The mechanism of mitochondrial damage, a key contributor to renal tubular cell death during acute kidney injury, remains largely unknown. Here, we have demonstrated a striking morphological change of mitochondria in experimental models of renal ischemia/reperfusion and cisplatin-induced nephrotoxicity. This change contributed to mitochondrial outer membrane permeabilization, release of apoptogenic factors, and consequent apoptosis. Following either ATP depletion or cisplatin treatment of rat renal tubular cells, mitochondrial fragmentation was observed prior to cytochrome c release and apoptosis. This mitochondrial fragmentation was inhibited by Bcl2 but not by caspase inhibitors. Dynamin-related protein 1 (Drp1), a critical mitochondrial fission protein, translocated to mitochondria early during tubular cell injury, and both siRNA knockdown of Drp1 and expression of a dominant-negative Drp1 attenuated mitochondrial fragmentation, cytochrome c release, caspase activation, and apoptosis. Further in vivo analysis revealed that mitochondrial fragmentation also occurred in proximal tubular cells in mice during renal ischemia/ reperfusion and cisplatin-induced nephrotoxicity. Notably, both tubular cell apoptosis and acute kidney injury were attenuated by mdivi-1, a newly identified pharmacological inhibitor of Drp1. This study demonstrates a rapid regulation of mitochondrial dynamics during acute kidney injury and identifies mitochondrial fragmentation as what we believe to be a novel mechanism contributing to mitochondrial damage and apoptosis in vivo in mouse models of disease.

Autophagy is induced in renal tubular cells during acute kidney injury, however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia-reperfusion induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule-specific autophagy-related gene 7 knockout mouse model shown to be defective in both basal and cisplatin induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.

Mitochondrial injury, characterized by outer membrane permeabilization and consequent release of apoptogenic factors, is a key to apoptosis of mammalian cells. Bax and Bak, two multidomain Bcl-2 family proteins, provide a requisite gateway to mitochondrial injury. However it is unclear how Bax and Bak cooperate to provoke mitochondrial injury and whether their roles are redundant. Here, we have identified a unique role of Bak in mitochondrial fragmentation, a seemingly morphological event that contributes to mitochondrial injury during apoptosis. We show that mitochondrial fragmentation is attenuated in Bak-deficient mouse embryonic fibroblasts, baby mouse kidney cells, and, importantly, also in primary neurons isolated from brain cortex of Bak-deficient mice. In sharp contrast, Bax deficiency does not prevent mitochondrial fragmentation during apoptosis. Bcl-2 and Bcl-XL inhibit mitochondrial fragmentation, and their inhibitory effects depend on the presence of Bak. Reconstitution of Bak into Bax/Bak doubleknockout cells restores mitochondrial fragmentation, whereas reconstitution of Bax is much less effective. Bak interacts with Mfn1 and Mfn2, two mitochondrial fusion proteins. During apoptosis, Bak dissociates from Mfn2 and enhances the association with Mfn1. Mutation of Bak in the BH3 domain prevents its dissociation from Mfn2 and diminishes its mitochondrial fragmentation activity. This study has uncovered a previously unrecognized function of Bak in the regulation of mitochondrial morphological dynamics during apoptosis. By this function, Bak may collaborate with Bax to permeabilize the outer membrane of mitochondria, unleashing the apoptotic cascade.Bax ͉ mitochondria ͉ Bcl-2 ͉ cytochrome c

Autophagy is a cellular process of bulk degradation of damaged organelles, protein aggregates and other macromolecules in the cytoplasm. It is thought to be a general response to stress contributing to cell death; alternatively it might act as a cytoprotective mechanism. Here we found that administration of cisplatin induced the formation of autophagic vesicles and autophagosomes in mouse kidneys. In cultured proximal tubular cells, the nephrotoxin caused autophagy in a dose- and time-dependent manner prior to apoptosis. Notably, autophagy occurred within hours of cisplatin administration but this was partially suppressed by the p53 inhibitor pifithrin-alpha, suggesting that p53 is involved in autophagic signaling. This cisplatin-induced autophagy was attenuated in renal cells stably transfected with Bcl-2, suggesting an anti-autophagic role for this well-known anti-apoptotic protein. Blockade of autophagy with pharmacological inhibitors (3-methyladenine or bafilomycin) or shRNA knockdown of the autophagic gene Beclin increased tubular cell apoptosis during cisplatin treatment. Our study has found that autophagy occurs in acute kidney injury and this may be an important protective mechanism for cell survival.

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