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The aim of this work was to test whether we can treat cholestasis with dietary approaches applied after the onset of the disease. The effects of intermittent fasting and dietary restriction on liver damage caused by common bile duct ligation (BDL) in rats were studied, with particular attention paid to changes in the activity of enzymes of energy metabolism and antioxidant protection. Morphological changes in liver tissue and serum markers of liver damage were assessed in rats with BDL kept for one month on ad libitum diet, intermittent fasting, or 35% dietary restriction. We studied parameters of glucose metabolism (activity of glycolysis and gluconeogenesis enzymes), TCA cycle, and indicators of oxidative stress and redox status of the liver tissue. Dietary restriction resulted in an increase in gluconeogenesis activity, antioxidant capacity, and autophagy activation. When implemented after BDL, none of the dietary restriction protocols reduced the level of oxidative stress, detrimental morphological and biochemical alterations, or the fibrosis progression. Thus, under severe damage and oxidative stress developing in cholestasis, dietary restrictions are not hepatoprotective and can only be used in a pre-treatment mode.

There is an increasing accumulation of data on the exceptional importance of mitochondria in the occurrence and treatment of cancer, and in all lines of evidence for such participation, there are both energetic and non-bioenergetic functional features of mitochondria. This analytical review examines three specific features of adaptive mitochondrial changes in several malignant tumors. The first feature is characteristic of solid tumors, whose cells are forced to rebuild their energetics due to the absence of oxygen, namely, to activate the fumarate reductase pathway instead of the traditional succinate oxidase pathway that exists in aerobic conditions. For such a restructuring, the presence of a low-potential quinone is necessary, which cannot ensure the conventional conversion of succinate into fumarate but rather enables the reverse reaction, that is, the conversion of fumarate into succinate. In this scenario, complex I becomes the only generator of energy in mitochondria. The second feature is the increased proliferation in aggressive tumors of the so-called mitochondrial (peripheral) benzodiazepine receptor, also called translocator protein (TSPO) residing in the outer mitochondrial membrane, the function of which in oncogenic transformation stays mysterious. The third feature of tumor cells is the enhanced retention of certain molecules, in particular mitochondrially directed cations similar to rhodamine 123, which allows for the selective accumulation of anticancer drugs in mitochondria. These three features of mitochondria can be targets for the development of an anti-cancer strategy.

The positive effects of female sex hormones, particularly estradiol and progesterone, have been observed in treatment of various pathologies. Acute kidney injury (AKI) is a common condition in hospitalized patients in which the molecular mechanisms of hormone action are poorly characterized. In this study, we investigated the influence of estradiol and progesterone on renal cells during ischemic injury. We performed both in vivo experiments on female and male rats and in vitro experiments on renal tubular cells (RTCs) obtained from the kidneys of intact animals of different sexes. Since mitochondria play an important role in the pathogenesis of AKI, we analyzed the properties of individual mitochondria in renal cells, including the area, roundness, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening time. We found that pre-treatment with progesterone or estradiol attenuated the severity of ischemia/reperfusion (I/R)-induced AKI in female rats, whereas in male rats, these hormones exacerbated renal dysfunction. We demonstrated that the mPTP opening time was higher in RTCs from female rats than that in those from male rats, which may be one of the reasons for the higher tolerance of females to ischemic injury. In RTCs from the kidneys of male rats, progesterone caused mitochondrial fragmentation, which can be associated with reduced cell viability. Thus, therapy with progesterone or estradiol displays quite different effects depending on sex, and could be only effective against ischemic AKI in females.

The development of drugs for the treatment of acute kidney injury (AKI) that could suppress the excessive inflammatory response in damaged kidneys is an important clinical challenge. Recently, synaptamide (N-docosahexaenoylethanolamine) has been shown to exert anti-inflammatory and neurogenic properties. The aim of this study was to investigate the anti-inflammatory effect of synaptamide in ischemic AKI. For this purpose, we analyzed the expression of inflammatory mediators and the infiltration of different leukocyte populations into the kidney after injury, evaluated the expression of the putative synaptamide receptor G-protein-coupled receptor 110 (GPR110), and isolated a population of CD11b/c+ cells mainly representing neutrophils and macrophages using cell sorting. We also evaluated the severity of AKI during synaptamide therapy and the serum metabolic profile. We demonstrated that synaptamide reduced the level of pro-inflammatory interleukins and the expression of integrin CD11a in kidney tissue after injury. We found that the administration of synaptamide increased the expression of its receptor GPR110 in both total kidney tissue and renal CD11b/c+ cells that was associated with the reduced production of pro-inflammatory interleukins in these cells. Thus, we demonstrated that synaptamide therapy mitigates the inflammatory response in kidney tissue during ischemic AKI, which can be achieved through GPR110 signaling in neutrophils and a reduction in these cells’ pro-inflammatory interleukin production.

The development of liver fibrosis is one of the most severe and life-threatening outcomes of chronic liver disease (CLD). For targeted therapy of CLD, it is highly needed to reveal molecular targets for normalizing metabolic processes impaired in damaged liver and associated with fibrosis. In this study, we investigated the morphological and biochemical changes in rat liver models of fibrosis induced by chronic administration of thioacetamide, carbon tetrachloride, bile duct ligation (BDL), and ischemia/reperfusion (I/R), with a specific focus on carbohydrate and energy metabolism. Changes in the levels of substrates and products, as well as enzyme activities of the major glucose metabolic pathways (glycolysis, glucuronidation, and pentose phosphate pathway) were examined in rat liver tissue after injury. We examined key markers of oxidative energy metabolism, such as the activity of the Krebs cycle enzymes, and assessed mitochondrial respiratory activity. In addition, pro- and anti-oxidative status was assessed in fibrotic liver tissue. We found that 6 weeks of exposure to thioacetamide, carbon tetrachloride, BDL or I/R resulted in a decrease in the activity of glycolytic enzymes, retardation of mitochondrial respiration, elevation of glucuronidation, and activation of pentose phosphate pathways, accompanied by a decrease in antioxidant activity and the onset of oxidative stress in rat liver. Resemblance and differences in the changes in the fibrosis models used are described, including energy metabolism alterations and antioxidant status in the used fibrosis models. The least pronounced changes in glucose metabolism and mitochondrial functions in the I/R and thioacetamide models were associated with the least advanced fibrosis. Ultimately, liver fibrosis significantly altered the metabolic profile in liver tissue and the flux of glucose metabolic pathways, which could be the basis for targeted therapy of liver fibrosis in CLD caused by toxic, cholestatic, or I/R liver injury.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the β-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and β-subunits, close to the anionic amino acid residues of the β-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

The ketogenic diet (KD) has been used as a treatment for epilepsy since the 1920s, and its role in the prevention of many other diseases is now being considered. In recent years, there has been an intensive investigation on using the KD as a therapeutic approach to treat acute pathologies, including ischemic ones. However, contradictory data are observed for the effects of the KD on various organs after ischemic injury. In this review, we provide the first systematic analysis of studies conducted from 1980 to 2022 investigating the effects and main mechanisms of the KD and its mimetics on ischemia–reperfusion injury of the brain, heart, kidneys, liver, gut, and eyes. Our analysis demonstrated a high diversity of both the composition of the used KD and the protocols for the treatment of animals, which could be the reason for contradictory effects in different studies. It can be concluded that a true KD or its mimetics, such as β-hydroxybutyrate, can be considered as positive exposure, protecting the organ from ischemia and its negative consequences, whereas the shift to a rather similar high-calorie or high-fat diet leads to the opposite effect.

Kidney diseases belong to a group of pathologies, which are most common among elderly people. With age, even outwardly healthy organisms start to exhibit some age-related changes in the renal tissue, which reduce the filtration function of kidneys and increase the susceptibility to injury. The therapy of acute kidney injury (AKI) is aggravated by the absence of targeted pharmacotherapies thus yielding high mortality of patients with AKI. In this study, we analyzed the protective effects of calorie restriction (CR) against ischemic AKI in senescence-accelerated OXYS rats. We observed that CR afforded OXYS rats with significant nephroprotection. To uncover molecular mechanisms of CR beneficial effects, we assessed the levels of anti- and proapoptotic proteins of the Bcl-2 family, COX IV, GAPDH, and mitochondrial deacetylase SIRT-3, as well as alterations in total protein acetylation and carbonylation, mitochondrial dynamics (OPA1, Fis1, Drp1) and kidney regeneration pathways (PCNA, GDF11). The activation of autophagy and mitophagy was analyzed by LC3 II/LC3 I ratio, beclin-1, PINK-1, and total mitochondrial protein ubiquitination. Among all considered protective pathways, the improvement of mitochondrial functioning may be suggested as one of the possible mechanisms for beneficial effects of CR.

One of the causes of death of patients infected by SARS-CoV-2 is the induced respiratory failure caused by excessive activation of the immune system, the so-called “cytokine storm”, leading to damage to lung tissue. In vitro models reproducing various stages of the disease can be used to explore the pathogenetic mechanisms and therapeutic approaches to treating the consequences of a cytokine storm. We have developed an in vitro test system for simulating damage to the pulmonary epithelium as a result of the development of a hyperinflammatory reaction based on the co-cultivation of pulmonary epithelial cells (A549 cells) and human peripheral blood mononuclear cells (PBMC) primed with lipopolysaccharide (LPS). In this model, after 24 h of co-cultivation, a sharp decrease in the rate of proliferation of A549 cells associated with the intrinsic development of oxidative stress and, ultimately, with the induction of PANoptotic death were observed. There was a significant increase in the concentration of 40 cytokines/chemokines in a conditioned medium, including TNF-α, IFN-α, IL-6, and IL-1a, which corresponded to the cytokine profile in patients with severe manifestation of COVID-19. In order to verify the model, the analysis of the anti-inflammatory effects of well-known substances (dexamethasone, LPS from Rhodobacter sphaeroides (LPS-RS), polymyxin B), as well as multipotent mesenchymal stem cells (MSC) and MSC-derived extracellular vesicles (EVs) was carried out. Dexamethasone and polymyxin B restored the proliferative activity of A549 cells and reduced the concentration of proinflammatory cytokines. MSC demonstrated an ambivalent effect through stimulated production of both pro-inflammatory cytokines and growth factors that regenerate lung tissue. LPS-RS and EVs showed no significant effect. The developed test system can be used to study molecular and cellular pathological processes and to evaluate the effectiveness of various therapeutic approaches for the correction of hyperinflammatory response in COVID-19 patients.

The decrease in the number of resident progenitor cells with age was shown for several organs. Such a loss is associated with a decline in regenerative capacity and a greater vulnerability of organs to injury. However, experiments evaluating the number of progenitor cells in the kidney during aging have not been performed until recently. Our study tried to address the change in the number of renal progenitor cells with age. Experiments were carried out on young and old transgenic nestin-green fluorescent protein (GFP) reporter mice, since nestin is suggested to be one of the markers of progenitor cells. We found that nestin+ cells in kidney tissue were located in the putative niches of resident renal progenitor cells. Evaluation of the amount of nestin+ cells in the kidneys of different ages revealed a multifold decrease in the levels of nestin+ cells in old mice. In vitro experiments on primary cultures of renal tubular cells showed that all cells including nestin+ cells from old mice had a lower proliferation rate. Moreover, the resistance to damaging factors was reduced in cells obtained from old mice. Our data indicate the loss of resident progenitor cells in kidneys and a decrease in renal cells proliferative capacity with aging.

Dietary restriction (DR) is believed to be one of the most promising approaches to extend life span of different animal species and to delay deleterious age-related physiological alterations and diseases. Among others, DR was shown to ameliorate acute kidney injury (AKI) and chronic kidney disease (CKD). However, to date, a comprehensive analysis of the mechanisms of the protective effect of DR specifically in kidney pathologies has not been carried out. The protective properties of DR are mediated by a range of signaling pathways associated with adaptation to reduced nutrient intake. The adaptation is accompanied by a number of metabolic changes, such as autophagy activation, metabolic shifts toward lipid utilization and ketone bodies production, improvement of mitochondria functioning, and decreased oxidative stress. However, some studies indicated that with age, the gain of DR-mediated positive remodeling gradually decreases. This may be an obstacle if we seek to translate the DR approach into a clinic for the treatment of kidney diseases as most patients with AKI and CKD are elderly. It is well known that aging is accompanied by impairments in a huge variety of organs and systems, such as hormonal regulation, stress sensing, autophagy and proteasomal activity, gene expression, and epigenome profile, increased damage to macromolecules and organelles including mitochondria. All these age-associated changes might be the reasons for the reduced protective potential of the DR during aging. We summarized the available mechanisms of DR-mediated nephroprotection and described ways to improve the effectiveness of this approach for an aged kidney.

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV.

The existence of niches of stem cells residence in the ventricular–subventricular zone and the subgranular zone in the adult brain is well-known. These zones are the sites of restoration of brain function after injury. Bioengineered scaffolds introduced in the damaged loci were shown to support neurogenesis to the injury area, thus representing a strategy to treat acute neurodegeneration. In this study, we explored the neuroprotective activity of the recombinant analog of Nephila clavipes spidroin 1 rS1/9 after its introduction into the ischemia-damaged brain. We used nestin–green fluorescent protein (GFP) transgenic reporter mouse line, in which neural stem/progenitor cells are easily visualized and quantified by the expression of GFP, to determine the alterations in the dentate gyrus (DG) after focal ischemia in the prefrontal cortex. Changes in the proliferation of neural stem/progenitor cells during the first weeks following photothrombosis-induced brain ischemia and in vitro effects of spidroin rS1/9 in rat primary neuronal cultures were the subject of the study. The introduction of microparticles of the recombinant protein rS1/9 into the area of ischemic damage to the prefrontal cortex leads to a higher proliferation rate and increased survival of progenitor cells in the DG of the hippocampus which functions as a niche of brain stem cells located at a distance from the injury zone. rS1/9 also increased the levels of a mitochondrial probe in DG cells, which may report on either an increased number of mitochondria and/or of the mitochondrial membrane potential in progenitor cells. Apparently, the stimulation of progenitor cells was caused by formed biologically active products stemming from rS1/9 biodegradation which can also have an effect upon the growth of primary cortical neurons, their adhesion, neurite growth, and the formation of a neuronal network. The high biological activity of rS1/9 suggests it as an excellent material for therapeutic usage aimed at enhancing brain plasticity by interacting with stem cell niches. Substances formed from rS1/9 can also be used to enhance primary neuroprotection resulting in reduced cell death in the injury area.

Uremic retention solutes are the compounds that accumulate in the blood when kidney excretory function is impaired. Some of these compounds are toxic at high concentrations and are usually known as “uremic toxins”. The cumulative detrimental effect of uremic toxins results in numerous health problems and eventually mortality during acute or chronic uremia, especially in end-stage renal disease. More than 100 different solutes increase during uremia; however, the exact origin for most of them is still debatable. There are three main sources for such compounds: exogenous ones are consumed with food, whereas endogenous ones are produced by the host metabolism or by symbiotic microbiota metabolism. In this article, we identify uremic retention solutes presumably of gut microbiota origin. We used database analysis to obtain data on the enzymatic reactions in bacteria and human organisms that potentially yield uremic retention solutes and hence to determine what toxins could be synthesized in bacteria residing in the human gut. We selected biochemical pathways resulting in uremic retention solutes synthesis related to specific bacterial strains and revealed links between toxin concentration in uremia and the proportion of different bacteria species which can synthesize the toxin. The detected bacterial species essential for the synthesis of uremic retention solutes were then verified using the Human Microbiome Project database. Moreover, we defined the relative abundance of human toxin-generating enzymes as well as the possibility of the synthesis of a particular toxin by the human metabolism. Our study presents a novel bioinformatics approach for the elucidation of the origin of both uremic retention solutes and uremic toxins and for searching for the most likely human microbiome producers of toxins that can be targeted and used for the therapy of adverse consequences of uremia.

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.

There has been an explosion of interest in the use of uncouplers of oxidative phosphorylation in mitochondria in the treatment of several pathologies, including neurological ones. In this review, we analyzed all the mechanisms associated with mitochondrial uncoupling and the metabolic and signaling cascades triggered by uncouplers. We provide a full set of positive and negative effects that should be taken into account when using uncouplers in experiments and clinical practice.

The use of stem cells is part of a strategy for the treatment of a large number of diseases. However, the source of the original stem cells for use is extremely important and determines their therapeutic potential. Mesenchymal stromal cells (MSC) have proven their therapeutic effectiveness when used in a number of pathological models. However, it remains an open question whether the chronological age of the donor organism affects the effectiveness of the use of MSC. The asymmetric division of stem cells, the result of which is some residential stem cells acquiring a non-senile phenotype, means that stem cells possess an intrinsic ability to preserve juvenile characteristics, implying an absence or at least remarkable retardation of senescence in stem cells. To test whether residential MSC senesce, we evaluated the physiological changes in the MSC from old rats, with a further comparison of the neuroprotective properties of MSC from young and old animals in a model of traumatic brain injury. We found that, while the effect of administration of MSC on lesion volume was minimal, functional recovery was remarkable, with the highest effect assigned to fetal cells; the lowest effect was recorded for cells isolated from adult rats and postnatal cells, having intermediate potency. MSC from the young rats were characterized by a faster growth than adult MSC, correlating with levels of proliferating cell nuclear antigen (PCNA). However, there were no differences in respiratory activity of MSC from young and old rats, but young cells showed much higher glucose utilization than old ones. Autophagy flux was almost the same in both types of cells, but there were remarkable ultrastructural differences in old and young cells.

Intestinal microbiota play a considerable role in the host’s organism, broadly affecting its organs and tissues. The kidney can also be the target of the microbiome and its metabolites (especially short-chain fatty acids), which can influence renal tissue, both by direct action and through modulation of the immune response. This impact is crucial, especially during kidney injury, because the modulation of inflammation or reparative processes could affect the severity of the resulting damage or recovery of kidney function. In this study, we compared the composition of rat gut microbiota with its outcome, in experimental acute ischemic kidney injury and named the bacterial taxa that play putatively negative or positive roles in the progression of ischemic kidney injury. We investigated the link between serum creatinine, urea, and a number of metabolites (acylcarnitines and amino acids), and the relative abundance of various bacterial taxa in rat feces. Our analysis revealed an increase in levels of 32 acylcarnitines in serum, after renal ischemia/reperfusion and correlation with creatinine and urea, while levels of three amino acids (tyrosine, tryptophan, and proline) had decreased. We detected associations between bacterial abundance and metabolite levels, using a compositionality-aware approach—Rothia and Staphylococcus levels were positively associated with creatinine and urea levels, respectively. Our findings indicate that the gut microbial community contains specific members whose presence might ameliorate or, on the contrary, aggravate ischemic kidney injury. These bacterial taxa could present perspective targets for therapeutical interventions in kidney pathologies, including acute kidney injury.

Electron microscopic study of cardiomyocytes taken from healthy Wistar and OXYS rats and naked mole rats (Heterocephalus glaber) revealed mitochondria in nuclei that lacked part of the nuclear envelope. The direct interaction of mitochondria with nucleoplasm is shown. The statistical analysis of the occurrence of mitochondria in cardiomyocyte nuclei showed that the percentage of nuclei with mitochondria was roughly around 1%, and did not show age and species dependency. Confocal microscopy of normal rat cardiac myocytes revealed a branched mitochondrial network in the vicinity of nuclei with an organization different than that of interfibrillar mitochondria. This mitochondrial network was energetically functional because it carried the membrane potential that responded by oscillatory mode after photodynamic challenge. We suggest that the presence of functional mitochondria in the nucleus is not only a consequence of certain pathologies but rather represents a normal biological phenomenon involved in mitochondrial/nuclear interactions.

We explored the neuroprotective properties of natural plant-derived antioxidants plastoquinone and thymoquinone (2-demethylplastoquinone derivative) modified to be specifically accumulated in mitochondria. The modification was performed through chemical conjugation of the quinones with penetrating cations: Rhodamine 19 or tetraphenylphosphonium. Neuroprotective properties were evaluated in a model of middle cerebral artery occlusion. We demonstrate that the mitochondria-targeted compounds, introduced immediately after reperfusion, possess various neuroprotective potencies as judged by the lower brain damage and higher neurological status. Plastoquinone derivatives conjugated with rhodamine were the most efficient, and the least efficiency was shown by antioxidants conjugated with tetraphenylphosphonium. Antioxidants were administered intraperitoneally or intranasally with the latter demonstrating a high level of penetration into the brain tissue. The therapeutic effects of both ways of administration were similar. Long-term administration of antioxidants in low doses reduced the neurological deficit, but had no effect on the volume of brain damage. At present, cationic decylrhodamine derivatives of plastoquinone appear to be the most promising anti-ischemic mitochondria-targeted drugs of the quinone family. We suggest these antioxidants could be potentially used for a stroke treatment.

Mitochondrial targeting is a novel strategy, which addresses pathologies originating from mitochondrial dysfunction. Here, one of the most potent therapeutics arises from the group of mitochondria-targeted antioxidants, which specifically quench mitochondrial reactive oxygen species (ROS). They show very high efficacy in the treatment of a diverse array of pathologies encountered in this Special Issue of Antioxidants. However, despite very encouraging results in the use of mitochondria-targeted antioxidants, the mechanistic principle of delivering these agents is, to some extent, counterproductive to the goal of selectively treating a population of damaged mitochondria. The main problem that arises is that injured mitochondria may carry a lower membrane potential when compared with normal ones and as a result, injured mitochondria are capable of taking up less therapeutic antioxidants than healthy mitochondria. Another problem is that the intracellular activity of mitochondrial ROS differs from cytosolic ROS in that they carry specific intracellular functions which are maintained at a delicate equilibrium and which may be disturbed under careless use of antioxidant doses. Consequently, understanding the overall benefit of targeting dysfunctional mitochondria in pathological tissue requires furthering the development of alternative techniques to target mitochondria.

Induced and frequently unwanted alterations in the mitochondrial structure and functions are a key component of the pathological cascade in many kidney pathologies, including those associated with acute damage. One of the principal pathogenic elements causing mitochondrial dysfunction in Acute Kidney Injury (AKI) is oxidative stress. After ischemia and nephrotoxic action of drugs, sepsis and systemic inflammation are the most frequent causes of AKI. As the kidney suffers from oxidative stress during sepsis, one of the most promising approaches to alleviate such damaging consequences is the use of antioxidants. Considering administration of lipopolysaccharide (LPS) as a model of sepsis, we demonstrate that the mitochondria of neonatal renal tissue are severely affected by LPS-induced AKI, with pathological ultrastructural changes observed in both the mitochondria of the renal tubular epithelium and the vascular endothelium. Upon mitochondrial damage, we evaluated the effect of the mitochondria-targeted antioxidant plastoquinol decylrhodamine 19 (SkQR1) on the development of acute renal failure in newborn rats associated with systemic inflammation induced by the administration of LPS. We found that SkQR1 administration 3 h before LPS led to decreased urinal expression of the AKI marker neutrophil gelatinase-associated lipocalin 2 (NGAL), in addition to a decrease in urea and creatinine levels in the blood. Additionally, an observed impairment of proliferative activity in the neonatal kidney caused by LPS treatment was also prevented by the treatment of rat pups with SkQR1. Thus, one of the key events for renal tissue damage in neonatal sepsis is an alteration in the structure and function of the mitochondria and the mitochondria-targeted antioxidant SkQR1 is an effective nephroprotective agent, which protects the neonatal kidney from sepsis-induced AKI.

Elucidation of molecular and cellular mechanisms of the uremic syndrome is a very challenging task. More than 130 substances are now considered to be “uremic toxins” and represent a very diverse group of molecules. The toxicity of these molecules affects many cellular processes, and expectably, some of them are able to disrupt mitochondrial functioning. However, mitochondria can be the source of uremic toxins as well, as the mitochondrion can be the site of complete synthesis of the toxin, whereas in some scenarios only some enzymes of the pathway of toxin synthesis are localized here. In this review, we discuss the role of mitochondria as both the target and source of pathological processes and toxic compounds during uremia. Our analysis revealed about 30 toxins closely related to mitochondria. Moreover, since mitochondria are key regulators of cellular redox homeostasis, their functioning might directly affect the production of uremic toxins, especially those that are products of oxidation or peroxidation of cellular components, such as aldehydes, advanced glycation end-products, advanced lipoxidation end-products, and reactive carbonyl species. Additionally, as a number of metabolic products can be degraded in the mitochondria, mitochondrial dysfunction would therefore be expected to cause accumulation of such toxins in the organism. Alternatively, many uremic toxins (both made with the participation of mitochondria, and originated from other sources including exogenous) are damaging to mitochondrial components, especially respiratory complexes. As a result, a positive feedback loop emerges, leading to the amplification of the accumulation of uremic solutes. Therefore, uremia leads to the appearance of mitochondria-damaging compounds, and consecutive mitochondrial damage causes a further rise of uremic toxins, whose synthesis is associated with mitochondria. All this makes mitochondrion an important player in the pathogenesis of uremia and draws attention to the possibility of reducing the pathological consequences of uremia by protecting mitochondria and reducing their role in the production of uremic toxins.

Mesenchymal stem cells (MSCs) have emerged as a potent therapeutic tool for the treatment of a number of pathologies, including immune pathologies. However, unwelcome effects of MSCs on blood coagulation have been reported, motivating us to explore the thrombotic properties of human MSCs from the umbilical cord. We revealed strong procoagulant effects of MSCs on human blood and platelet-free plasma using rotational thromboelastometry and thrombodynamic tests. A similar potentiation of clotting was demonstrated for MSC-derived extracellular vesicles (EVs). To offer approaches to avoid unwanted effects, we studied the impact of a heparin supplement on MSC procoagulative properties. However, MSCs still retained procoagulant activity toward blood from children receiving a therapeutic dose of unfractionated heparin. An analysis of the mechanisms responsible for the procoagulant effect of MSCs/EVs revealed the presence of tissue factor and other proteins involved in coagulation-associated pathways. Also, we found that some MSCs and EVs were positive for annexin V, which implies the presence of phosphatidylserine on their surfaces, which can potentiate clot formation. Thus, we revealed procoagulant activity of MSCs/EVs associated with the presence of phosphatidylserine and tissue factor, which requires further analysis to avoid adverse effects of MSC therapy in patients with a risk of thrombosis.

Thirty-five years ago, we described fragmentation of the mitochondrial population in a living cell into small vesicles (mitochondrial fission). Subsequently, this phenomenon has become an object of general interest due to its involvement in the process of oxidative stress-related cell death and having high relevance to the incidence of a pathological phenotype. Tentatively, the key component of mitochondrial fission process is segregation and further asymmetric separation of a mitochondrial body yielding healthy (normally functioning) and impaired (incapable to function in a normal way) organelles with subsequent decomposition and removal of impaired elements through autophagy (mitophagy). We speculate that mitochondria contain cytoskeletal elements, which maintain the mitochondrial shape, and also are involved in the process of intramitochondrial segregation of waste products. We suggest that perturbation of the mitochondrial fission/fusion machinery and slowdown of the removal process of nonfunctional mitochondrial structures led to the increase of the proportion of impaired mitochondrial elements. When the concentration of malfunctioning mitochondria reaches a certain threshold, this can lead to various pathologies, including aging. Overall, we suggest a process of mitochondrial fission to be an essential component of a complex system controlling a healthy cell phenotype. The role of reactive oxygen species in mitochondrial fission is discussed.

Dietary restriction (DR) is one of the most efficient approaches ameliorating the severity of different pathological conditions including aging. We investigated the protective potential of short-term DR in the model of acute kidney injury (AKI) in young and old rats. In kidney tissue, the levels of autophagy and mitophagy were examined, and proliferative properties of renal cells obtained from rats of different age were compared. DR afforded a significant nephroprotection to ischemic kidneys of young rats. However, in old rats, DR did not provide such beneficial effect. On the assessment of the autophagy marker, the LC3 II/LC3 I ratio, and after staining the tissue with LysoTracker Green, we concluded that in old rats activity of the autophagic-lysosomal system decreased. Mitophagy, as assessed by the levels of PINK-1, was also deteriorated in old animals. Renal cells from old rats showed impaired proliferative capacity, a worse rate of recovery after ischemic injury, increased levels of oxidative stress, accumulation of lipofuscin granules and lower mitochondria membrane potential. The results suggest that the loss of DR benefits in old animals could be due to deterioration in the autophagy/mitophagy flux.

Neonatal sepsis is one of the major causes of mortality and morbidity in newborns, greatly associated with severe acute kidney injury (AKI) and failure. Handling of newborns with kidney damage can be significantly different compared to adults, and it is necessary to consider the individuality of an organism’s response to systemic inflammation. In this study, we used lipopolysaccharide (LPS)-mediated acute kidney injury model to study mechanisms of kidney cells damage in neonatal and adult rats. We found LPS-associated oxidative stress was more severe in adults compared to neonates, as judged by levels of carbonylated proteins and products of lipids peroxidation. In both models, LPS-mediated septic simulation caused apoptosis of kidney cells, albeit to a different degree. Elevated levels of proliferating cell nuclear antigen (PCNA) in the kidney dropped after LPS administration in neonates but increased in adults. Renal fibrosis, as estimated by smooth muscle actin levels, was significantly higher in adult kidneys, whereas these changes were less profound in LPS-treated neonatal kidneys. We concluded that in LPS-mediated AKI model, renal cells of neonatal rats were more tolerant to oxidative stress and suffered less from long-term pathological consequences, such as fibrosis. In addition, we assume that by some features LPS administration simulates the conditions of accelerated aging.

Neonatal hypoxia–ischemia is one of the main causes of mortality and disability of newborns. To study the mechanisms of neonatal brain cell damage, we used a model of neonatal hypoxia–ischemia in seven-day-old rats, by annealing of the common carotid artery with subsequent hypoxia of 8% oxygen. We demonstrate that neonatal hypoxia–ischemia causes mitochondrial dysfunction associated with high production of reactive oxygen species, which leads to oxidative stress. Targeted delivery of antioxidants to the mitochondria can be an effective therapeutic approach to treat the deleterious effects of brain hypoxia–ischemia. We explored the neuroprotective properties of the mitochondria-targeted antioxidant SkQR1, which is the conjugate of a plant plastoquinone and a penetrating cation, rhodamine 19. Being introduced before or immediately after hypoxia–ischemia, SkQR1 affords neuroprotection as judged by the diminished brain damage and recovery of long-term neurological functions. Using vital sections of the brain, SkQR1 has been shown to reduce the development of oxidative stress. Thus, the mitochondrial-targeted antioxidant derived from plant plastoquinone can effectively protect the brain of newborns both in pre-ischemic and post-stroke conditions, making it a promising candidate for further clinical studies.

A recently discovered key role of reactive oxygen species (ROS) in mitochondrial traffic has opened a wide alley for studying the interactions between cells, including stem cells. Since its discovery in 2006, intercellular mitochondria transport has been intensively studied in different cellular models as a basis for cell therapy, since the potential of replacing malfunctioning organelles appears to be very promising. In this study, we explored the transfer of mitochondria from multipotent mesenchymal stem cells (MMSC) to neural cells and analyzed its efficacy under normal conditions and upon induction of mitochondrial damage. We found that mitochondria were transferred from the MMSC to astrocytes in a more efficient manner when the astrocytes were exposed to ischemic damage associated with elevated ROS levels. Such transport of mitochondria restored the bioenergetics of the recipient cells and stimulated their proliferation. The introduction of MMSC with overexpressed Miro1 in animals that had undergone an experimental stroke led to significantly improved recovery of neurological functions. Our data suggest that mitochondrial impairment in differentiated cells can be compensated by receiving healthy mitochondria from MMSC. We demonstrate a key role of Miro1, which promotes the mitochondrial transfer from MMSC and suggest that the genetic modification of stem cells can improve the therapies for the injured brain.