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

We describe here the development of a carbohydrate-based microarray to extend the scope of biomedical research on carbohydrate-mediated molecular recognition and anti-infection responses. We have demonstrated that microbial polysaccharides can be immobilized on a surface-modified glass slide without chemical conjugation. With this procedure, a large repertoire of microbial antigens (approximately 20,000 spots) can be patterned on a single micro-glass slide, reaching the capacity to include most common pathogens. Glycoconjugates of different structural characteristics are shown here to be applicable for microarray fabrication, extending the repertoires of diversity and complexity of carbohydrate microarrays. The printed microarrays can be air-dried and stably stored at room temperature for long periods of time. In addition, the system is highly sensitive, allowing simultaneous detection of a broad spectrum of antibody specificities with as little as a few microliters of serum specimen. Finally, the potential of carbohydrate microarrays is demonstrated by the discovery of previously undescribed cellular markers, Dex-Ids.

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.

Conflict of interest: GWB, AF, and SM are inventors on pending or issued patents (US 10,183,038 and US 10,052,345) aimed at diagnosing or treating proteinuric kidney diseases. They stand to gain royalties from the future commercialization of these patents. AF is Chief Scientific Officer of L&F Health LLC and is a consultant for Variant Pharmaceuticals. Variant Pharmaceuticals has licensed worldwide rights from L&F Research to develop and commercialize hydroxypropyl-beta-cyclodextrin for the treatment of kidney disease. AF is Chief Medical Officer of LipoNexT LLC. SM holds equity interest in a company presently commercializing the form of cyclodextrin referenced in this paper. AF and SM are supported by Hoffman-La Roche. AF is supported by Boehringer Ingelheim.

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.

This study shows that pretreatment with both SS-31 and SS-20 significantly reduced myocardial lipid peroxidation and infarct size in ischemia-reperfusion injury, and suggests that the cardioprotective properties of 2',6'-dimethyl-tyrosine-D-Arg-Phe-Lys-NH2 was primarily mediated by its antioxidant properties. As SS-20 does not scavenge reactive oxygen species, it most likely reduces reactive oxygen species production during ischemia-reperfusion.

Microvascular rarefaction, or loss of microvascular density, is increasingly implicated in the progression from acute ischemic kidney injury to chronic kidney disease. Microvascular dropout results in chronic tissue hypoxia, interstitial inflammation, and fibrosis. There is currently no therapeutic intervention for microvascular rarefaction. We hypothesize that capillary dropout begins with ischemic damage to endothelial mitochondria due to cardiolipin peroxidation, resulting in loss of cristae and the failure to regenerate ATP upon reperfusion. SS-31 is a cell-permeable peptide that targets the inner mitochondrial membrane and binds selectively to cardiolipin. It was recently shown to inhibit cardiolipin peroxidation by cytochrome c peroxidase activity, and it has been shown to protect mitochondrial cristae in proximal tubular cells during ischemia, and accelerated ATP recovery upon reperfusion. We found mitochondrial swelling and loss of cristae membranes in endothelial and medullary tubular epithelial cells after 45-min ischemia in the rat. The loss of cristae membranes limited the ability of these cells to regenerate ATP upon reperfusion and led to loss of vascular integrity and to tubular cell swelling. SS-31 prevented mitochondria swelling and protected cristae membranes in both endothelial and epithelial cells. By minimizing endothelial and epithelial cell injury, SS-31 prevented "no-reflow" after ischemia and significantly reduced the loss of peritubular capillaries and cortical arterioles, interstitial inflammation, and fibrosis at 4 wk after ischemia. These results suggest that mitochondria protection represents an upstream target for pharmacological intervention in microvascular rarefaction and fibrosis.