Cell death and inflammation in the proximal tubules are the hallmarks of cisplatin-induced AKI, but the mechanisms underlying these effects have not been fully elucidated. Here, we investigated whether necroptosis, a type of programmed necrosis, has a role in cisplatin-induced AKI. We found that inhibition of any of the core components of the necroptotic pathway-receptor-interacting protein 1 (RIP1), RIP3, or mixed lineage kinase domain-like protein (MLKL)-by gene knockout or a chemical inhibitor diminished cisplatin-induced proximal tubule damage in mice. Similar results were obtained in cultured proximal tubular cells. Furthermore, necroptosis of cultured cells could be induced by cisplatin or by a combination of cytokines (TNF-a, TNF-related weak inducer of apoptosis, and IFN-g) that were upregulated in proximal tubules of cisplatin-treated mice. However, cisplatin induced an increase in RIP1 and RIP3 expression in cultured tubular cells in the absence of cytokine release. Correspondingly, overexpression of RIP1 or RIP3 enhanced cisplatin-induced necroptosis in vitro. Notably, inflammatory cytokine upregulation in cisplatintreated mice was partially diminished in RIP3-or MLKL-deficient mice, suggesting a positive feedback loop involving these genes and inflammatory cytokines that promotes necroptosis progression. Thus, our data demonstrate that necroptosis is a major mechanism of proximal tubular cell death in cisplatin-induced nephrotoxic AKI.

Pyroptosis is a proinflammatory form of cell death that is associated with pathogenesis of many chronic inflammatory diseases. Melatonin is substantially reported to possess anti-inflammatory properties by inhibiting inflammasome activation. However, the effects of melatonin on inflammasome-induced pyroptosis in adipocytes remain elusive. Here, we demonstrated that melatonin alleviated lipopolysaccharides (LPS)-induced inflammation and NLRP3 inflammasome formation in mice adipose tissue. The NLRP3 inflammasome-mediated pyroptosis was also inhibited by melatonin in adipocytes. Further analysis revealed that gasdermin D (GSDMD), the key executioner of pyroptosis, was the target for melatonin inhibition of adipocyte pyroptosis. Importantly, we determined that nuclear factor κB (NF-κB) signal was required for the GSDMD-mediated pyroptosis in adipocytes. We also confirmed that melatonin alleviated adipocyte pyroptosis by transcriptional suppression of GSDMD. Moreover, GSDMD physically interacted with interferon regulatory factor 7 (IRF7) and subsequently formed a complex to promote adipocyte pyroptosis. Melatonin also attenuated NLRP3 inflammasome activation and pyroptosis, which was induced by LPS or obesity. In summary, our results demonstrate that melatonin alleviates inflammasome-induced pyroptosis by blocking NF-κB/GSDMD signal in mice adipose tissue. Our data reveal a novel function of melatonin on adipocyte pyroptosis, suggesting a new potential therapy for melatonin to prevent and treat obesity caused systemic inflammatory response.

Zhang et al. show that Golgi-mediated protein kinase D (PKD) signaling is required and sufficient for NLRP3 inflammasome activation. PKD at the Golgi phosphorylates NLRP3 to release it from mitochondria-associated endoplasmic reticulum membranes, allowing for assembly of the mature inflammasome in the cytosol.

Spinal cord injury (SCI) is one of the most common devastating injuries, which causes permanent disabilities such as paralysis and loss of movement or sensation. The precise pathogenic mechanisms of the disease remain unclear, and, as of yet, there is no effective cure. Mesenchymal stem cells (MSCs) show promise as an effective therapy in the experimental models of SCI. MSCs secrete various factors that can modulate a hostile environment, which is called the paracrine effect. Among these paracrine molecules, exosome is considered to be the most valuable therapeutic factor. Thus, exosomes from MSCs (MSCs-exosomes) can be a potential candidate of therapeutic effects of stem cells. The present study was designed to investigate the effect of whether systemic administration of exosomes generated from MSCs can promote the function recovery on the rat model of SCI in vivo. In the present study, we observed that systemic administration of MSCs-exosomes significantly attenuated lesion size and improved functional recovery post-SCI. Additionally, MSCs-exosomes treatment attenuated cellular apoptosis and inflammation in the injured spinal cord. Expression levels of proapoptotic protein (Bcl-2-associated X protein) and proinflammatory cytokines (tumor necrosis factor alpha and interleukin [IL]-1β) were significantly decreased after MSCs-exosomes treatment, whereas expression levels of antiapoptotic (B-cell lymphoma 2) and anti-inflammatory (IL-10) proteins were upregulated. Further, administration of MSCs-exosomes significantly promoted angiogenesis. These results show, for the first time, that systemic administration of MSCs-exosomes attenuated cell apoptosis and inflammation, promoted angiogenesis, and promoted functional recovery post-SCI, suggesting that MSCs-exosomes hold promise as a novel therapeutic strategy for treating SCI.

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Necroptosis predominates functionally over apoptosis in the pathophysiology of renal ischemia-reperfusion injury (IRI). Inhibition of the core components of the necroptotic pathway—receptor-interacting protein kinase 1 (RIPK1), RIPK3 or mixed lineage kinase domain-like protein (MLKL) reduced renal injury after ischemia/reperfusion (IR). Necrosis can initiate inflammation, which enhances necrosis in a positive feedback loop, subsequently leading to triggering more inflammation, termed as necroinflammation. However, the mechanisms underlying necroinflammation driven by renal tubular cell necroptosis in progression of AKI to CKD are still largely unknown. Here we showed that the upregulated expression and interactions between RIPK3 and MLKL induced necroptosis of renal proximal tubular cells and contributed to NLRP3 inflammasome activation under the conditions of IRI. Gene deletion of Ripk3 or Mlkl ameliorated renal tubular cell necroptosis, macrophage infiltration and NLRP3 inflammasome activation with a reduction in caspase-1 activation and maturation of IL-1β, and then finally reduced interstitial fibrogenesis in the long term after IRI. Bone marrow chimeras confirmed that RIPK3-MLKL-dependent necroptosis is responsible for the initiation of the early renal injury after IRI, and then necroptosis triggered NLRP3 inflammasome activation, which subsequently accelerates necroptosis and triggers more inflammation in an auto-amplification loop. These data indicate that necroinflammation driven by RIPK3-MLKL-dependent necroptosis plays a crucial role in the progression of IRI to CKD.

Tubular epithelial loss has been shown to be responsible for the formation of atubular glomeruli leading to nephron decomposition and interstitial fibrosis in obstructive uropathy. Cells undergoing apoptosis and autophagic cell death play an important role in this process, yet the mechanisms are not fully understood. In this study, we aimed to investigate whether autophagy cooperating with apoptosis is associated with mitochondrial damage and whether oxidative stress plays an important role in the loss of tubular epithelium following unilateral ureteral obstruction. In this model, we demonstrated that there is coexistence of autophagy and apoptosis with tubular atrophy in obstructed proximal tubules. After unilateral ureteral obstruction (UUO), autophagy in proximal tubular cells was enhanced steadily up to 7 days in the obstructed kidney and declined thereafter, while apoptosis was induced in a time-dependent manner from 3 to 14 days. Mitochondrial structure and number also changed during UUO. Lipid peroxidation products, NOX4, and NADPH oxidase activity were also increased in the obstructed renal cortex, and peaked at 7 days. In vitro, we showed that H2O2 induced mitochondrial injury leading to autophagy and apoptosis through the Beclin 1 pathway and interference with Bcl-2 expression. Thus, our data demonstrate that oxidative stress leading to mitochondrial damage and driven autophagy-dependent cell death and apoptosis are important mechanisms of tubular decomposition in obstructive nephropathy.

Abstract. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that regulates cell growth, differentiation, apoptosis and autophagy in various cell types. It has been shown that TGF-β1-driven autophagy represents a novel mechanism of tubular decomposition, leading to renal interstitial fibrosis. However, the exact mechanism by which TGF-β1 regulates autophagy is still poorly understood. In the present study, we investigated the effects of exogenous TGF-β1 on cultured human renal proximal tubular epithelial cells (HRPTEpiCs). Presence of TGF-β1 in the medium induced accumulation of autophagosomes in a time-and dose-dependent manner as seen by monitoring the marker LC3 by confocal fluorescence microscopy and immunoblotting. In addition, TGF-β1 induced upregulation of autophagy-related genes, Atg5, Atg7 and Beclin1. Importantly, increased generation of reactive oxygen species (ROS) and enhanced expression of NADPH oxidases were found to be associated with the TGF-β1-induced autophagy. Conversely, treatment with inhibitors of NADPH oxidase markedly reversed the autophagic effects of TGF-β1. Apoptotic effects were evaluated by the TUNEL assay, measuring mitochondrial membrane potential and monitoring expression of the pro-and anti-apoptotic genes, Bim and Bcl-2, respectively. Transcriptional silencing of the above three autophagy-related genes in HRPTEpiCs caused attenuation of TGF-β1-mediated apoptosis. Similarly, when autophagy was prevented at an early stage by application of 3-methyladenine, the pro-apoptotic effects of TGF-β1 were attenuated. These observations suggest that in HRPTEpiCs TGF-β1 promotes autophagy through the generation of ROS, which contributes to its proapoptotic effect. IntroductionAutophagy and apoptosis are two processes, through which injured/aged cells or organelles are eliminated (1,2). Autophagy, or the 'self-eating' function, is characterized by the presence of abundant double-membraned vacuoles called autophagosomes that sequester cytoplasm and cytosolic organelles, such as mitochondria and endoplasmic reticulum. Subsequently, the autophagosome fuses to a lysosome and its contents and inner membrane are degraded and recycled (3). Autophagy is usually regarded as a protective mechanism for cell survival under various conditions including nutrient deprivation and hypoxia (4). However, increasing evidence suggests that in response to excessive stress autophagy may be detrimental and can lead to cell death (5). Excessive autophagic activity may lead to cellular dysfunctions and induce death by destroying a large proportion of the cytosol and organelles, especially the mitochondria and the endoplasmic reticulum (ER) (6,7). The contribution of autophagy to cell death depends on the threshold of the stimuli. It either constitutes a stress adaptation aimed at suppressing apoptosis or conversely provides an alternative pathway to cell death (2,8).Tubular injury is the major contributor to reduction of renal function. Nephron loss can initiate from the tubular decomposition, follo...