**3. Necroptosis in AKI**

#### **3.1. The signaling pathways of necroptosis**

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 of MLKL in necroptosis.

time of the renal pedicles in these studies might result in the alterations of the magnitude of apoptotic and necroptotic cell death, which subsequently leads to a changed treatment effect. In addition, the time courses of the zVAD administration were also different. The profiles of cellular death kidneys will continuously evolve during AKI and apoptosis does not occur immediately after the onset of ischemia [10]. Therefore, application of zVAD just 15 min before ischemia might diminish its therapeutic effect. In accordance with this research, the protection of Nec-1 in rat and human renal tubular epithelia cells (TECs) against ischemic insults was confirmed by other investigators *in vitro* [41, 42]. Importantly, more convincing evidence of necroptosis in renal IRI was provided by using the *Rip3*-knockout mice in the following study. Linkermann et al. exhibited that *Rip3*-knockout mice improved kidney damages in contrast to wild-type mice, and Nec-1 administration in *Rip3*-knockout mice could not

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

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

7

Besides IRI, accumulating evidence demonstrated necroptosis also contributes to AKI induced by nephrotoxic agents, including cisplatin, cyclosporin A (CsA) and imaging contrast. Tristao et al. found that Nec-1 can provide additional protection against the cisplatin-associated damage on human renal TECs on the basis of zVAD treatment [44]. Later, Linkermann [43] and Xu [45] further provided more reliable evidence to confirm the role of necroptosis in cisplatininduced AKI with *Rip3*- and *Mlkl-*knockout mice models respectively. Ouyang et al. showed that rat TECs subjected to CsA, a widely used immunosuppressive drug for organ transplantation and other autoimmune diseases, were effectively protected by Nec-1 and knockdown of *Rip3,* indicating a role of necroptosis in the pathologic process of CsA-related AKI [46]. Furthermore, Nec-1 was similarly shown to prevent from contrast-induced AKI in a following study [47].

The existence of necroptosis was also indicated in a glycerol-induced rhabdomyolysis model [48]. The authors found that treatment with Nec-1 led to a reduced tubular necrosis, underscoring the importance of TNF-alpha-mediated tubular necroptosis in this model [48].

Taken together, these findings demonstrated that necroptosis is of vital importance for the pathogenesis of various types of AKI, suggesting a potential therapeutic checkpoint which

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

tion 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+

F1

ATP synthase that maintains the inactiva-

along with ATP can be further consumed in the

to cytosol and mito-

lead to addictive protection [43].

invite further investigations basically and clinically.

[50]. CypD interacts with another regulator the F0

chondrial potential disruption [51]. NAD+

**4. Other regulated necrosis in AKI**

**4.1. MPT-RN**

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

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 time of the renal pedicles in these studies might result in the alterations of the magnitude of apoptotic and necroptotic cell death, which subsequently leads to a changed treatment effect. In addition, the time courses of the zVAD administration were also different. The profiles of cellular death kidneys will continuously evolve during AKI and apoptosis does not occur immediately after the onset of ischemia [10]. Therefore, application of zVAD just 15 min before ischemia might diminish its therapeutic effect. In accordance with this research, the protection of Nec-1 in rat and human renal tubular epithelia cells (TECs) against ischemic insults was confirmed by other investigators *in vitro* [41, 42]. Importantly, more convincing evidence of necroptosis in renal IRI was provided by using the *Rip3*-knockout mice in the following study. Linkermann et al. exhibited that *Rip3*-knockout mice improved kidney damages in contrast to wild-type mice, and Nec-1 administration in *Rip3*-knockout mice could not lead to addictive protection [43].

Besides IRI, accumulating evidence demonstrated necroptosis also contributes to AKI induced by nephrotoxic agents, including cisplatin, cyclosporin A (CsA) and imaging contrast. Tristao et al. found that Nec-1 can provide additional protection against the cisplatin-associated damage on human renal TECs on the basis of zVAD treatment [44]. Later, Linkermann [43] and Xu [45] further provided more reliable evidence to confirm the role of necroptosis in cisplatininduced AKI with *Rip3*- and *Mlkl-*knockout mice models respectively. Ouyang et al. showed that rat TECs subjected to CsA, a widely used immunosuppressive drug for organ transplantation and other autoimmune diseases, were effectively protected by Nec-1 and knockdown of *Rip3,* indicating a role of necroptosis in the pathologic process of CsA-related AKI [46]. Furthermore, Nec-1 was similarly shown to prevent from contrast-induced AKI in a following study [47].

The existence of necroptosis was also indicated in a glycerol-induced rhabdomyolysis model [48]. The authors found that treatment with Nec-1 led to a reduced tubular necrosis, underscoring the importance of TNF-alpha-mediated tubular necroptosis in this model [48].

Taken together, these findings demonstrated that necroptosis is of vital importance for the pathogenesis of various types of AKI, suggesting a potential therapeutic checkpoint which invite further investigations basically and clinically.
