**5.1. Combination therapy**

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 regulated cell death in the context of AKI.

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 for AKI. But further investigations are still needed.

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

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 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 kidney diseases.
