to the final However, the proposed inhibitory effect of RIP1 needs further
execution of necroptosis.24 validation.
Other factors beyond TNF-α could also initiate RIP3-
dependent necroptosis. It has been reported that activation cIAPs, LUBAC and CYLD. In fact, not only necroptosis but
of TLR3 or TLR4 by polyinosine–polycytidylic acid (poly(I:C)) also apoptosis and NF-κB pathways can be triggered by the
and lipopolysaccharide (LPS), respectively, in the presence of engagement of TNFR1. During this process, cellular inhibitor
zVAD (a pan-caspase inhibitor) leads to necroptosis through of apoptosis proteins (cIAPs), linear ubiquitin chain assembly
the adaptor protein TRIF, which contains RHIM domain to complex (LUBAC) and cylindromatosis (CYLD) are reported
interact with RIP3.25,26 Additionally, both type I (α/β) and type II to have crucial roles in deciding the switch between different
(γ) IFNs were demonstrated to trigger necroptosis when FADD cellular outcomes.33–37 Briefly, TNFR1 signaling leads to the
and caspase-8 are absent. IFNs-induced necroptosis pro- formation of distinct types of complexes with different
ceeds by upregulating the expression of protein kinase functions. Polyubiquitination of RIP1 by cIAPs enables
R (PKR), which relies on JAK-STAT pathway. The PKR recruitment of LUBAC, which in turn stabilizes a so-called
interacts with RIP1 and promotes the formation of the ‘PKR prosurvival complex (complex I) by generating the linear
necrosome’ consisting of PKR, RIP1 and RIP3 to implement ubiquitin chains on RIP1. Subsequently, complex I leads to
necroptosis.27 It should be noted that type I IFNs have been the well-known NF-κB signaling. Conversely, deubiquitination
recently shown to have a predominant role in sustaining the of RIP1 by CYLD or the absence of cIAPs and LUBAC
activation of the RIP1–RIP3 complex.28 Macrophage deficient renders complex I unstable and facilitates other complexes
in IFN-α receptor (IFNAR) 1 was found to be resistant to assembled to initiate apoptosis or necroptosis.
LPS–TLR4-, poly(I:C)–TLR3- and TNF–TNFR1-mediated
necroptosis in the presence of caspase inhibitors. Further- Complex of RIP1, FADD, caspase-8 and cFLIP isoforms.
more, it is proposed that LPS-, poly(I:C)-, TNF- and When cIAPs are absent, RIP1, FADD, caspase-8/10 and
IFN-β-induced necroptosis in macrophages proceed FADD-like interleukin (IL)-1β-converting enzyme (FLICE)-inhi-
through IFN regulatory factors-dependent de novo transcrip- bitory protein (cFLIP) isoforms assemble an intracellular
tion of IFNs to facilitate the activation of necrosome. Also, complex referred to as ripoptosome.38 Within the complex,
the autocrine loops via de novo IFNs/IFNAR1 amplifies the cFLIP forms heterodimer with caspase-8, and controls the
initiating signals of necroptosis. Therefore, it is likely that type caspase activity. Depending on the isoforms of cFLIP, ripopto-
I IFNs provide a positive feedback to license the final execution some could lead to either apoptosis or necroptosis.38–40 cFLIPL
of necroptosis. However, the ‘license’ role of type I IFNs in (the long isoform of cFLIP)-caspase-8 heterodimer has
other cells need to be further validated. restricted enzymatic activity that could inactivate RIP1 and
Besides extrinsic pathways, intracellular signaling can also RIP3 through cleavage, and consequently inhibits necroptosis
lead to necroptotic cell death. Viral infection-induced expres- and favors apoptosis; conversely, the heterodimer of caspase-8
sion of cytosolic DAI could interact with RIP3 by RHIM domain and cFLIPS (the short isoform of cFLIP) lacks such catalytic
and forms DAI–RIP3 complex with initiating function analo- activity and sensitizes cells to TLR- and Fas-induced
gous to the RIP1–RIP3 necrosome or TRIF–RIP3 complex.29 necroptosis.38,41 In the absence of cIAPs, RIP1 dissociates
These studies indicate that there exist diverse upstream from complex I and forms an assembly consisting of RIP1,
pathways dependent on different stimuli. Various upstream FADD, caspase-8 and long isoform of FADD-like interleukin

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Figure 2 Timeline for major events in the studies of necroptosis and its role in AKI. Orange boxes represent the findings in the discovery of necroptotic pathway; purple boxes
demonstrate the breakthroughs in researches of necroptosis in AKI

(IL)-1β- converting enzyme (FLICE)-inhibitory protein (FLIPL), another influence. It is known that apoptosis does not occur
which favors apoptosis and inhibits spontaneous necroptosis. immediately after the onset of ischemia.11 Thus, it cannot be
Consistently, Caspase-8-deficient mice die as embryos with ruled out the possibility that application of zVAD just 15 min
heart defects,42 and this embryonic lethality can be rescued by before ischemia might diminish its therapeutic effect. Both
Rip3 deletion.43 This phenomenon implies the negatively factors could be partially responsible for the ‘ineffectiveness’
regulatory role of caspase-8 in necroptosis pathways. Of note, of zVAD observed in this research. The role of necroptosis in
caspase-8 seems to inhibit necroptosis by forming a hetero- ischemic kidney injury was further suggested in subsequent
dimer with FLIP39,40
L. High-expression level of FLIPL facilitates studies. Consistently, two researches confirmed the protection
the formation of caspase-8–FLIPL complex, which cleaves and of Nec-1 in rat and human renal tubular epithelia cells (TECs),
inactivates RIP1. Moreover, embryonic death of Fadd-deficient respectively, in a model of TNF-α stimulation and ATP
mice can also be prevented by Rip3 deletion, indicating that depletion that mimics the renal ischemic injury in vitro.49,50
FADD also plays an inhibitory role.44,45 Complex of RIP1, Furthermore, genetic model provided more convincing evi-
FADD, caspase-8 and FLIPL will divert to necrosome and dence of necroptosis in renal IRI. Protection from ischemic
necroptosis will be induced when caspase-8-FLIPL heterodimer damage was exhibited in Rip3-knockout mice, and further
is functionally defective or FADD is absent. amelioration by addictive Nec-1 administration was not
observed, implicating that Rip3-dependent necroptosis path-
A20. RIP3 is required to be ubiquitinated at Lys5 to facilitate way participates in ischemic AKI as a crucial mediator.51
the formation of necrosome during necroptosis, while A20 Rip3-deficient mice demonstrate a normal phenotype except
could inhibit necroptosis by targeting this process.46 This for a slight failure to gain weight.52,53 Notably, on the other
theory is further validated by the phenomenon that loss of hand, Rip3-knockout mice might also exert addictive protec-
Rip3 reverses the lethality of A20-deficient mice.46 tion due to its obviously increased peritubular diameters than
wild-type mice.53 Therefore, tissue-specific Rip3-knockout
models are needed to provide more explicit evidence. Taken
Necroptosis in AKI: evidence and implications
together, these findings suggested that necroptosis is of
As shown in Figure 2, the presence of necroptosis in AKI was crucial importance for the pathologic processes of renal IRI
first determined in a murine model of renal ischemia– and targeting necroptotic pathway molecular emerges as a
reperfusion injury (IRI) by Linkermann et al.47 who evaluated promising treatment option to improve the prognosis of
the protective effect of necrostatin-1 (Nec-1), a chemical ischemic AKI. However, the mechanism by which necroptosis
inhibitor of RIP1. Nec-1 was shown to significantly alleviate the contributes to renal IRI has not be fully delineated, which
renal dysfunction and tissue damage, supporting the vital role invites further investigations.
of necroptosis in the pathogenesis of ischemic AKI. Surpris- In addition to renal ischemic injury, necroptosis also
ingly, compared with Nec-1, treatment with the pan-caspase contributes to AKI induced by cisplatin, a widely used
inhibitor zVAD to block apoptosis did not provide any chemotherapy agent with nephrotoxic effect. Tristao et al.54
detectable protection. This finding contradicts with a previous found that zVAD prevents the cisplatin-associated damage on
report that demonstrated the protective effect of zVAD in the human renal TECs in vitro and combined use of zVAD
context of IRI.48 One possible reason is the incontinence in the and Nec-1 can provide additional protection. In agreement,
clamping time of the renal pedicles adopted in these studies, Linkermann et al.51 showed an evident protection in
which may result in diverse profiles of cellular death in kidneys. Rip3-knockout mice in the background of cisplatin-induced
Besides, the different timing of zVAD administration may be AKI. Importantly, a more recent study by Xu et al.55 used

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Table 1 Summary of necroptosis in different AKI models in vitro

Year Cell line/type Induction Therapeutic intervention Effect of zVAD Reference

2012 Mice tubular cell line TKPTS, mice glo- TNF-α/cycloheximide in the pre- Nec-1; Rip1 siRNA; Rip3 NA 47
merular endothelial cell line glENDp54 sence of zVAD siRNA
2012 Human proximal tubular cell HK-2 Cisplatin Nec-1; Nec-1+zVAD Protective 54
2012 Rat tubular cell line NRK-52E CsA Nec-1; Rip3 siRNA NA 57
2013 Rat tubular cell line NRK-52E TNF-α/antimycin A Nec-1 NA 49
2013 Mice renal proximal tubular cells TNF-α/TWEAK/IFN-γ Nec-1 Harmful 51
2014 Human proximal tubular cell HK-2 TNF-α/antimycin A in the pre- Nec-1 NA 50
sence of zVAD
2015 Mice renal proximal tubular cells Cisplatin Nec-1; Rip3-KO; Mlkl-KO; No effect 55
Rip1 shRNA

Abbreviations: CsA, cyclosporin A; IFN, interferon; TNF, tumor necrosis factor; TWEAK, TNF-related weak inducer of apoptosis.

Table 2 Summary of necroptosis in different AKI models in vivo

Year Model Possible initiating Therapeutic intervention Effect of zVAD Reference

molecules

2012 Renal ischemia/reperfusion injury NA Nec-1 No effect 47

2013 Renal ischemia/reperfusion injury; NA Nec-1; Rip3-KO Protective in cisplatin- 51
cisplatin-induced AKI induced AKI
2013 Contrast-induced AKI NA Nec-1 No effect 58
2015 Cisplatin-induced AKI TNF-α, TWEAK, IFN-γ Nec-1; Rip3-KO; Mlkl-KO NA 55
2015 Cardiorenal injury after glycerol-induced TNF-α Infliximab (TNF-α antibody); NA 59
rhabdomyolysis Nec-1

Abbreviations: AKI, acute kidney injury; IFN, interferon; Nec-1, necrostatin-1; TNF, tumor necrosis factor; TWEAK, TNF-related weak inducer of apoptosis.

Mlkl-knockout mice to investigate the role of necroptosis in AKI unexpected effect on renal peritubular diameters. Thus, it
for the first time, providing more reliable evidence due to the cannot be excluded that the protection of Nec-1 is due to
indispensable role of MLKL in necroptotic pathway. Notably, it affecting renal blood flow but not blocking necroptosis
is shown that the increased production of TNF-α, TNF-related pathways in this setting.
weak inducer of apoptosis (TWEAK) and IFN-γ (also known as Recently, the contribution of necroptosis to AKI was
TTI) in cisplatin-induced AKI may be an important promoter of determined in a glycerol-induced rhabdomyolysis model.59
necroptosis.55 However, whether TTI induces necroptosis of The authors identified necroptosis, which is mainly mediated
renal tissue in vivo directly in the background of cisplatin- by increased circulating TNF, as the predominant form of
associated AKI was not answered in this research. The tubular injury in this background. Meanwhile, cardiac injury
nephrotoxicity of cisplatin remains a severe obstacle in its was also observed in this model. Oppositely, necroptosis was
clinical application as an anticancer agent. One of the detected to be of minor importance and apoptosis acts as a
characteristics of cisplatin-induced AKI is the upregulation of crucial mediator in the cardiac damage during rhabdomyo-
proinflammatory cytokines.56 But to what extent these proin- lysis. This study exemplified the difference of organ-specific
flammatory cytokines are related to necroptosis activation is susceptibility to necroptotic cell death in the same circum-
unknown. More researches to address this question are stance. The overexpression of FLIP in kidneys may help drive
urgently needed, which can optimize a combined therapeutic the TNF signaling pathway towards necroptosis, but the exact
strategy to improve the efficacy of blocking necroptotic mechanism remains to be illustrated.
signaling. Collectively, accumulating evidence has been extensively
Likewise, blocking necroptosis pathway by chemical inhi- reported in recent years to demonstrate that necroptosis plays
an crucial role in different types of AKI, suggesting a potential
bitor Nec-1 or gene knockout of key mediators can protect
therapeutic target for this medical condition that needs to be
from AKI initiated by other nephrotoxic agents. For example,
clinically validated in the future (Tables 1 and 2).
cyclosporin A (CsA) is a widely used immunosuppressive drug
for organ transplantation and other autoimmune diseases.
Necroptosis was also suggested in CsA-associated tubular Open questions
injury with an in vitro model.57 The authors demonstrated
obvious therapeutic effects of Nec-1 and knockdown of Rip3 in How to monitor necroptosis in vivo?. Substantial progress
rat TECs exposed to CsA, indicating that necroptosis might made in the past decade brought insights into the molecular
also be implicated in the pathologic process of CsA-related mechanism of necroptosis, as well as its contribution to the
AKI. Furthermore, Nec-1 was similarly shown to prevent from pathogenesis of AKI. Until now, however, these studies have
contrast-induced AKI in another study.58 Unfortunately, Nec-1 confined to preclinical models, and the diagnosis of necrop-
showed no relevance with cell death in this model but an tosis has still largely relied on detecting the protective effect

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of Nec-1 or knockout of Rip3 and Mlkl. However, protection of level of RIP3 could serve as a predictor for cellular sensitivity
Nec-1 alone cannot provide a definite identification of towards necroptosis, whereas the exact regulating mechan-
necroptosis as its nonspecific functions on renal peritubular ism remains unclear. Understanding the precise susceptibility
diameters, indolamin-2, 3-dioxygenase (IDO) and ferropto- profiles of renal cells could help develop more efficient
sis;58,60,61 and one, therefore, should be cautious when the treatment aiming at certain cell types that are more vulnerable
presence of necroptosis is based only on the effect of Nec-1. to necroptosis. More researches are required and tissue-
Exploring the role of necroptosis in vivo requires reliable and specific gene deletion model should be used for better
feasible detection approaches. Antibodies that target phos- investigation of exact role of necroptosis in various renal cells
phorylated MLKL through immunostaining or immunoblotting during AKI.
methods emerge as promising biomarkers to directly detect
necroptosis. RIP3 could phosphorylate the activation loop of
murine MLKL at Ser345, Ser347 and Thr349.62 A recent
How does necroptosis interplay with inflammation
study from Rodriguez et al.63 indicated that phosphorylation
specifically during AKI?. Unlike apoptosis, necroptosis
of Ser345 is critical for RIP3-mediated necroptosis, during
results in the release of unprocessed intracellular contents,
both the processes of MLKL activation and execution. They
also known as damage-associated molecular pattern
further generated a specific monoclonal antibody to detect
(DAMP).22,66 Subsequently, DAMP activates the innate
phospho-Ser345 in murine cells.63 Distinctly, RIP3 phosphor-
ylates human MLKL at the Thr357 and Ser358 sites.21 Wang immunity to produce more proinflammatory cytokines, which
et al.64 found that phosphorylation of both sites are essential in turn leads to more necrosis in renal tissues. This
for the engagement of necroptosis, and developed a autoamplification loop of inflammation is termed as necroin-
monoclonal antibody that specifically recognizes human flammation;67,68 and the conceptual advance very well
phospho-MLKL. This antibody has been applied to detect delineates the process of immune cascades induced by
necroptosis in situ based on human liver biopsy samples from necroptosis as well as other necrotic cell deaths. Specially, it
patients with drug-induced liver injury,64 although further is reported that necroptotic cells are characterized with
validation in other disease models is required before its increasing secretory IL-33.69 IL-33 is a pleiotropic cytokine of
clinical application. To measure specific phosphorylation site the IL-1 family and has an important role in tissue
of MLKL, other than to detect expression levels of pathway homeostasis.70 Increased level of extracellular IL-33 indi-
molecules, seems more efficient and accurate. It is inspired cated that necroptosis might not be such proinflammatory as
that commercial mAbs against murine and human phos- originally assumed. A recent study showed that liver-resident
phorylated MLKL are available now. Moreover, besides macrophages experience necroptosis during bacterial
biopsy samples, biomarkers derived from blood or urine infection.71 Considering the abundance of tissue-resident
may also promise to offer clinically feasible tools to evaluate immune cells within the kidney,72–74 whether they occur
the dynamics of necroptosis in vivo. For instance, measure- necroptosis upon various initiating stimuli remains an
ment of graft-derived circulating cell-free DNA has shown intriguing question. Furthermore, how does necroptosis
promise as a way to detect severe graft injury using droplet orchestrate the dynamics of both resident and infiltrating
digital PCR.65 Hence, development of specific biomarkers is immune cells? And how necroptosis is connected with the
urgently needed in the future. phased transition of immune cells from destructive to
reparative role during AKI? All these questions need more
Necroptosis in AKI: when and where?. Some basic studies in depth in the future.
questions about necroptosis in AKI remain to be addressed.
For example, whether and how necroptosis relates to
different stages of AKI are still unknown. AKI consists of
What is the relative contribution of necroptosis and
several mutually overlapping stages: initiation, extension,
apoptosis in AKI?. Undoubtedly, necroptosis and apoptosis
maintenance and recovery phases. Necroptosis may con-
coexist in the pathophysiological process of AKI. However,
tribute to a variable degree to tissue injury and regeneration
whether necroptosis is relevant to the damage of kidney
in various phases. Understanding this question enables
better therapeutic strategies for AKI targeting at necroptosis. function during AKI are challenged recently. Despite some
Recent knowledge recognizes necroptosis as a crucial limitations in the detection techniques for apoptosis,11,75 the
participant in the early stage of AKI. In a previous study, effectiveness of anti-apoptosis therapeutic interventions in
furthermore, extra doses of the Nec-1 at 2 and 4 h after renal previous studies have proven the contribution of apoptosis to
reperfusion followed ischemia that demonstrated no addictive AKI,11 which should not be neglected even from current
protective effect.47 Despite of this observation, it cannot be perspective. In fact, necroptosis, apoptosis and other modes
concluded that necroptosis occurs in a snapshot fashion. The of regulated cell death orchestrate the pathogenesis of AKI
time course of necroptosis in various types of AKI is worthy of together; and the relative contribution of each cell death to
being further delineated. AKI depends on the type and severity of the injury. The
Another question involves in the cell-specific sensitivity to relative contribution of each one to the renal dysfunction in
necroptosis within kidney and its underlying mechanism. AKI remains elusive, and therefore exploring in more detail
Compared with mesangial cells and podocytes, glomerular the contribution of necroptosis, apoptosis as well as other
endothelial cells and tubular cell are more susceptible to types of cell death in certain conditions of AKI will optimize
TNF-triggered necroptosis in vitro.47 It is likely the expression specific therapeutic modalities.

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Therapeutic implications presently used for treatment of necroptosis-associated dis-

eases in clinic. Although necroptosis inhibitors have entered
Identification of chemicals inhibiting the critical checkpoints
clinical trials, several considerations should be seriously taken
makes it possible to manipulate necroptotic pathway. Nec-1,
into account. Current data supported that an early intervention
originally identified as the RIP1 inhibitor, has been widely used
might exert a satisfying therapeutic effects, which is also
to treat necroptosis-associated diseases. However, a recent
implied by the necroinflammation theory. Unfortunately, such
study has observed that Nec-1 could protect Rip1− / − cells
strategy might not be practical in a large fraction of cases, for
from ferroptosis, indicating potential off-target effects of Nec-1
AKI is usually asymptomatic at the early stage. This is why the
on ferroptosis.61 Besides, Nec-1 has yet unrecognized effects
time course of necroptosis discussed previously in the ‘open
on renal peritubular diameters as mentioned above,58 and an
question’ section is particularly important. Because necropto-
unexpected inhibitory role on IDO.60 All these nonspecific
sis can hardly be transient in AKI, the therapeutic window
functions of Nec-1 as well as the relatively short half-life
might be wide enough, which of course requires further
period60 hamper its final clinical application. In spite of
validation. The establishment of such clinical trials in patients
possible similar pharmacokinetic properties, a more-specific
who have some certain predictable risk factors for AKI may be
variant Nec-1 s that does not affect either ferroptosis or
much easier, for example, kidney transplantation recipients.
IDO provides a better alternative.60,61 Beyond Nec-1(s),
Some iatrogenic factors can also cause AKI, such as cardiac
a series of necroptosis inhibitors have been reported
surgery. AKI has high incidence under these conditions,
recently.21,26,63,76–81 Particularly, two independent groups
particularly among those with preexisting renal comorbidities
performed screens with a range of FDA-approved agents
(high-risk patients).82,83 Therefore, prophylactic strategies are
and identified three of them as potential drugs to block recommended under these situations. Meanwhile, the pro-
necroptosis. Dabrafenib was recognized as a selective phylactic strategy and early-onset intervention could be used
inhibitor for RIP3,79 pazopanib for RIP1, and surprisingly, in combination on a case-by-case basis, because the
ponatinib for both RIP1 and RIP3.76 Given that the toxicities of intensive management for these patients provides better
pharmacological candidates are critical considerations in the detection for AKI in the early phase. It is reported that recipient
clinical application, these drugs are more advantageous mice receiving kidneys from Rip3-/- donors have longer
regarding this issue, for all of them are anticancer agents in survival and improved renal function.84 However, in clinic, it
current clinical use with well-documented side effects. How- is not ethically or practically feasible to treat either living or
ever, whether such side effects are specifically acceptable for deceased donors in kidney transplantation. Fortunately, cold
patients with necroptosis-associated diseases remains ischemia time offers an ideal therapeutic window for pre-
another question that warrants further elaborate evaluation. conditioning allografts with necroptosis inhibitors ex vivo. The
Considering the multiple initiating pathways at upstream trial designers should be precautious about the possible
levels, manipulating downstream mediators of necroptosis synergistic effects of necroptosis blockage and immunosup-
such as MLKL may be more effective. A direct inhibitor of pression on acute severe infection.
human MLKL, necrosulfonamide, holds the promise to serve A safety concern related to the increased risk of viral
as a therapeutic drug.21 However, as many inhibitors interfer- infection should be specially considered in the establishment
ing necroptosis have not been extensively explored to date, of ‘anti-necroptosis’ clinical trials. The prevailing theory
the efficacy and safety of these potential drugs should be believes that necroptosis is a defensive mechanism against
further carefully validated. virus in physiological conditions.85 As a classic example,
In addition to inhibitors of key mediators, it is also possible to Rip3-deficient mice died during vaccinia virus infection.18
block necroptosis at the level of receptors. But the contribution Although these are no sufficient evidence indicating that
of each receptor to differently induced AKI is uncertain, which necroptosis is crucial in the control of whole spectrum of
complicates the development of a feasible and effective viruses and pharmacological inhibitors of necroptosis could
therapy. Thus the role of different receptors in various type of result in viral infection, the safety concern regarding this
AKI must be clearly described in the future. Moreover, problem remains a critical issue. Information on this potential
understanding the exact executive mechanism in the down- risk should be included in the informed consent processes for
stream of MLKL might lead to novel potential therapeutic participants in the clinical trials. In addition, careful screening
checkpoints. for potential infections should be employed during anti-
One should be cautious about the unwanted effect of necroptosis treatment, especially for the recipients of renal
necroptosis blockade on other forms of cell death or vice transplantation.
versa. For example, molecular pathways of necroptosis and Finally, given the fact that there are various and complex
apoptosis could crosstalk at various levels and therefore could mechanisms contributing to AKI pathogenesis, it is reasonable
mutually impact each other. This theory can be exemplified by to adopt a combination strategy. Blocking necroptosis in
zVAD that is shown to shift apoptosis to necroptosis.51 conjunction with current clinical treatment can be applied to
Therefore, more preclinical animal model researches are improve the therapeutic efficacy and reduce side effects.
needed to clarify any possible reciprocal effects between
necroptosis and other forms of cell death.
Conclusions
Theoretically, blocking necroptosis not only ameliorates cell
death, but also reduces release of DAMPs as well as the Taken together, necroptosis has been suggested to serve
subsequent inflammation, thus providing a promising ther- as a crucial role in AKI. The improving understanding of
apeutic option. However, no necroptosis inhibitors are the underlying mechanism of necroptosis reveals a novel

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