Targeting Inflammation and Oxidative Stress as a Therapy for Ischemic Kidney Injury.

Inflammation and oxidative stress are the main pathological processes that accompany ischemic injury of kidneys and other organs. Based on this, these factors are often chosen as a target for treatment of acute kidney injury (AKI) in a variety of experimental and clinical studies. Note, that since these two components are closely interrelated during AKI development, substances that treat one of the processes often affect the other. The review considers several groups of promising nephroprotectors that have both anti-inflammatory and antioxidant effects. For example, many antioxidants, such as vitamins, polyphenolic compounds, and mitochondria-targeted antioxidants, not only reduce production of the reactive oxygen species in the cell but also modulate activity of the immune cells. On the other hand, immunosuppressors and non-steroidal anti-inflammatory drugs that primarily affect inflammation also reduce oxidative stress under some conditions. Another group of therapeutics is represented by hormones, such as estrogens and melatonin, which significantly reduce severity of the kidney damage through modulation of both these processes. We conclude that drugs with combined anti-inflammatory and antioxidant capacities are the most promising agents for the treatment of acute ischemic kidney injury.

Linking 7-Nitrobenzo-2-oxa-1,3-diazole (NBD) to Triphenylphosphonium Yields Mitochondria-Targeted Protonophore and Antibacterial Agent.

Appending lipophilic cations to small molecules has been widely used to produce mitochondria-targeted compounds with specific activities. In this work, we obtained a series of derivatives of the well-known fluorescent dye 7-nitrobenzo-2-oxa-1,3-diazole (NBD). According to the previous data [Denisov et al. (2014) Bioelectrochemistry, 98, 30-38], alkyl derivatives of NBD can uncouple isolated mitochondria at concentration of tens of micromoles despite a high pKa value (~11) of the dissociating group. Here, a number of triphenylphosphonium (TPP) derivatives linked to NBD via hydrocarbon spacers of varying length (C5, C8, C10, and C12) were synthesized (mitoNBD analogues), which accumulated in the mitochondria in an energy-dependent manner. NBD-C10-TPP (C10-mitoNBD) acted as a protonophore in artificial lipid membranes (liposomes) and uncoupled isolated mitochondria at micromolar concentrations, while the derivative with a shorter linker (NBD-C5-TPP, or C5-mitoNBD) exhibited no such activities. In accordance with this data, C10-mitoNBD was significantly more efficient than C5-mitoNBD in suppressing the growth of Bacillus subtilis. C10-mitoNBD and C12-mitoNBD demonstrated the highest antibacterial activity among the investigated analogues. C10-mitoNBD also exhibited the neuroprotective effect in the rat model of traumatic brain injury.

Effect of Silk Fibroin on Neuroregeneration After Traumatic Brain Injury.

Traumatic brain injury is one of the leading causes of disability among the working-age population worldwide. Despite attempts to develop neuroprotective therapeutic approaches, including pharmacological or cellular technologies, significant advances in brain regeneration have not yet been achieved. Development of silk fibroin-based biomaterials represents a new frontier in neuroregenerative therapies after brain injury. In this study, we estimated the short and long-term effects of silk fibroin scaffold transplantation on traumatic brain injury and biocompatibility of this biomaterial within rat neuro-vascular cells. Silk fibroin microparticles were injected into a brain damage area 1 day after the injury. Silk fibroin affords neuroprotection as judged by diminished brain damage and recovery of long-term neurological functions. We did not detect considerable toxicity to neuro-vascular cells cultured on fibroin/fibroin-gelatin microparticles in vitro. Cultivation of primary cell cultures of neurons and astrocytes on silk fibroin matrices demonstrated their higher viability under oxygen-glucose deprivation compared to 2D conditions on plastic plates. Thus, we conclude that scaffolds based on silk fibroin can become the basis for the creation of constructs aimed to treat brain regeneration after injury.

Rapamycin Is Not Protective against Ischemic and Cisplatin-Induced Kidney Injury.

Autophagy plays an important role in the pathogenesis of acute kidney injury (AKI). Although autophagy activation was shown to be associated with an increased lifespan and beneficial effects in various pathologies, the impact of autophagy activators, particularly, rapamycin and its analogues on AKI remains obscure. In our study, we explored the effects of rapamycin treatment in in vivo and in vitro models of ischemic and cisplatin-induced AKI. The impact of rapamycin on the kidney function after renal ischemia/reperfusion (I/R) or exposure to the nephrotoxic agent cisplatin was assessed by quantifying blood urea nitrogen and serum creatinine and evaluating the content of neutrophil gelatinase-associated lipocalin, a novel biomarker of AKI. In vitro experiments were performed on the primary culture of renal tubular cells (RTCs) that were subjected to oxygen-glucose deprivation (OGD) or incubated with cisplatin under various rapamycin treatment protocols. Cell viability and proliferation were estimated by the MTT assay and real-time cell analysis using an RTCA iCELLigence system. Although rapamycin inhibited mTOR (mammalian target of rapamycin) signaling, it failed to enhance the autophagy and to ameliorate the severity of AKI caused by ischemia or cisplatin-induced nephrotoxicity. Experiments with RTCs demonstrated that rapamycin exhibited the anti-proliferative effect in primary RTCs cultures but did not protect renal cells exposed to OGD or cisplatin. Our study revealed for the first time that the mTOR inhibitor rapamycin did not prevent AKI caused by renal I/R or cisplatin-induced nephrotoxicity and, therefore, cannot be considered as an ideal mimetic of the autophagy-associated nephroprotective mechanisms (e.g., those induced by caloric restriction), as it had been suggested earlier. The protective action of such approaches like caloric restriction might not be limited to mTOR inhibition and can proceed through more complex mechanisms involving alternative autophagy-related targets. Thus, the use of rapamycin and its analogues for the treatment of various AKI forms requires further studies in order to understand potential protective or adverse effects of these compounds in different contexts.

Bacterial Therapy and Mitochondrial Therapy.

Current methods for treatment of cellular and organ pathologies are extremely diverse and constantly evolving, going beyond the use of drugs, based on chemical interaction with biological targets to normalize the functions of the system. Because pharmacological approaches are often untenable, recent strategies in the therapy of different pathological conditions are of particular interest through introducing into the organism of some living system or its components, in particular, bacteria or isolated subcellular structures such as mitochondria. This review describes the most interesting and original examples of therapy using bacteria and mitochondria, which in perspective can dramatically change our views on the principles for the treatment of many diseases. Thus, we analyze such therapeutic effects from the perspective of the similarities between mitochondria and bacteria as the evolutionary ancestors of mitochondria.

Molecular and Cellular Interactions between Mother and Fetus. Pregnancy as a Rejuvenating Factor.

Aging is associated with a decline of various body functions, including ability to regenerate. Over recent decades, it has been demonstrated that some of these changes could be reversed in response to factors originating from a young organism, for example, fetal stem cells or "young blood" in models of heterochronic parabiosis. Pregnancy might be considered as parabiotic model of the interaction between two organisms of different age. In this work, we analyzed and summarized data on the effects of pregnancy on the maternal organism that confirm the hypothesis that pregnancy rejuvenates the mother's organism or slows its aging.

Mechanisms of Inflammatory Injury of Renal Tubular Cells in a Cellular Model of Pyelonephritis.

Previously, we have assembled a cellular model of pyelonephritis which contains a primary culture of renal tubular epithelial cells, mononuclear leukocytes, and bacterial lysate or lipopolysaccharide. After cocultivation of renal cells with leukocytes and bacterial lysate, proinflammatory changes were observed in the renal cells, followed by nitrosative and oxidative stress and cell death. The interaction of bacterial antigens not only with leukocytes, but also with epithelial cells of the renal tubules, was partially mediated by signaling pathways involving Toll-like receptors (TLR2 and TLR4). Activation of these receptors led to increased levels of oxidative stress and synthesis of proinflammatory cytokines (TNF, IL-6, IL-1α) in the renal epithelium, while TLR4 blockade decreased the severity of these processes. Apart from the fact that activation of inflammatory signaling in response to bacterial antigens is observed directly in the renal cells, the presence of leukocytes significantly amplifies the inflammatory response as measured by the level of cytokines generated in the ensemble. In the presence of activated leukocytes, higher expression of TLR2 on the surface of renal cells was observed in response to exposure to bacterial components, which might explain the increased inflammatory response in the presence of leukocytes. The synthesis of IL-1α in the epithelial cells of the renal tubules in this inflammatory model leads to its accumulation in the nuclei, which has been reduced by the TLR4 antagonist polymyxin. TLR2 agonists also led to increased levels of IL-1α. The elevation in the content of IL-1α in nuclei was accompanied by increased acetylation of nuclear proteins, which has been reduced to control values after exposure to protective agents (Trolox, mitochondria-targeted antioxidant SkQR1 or LiCl). The high level of acetylation of histones is probably regulated by proinflammatory cytokines, and to some extent it is a marker of inflammation, which can indirectly be reduced by protective agents.

Dysfunction of Kidney Endothelium after Ischemia/Reperfusion and Its Prevention by Mitochondria-Targeted Antioxidant.

One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48 h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen-glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.

Do Mitochondria Have an Immune System?

The question if mitochondria have some kind of immune system is not trivial. The basis for raising this question is the fact that bacteria, which are progenitors of mitochondria, do have an immune system. The CRISPR system in bacteria based on the principle of RNA interference serves as an organized mechanism for destroying alien nucleic acids, primarily those of viral origin. We have shown that mitochondria are also a target for viral attacks, probably due to a related organization of genomes in these organelles and bacteria. Bioinformatic analysis performed in this study has not given a clear answer if there is a CRISPR-like immune system in mitochondria. However, this does not preclude the possibility of mitochondrial immunity that can be difficult to decipher or that is based on some principles other than those of CRISPR.

Intercellular Transfer of Mitochondria.

Recently described phenomenon of intercellular transfer of mitochondria attracts the attention of researchers in both fundamental science and translational medicine. In particular, the transfer of mitochondria results in the initiation of stem cell differentiation, in reprogramming of differentiated cells, and in the recovery of the lost mitochondrial function in recipient cells. However, the mechanisms of mitochondria transfer between cells and conditions inducing this phenomenon are studied insufficiently. It is still questionable whether this phenomenon exists in vivo. Moreover, it is unclear, how the transfer of mitochondria into somatic cells is affected by the ubiquitination system that, for example, is responsible for the elimination of "alien" mitochondria of the spermatozoon in the oocyte during fertilization. Studies on these processes can provide a powerful incentive for development of strategies for treatment of mitochondria-associated pathologies and give rise a new avenue for therapeutic approaches based on "mitochondrial transplantation".

Mitodiversity.

Here, in addition to the previously coined term "mitobiota", we introduce the term "mitodiversity" for various phenotypic and genetic heterogeneities of mitochondria within the same cell or organ. Based on data on the mitochondrial transmembrane potential determined both in situ and in vitro under normal conditions and after organ ischemia/reperfusion, such heterogeneity is most evident under pathologic conditions. Herein, a part of the mitochondrial population with transmembrane potential typical of the normal state is sustained even under a pathological condition that, perhaps, underlies the development of ways of reversing pathology back to the normal state. The membrane potentials of isolated mitochondria were shown to directly correlate with the magnitude of side-scattered light depicting internal structure of mitochondria. We analyzed possible interpretations of data on mitochondrial membrane potential obtained using fluorescent probes. We suggest a possible mechanism underlying retention of fluorescent probes inside the cells and mitochondria.

Microbiota and mitobiota. Putting an equal sign between mitochondria and bacteria.

The recent revival of old theories and setting them on modern scientific rails to a large extent are also relevant to mitochondrial science. Given the widespread belief that mitochondria are symbionts of ancient bacterial origin, the processes inherent to mitochondrial physiology can be revised based on their comparative analysis with possible involvement of bacteria. Such comparison combined with discussion of the role of microbiota in pathogenesis allows discussion of the role of "mitobiota" (we introduce this term) as the combination of different phenotypic manifestations of mitochondria in the organism reflecting pathological changes in the mitochondrial genome. When putting an equal sign between mitochondria and bacteria, we find similarity between the mitochondrial and bacterial theories of cancer. The presence of the term "bacterial infection" suggests "mitochondrial infection", and mitochondrial (oxidative) theory of aging can in some way be transformed into a "bacterial theory of aging". The possible existence of such processes and the data confirming their presence are discussed in this review. If such a comparison has the right to exist, the homeostasis of "mitobiota" is of not lesser physiological importance than homeostasis of microbiota, which has been so intensively discussed recently.

Lithium salts -- simple but magic.

For many decades pharmacological drugs based on lithium salts have been successfully used in psychiatry to treat bipolar disorder, and they remain the "gold standard" of pharmacological therapy of patients with this disease. At the same time, over recent years in experiments in vitro and in vivo a plethora of evidence has accumulated on a positive effect of lithium ions in other areas including their neuro-, cardio-, and nephroprotective properties, regulation of stem cells functions, regulation of inflammation, and others. Numerous studies have shown that the effect of lithium ions involves several mechanisms; however, one of its main targets in the implementation of most of the effects is glycogen synthase kinase 3ß, a key enzyme in various pathological and protective signaling pathways in cells. However, one of the main limitations of the use of lithium salts in clinics is their narrow therapeutic window, and the risk of toxic side effects. This review presents the diversity of effects of lithium ions on the organism emphasizing their potential clinical applications with minimal undesirable side effects. In the end, we present a schematic "Lithiometer", comparing the range of Li(+) concentrations that might be used for the treatment of acute pathologies with possible toxic effects of Li(+).

[Pathological changes after brain ischemia are similar to those observed in Alzheimer disease].

We studied the influence of ischemia/reperfusion of the middle cerebral artery in the rat's brain on the deferred violation of cognitive functions of the brain which are similar to main symptoms observed in the development of Alzheimer's disease. Using 8-hose radial labyrinth we demonstrated that 6 months after incidence of cerebral ischemia a significant impairment of working memory and a decrease in animals the ability to learn are developed. 7 months after focal cerebral ischemia we could observe the accumulation of a mature amyloid peptide and hyperphosphorylated form of the Tau pro- tein in ipsilateral cerebral hemisphere and of the the beta-amyloid peptide precursor in the contralateral hemisphere. Thus, after an experimental stroke in the brain pathological chanres occur as those typical of Alzheimer's disease.

Perspectives of mitochondrial medicine.

Mitochondrial medicine was established more than 50 years ago after discovery of the very first pathology caused by impaired mitochondria. Since then, more than 100 mitochondrial pathologies have been discovered. However, the number may be significantly higher if we interpret the term "mitochondrial medicine" more widely and include in these pathologies not only those determined by the genetic apparatus of the nucleus and mitochondria, but also acquired mitochondrial defects of non-genetic nature. Now the main problems of mitochondriology arise from methodology, this being due to studies of mitochondrial activities under different models and conditions that are far from the functioning of mitochondria in a cell, organ, or organism. Controversial behavior of mitochondria ("friends and foes") to some extent might be explained by their bacterial origin with possible preservation of "egoistic" features peculiar to bacteria. Apparently, for normal mitochondrial functioning it is essential to maintain homeostasis of a number of mitochondrial elements such as mitochondrial DNA structure, membrane potential, and the system of mitochondrial quality control. Abrogation of these elements can cause a number of pathologies that have become subjects of mitochondrial medicine. Some approaches to therapy of mitochondrial pathologies are discussed.

Role of oxidative stress and mitochondria in onset of urinary bladder dysfunction under acute urine retention.

Acute urine retention is a frequent complication in patients with benign hyperplasia of the prostate gland. According to studies made on experimental animals and people, it is accompanied by the deterioration of the bladder blood supply. This study attempts to explore the relationship of intramural blood flow, production of reactive oxygen species, and functional state of the bladder detrusor in modeling of acute urine retention in rats, as well as the impact of mitochondria-targeted antioxidants (which are supposed to alleviate the effects of oxidative stress induced by experimental ischemia) on these parameters. The study showed beneficial effects of mitochondria-targeted antioxidant SkQR1 in preventing damage of the bladder caused by acute urinary retention, which suggests the therapeutic use of this type of compounds for the treatment of ischemic abnormalities of the urinary bladder.

Decreased renal expression of PAQR5 is associated with the absence of a nephroprotective effect of progesterone in a rat UUO model.

Fibrosis is a severe complication of chronic kidney disease (CKD). Progesterone, like other sex hormones, plays an important role in renal physiology, but its role in CKD is poorly understood. We investigated progesterone effect on renal fibrosis progression in the rat model of unilateral ureteral obstruction (UUO). Female rats were exposed to UUO, ovariectomy and progesterone administration after UUO with ovariectomy. Expression of key fibrosis markers, proinflammatory cytokines, levels of membrane-bound (PAQR5) and nuclear (PGR) progesterone receptors, and matrix metalloproteinase (MMP) activity were analyzed in the obstructed and intact rat kidney. In all groups exposed to UUO, decreased PAQR5 expression was observed in the obstructed kidney while in the contralateral kidney, it remained unaffected. We found increased mRNA levels for profibrotic COL1A1, FN1, MMP2, TIMP1, TIMP2, proinflammatory IL1α, IL1ß, and IL18, as well as elevated α-SMA and MMP9 proteins, collagen deposition, and MMP2 activity in all UUO kidneys. Progesterone had slight or no effect on the change in these markers. Thus, we demonstrate for the first time diminished sensitivity of the kidney to progesterone associated with renal fibrosis due to a severe decrease in PAQR5 expression that was accompanied by the lack of nephroprotection in a rat UUO model.

Effects of caloric restriction and ketogenic diet on renal fibrosis after ischemia/reperfusion injury.

The beneficial effects of caloric restriction (CR) and a ketogenic diet (KD) have been previously shown when performed prior to kidney injury. We investigated the effects of CR and KD on fibrosis development after unilateral kidney ischemia/reperfusion (UIR). Post-treatment with CR significantly (p < 0.05) affected blood glucose (2-fold decrease), ketone bodies (3-fold increase), lactate (1.5-fold decrease), and lipids (1.4-fold decrease). In the kidney, CR improved succinate dehydrogenase and malate dehydrogenase activity by 2-fold each, but worsened fibrosis progression. Similar results were shown for the KD, which restored the post-UIR impaired activities of succinate dehydrogenase, malate dehydrogenase, and α-ketoglutarate dehydrogenase (which was decreased 2-fold) but had no effect on fibrosis progression. Thus, our study shows that the use of CR or KD after UIR did not reduce the development of fibrosis, as shown by hydroxyproline content, western-blotting, and RT-PCR, whereas it caused significant metabolic changes in kidney tissue after UIR.

Mechanisms of nephroprotective effect of mitochondria-targeted antioxidants under rhabdomyolysis and ischemia/reperfusion.

Oxidative stress-related renal pathologies apparently include rhabdomyolysis and ischemia/reperfusion phenomenon. These two pathologies were chosen for study in order to develop a proper strategy for protection of the kidney. Mitochondria were found to be a key player in these pathologies, being both the source and the target for excessive production of reactive oxygen species (ROS). A mitochondria-targeted compound which is a conjugate of a positively charged rhodamine molecule with plastoquinone (SkQR1) was found to rescue the kidney from the deleterious effect of both pathologies. Intraperitoneal injection of SkQR1 before the onset of pathology not only normalized the level of ROS and lipid peroxidized products in kidney mitochondria but also decreased the level of cytochrome c in the blood, restored normal renal excretory function and significantly lowered mortality among animals having a single kidney exposed to ischemia/reperfusion. The SkQR1-derivative missing plastoquinone (C12R1) possessed some, although limited nephroprotective properties and enhanced animal survival after ischemia/reperfusion. SkQR1 was found to induce some elements of nephroprotective pathways providing ischemic tolerance such as an increase in erythropoietin levels and phosphorylation of glycogen synthase kinase 3ß in the kidney. SkQR1 also normalized renal erythropoietin level lowered after kidney ischemia/reperfusion and injection of a well-known nephrotoxic agent gentamicin.

Mitochondria-targeted antioxidant SkQR1 ameliorates gentamycin-induced renal failure and hearing loss.

The influence of the mitochondria-targeted antioxidant SkQR1 on gentamycin-induced nephrotoxicity and ototoxicity has been analyzed. SkQR1 reduces the death of kidney epithelium cells and decreases the severity of renal failure caused by gentamycin application and also lowers the animals' mortality. Treatment with SkQR1 also decreases gentamycin-induced hearing loss. Mitochondria-targeted antioxidants, such as SkQR1, are new promising agents for preventing negative consequences of therapy with antibiotics.