Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N 2 A cells (EC 50 in nM range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca 2؉ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.
Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemiareperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane. Ischemic AKI occurs in many clinical settings, including shock, sepsis, and cardiovascular surgery, and it leads to increased mortality in critically ill patients. 1 Ischemia-reperfusion injury is also a critical issue in organ transplantation, where it can result in delayed graft function, and is a major risk factor for chronic allograft nephropathy. 2,3 Tissue injury occurs during ischemia as a result of ATP depletion. The rapid drop in ATP leads to cytoskeletal changes in tubular epithelial cells, because ATP is required for actin polymerization, 4 resulting in breakdown of the brush border, loss of cell-cell contact, disruption of barrier function, and cell detachment. 5 These cytoskeletal changes are reversible if the duration of ischemia is brief and ATP recovery occurs rapidly on reperfusion. Mitochondrial function is pivotal to the recovery of ATP in proximal tubular cells, because they have minimal glycolytic capacity and must rely on oxidative phosphorylation for ATP synthesis. However, ATP recovery is often delayed on reperfusion, because ischemia results in loss of cristae membranes and mitochondrial swelling. 6,7 The recovery of ATP can be further compromised by mitochondrial permeability transition (MPT) during
The burst of reactive oxygen species (ROS) during reperfusion of ischemic tissues can trigger the opening of the mitochondrial permeability transition (MPT) pore, resulting in mitochondrial depolarization, decreased ATP synthesis, and increased ROS production. Rapid recovery of ATP upon reperfusion is essential for survival of tubular cells, and inhibition of oxidative damage can limit inflammation. SS-31 is a mitochondria-targeted tetrapeptide that can scavenge mitochondrial ROS and inhibit MPT, suggesting that it may protect against ischemic renal injury. Here, in a rat model of ischemia-reperfusion (IR) injury, treatment with SS-31 protected mitochondrial structure and respiration during early reperfusion, accelerated recovery of ATP, reduced apoptosis and necrosis of tubular cells, and abrogated tubular dysfunction. In addition, SS-31 reduced medullary vascular congestion, decreased IR-mediated oxidative stress and the inflammatory response, and accelerated the proliferation of surviving tubular cells as early as 1 day after reperfusion. In summary, these results support MPT as an upstream target for pharmacologic intervention in IR injury and support early protection of mitochondrial function as a therapeutic maneuver to prevent tubular apoptosis and necrosis, reduce oxidative stress, and reduce inflammation. SS-31 holds promise for the prevention and treatment of acute kidney injury. Acute kidney injury (AKI) develops in 5% of hospitalized patients and is associated with significant morbidity. Ischemia is the most common cause of AKI. Despite our current knowledge of the pathophysiology underlying renal ischemia-reperfusion (IR) injury, pharmacologic interventions have not reduced the mortality and morbidity associated with AKI.Rapid recovery of ATP after ischemia is essential for cell survival after IR injury. A profound reduction in intracellular ATP occurs early after onset of ischemia and leads to cytoskeletal derangements, membrane alterations, and cell death by apoptosis and necrosis. 1 Disruption of the cytoskeleton leads to redistribution of integrins and Na ϩ ,K ϩ -ATPase from the basal membrane, resulting in detachment of viable cells from the basement membrane and impairment of Na ϩ reabsorption. The mode of cell death depends on the duration of ischemia and the region of the nephron. Cell death is usually restricted to the outer medullary region where oxygen tension drops precipitously at the corticomedullary junction. 2 The proximal tubules are particularly susceptible to IR injury because they have minimal glycolytic capacity and must rely on mitochondrial metabolism for ATP synthesis. [3][4][5] Ischemia causes damage to all components of the mitochondrial electron transport chain (ETC), resulting in decreased oxidative phosphorylation upon reperfusion. 6 In addition, mitochondria are the primary source of reactive oxygen species (ROS). 6,7 Mitochondria can undergo further damage upon reperfusion because of mitochondrial permeability transition (MPT). During ischemia, elevated mitocho...
Obesity is a major risk factor for the development of chronic kidney disease, even independent of its association with hypertension, diabetes, and dyslipidemia. The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis. Proposed mechanisms leading to renal pathology include abnormal lipid metabolism, lipotoxicity, inhibition of AMP kinase, and endoplasmic reticulum stress. Here we report dramatic changes in mitochondrial structure in glomerular endothelial cells, podocytes, and proximal tubular epithelial cells after 28 weeks of a high-fat diet in C57BL/6 mice. Treatment with SS-31, a tetrapeptide that targets cardiolipin and protects mitochondrial cristae structure, during high-fat diet preserved normal mitochondrial structure in all kidney cells, restored renal AMP kinase activity, and prevented intracellular lipid accumulation, endoplasmic reticulum stress, and apoptosis. SS-31 had no effect on weight gain, insulin resistance or hyperglycemia. However, SS-31 prevented loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory (TNF-α, MCP-1, NF-κB) and profibrotic (TGF-β) cytokines. Thus, mitochondria protection can overcome lipotoxicity in the kidney and represent a novel upstream target for therapeutic development.
The innate immune system has been implicated in both AKI and CKD. Damaged mitochondria release danger molecules, such as reactive oxygen species, DNA, and cardiolipin, which can cause NLRP3 inflammasome activation and upregulation of IL-18 and IL-1 It is not known if mitochondrial damage persists long after ischemia to sustain chronic inflammasome activation. We conducted a 9-month study in Sprague-Dawley rats after 45 minutes of bilateral renal ischemia. We detected glomerular and peritubular capillary rarefaction, macrophage infiltration, and fibrosis at 1 month. Transmission electron microscopy revealed mitochondrial degeneration, mitophagy, and deformed foot processes in podocytes. These changes progressed over the study period, with a persistent increase in renal cortical expression of IL-18, IL-1, and TGF-, despite a gradual decline in TNF- expression and macrophage infiltration. Treatment with a mitoprotective agent (SS-31; elamipretide) for 6 weeks, starting 1 month after ischemia, preserved mitochondrial integrity, ameliorated expression levels of all inflammatory markers, restored glomerular capillaries and podocyte structure, and arrested glomerulosclerosis and interstitial fibrosis. Further, helium ion microscopy vividly demonstrated the restoration of podocyte structure by SS-31. The protection by SS-31 was sustained for ≥6 months after treatment ended, with normalization of IL-18 and IL-1 expression. These results support a role for mitochondrial damage in inflammasome activation and CKD and suggest mitochondrial protection as a novel therapeutic approach that can arrest the progression of CKD. Notably, SS-31 is effective when given long after AKI and provides persistent protection after termination of drug treatment.
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