**4.1. MPT-RN**

**3. Necroptosis in AKI**

of MLKL in necroptosis.

**3.2. The contribution of necroptosis to AKI**

**3.1. The signaling pathways of necroptosis**

6 Current Understanding of Apoptosis - Programmed Cell Death

Necrostatin-1 (Nec-1) was identified as a specific inhibitor of receptor-interacting protein kinase 1 (RIP1) in 2008. Since then, the molecular pathways of necroptosis have been extensively studied [26, 27]. As the best-characterized regulated necrosis, necroptosis is shown to be initiated by the engagement of death receptors, Toll-like receptors (TLRs), interferon signals, as well as intracellular stimuli from protein DNA-dependent activator of IFN regulatory factors (DAI) [28]. The details about the signaling of necroptotic cell death have been already reviewed [7, 28], and in this character, we only present the molecular pathways of TNF-α-induced necroptosis in the absence of functional caspases. Upon the binding of TNF-α to TNF receptor (TNFR)1, the adaptor molecules Fas-associated death domain (FADD) and TNF-receptor-associated death domain (TRADD) are recruited to the ligated TNFR1 successively. These adaptor molecules then bind to RIP1. Subsequently, RIP1 combines with RIP3 to assemble a complex termed "necrosome" via the interaction of RIP homotypic interaction motif (RHIM) domain on both RIP1 and RIP3 [29–32]. RIP3 goes through autophosphorylation within necrosome, which leads to the activation of RIP3 [30, 31]. Activated RIP3 subsequently recruits and phosphorylates the downstream MLKL, which is believed as the executor of necroptosis [33, 34]. The exact mechanisms underlying the execution activity of MLKL are not totally delineated yet. It is thought that phosphorylated MLKL goes through a molecular switch to translocate to the membrane and consequently disrupt the integrity of plasma membrane to finish the action of necroptosis [35, 36]. In addition, study showed that MLKL could also induce mitochondria fission via the action of phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp-1) [37]. As mitochondria play an important role in apoptosis, this result suggests a broad involvement of mitochondria in different types of cell death. But the relative contribution of mitochondria-mediated damages in the background of necroptosis needs further confirmation [38]. It is notable that other necroptotic pathways mediated by TLRs, interferon signals and DAI converge on the RIP3 and share the same downstream executing pathway, indicating the indispensable role

In 2012, Linkermann and colleagues found the protective effect of Nec-1, the first-generation of necrostatins, in a murine model of renal IRI, providing the first evidence of the presence of necroptosis in AKI [39]. In this study, Nec-1 was shown to prevent ischemic kidneys from renal dysfunction and tissue damage, indicating both functional and histological relevance of necroptosis in the pathogenesis of AKI. The pan-caspase inhibitor zVAD that was used to inhibit apoptosis in treatment of kidney diseases, surprisingly, was demonstrated nonprotective in the same research. The reasons of this conflict result compared with a previous report that demonstrated the protective effect of zVAD in the context of IRI need further investigations [40]. The different methods adopted in these two independent researches may partially explain the incontinence of the therapeutic effects of zVAD. First, different clamping

Mitochondrial permeability transition (MPT) mediated regulated necrosis (MPT-RN) is featured by the opening of a trans-mitochondrial membrane pore, namely the MPT pore (MPTP) [49]. CypD is identified as a controller of MPTP, which promotes the opening of this channel [50]. CypD interacts with another regulator the F0 F1 ATP synthase that maintains the inactivation of MPTP. Although the upstream pathways that initiate MPT-RN and the exact mechanisms to modulate the activity of CypD and F0F1ATP synthase remain elusive, it is believed that the opening of MPTP is capable to result in translocation of NAD+ to cytosol and mitochondrial potential disruption [51]. NAD+ along with ATP can be further consumed in the process of NAD+ glycohydrolases. The final result of these physiopathological alterations is the occurrence of regulated necrosis. Therapeutically, MPT-RN can be inhibited by sanglifehrin A and cyclosporin A [52].

type cell death that were distinct from either apoptosis, necroptosis, or other known regulated necrosis. This form of cell death, characterized by perioxidation, relies on accessible intracellular iron, and is therefore named as ferroptosis (ferro, ferrous ion) [70]. Erastin is believed to inhibit the system Xc- cystine/glutamate antiporter, which plays a key role in the exchange of extracellular cystine and intracellular glutamate. Cystine is required for synthesizing glutathione (GSH). Glutathione peroxidase 4 (GPX4) is an indispensable enzyme maintaining intracellular homeostasis by prevent reactive oxygen species accumulation and lipid peroxidation. Importantly, GPX4 is determined as a key inhibitor of ferroptosis, and its function is dependent on intracellular levels of GSH [71]. Therefore, inhibition of the system Xc- cystine/glutamate antiporter could result in a catastrophic decrease of GSH and thereby functional ablation of GPX4. More details about the emerging signaling of ferroptosis are provided by Yang and Stockwell [72]. Upon the introduction of ferroptosis, Dixon and Stockwell also identified a small molecule—ferrostatin-1—as an inhibitor of ferroptosis, namely the first-generation of ferrostatins, which serves as a crucial tool in the research of ferroptosis thereafter [70]. Due to the pharmacological instability of ferrostatin-1, second- and third-generation ferrostatins have been developed with promising therapeutic outlooks.

From Apoptosis to Regulated Necrosis: An Evolving Understanding of Acute Kidney Injury

http://dx.doi.org/10.5772/intechopen.74816

9

In 2014, three different teams from German and the United States reported that ferroptosis served as a crucial participant in the pathologic process of renal injuries. Friedmann Angeli and colleagues used inducible Gpx4 knockout mice to exhibit that deletion of Gpx4 led to ferroptosis-related acute renal failure and associated death. They also confirmed that Gpx4 ablation could cause extra-mitochondrial lipid peroxidation which thereby triggered ferroptosis. Furthermore, Liproxstatin-1, a spiroquinoxalinamine derivative, was demonstrated to inhibit ferroptosis *in vitro* and *in vivo* [73]. Rhabdomyolysis, as a severe and common clinical condition, is regarded as one of the main causes of AKI and rhabdomyolysis-induced AKI accounts for ~10% of all AKI cases. Rhabdomyolysis is the disruption of striped muscle followed by massive releases of intracellular molecules, in particular electrolytes and myoglobin, which induces oxidative damages and cell death. Skouta et al. subjected freshly isolated mouse kidney proximal tubules to an *ex vivo* model of rhabdomyolysis-induced AKI with or without classic ferroptosis inhibitor Ferrostatin-1 and found that Ferrostatin-1 could effectively prevent cell death [74]. Linkermann et al. have found that ferroptosis contributed to the synchronized necrosis of freshly isolated renal tubules in the context of IRI and oxalate crystal-induced acute kidney injury and Ferrostatin-1could alleviate the synchronized necrosis. Linkermann and colleagues also developed a third generation ferrostatin 16–86 with a more stable biochemical and metabolic feature, which were able to protect mice from severe IRI [75]. These reports provide direct evidence for the vital importance of ferroptosis in the pathogenesis of several types of

AKI, indicating a potential therapeutic checkpoint in treating renal diseases.

Pyroptosis was initially referred to a certain kind of highly inflammatory cell death of infected macrophages [76]. Later, the cellular profile of pyroptosis has expanded from macrophages to other cell types. It is notable that a distinct feature of pyroptosis is the active release of IL-1b and IL-18 during pyroptotic cell death process, which contributes greatly to the high immunogenicity of pyroptosis [77]. Although the signaling pathway of pyroptosis, especially

**4.4. Pyroptosis**

Several independent groups of investigators have demonstrated the role of MPT-RN in AKI by detecting the contribution of CypD in the pathogenesis of kidney injuries. In 2009, Devalaraja-Narashimha et al. found that renal function, as well as the magnitude of erythrocyte trapping, tubular cell necrosis, tubular dilatation, and neutrophil infiltration in kidney histology improved significantly in CypD-deficient mice in the background of renal ischemia–reperfusion injury compared with wild-type mice [53]. Later, Hu et al. showed that knockdown of CypD by RNA interference could also protects rats from renal IRI [54]. The protective effects of CypD inhibition against kidney IRI *in vitro* and *in vivo* were further confirmed by Park et al. using a mouse model null for *Ppif*, the gene encoding CyPD [55]. Linkermann et al. evaluated CypD-deficient mice and RIP3-deficient mice in renal IRI and found that RIP3 deletion seemed to offer a better protection, providing a direct comparison between the selective contributions of MPT-RN and necroptosis to renal IRI [43]. More importantly, the researchers also showed CypD-RIPK3 double-knockout or combined application of Nec-1 and sanglifehrin A were more protective than inhibiting either of these two genes alone, indicating the coexistence of independent regulated necrosis in the same physiopathologic process.

#### **4.2. Parthanatos**

Parthanatos is the poly(ADP-ribose) polymerase 1 (PARP1)-dependent regulated necrosis [56]. PARPs cause the poly(ADP-ribosyl)ation (PARylation) of target proteins and thereby regulate various cellular bioactivities [57]. Different stimuli such as DNA breaks and Ca2+ signaling can activate PARP1, which induces the accumulation of PAR polymers. Both PARP1 and PAR polymers are able to delete NAD+ and ATP via their PARylation [58, 59].

Increasing body of researches have demonstrated that parthanatos plays an important role in the pathogenesis of various types of AKI. By using genetic knockout models or chemical inhibitors of PARP1, several studies provided direct evidence that PARP1-dependent parthanatos was functionally related to renal IRI and showed that inhibition of PARP1 could effectively improve renal injuries [60–62]. Besides *in vivo* models, upregulated PARP1 were also detected in cultured renal tubular epithelial cells that were subjected to H2 O2 [60, 63]. In addition to renal ischemic injury, parthanatos also contributes to AKI induced by various nephrotoxic agents [64–66]. Furthermore, the contribution of parthanatos to AKI was determined in a LPS-induced sepsis-related kidney injury model [67–69]. Taken together, these studies exhibit that parthanatos is an important participant in different forms of AKI, indicating a promising therapeutic target in clinical routines.

#### **4.3. Ferroptosis**

Ferroptosis was discovered during a pharmacological intervention in highly resistant RAStransformed tumor cells with application of erastin by Dixon et al. [70]. Erastin, a lethal small molecule, was originally screened to eliminate cancer cells and was found to cause an unrecognized type cell death that were distinct from either apoptosis, necroptosis, or other known regulated necrosis. This form of cell death, characterized by perioxidation, relies on accessible intracellular iron, and is therefore named as ferroptosis (ferro, ferrous ion) [70]. Erastin is believed to inhibit the system Xc- cystine/glutamate antiporter, which plays a key role in the exchange of extracellular cystine and intracellular glutamate. Cystine is required for synthesizing glutathione (GSH). Glutathione peroxidase 4 (GPX4) is an indispensable enzyme maintaining intracellular homeostasis by prevent reactive oxygen species accumulation and lipid peroxidation. Importantly, GPX4 is determined as a key inhibitor of ferroptosis, and its function is dependent on intracellular levels of GSH [71]. Therefore, inhibition of the system Xc- cystine/glutamate antiporter could result in a catastrophic decrease of GSH and thereby functional ablation of GPX4. More details about the emerging signaling of ferroptosis are provided by Yang and Stockwell [72]. Upon the introduction of ferroptosis, Dixon and Stockwell also identified a small molecule—ferrostatin-1—as an inhibitor of ferroptosis, namely the first-generation of ferrostatins, which serves as a crucial tool in the research of ferroptosis thereafter [70]. Due to the pharmacological instability of ferrostatin-1, second- and third-generation ferrostatins have been developed with promising therapeutic outlooks.

In 2014, three different teams from German and the United States reported that ferroptosis served as a crucial participant in the pathologic process of renal injuries. Friedmann Angeli and colleagues used inducible Gpx4 knockout mice to exhibit that deletion of Gpx4 led to ferroptosis-related acute renal failure and associated death. They also confirmed that Gpx4 ablation could cause extra-mitochondrial lipid peroxidation which thereby triggered ferroptosis. Furthermore, Liproxstatin-1, a spiroquinoxalinamine derivative, was demonstrated to inhibit ferroptosis *in vitro* and *in vivo* [73]. Rhabdomyolysis, as a severe and common clinical condition, is regarded as one of the main causes of AKI and rhabdomyolysis-induced AKI accounts for ~10% of all AKI cases. Rhabdomyolysis is the disruption of striped muscle followed by massive releases of intracellular molecules, in particular electrolytes and myoglobin, which induces oxidative damages and cell death. Skouta et al. subjected freshly isolated mouse kidney proximal tubules to an *ex vivo* model of rhabdomyolysis-induced AKI with or without classic ferroptosis inhibitor Ferrostatin-1 and found that Ferrostatin-1 could effectively prevent cell death [74]. Linkermann et al. have found that ferroptosis contributed to the synchronized necrosis of freshly isolated renal tubules in the context of IRI and oxalate crystal-induced acute kidney injury and Ferrostatin-1could alleviate the synchronized necrosis. Linkermann and colleagues also developed a third generation ferrostatin 16–86 with a more stable biochemical and metabolic feature, which were able to protect mice from severe IRI [75]. These reports provide direct evidence for the vital importance of ferroptosis in the pathogenesis of several types of AKI, indicating a potential therapeutic checkpoint in treating renal diseases.

#### **4.4. Pyroptosis**

process of NAD+

**4.2. Parthanatos**

**4.3. Ferroptosis**

and PAR polymers are able to delete NAD+

ing a promising therapeutic target in clinical routines.

rin A and cyclosporin A [52].

8 Current Understanding of Apoptosis - Programmed Cell Death

glycohydrolases. The final result of these physiopathological alterations is

the occurrence of regulated necrosis. Therapeutically, MPT-RN can be inhibited by sanglifeh-

Several independent groups of investigators have demonstrated the role of MPT-RN in AKI by detecting the contribution of CypD in the pathogenesis of kidney injuries. In 2009, Devalaraja-Narashimha et al. found that renal function, as well as the magnitude of erythrocyte trapping, tubular cell necrosis, tubular dilatation, and neutrophil infiltration in kidney histology improved significantly in CypD-deficient mice in the background of renal ischemia–reperfusion injury compared with wild-type mice [53]. Later, Hu et al. showed that knockdown of CypD by RNA interference could also protects rats from renal IRI [54]. The protective effects of CypD inhibition against kidney IRI *in vitro* and *in vivo* were further confirmed by Park et al. using a mouse model null for *Ppif*, the gene encoding CyPD [55]. Linkermann et al. evaluated CypD-deficient mice and RIP3-deficient mice in renal IRI and found that RIP3 deletion seemed to offer a better protection, providing a direct comparison between the selective contributions of MPT-RN and necroptosis to renal IRI [43]. More importantly, the researchers also showed CypD-RIPK3 double-knockout or combined application of Nec-1 and sanglifehrin A were more protective than inhibiting either of these two genes alone, indicating the

coexistence of independent regulated necrosis in the same physiopathologic process.

Parthanatos is the poly(ADP-ribose) polymerase 1 (PARP1)-dependent regulated necrosis [56]. PARPs cause the poly(ADP-ribosyl)ation (PARylation) of target proteins and thereby regulate various cellular bioactivities [57]. Different stimuli such as DNA breaks and Ca2+ signaling can activate PARP1, which induces the accumulation of PAR polymers. Both PARP1

Increasing body of researches have demonstrated that parthanatos plays an important role in the pathogenesis of various types of AKI. By using genetic knockout models or chemical inhibitors of PARP1, several studies provided direct evidence that PARP1-dependent parthanatos was functionally related to renal IRI and showed that inhibition of PARP1 could effectively improve renal injuries [60–62]. Besides *in vivo* models, upregulated PARP1 were

In addition to renal ischemic injury, parthanatos also contributes to AKI induced by various nephrotoxic agents [64–66]. Furthermore, the contribution of parthanatos to AKI was determined in a LPS-induced sepsis-related kidney injury model [67–69]. Taken together, these studies exhibit that parthanatos is an important participant in different forms of AKI, indicat-

Ferroptosis was discovered during a pharmacological intervention in highly resistant RAStransformed tumor cells with application of erastin by Dixon et al. [70]. Erastin, a lethal small molecule, was originally screened to eliminate cancer cells and was found to cause an unrecognized

also detected in cultured renal tubular epithelial cells that were subjected to H2

and ATP via their PARylation [58, 59].

O2

[60, 63].

Pyroptosis was initially referred to a certain kind of highly inflammatory cell death of infected macrophages [76]. Later, the cellular profile of pyroptosis has expanded from macrophages to other cell types. It is notable that a distinct feature of pyroptosis is the active release of IL-1b and IL-18 during pyroptotic cell death process, which contributes greatly to the high immunogenicity of pyroptosis [77]. Although the signaling pathway of pyroptosis, especially the execution mechanisms, still remains elusive now, it has been documented that pyroptotic cell death results from caspase 1-dependent formation of transmembrane channels and subsequent osmotic pressure disruption [78]. In addition to caspase 1, caspase 11 are further identified as another crucial mediator of pyroptosis [79, 80]. Pyroptosis can be suppressed by chemical inhibitors VX-740, VX-765 as well as virus-derived molecule cytokine response modifier A (CrmA) [4, 76]. Few researches have focused on pyroptosis in the context of AKI. Yang et al. found that the expressions of pyroptosis-associated markers caspase 1 and caspase 11 were both significantly upregulated in a rat model of IRI and pyroptosis could also be observed in an *in vitro* model of hypoxia-reoxygenation, suggesting the existence of pyroptosis in kidney IRI [81]. Additionally, the authors demonstrated a possible regulation of endoplasmic reticulum (ER) stress on pyroptosis. But this interesting report provided no direct evidence for the functional responsibility of pyroptosis in renal injuries. The underlying physiological and pathological relevance of pyroptosis in kidneys, therefore, still remains unclear and needs intensive investigations urgently in the future [82].

because Nec-1 could also protect *Rip1−/−* cells from ferroptosis, indicating an inhibitory effect of Nec-1 on ferroptosis [73]. More seriously, unexpected side effects of Nec-1 were observed on renal peritubular diameters [47], and on the action of indolamin-2, 3-dioxygenase (IDO) [83]. Besides, a relatively short half-life period of Nec-1 also hampers its final clinical application [83]. Thus, Nec-1 is a typical example of the original edition of regulated necrosis inhibitors that are prevailingly nonspecific and pharmacologically unstable. Great efforts have been made to searching for more effective and reliable inhibitors and a series of new inhibitors have been reported recently [34, 73, 75, 83–91]. It is remarkable that some researchers performed screens in the FDAapproved agent pools to identify effective drugs to suppress necroptosis, providing a helpful screening strategy [84, 87]. Considering that FDA-approved drugs have already been carefully evaluated in critical procedures before the clinical application, their pharmacological features and side effects are well documented. Most "new" inhibitors have not been extensively evaluated and therefore need elaborate investigations in the near future. It is indeed exciting that increasing agents targeting regulated cell death have entered clinical trials for the treatment of AKI or other

From Apoptosis to Regulated Necrosis: An Evolving Understanding of Acute Kidney Injury

http://dx.doi.org/10.5772/intechopen.74816

11

There has been an interesting finding published previously that the application of zVAD, a pan-caspase inhibitor, could shift the paradigm of cell death from apoptosis to necroptosis [43]. Researches have demonstrated that apoptosis and regulated necrosis could crosstalk at various molecular levels and therefore could mutually impact each other in some certain conditions. Therefore, researchers and clinicians should be cautious about the unwanted effect in designing cell death inhibition strategies. On the other side, however, it is also reasonable to consider whether the cell death paradigm shifting is a feasible therapeutic modality in AKI treatment. Theoretically, regulated necrosis, unlike apoptosis, can cause the massive release of DAMPs and are thus more inflammatory. Manipulating the cell death profile in favor of reducing structural and functional loss of individuals may provide an optimized treatment effect. This hypothesis, of course, warrants further investigations in the

Taken together, the programmed forms of cell death in AKI consist of apoptosis as well as regulated necrosis that both serve as crucial contributors in renal injuries. An updated and better understanding of the underlying mechanism of regulated cell death provides potential "checkpoints" for AKI treatment. Therapeutic regimens, targeting the regulated cell death,

This study was supported by National Natural Science Foundation of China (Grants 81400752;

kidney diseases.

following studies.

**6. Conclusions**

**Acknowledgements**

81770746 to CY).

warrant intensive investigations in the near future.

**5.3. Paradigm shift of cell death**
