Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds.

Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.

SS-31 as a Mitochondrial Protectant in the Treatment of Tendinopathy: Evaluation in a Murine Supraspinatus Tendinopathy Model.

BACKGROUND: Prior studies have demonstrated mitochondrial dysfunction in tendinopathy. The objective of this investigation was to explore the potential of SS-31 (elamipretide), a mitochondrial protectant, to improve mitochondrial function and promote tendon healing in a murine supraspinatus tendinopathy model. METHODS: One hundred and twenty-six mice (252 limbs) were divided into 6 groups (42 limbs/group) that received (I) 4 weeks of impingement; (II) 8 weeks of impingement; (III) 8 weeks of impingement including 4 weeks of SS-31 treatment (5 mg/kg/d) starting after 4 weeks of impingement; (IV) 4 weeks of impingement ending with clip removal, followed by harvesting 4 weeks later; and (V) 4 weeks of impingement ending with clip removal, followed by 4 weeks of SS-31 treatment and harvesting; and a control group. Specimens were prepared for biomechanical testing, histological analysis, transmission electron microscopy, measurement of superoxidative dismutase (SOD) activity, and measurement of gene expression. RESULTS: Failure force decreased after impingement, compared with the intact tendon, and the decrease was partially reversed after clip removal, SS-31 treatment, and the 2 treatments combined. A similar pattern was observed for stiffness. Histological analysis demonstrated higher modified Bonar scores in the impingement groups; however, the changes in tendon morphology were partially reversed following all treatments, especially the combined treatment. Decreased mitochondrial number and altered organization and density of cristae were observed in the impingement groups. Mitochondrial structure and number became more normal, with improvement in morphology of the cristae, after clip removal and/or SS-31 treatment. SOD activity decreased after impingement, compared with the control group, then increased significantly again after treatment, especially in the combined treatment group. Mitochondria-related gene expression decreased in the impingement groups and increased again after treatment. CONCLUSIONS: The mitochondrial protectant SS-31 improved mitochondrial function, promoting tendon healing, especially when combined with removal of subacromial impingement. CLINICAL RELEVANCE: Improving mitochondrial function with agents such as SS-31 may represent an effective treatment to promote healing in the setting of supraspinatus tendinopathy.

Evaluation of SS-31 as a Potential Strategy for Tendinopathy Treatment: An In Vitro Model.

BACKGROUND: Studies in our laboratory have demonstrated mitochondrial dysfunction in human and animal models of supraspinatus tendinopathy. SS-31 (elamipretide) has been reported to improve mitochondrial function and to be effective in clinical trials for several diseases. The potential of SS-31 in treating tendinopathy has not been explored. HYPOTHESIS: SS-31 would improve mitochondrial function in human tenocytes sampled from patients with tendinopathy. STUDY DESIGN: Controlled laboratory study. METHODS: Healthy tenocytes were obtained from normal hamstring tendon biopsy specimens in 9 patients undergoing anterior cruciate ligament reconstruction, and tenocytes were collected from degenerative supraspinatus tendon biopsy specimens in 9 patients undergoing rotator cuff repair. Tenocytes were cultured, used at passage 1, and assigned to 4 groups: healthy tenocytes, healthy tenocytes with 1µM SS-31 treatment for 72 hours, degenerative tenocytes, and degenerative tenocytes with 1µM SS-31 treatment for 72 hours. The outcomes included measurements of mitochondrial potential, mitochondrial morphology by transmission electron microscopy imaging, reactive oxygen species and superoxidative dismutase activity, gene expression, and cell viability. RESULTS: An increase in the cell fraction with depolarized mitochondria was found in degenerative tenocytes (P = .014), followed by a decrease after SS-31 treatment (P = .018). Transmission electron microscopy images demonstrated morphological changes with a decreased number and size of mitochondria per cell in the degenerative tenocytes (P = .018) and with improvement after SS-31 treatment. There was no significant difference in the level of reactive oxygen species between healthy and degenerative tenocytes in culture, but superoxidative dismutase activity was significantly decreased in the degenerative group (P = .006), which then increased after SS-31 treatment (P = .012). These findings suggested that mitochondrial dysfunction may be reversed by SS-31 treatment. The gene expression of matrix metalloproteinase-1 (matrix remodeling, P = .029) and fatty acid-binding protein 4 (fatty infiltration, P = .046) was significantly upregulated in the degenerative tenocytes and reduced by SS-31 treatment (P = .048; P = .007). Gene expression for hypoxia-inducible factor1 α and the proapoptotic regulator Bcl-2-associated X protein was increased in the degenerative tenocytes. There was a significant decrease in cell viability in degenerative tenocytes as compared with the healthy tenocytes, with small improvement after treatment with SS-31. CONCLUSION: There are changes in mitochondrial structure and function in tenocytes derived from degenerative tendons, and SS-31, as a mitochondrial protectant, could improve mitochondrial function and promote the healing of tendinopathy. CLINICAL RELEVANCE: Mitochondrial dysfunction appears to play a role in the development of tendinopathy, and SS-31, as a mitochondrial protective agent, may be a therapeutic agent in the treatment of tendinopathy.

Targeting Mitochondria by SS-31 Ameliorates the Whole Body Energy Status in Cancer- and Chemotherapy-Induced Cachexia.

Objective: Cachexia is a complex metabolic syndrome frequently occurring in cancer patients and exacerbated by chemotherapy. In skeletal muscle of cancer hosts, reduced oxidative capacity and low intracellular ATP resulting from abnormal mitochondrial function were described. Methods: The present study aimed at evaluating the ability of the mitochondria-targeted compound SS-31 to counteract muscle wasting and altered metabolism in C26-bearing (C26) mice either receiving chemotherapy (OXFU: oxaliplatin plus 5-fluorouracil) or not. Results: Mitochondrial dysfunction in C26-bearing (C26) mice associated with alterations of cardiolipin fatty acid chains. Selectively targeting cardiolipin with SS-31 partially counteracted body wasting and prevented the reduction of glycolytic myofiber area. SS-31 prompted muscle mitochondrial succinate dehydrogenase (SDH) activity and rescued intracellular ATP levels, although it was unable to counteract mitochondrial protein loss. Progressively increased dosing of SS-31 to C26 OXFU mice showed transient (21 days) beneficial effects on body and muscle weight loss before the onset of a refractory end-stage condition (28 days). At day 21, SS-31 prevented mitochondrial loss and abnormal autophagy/mitophagy. Skeletal muscle, liver and plasma metabolomes were analyzed, showing marked energy and protein metabolism alterations in tumor hosts. SS-31 partially modulated skeletal muscle and liver metabolome, likely reflecting an improved systemic energy homeostasis. Conclusions: The results suggest that targeting mitochondrial function may be as important as targeting protein anabolism/catabolism for the prevention of cancer cachexia. With this in mind, prospective multi-modal therapies including SS-31 are warranted.

APOL1 risk variants affect podocyte lipid homeostasis and energy production in focal segmental glomerulosclerosis.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Pharmacological targeting of mitochondrial function and reactive oxygen species production prevents colon 26 cancer-induced cardiorespiratory muscle weakness.

Cancer cachexia is a syndrome characterized by profound cardiac and diaphragm muscle wasting, which increase the risk of morbidity in cancer patients due to failure of the cardiorespiratory system. In this regard, muscle relies greatly on mitochondria to meet energy requirements for contraction and mitochondrial dysfunction can result in muscle weakness and fatigue. In addition, mitochondria are a major source of reactive oxygen species (ROS) production, which can stimulate increased rates of muscle protein degradation. Therefore, it has been suggested that mitochondrial dysfunction may be an underlying factor that contributes to the pathology of cancer cachexia. To determine if pharmacologically targeting mitochondrial dysfunction via treatment with the mitochondria-targeting peptide SS-31 would prevent cardiorespiratory muscle dysfunction, colon 26 (C26) adenocarcinoma tumor-bearing mice were administered either saline or SS-31 daily (3 mg/kg/day) following inoculation. C26 mice treated with saline demonstrated greater ROS production and mitochondrial uncoupling compared to C26 mice receiving SS-31 in both the heart and diaphragm muscle. In addition, saline-treated C26 mice exhibited a decline in left ventricular function which was significantly rescued in C26 mice treated with SS-31. In the diaphragm, muscle fiber cross-sectional area of C26 mice treated with saline was significantly reduced and force production was impaired compared to C26, SS-31-treated animals. Finally, ventilatory deficits were also attenuated in C26 mice treated with SS-31, compared to saline treatment. These data demonstrate that C26 tumors promote severe cardiac and respiratory myopathy, and that prevention of mitochondrial dysfunction is sufficient to preclude cancer cachexia-induced cardiorespiratory dysfunction.

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice.

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

The mitochondria-targeted peptide SS-31 binds lipid bilayers and modulates surface electrostatics as a key component of its mechanism of action.

Mitochondrial dysfunction underlies many heritable diseases, acquired pathologies, and aging-related declines in health. Szeto-Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that are efficacious toward a wide array of mitochondrial disorders and are believed to target mitochondrial membranes because they are enriched in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, using biophysical and computational approaches, we have analyzed the interactions of the lead compound SS-31 (elamipretide) with model and mitochondrial membranes. Our results show that this polybasic peptide partitions into the membrane interfacial region with an affinity and a lipid binding density that are directly related to surface charge. We found that SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it did cause saturable alterations in lipid packing. Most notably, SS-31 modulated the surface electrostatics of both model and mitochondrial membranes. We propose nonexclusive mechanisms by which the tuning of surface charge could underpin the mitoprotective properties of SS-31, including alteration of the distribution of ions and basic proteins at the interface, and/or modulation of bilayer physical properties. As a proof of concept, we show that SS-31 alters divalent cation (calcium) distribution within the interfacial region and reduces the energetic burden of calcium stress in mitochondria. The mechanistic details of SS-31 revealed in this study will help inform the development of future compound variants with enhanced efficacy and bioavailability.

Mitoprotective therapy prevents rapid, strain-dependent mitochondrial dysfunction after articular cartilage injury.

Posttraumatic osteoarthritis (PTOA) involves the mechanical and biological deterioration of articular cartilage that occurs following joint injury. PTOA is a growing problem in health care due to the lack of effective therapies combined with an aging population with high activity levels. Recently, acute mitochondrial dysfunction and altered cellular respiration have been associated with cartilage degeneration after injury. This finding is particularly important because recently developed mitoprotective drugs, including SS peptides, can preserve mitochondrial structure and function after acute injury in other tissues. It is not known, however, if cartilage injury induces rapid structural changes in mitochondria, to what degree mitochondrial dysfunction in cartilage depends on the mechanics of injury or the time frame over which such dysfunction develops. Similarly, it is unknown if SS-peptide treatment can preserve mitochondrial structure and function after cartilage injury. Here, we combined fast camera elastography, longitudinal fluorescence assays, and computer vision techniques to track the fates of thousands of individual cells. Our results show that impact induces mechanically dependent mitochondrial depolarization within a few minutes after injury. Electron microscopy revealed that impact causes rapid structural changes in mitochondria that are related to reduced mitochondrial function, namely, fission and loss of cristae structure. We found that SS-peptide treatment prior to impact protects the mitochondrial structure and preserves mitochondrial function at levels comparable with that of unimpacted control samples. Overall, this study reveals the vital role of mitochondria in mediating cartilage's peracute (within minutes) response to traumatic injury and demonstrates mitoprotection as a promising therapeutic strategy for injury-induced cartilage damage.

Doxorubicin-induced oxidative stress differentially regulates proteolytic signaling in cardiac and skeletal muscle.

Doxorubicin (DOX) is a highly effective antineoplastic agent used in cancer treatment. Unfortunately, clinical use of DOX is limited due to the development of dose-dependent toxicity to cardiac and respiratory (i.e., diaphragm) muscles. After administration, DOX preferentially localizes to the inner mitochondrial membrane, where it promotes cellular toxicity via enhanced mitochondrial reactive oxygen species (ROS) production. Although recent evidence suggests that amelioration of mitochondrial ROS emission preserves cardiorespiratory muscle function following DOX treatment, the mechanisms responsible for this protection remain unknown. Therefore, we tested the hypothesis that DOX-induced mitochondrial ROS production is required to stimulate pathological signaling by the autophagy/lysosomal system (ALS), the ubiquitin-proteasome pathway (UPP), and the unfolded protein response (UPR). Cause and effect were determined by administration of the mitochondria-targeted peptide SS-31 to DOX-treated animals. Interestingly, while SS-31 abrogated aberrant ROS emission in cardiorespiratory muscles of DOX-treated animals, our results revealed muscle-specific regulation of effector pathways. In the heart, SS-31 prevented DOX-induced proteolytic signaling through the ALS and UPP. In contrast, ALS signaling was inhibited by SS-31 in the diaphragm, but the UPP was not affected. UPR signaling was activated in both muscles at eukaryotic translation initiation factor 2α (eIF2α) S51 in the heart and diaphragm of DOX-treated animals and was attenuated with SS-31 treatment in both tissues. However, downstream signaling of eIF2α (activating transcription factor 4 and CCAAT/enhancer-binding protein homologous protein) was diminished in the heart but upregulated in the diaphragm with DOX. Collectively, these results show that DOX-induced ROS production plays distinct roles in the regulation of cardiac and diaphragm muscle proteolysis.

ATP-binding cassette A1 deficiency causes cardiolipin-driven mitochondrial dysfunction in podocytes.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1fl/fl) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1fl/fl mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice.

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

Stealth Peptides Target Cellular Powerhouses to Fight Rare and Common Age-Related Diseases.

BACKGROUND: Mitochondria are the primary source of energy in most tissues. Mitochondrial dysfunction results in cellular energy deficiency, triggers the production of reactive oxygen species, and initiates various cell death and inflammatory pathways. Several cell-permeable peptides (SS peptides) have been described that selectively target cardiolipin on the inner mitochondrial membrane and promote efficiency of the electron transport chain to produce more ATP. CONCLUSION: In preclinical disease models, these peptides have been shown to repair damaged mitochondria and promote cellular repair and restore function. By mitigating cell injury, these peptides prevent inflammatory responses that can result in chronic inflammation and tissue remodeling. This peptide technology platform represents a paradigm shift in targeting the fundamental cause of cellular energy failure for age-related degenerative diseases.

Cardiolipin-targeted peptides rejuvenate mitochondrial function, remodel mitochondria, and promote tissue regeneration during aging.

It has been proposed that a loss of bioenergetic capacity of cells contributes to the progressive loss of biological function with age. Aging is associated with loss of mitochondrial cristae membranes and inhibition of ATP production. Despite the many approaches being pursued for improving mitochondrial function, none of them directly targets the electron transport chain to improve ATP production. Recent studies have brought attention to cardiolipin as a unique target for promoting mitochondrial efficiency. Cardiolipin is important for cristae curvatures and is necessary for optimal activity of the respiratory complexes and the assembly of supercomplexes. Here we describe the discovery of a class of cell-penetrating aromatic-cationic tetrapeptides that selectively target cardiolipin and increase coupling efficiency while reducing reactive oxygen species production. These compounds can rejuvenate mitochondrial bioenergetics, remodel mitochondrial cristae structure, repair cellular structure, and restore organ function during aging.

Protective Effect of a Mitochondria-Targeted Peptide against the Development of Chemotherapy-Induced Peripheral Neuropathy in Mice.

Several chemotherapeutic agents used for cancer treatment induce dose-limiting peripheral neuropathy that compromises patients' quality of life and limits cancer treatment. Recently, mitochondrial dysfunction has been shown to be involved in the mechanism of chemotherapy-induced peripheral neuropathy. SS-20 is a mitochondria-targeted peptide that promotes mitochondrial respiration and restores mitochondrial bioenergetics. In the present study, we examined the protective effect of SS-20 against the development of chemotherapy-induced peripheral neuropathy utilizing a murine model of peripheral neuropathy induced by oxaliplatin, a first-line chemotherapy agent for colon cancer. Weekly administrations of oxaliplatin induced peripheral neuropathy as demonstrated by the development of neuropathic pain and loss of intraepidermal nerve fibers in the hind paw. Continuous administration of SS-20 protected against the development of oxaliplatin-induced neuropathic pain and mitigated the loss of intraepidermal nerve fibers to normal levels. Our findings suggest that SS-20 may be a drug candidate for the prevention of chemotherapy-induced peripheral neuropathy.

Crosstalk between autophagy and oxidative stress regulates proteolysis in the diaphragm during mechanical ventilation.

Mechanical ventilation (MV) results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD). While the mechanisms responsible for VIDD are not fully understood, recent data reveal that prolonged MV activates autophagy in the diaphragm, which may occur as a result of increased cellular reactive oxygen species (ROS) production. Therefore, we tested the hypothesis that (1) accelerated autophagy is a key contributor to VIDD; and that (2) oxidative stress is required to increase the expression of autophagy genes in the diaphragm. Our findings reveal that targeted inhibition of autophagy in the rat diaphragm prevented MV-induced muscle atrophy and contractile dysfunction. Attenuation of VIDD in these animals occurred as a result of increased diaphragm concentration of the antioxidant catalase and reduced mitochondrial ROS emission, which corresponded to reductions in the activity of calpain and caspase-3. To determine if increased ROS production is required for the upregulation of autophagy biomarkers in the diaphragm, rats that were administered the mitochondrial-targeted peptide SS-31 during MV. Results from this study demonstrated that mitochondrial ROS production in the diaphragm during MV is required for the increased expression of key autophagy genes (i.e. LC3, Atg7, Atg12, Beclin1 and p62), as well as for increased activity of cathepsin L. Together, these data reveal that autophagy is required for VIDD, and that autophagy inhibition reduces MV-induced diaphragm ROS production and prevents a positive feedback loop whereby increased autophagy is stimulated by oxidative stress, resulting in further increases in ROS and autophagy.

Pharmacologic Approaches to Improve Mitochondrial Function in AKI and CKD.

AKI is associated with high morbidity and mortality, and it predisposes to the development and progression of CKD. Novel strategies that minimize AKI and halt the progression of CKD are urgently needed. Normal kidney function involves numerous different cell types, such as tubular epithelial cells, endothelial cells, and podocytes, working in concert. This delicate balance involves many energy-intensive processes. Fatty acids are the preferred energy substrates for the kidney, and defects in fatty acid oxidation and mitochondrial dysfunction are universally involved in diverse causes of AKI and CKD. This review provides an overview of ATP production and energy demands in the kidney and summarizes preclinical and clinical evidence of mitochondrial dysfunction in AKI and CKD. New therapeutic strategies targeting mitochondria protection and cellular bioenergetics are presented, with emphasis on those that have been evaluated in animal models of AKI and CKD. Targeting mitochondrial function and cellular bioenergetics upstream of cellular damage may offer advantages compared with targeting downstream inflammatory and fibrosis processes.

The mitochondrial-targeted peptide, SS-31, improves glomerular architecture in mice of advanced age.

Although age-associated changes in kidney glomerular architecture have been described in mice and man, the mechanisms are unknown. It is unclear if these changes can be prevented or even reversed by systemic therapies administered at advanced age. Using light microscopy and transmission electron microscopy, our results showed glomerulosclerosis with injury to mitochondria in glomerular epithelial cells in mice aged 26 months (equivalent to a 79-year-old human). To test the hypothesis that reducing mitochondrial damage in late age would result in lowered glomerulosclerosis, we administered the mitochondrial targeted peptide, SS-31, to aged mice. Baseline (24-month-old) mice were randomized to receive 8 weeks of SS-31, or saline, and killed at 26 months of age. SS-31 treatment improved age-related mitochondrial morphology and glomerulosclerosis. Assessment of glomeruli revealed that SS-31 reduced senescence (p16, senescence-associated-ß-Gal) and increased the density of parietal epithelial cells. However, SS-31 treatment reduced markers of parietal epithelial cell activation (Collagen IV, pERK1/2, and α-smooth muscle actin). SS-31 did not impact podocyte density, but it reduced markers of podocyte injury (desmin) and improved cytoskeletal integrity (synaptopodin). This was accompanied by higher glomerular endothelial cell density (CD31). Thus, despite initiating therapy in late-age mice, a short course of SS-31 has protective benefits on glomerular mitochondria, accompanied by temporal changes to the glomerular architecture. This systemic pharmacological intervention in old-aged animals limits glomerulosclerosis and senescence, reduces parietal epithelial cell activation, and improves podocyte and endothelial cell integrity.

Mitochondria Protection after Acute Ischemia Prevents Prolonged Upregulation of IL-1ß and IL-18 and Arrests CKD.

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-1ß 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-1ß, and TGF-ß, despite a gradual decline in TNF-α 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-1ß 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.