Metabolic properties of murine kidney mitochondria

We show that mitochondria from the kidney of mice (MKM), rat brain (RBM), and heart (RHM) oxidize long-chain fatty acids at high rates in all metabolic states only in the presence of any other mitochondrial metabolites: succinate, glutamate, or pyruvate. All supporting substrates increased several folds the respiration rates in State 4 and State 3. The stimulations of the State 3 respiration with palmitoyl-carnitine + malate oxidation (100%) were: with succinate in MKM 340%, RBM 370%, and RHM 340%; with glutamate - MKM 200%, RBM 270%, and RHM 270%; and with pyruvate - MKM 150%, RBM 260%, and RHM 280%. The increases in O2 consumption in State 4 were due to increased leakage of electrons to produce superoxide radicals (O2•). Earlier, we have shown that the brain and heart mitochondria possess a strong intrinsic inhibition of succinate oxidation to prevent the excessive O2• production at diminished functional loads. We show that kidney mitochondria lack the intrinsic inhibition of SDH. The new methodology to study β-oxidation of LCFAs opens the opportunity to study energy metabolism under normal and pathological conditions, particularly in the organs that utilize LCFAs as the main energy source.

Hypolipidemic and Antioxidant Effects of Guishe Extract from Agave lechuguilla, a Mexican Plant with Biotechnological Potential, on Streptozotocin-Induced Diabetic Male Rats

In the present study, we used a by-product from Agave lechuguilla (guishe) to test its antidiabetic effect, hypolipidemic activity, and capacity to mitigate the oxidative stress in kidney mitochondria from streptozotocin-induced diabetic rats. Orally, a crude aqueous extract from lyophilized guishe was administered over 5 weeks at different doses. Blood glucose and body weight were monitored. Also, blood chemistry, bilirubin, and alanine aminotransferase were assayed. Furthermore, the activity of catalase, thiobarbituric acid reactive species, mitochondrial superoxide dismutase, glutathione and glutathione peroxidase were determined in isolated kidney mitochondria. Our results show that guishe extracts have no antidiabetic properties at any dose. Nevertheless, it was able to diminish serum triglyceride levels and regulate the oxidative stress observed in isolated kidney mitochondria. These observations indicate that the aqueous extract from guishe can be used to treat abnormalities in serum lipids, as a hypolipidemic, and mitigate the oxidative stress, as an antioxidant, occurring during diabetes.

Influence of a B16/F10 melanoma variant on the Вcl-2 levels in mitochondria in various organs of female mice

Aim. To study the Bcl-2 level in mitochondria of various organs in female mice with standard and stimulated growth of an experimental B16/F10 melanoma.Materials and methods. The study included С57ВL/6 female mice (n = 168). The experimental animals were divided into the following groups: an intact group (n = 21), a group with modelled chronic neuropathic pain (CNP) (n = 21), an M group with B16/F10 melanoma (n = 63), and a CNP + M group (n = 63). The Bcl-2 concentration (ng / mg protein) in mitochondrial samples was determined by ELISA (Thermo Fisher Scientific, Austria). Statistical analysis of the results was carried out using Statistica 10.0.Results. Compared to the Bcl-2 levels in the intact animals, CNP decreased this parameter in the cardiac mitochondria by 1.3 times, while increasing it by 5.9 times in the skin mitochondria. In the dynamics of standard melanoma growth, the Bcl-2 content changed compared with the corresponding intact values in the mitochondria of the brain, heart, and skin, but did not change in the liver and kidneys. In the mitochondria in melanoma, the Bcl-2 levels were high throughout the entire period of standard tumor growth in comparison with the intact skin. The stimulated melanoma growth in CNP was involving more organs into the pathological process as the tumor was growing. Thus, in comparison with the values in the CNP group, the mitochondrial Bcl-2 levels changed in the heart at week 1; in the heart and skin – at week 2; in the heart, skin, and brain – at week 3. The Bcl-2 levels did not change in the liver and kidney mitochondria. In the mitochondria in the CNP-stimulated melanoma, the Bcl-2 levels were lower than in the skin mitochondria in CNP throughout the entire tumor growth period.Conclusion. The liver and kidney mitochondria are somewhat Bcl-2 stable in both standard and stimulated tumor growth. It is assumed that different Bcl-2 dynamics in the mitochondria in melanoma depending on the variant of tumor development reflects the modulating effect of CNP and the ability to change the Bcl-2 levels according to the growth phase.

Alpha-Lipoic Acid: Antioxidant Activity Against Non-Enzymatic Peroxidation of Rat Kidney and Liver Mitochondria

Reactive Oxygen Species (ROS) participate in the induction and progression of damage in many human pathologies, such as: heart attack, cerebral ischemia, diabetic neuropathy and Alzheimer's disease, among others. Alpha Lipoic Acid (ALA, also called thioctic acid) is a sulfur compound that acts as a growth factor in some microorganisms and as a coenzyme or prosthetic group in mammalian tissues. The beneficial action of ALA is due to its high antioxidant power that allows it to capture numerous free radicals such as Hydroxyl Radicals (OH•), Hypochlorous (HClO-) and oxygen (O•). ALA easily crosses cell membranes acting in both lipophilic and hydrophilic media, so it can act against oxidative stress and prevent cell damage at many levels. In the study reported here the effect of ALA on chemiluminescence of mitochondria isolated from liver and kidney rat was analyzed. After incubation of both mitochondria in an ascorbate (0.4mM)-Fe++ (2.15μM) system (120min at 37°C), non-enzymatic peroxidation, it was observed that the total cpm/mg protein originated from light emission: chemiluminescence was lower in liver and kidney mitochondria obtained from ALA group than in the control group (without ALA). Moreover, it was observed that the ALA was reduced, concentration dependent (0.05 mg, 0.15 mg and 0.25 mg of solution), of chemiluminescence, measured as total cpm. The analyses of chemiluminescence indicate that ALA may act as antioxidant protecting rat liver and kidney mitochondria from peroxidative damage.

Overexpression of lipoic acid synthase gene alleviates diabetic nephropathy of Leprdb/db mice

IntroductionDiabetic nephropathy (DN) develops in about 40% of patients with type 2 diabetes and remains the leading cause of end-stage renal disease. The mechanisms of DN remain to be elucidated. Oxidative stress is thought to be involved in the development of DN but antioxidant therapy has produced conflicting results. Therefore, we sought to define the role of antioxidant in retarding the development of DN in this study.Research design and methodsWe generated a new antioxidant/diabetes mouse model, LiasH/HLeprdb/db mice, by crossing db/db mice with LiasH/H mice, which have overexpressed Lias gene (~160%) compared with wild type, and also correspondingly increased endogenous antioxidant capacity. The new model was used to investigate whether predisposed increased endogenous antioxidant capacity was able to retard the development of DN. We systemically and dynamically examined main pathological alterations of DN and antioxidant biomarkers in blood and kidney mitochondria.ResultsLiasH/HLeprdb/db mice alleviated major pathological alterations in the early stage of DN, accompanied with significantly enhanced antioxidant defense. The model targets the main pathogenic factors by exerting multiple effects such as hypoglycemic, anti-inflammation, and antioxidant, especially protection of mitochondria.ConclusionThe antioxidant animal model is not only very useful for elucidating the underlying mechanisms of DN but also brings insight into a new therapeutic strategy for clinical applications.

Caffeic Acid Phenethyl Ester Protects Kidney Mitochondria against Ischemia/Reperfusion Induced Injury in an In Vivo Rat Model

To improve ischemia/reperfusion tolerance, a lot of attention has been focused on natural antioxidants. Caffeic acid phenethyl ester (CAPE), an active component of the resinous exudates of the buds and young leaves of Populus nigra L., Baccharis sarothroides A., etc., and of propolis, possesses unique biological activities such as anti-inflammatory, antioxidant, immunomodulating, and cardioprotective effects, among others. There is a lack of studies showing a link between the antioxidant potential of CAPE and the mechanism of protective action of CAPE at the level of mitochondria, which produces the main energy for the basic functions of the cell. In the kidney, ischemia/reperfusion injury contributes to rapid kidney dysfunction and high mortality rates, and the search for biologically active protective compounds remains very actual. Therefore, the aim of this study was to identify the antioxidant potential of CAPE and to investigate whether CAPE can protect rat kidney mitochondria from in vivo kidney ischemia/reperfusion induced injury. We found that CAPE (1) possesses antioxidant activity (the reducing properties of CAPE are more pronounced than its antiradical properties); CAPE effectively reduces cytochrome c; (2) protects glutamate/malate oxidation and Complex I activity; (3) preserves the mitochondrial outer membrane from damage and from the release of cytochrome c; (4) inhibits reactive oxygen species (ROS) generation in the Complex II (SDH) F site; (5) diminishes ischemia/reperfusion-induced LDH release and protects from necrotic cell death; and (6) has no protective effects on succinate oxidation and on Complex II +III activity, but partially protects Complex II (SDH) from ischemia/reperfusion-induced damage. In summary, our study shows that caffeic acid phenethyl ester protects kidney mitochondrial oxidative phosphorylation and decreases ROS generation at Complex II in an in vivo ischemia/reperfusion model, and shows potential as a therapeutic agent for the development of pharmaceutical preparations against oxidative stress-related diseases.

Methodological Issue of Mitochondrial Isolation in Acute-Injury Rat Model: Asphyxia Cardiac Arrest and Resuscitation

Background: Identification of the mechanisms underlying mitochondrial dysfunction is key to understanding the pathophysiology of acute injuries such as cardiac arrest (CA); however, effective methods for measurement of mitochondrial function associated with mitochondrial isolation have been debated for a long time. This study aimed to evaluate the dysregulation of mitochondrial respiratory function after CA while testing the sampling bias that might be induced by the mitochondrial isolation method.Materials and Methods: Adult rats were subjected to 10-min asphyxia-induced CA. 30 min after resuscitation, the brain and kidney mitochondria from animals in sham and CA groups were isolated (n = 8, each). The mitochondrial quantity, expressed as protein concentration (isolation yields), was determined, and the oxygen consumption rates were measured. ADP-dependent (state-3) and ADP-limited (state-4) respiration activities were compared between the groups. Mitochondrial quantity was evaluated based on citrate synthase (CS) activity and cytochrome c concentration, measured independent of the isolation yields.Results: The state-3 respiration activity and isolation yield in the CA group were significantly lower than those in the sham group (brain, p < 0.01; kidney, p < 0.001). The CS activity was significantly lower in the CA group as compared to that in the sham group (brain, p < 0.01; kidney, p < 0.01). Cytochrome c levels in the CA group showed a similar trend (brain, p = 0.08; kidney, p = 0.25).Conclusions: CA decreased mitochondrial respiration activity and the quantity of mitochondria isolated from the tissues. Owing to the nature of fragmented or damaged mitochondrial membranes caused by acute injury, there is a potential loss of disrupted mitochondria. Thus, it is plausible that the mitochondrial function in the acute-injury model may be underestimated as this loss is not considered.

Effects of selected Taiwanese endemic plants on anti-lipid peroxidation and antibacterial activities against clinically isolated extended-spectrum-β-lactamase producing Klebsiella pneumoniae and Escherichia coli

It has been a challenge for many clinicians to treat a complicated extended-spectrum-β-lactamase (ESBL) producing Klebsiella Pneumoniae (Kp) and Escherichia coli (E. coli) infection due to widespread antibiotic abuse with renal damage as one of its common side effects. Therefore, this study aimed to assess the antibacterial activity of extracts from several Taiwanese folk medicinal plants against ESBL- Kp and E. coli. with renal protecting ability against lipid peroxidation (LPO) on mice kidney mitochondria. Preliminary antibacterial activities of ethanol extracts from twenty (20) Taiwanese folk medicinal plants were measured by agar-dilution method against standard ESBL strains of E. coli (ATCC 25922, ATCC 35218) and Kp (ATCC 23856, ATCC 700603). Rhus semialata var. roxburghiana DC. (RSR) exerted the most inhibitory effect and then further extracted with n-hexane, ethyl acetate, acetone, ethanol, and water, respectively. Each extract also evaluated against the four standard ATCC microorganisms. Their MIC50, MIC90, and time kill assay were adapted with detecting the maximum inhibitory activities and the antibacterial spectrum range of each extract was measured against twenty-four (24) kinds of microbes. Which were used including gram-positive, gram-negative bacteria and fungus by agar dilution method. Finally, renal protective ability was detected inhibitory effect of ferrous induced lipid peroxidation on mice mitochondria. Among 20 Taiwanese folk medicinal plants tested, Rhus semialata var. roxburghiana DC. (RSR) exhibited maximum inhibition against clinical ESBL-producing Kp and E. coli strains with acetone extracts showing MIC50/MIC90 values at 1000 µg/mL, the course of antimicrobial action was bacteriostatic and with inhibitions to all 24 kinds of microbial including Gram positive and negative bacteria and fungi. Furthermore, result of thiobarbituric acid reactive substances (TBARS) assay from this extract showed high lipid peroxidative (LPO) protective capability on mice kidney mitochondria (IC50: 29.29 ± 0.35µg/mL). RSR acetone extract, with its maximum activity against clinical isolated ESBL-producing Kp and E. coli, antimicrobial effect against other wide spectral range bacteria and relatively high LPO protective ability on mice kidney mitochondria, is a potential source, albeit further studies have yet to be conducted, to develop an antimicrobial drug against ESBL-Kp and E. coli.

Methodological Issue of Isolating Mitochondria in Acute Injury: A Rat Cardiac Arrest Model

Background Mitochondrial studies are key to understanding the pathophysiology of cardiac arrest (CA), however there is a potential risk of sampling bias during the mitochondrial isolation process. This study aimed to evaluate the dysregulation of mitochondrial respiratory function after CA while testing the sampling bias induced by the mitochondrial isolation method.Methods and Results Adult rats were subjected to 10-minunte asphyxia-induced CA. Thirty minutes after resuscitation, brain and kidney mitochondria from sham and CA group animals were isolated (n=8, each). The mitochondria quantity, expressed as protein concentrations (isolation yields), was determined and then oxygen consumption rates were measured. ADP-dependent (state-3) and ADP-limited (state-4) respiration activity were compared between the groups. The mitochondrial quantity was evaluated by citrate synthase (CS) activity and cytochrome c concentration measured independently from isolation yields. The state-3 respiration activity and isolation yield in the CA group declined significantly as compared to those in the sham group (brain, p < 0.01; kidney, p < 0.001). The CS activity in the CA group declined significantly as compared to that of the sham group (brain, p < 0.01; kidney, p < 0.01). Likewise, cytochrome c levels in the CA group had decreasing trends (brain, p = 0.08; kidney, p = 0.25).Conclusions CA decreased mitochondrial respiration activity and the quantity of mitochondria isolated from the tissues. Due to the nature of fragmented or damaged mitochondria membranes caused by this acute injury model, it is plausible that the mitochondrial function measured in the acute injury animal model might be underestimated.