**5.3. Paradigm shift of cell death**

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

Anti-apoptosis-based therapeutic strategies have been intensively explored for the treatment of AKI prior to the recognition of regulated necrosis. However, few anti-apoptosis interventions have been widely applied in clinical practice, despite the promising results obtained in animal models, which might be, at least partially, ascribed to our limited understanding of the

Thanks to the improved interpretation of the roles of regulated necrosis in kidney diseases, it is now possible to manipulate the apoptotic and regulated necrotic signaling simultaneously. In an interesting study, Tristao et al. found that the combined use of apoptosis and necroptosis inhibitors could provide additional protection in AKI, suggesting that the combination therapy targeting apoptosis and regulated necrosis might provide optimized therapeutic alternatives [44]. Moreover, combined inhibition of different regulated necrosis is also effective. Linkermann et al. demonstrated that the third-generation ferroptosis inhibitor 16–86 was able to further enhance the protective effect on IRI via combined treatment with necrostatins and MPT-RN inhibitor [75]. These results indicate that the combined blocking of several different regulated cell deaths hold the great promise to improve the current treatment

Searching for chemical inhibitors or generating novel compounds targeting the critical checkpoints makes it possible to manipulate regulated cell death more efficiently. Compared with apoptosis whose inhibitors have been widely explored, the inhibitors of regulated necrosis warrant more explorations in the future. For example, Nec-1 is the first-generation of necrostatins that initially identified as the RIP1 inhibitor, and later widely used as a tool to distinguish necroptosis. However, a recent study demonstrated that Nec-1 was not a specific inhibitor against necroptosis

unclear and needs intensive investigations urgently in the future [82].

**5. Therapeutic implications**

10 Current Understanding of Apoptosis - Programmed Cell Death

regulated cell death in the context of AKI.

for AKI. But further investigations are still needed.

**5.2. Screening for next-generation inhibitors**

**5.1. Combination therapy**

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 following studies.
