A. V. Kazachenko

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and Age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke)

Effects of 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6′-plastoquinonyl) decylrhod-amine 19 (SkQR1) on rat models of H2O2- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H2O2 or by ischemia/reperfusion. Higher SkQ1 (125–250 nmol/kg per day for 2–3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2–4 days, whereas one injection of SkQ1 or SkQR1 (1 μmol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 μmol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound.

Experimental intravenous cell therapy of acute and chronic renal failure

The therapeutic effect of intravenous injection of human fetal bone marrow mesenchymal stem cells or summary culture of kidney cells were studied on models of chronic or acute renal failure in outbred albino rats. Both cell types promoted improvement and normalization of the renal function in rats with stable chronic renal insufficiency (2 weeks after kidney cell injection, 1 month after bone marrow mesenchymal stem cell injection). Renal function remained normal or subnormal during the delayed period (3–3.5 months after injection). In rats with latent stage of chronic renal insufficiency, exacerbation was induced by additional 40-min ischemia. All rats receiving intravenous injection of saline died. Improvement of the functional parameters started 2 weeks after injection of kidney cells or bone marrow mesenchymal stem cells, and normalization was observed after 1.1–5 months. During the delayed period (after 3–4 months), functional parameters retained at normal or subnormal levels. In experimental series III, all rats with acute renal failure intravenously injected with saline (control) died from uremia on days 2–4. After injection of kidney cells 50% rats survived and renal function in these animals returned to normal after 2 weeks. After injection of bone marrow mesenchymal stem cells 83% rats survived, functional parameters returned to normal after 3 weeks.

Functional aftereffects of intraparenchymatous injection of human fetal stem and progenitor cells to rats with chronic and acute renal failure.

Chronic renal insufficiency was modeled in rats by unilateral nephrectomy and electrocoagulation of both poles of the remaining kidney; acute renal failure was induced by 90-min clamping of the vascular pedicle of the only kidney. Injection of unfractionated culture of human fetal kidney cells or bone marrow mesenchymal stem cells into damaged kidney restored its function in rats with chronic renal insufficiency (observation period up to 2 months). After 2.5 months a relapse of chronic renal insufficiency was observed in 1 of 3 rats receiving human fetal kidney cells and in 1 of 2 animals receiving bone marrow mesenchymal stem cell culture. Injection of bone marrow mesenchymal stem cell culture to rats with acute renal failure improved recovery of renal function and prevented the death from uremia, while injection of total culture of human fetal kidney cells had virtually no effect on the course of acute renal failure.

Inhibition of GSK-3β decreases the ischemia-induced death of renal cells.

Pharmacological preconditioning with insulin and lithium ions prevented the death of renal cells under conditions of ischemia/reperfusion. Preincubation of cells with insulin or lithium ions decreased production of reactive oxygen species after ischemia/reoxygenation. These agents also prevented the development of mitochondrial dysfunction in renal cells induced by ischemia/reoxygenation. It was hypothesized that the protective effects of these agents are related to inhibition of glycogen synthase kinase-3(. This enzyme is inactivated upon phosphorylation of serine residue in position 9. We found that in vivo administration of lithium ions to animals before renal ischemia prevents the development of kidney failure.

Effects of Ischemic and Hypoxic Preconditioning on the State of Mitochondria and Function of Ischemic Kidneys

Laser confocal microscopy showed that fluorescence of tetramethylrhodamine ethyl ether probe specifically accumulating in energized mitochondria significantly decreased in renal tubular epithelium after 40-min thermal ischemia, while fluorescence of dichlorodihydrofluorescein and diaminofluorescein probes in the same structures increased under these conditions, which attests to increased generation of ROS and NO, respectively. These forms were generated predominantly in mitochondria of tubular epitheliocytes. Hypoxic preconditioning (a series of sessions of breathing hypoxic mixture) preserved functional activity of mitochondria and prevented activation of ROS and NO generation. Ischemic preconditioning of the kidney consisting of three preliminary episodes of vascular clamping (5 min with 5 min reperfusion periods) also increased the percentage of functionally active mitochondria and prevented activation of NO synthesis without appreciably modifying ROS production. Both protective methods significantly reduced the severity of postischemic dysfunction of the kidney.

Morphological Changes in the Kidneys of Rats with Postischemic Acute Renal Failure after Intrarenal Administration of Fetal Mesenchymal Stem Cells from Human Bone Marrow

Chronic experiments on outbred albino rats were performed to compare the dynamics of histological signs for postischemic renal injury (90-min thermal ischemia) after intraparenchymal injection of cultured fetal MSC from human bone marrow. Functional indexes of the ischemic kidney were predetermined. In the early period after ischemia (day 4), administration of human bone marrow MSC was followed by the increase in blood flow in the microcirculatory bed and decrease in the degree of alteration in renal tubules. An increase in the area of zones with histological signs for normal function of tubules was accompanied by the improvement of biochemical indexes for renal function. In the delayed period, a protective effect of cell therapy was manifested in the prevention of death of renal tubules. Mild calcification of the necrotic tubular epithelium served as a marker of this process. Human bone marrow MSC were labeled with the fluorescent probe Calcein. These cells migrated from the site of injection, spread in the interstitium, and retained viability for 7 days. During this period, some cells were incorporated into the lumen of renal tubules.

Mitochondrial regulation of production of reactive oxygen species and nitrogen in rat cells of kidney during ischemia/reperfusion

Injuries caused by limited blood supply may occur in all organs during different surgical operations. In particular, induced ischemia of kidney is a risk factor, especially during organ transplantation and operations on organs of abdominal cavity, as well as during strong arterial hypotension. Often under natural (in thrombosis) and always under artificial conditions (during surgical operation) ischemia is followed by reperfusion and reoxygenation of tissues, which occurs during blood supply recovery. Such alternation of hypoxia and reoxygenation leads to the state of oxidative stress, which has been well described for brain tissues [1], myocardium [2], and other organs. Oxidative stress is an acute and substantially irreversible destructive process that severely limits the strategy of intervention in the vital functions of organs. Postischemic injuries of kidneys result in destruction of tubules, reversed flow of filtrate from the tubule lumen [3], and perturbation of filtration in the glomerules [4].

Enhancement of the anti-ischemic kidney resistance by adaptive hypoxic preconditioning and drug therapy

Hypoxic preconditioning and pretreatment with pentoxifylline, α-tocopherol, or their combination promotes stabilization of Po2 in rabbit kidney cortex in the early reperfusion period (in experiments with α-tocopherol at a significantly lower level), while after 30-min reperfusion, the protective effect of α-tocopherol (especially in combination with pentoxifylline) markedly surpasses that of pentoxifylline and hypoxic preconditioning. α-Tocopherol sharply inhibits LPO in the reperfusion period, while pentoxifylline exhibits only weak antioxidant activity and hypoxic preconditioning was ineffective.