Tissue damage by ischemia/reperfusion (I/R) results from a temporary cessation of blood flow followed by the restoration of circulation. The injury depresses mitochondrial respiration, increases the production of reactive oxygen species (ROS), decreases the mitochondrial transmembrane potential, and stimulates invasion by inflammatory cells. The primary objective of this work was to address the potential use of bone marrow stem cells (BMSCs) to preserve and restore mitochondrial function in the kidney after I/R. Mitochondria from renal proximal tubule cells were isolated by differential centrifugation from rat kidneys subjected to I/R (clamping of renal arteries followed by release of circulation after 30 min), without or with subcapsular administration of BMSCs. Respiration starting from mitochondrial complex II was strongly affected following I/R. However, when BMSCs were injected before ischemia or together with reperfusion, normal electron fluxes, electrochemical gradient for protons, and ATP synthesis were almost completely preserved, and mitochondrial ROS formation occurred at a low rate. In homogenates from cultured renal cells transiently treated with antimycin A, the coculture with BMSCs induced a remarkable increase in protein S-nitrosylation that was similar to that found in mitochondria isolated from I/R rats, evidence that BMSCs protected against both superoxide anion and peroxynitrite. Labeled BMSCs migrated to damaged tubules, suggesting that the injury functions as a signal to attract and host the injected BMSCs. Structural correlates of BMSC injection in kidney tissue included stimulus of tubule cell proliferation, inhibition of apoptosis, and decreased inflammatory response. Histopathological analysis demonstrated a score of complete preservation of tubular structures by BMSCs, associated with normal plasma creatinine and urinary osmolality. These key findings shed light on the mechanisms that explain, at the mitochondrial level, how stem cells prevent damage by I/R. The action of BMSCs on mitochondrial functions raises the possibility that autologous BMSCs may help prevent I/R injuries associated with transplantation and acute renal diseases.
The alkylating agent 3-Bromopyruvate (3-BrPA) has been used as an anti-tumoral drug due to its anti-proliferative property in hepatomas cells. This propriety is believed to disturb glycolysis and respiration, which leads to a decreased rate of ATP synthesis. In this study, we evaluated the effects of the alkylating agent 3-BrPA on the respiratory states and the metabolic steps of the mitochondria of mice liver, brain and in human hepatocarcinoma cell line HepG2. The mitochondrial membrane potential (ΔΨ(m)), O(2) consumption and dehydrogenase activities were rapidly dissipated/or inhibited by 3-BrPA in respiration medium containing ADP and succinate as respiratory substrate. 3-BrPA inhibition was reverted by reduced glutathione (GSH). Respiration induced by yeast soluble hexokinase (HK) was rapidly inhibited by 3-BrPA. Similar results were observed using mice brain mitochondria that present HK naturally bound to the outer mitochondrial membrane. When the adenine nucleotide transporter (ANT) was blocked by the carboxyatractiloside, the 3-BrPA effect was significantly delayed. In permeabilized human hepatoma HepG2 cells that present HK type II bound to mitochondria (mt-HK II), the inhibiting effect occurred faster when the endogenous HK activity was activated by 2-deoxyglucose (2-DOG). Inhibition of mt-HK II by glucose-6-phosphate retards the mitochondria to react with 3-BrPA. The HK activities recovered in HepG2 cells treated or not with 3-BrPA were practically the same. These results suggest that mitochondrially bound HK supporting the ADP/ATP exchange activity levels facilitates the 3-BrPA inhibition reaction in tumors mitochondria by a proton motive force-dependent dynamic equilibrium between sensitive and less sensitive SDH in the electron transport system.
Perinatal asphyxia remains a significant cause of neonatal mortality and is associated with long-term neurodegenerative disorders. In the present study, we evaluated cellular and subcellular damages to brain development in a model of mild perinatal asphyxia. Survival rate in the experimental group was 67%. One hour after the insult, intraperitoneally injected Evans blue could be detected in the fetuses' brains, indicating disruption of the blood-brain barrier. Although brain mass and absolute cell numbers (neurons and non-neurons) were not reduced after perinatal asphyxia immediately and in late brain development, subcellular alterations were detected. Cortical oxygen consumption increased immediately after asphyxia, and remained high up to 7 days, returning to normal levels after 14 days. We observed an increased resistance to mitochondrial membrane permeability transition, and calcium buffering capacity in asphyxiated animals from birth to 14 days after the insult. In contrast to ex vivo data, mitochondrial oxygen consumption in primary cell cultures of neurons and astrocytes was not altered after 1% hypoxia. Taken together, our results demonstrate that although newborns were viable and apparently healthy, brain development is subcellularly altered by perinatal asphyxia. Our findings place the neonate brain mitochondria as a potential target for therapeutic protective interventions.
Acute kidney injury due to ischemia followed by reperfusion (IR) is a severe clinical condition with high death rates. IR affects the proximal tubule segments due to their predominantly oxidative metabolism and profoundly altered mitochondrial functions. We previously described the impact of IR on oxygen consumption, the generation of membrane potential (ΔΨ), and formation of reactive oxygen species, together with inflammatory and structural alterations. We also demonstrated the benefits of bone marrow mononuclear cells (BMMC) administration in these alterations. The objective of the present study has been to investigate the effect of IR and the influence of BMMC on the mechanisms of Ca2+ handling in mitochondria of the proximal tubule cells. IR inhibited the rapid accumulation of Ca2+ (Ca2+ green fluorescence assays) and induced the opening of the cyclosporine A-sensitive permeability transition pore (PTP), alterations prevented by BMMC. IR accelerated Ca2+-induced decrease of ΔΨ (Safranin O fluorescence assays), as evidenced by decreased requirement for Ca2+ load and t1/2 for complete depolarization. Addition of BMMC and ADP recovered the normal depolarization profile, suggesting that stabilization of the adenine nucleotide translocase (ANT) in a conformation that inhibits PTP opening offers a partial defense mechanism against IR injury. Moreover, as ANT forms a complex with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, it is possible that this complex is also a target for IR injury—thus favoring Ca2+ release, as well as the supramolecular structure that BMMC protects. These beneficial effects are accompanied by a stimulus of the citric acid cycle—which feed the mitochondrial complexes with the electrons removed from different substrates—as the result of accentuated stimulus of citrate synthase activity by BMMC.
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