scholarly journals Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments

2020 ◽  
Vol 318 (5) ◽  
pp. F1237-F1245
Author(s):  
Allison McCrimmon ◽  
Mark Domondon ◽  
Regina F. Sultanova ◽  
Daria V. Ilatovskaya ◽  
Krisztian Stadler
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Respiratory Function ◽  
Rapid Assessment ◽  
Acute Injury ◽  
Mitochondrial Oxygen Consumption ◽  
Isolated Mitochondria ◽  
Proximal Tubular ◽  
Mitochondrial Respiratory

Changes in mitochondrial function are central to many forms of kidney disease, including acute injury, diabetic nephropathy, hypertension, and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close to in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared with in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here, we report the development and validation of a new approach for the rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular fragments using Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models: the C57BL/6J mouse, the diabetic db/ db mouse and matching db/+ control mouse, and the Dahl salt-sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for the rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator’s choice. The isolation methods presented here ensure viable and functional proximal tubular fragments and glomeruli, with a preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.

scholarly journals Comparison of in vivo postexercise phosphocreatine recovery and resting ATP synthesis flux for the assessment of skeletal muscle mitochondrial function

2010 ◽  
Vol 299 (5) ◽  
pp. C1136-C1143 ◽  
Author(s):  
N. M. A. van den Broek ◽  
J. Ciapaite ◽  
K. Nicolay ◽  
J. J. Prompers
Keyword(s):  
Skeletal Muscle ◽  
Oxygen Consumption ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Function ◽  
Atp Synthesis ◽  
Resonance Spectroscopy ◽  
Isolated Mitochondria ◽  
Significant Difference

31P magnetic resonance spectroscopy (MRS) has been used to assess skeletal muscle mitochondrial function in vivo by measuring 1) phosphocreatine (PCr) recovery after exercise or 2) resting ATP synthesis flux with saturation transfer (ST). In this study, we compared both parameters in a rat model of mitochondrial dysfunction with the aim of establishing the most appropriate method for the assessment of in vivo muscle mitochondrial function. Mitochondrial dysfunction was induced in adult Wistar rats by daily subcutaneous injections with the complex I inhibitor diphenyleneiodonium (DPI) for 2 wk. In vivo 31P MRS measurements were supplemented by in vitro measurements of oxygen consumption in isolated mitochondria. Two weeks of DPI treatment induced mitochondrial dysfunction, as evidenced by a 20% lower maximal ADP-stimulated oxygen consumption rate in isolated mitochondria from DPI-treated rats oxidizing pyruvate plus malate. This was paralleled by a 46% decrease in in vivo oxidative capacity, determined from postexercise PCr recovery. Interestingly, no significant difference in resting, ST-based ATP synthesis flux was observed between DPI-treated rats and controls. These results show that PCr recovery after exercise has a more direct relationship with skeletal muscle mitochondrial function than the ATP synthesis flux measured with 31P ST MRS in the resting state.

Proximal tubular Na, Cl, and HCO3 reabsorption and renal oxygen consumption

1984 ◽  
Vol 247 (1) ◽  
pp. F151-F157 ◽  
Author(s):  
S. W. Weinstein ◽  
R. Klose ◽  
J. Szyjewicz
Keyword(s):  
Oxygen Consumption ◽  
Ion Transport ◽  
Rat Kidney ◽  
Fluid Reabsorption ◽  
Bicarbonate Reabsorption ◽  
Renal Oxygen Consumption ◽  
Proximal Tubular ◽  
Tubular Fluid Reabsorption ◽  
Renal Oxygen

The majority of the oxygen consumed by the rat kidney appears to occur in the proximal tubule. Therefore changes in metabolically linked ion transport in this segment of the nephron should result in changes in renal oxygen consumption. To study the role of bicarbonate reabsorption in metabolically linked proximal tubular ion transport a series of micropuncture-clearance-extraction experiments were performed comparing the effects of the carbonic anhydrase inhibitor benzolamide and of hypertonic sodium bicarbonate infusion with control conditions in the rat. End-proximal tubular fluid and chloride reabsorption were measured. From these, the rates of sodium and bicarbonate reabsorption were estimated. Simultaneously with the tubular fluids, extraction collections were obtained for determination of renal oxygen consumption. Both benzolamide and hypertonic bicarbonate reduced proximal tubular fluid reabsorption while concomitantly reducing the transepithelial gradient for chloride. The mean rate of renal oxygen consumption did not differ from the control rate in either experimental group and could be dissociated from the calculated net rates of proximal tubular sodium, chloride, and bicarbonate reabsorption. We interpret these data as evidence that proximal tubular hydrogen ion secretion supporting bicarbonate reabsorption requires at most small amounts of oxidative energy, less than detectable by these techniques. The data, in contrast, support the conclusion that the chloride-bicarbonate transepithelial gradient appears to be an important passive driving force in vivo for proximal tubular fluid reabsorption.

scholarly journals A Multivariate Metabolomics Method for Estimating Platelet Mitochondrial Oxygen Consumption Rates in Patients with Sepsis

Metabolites ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 139
Author(s):  
Marc R. McCann ◽  
Cora E. McHugh ◽  
Maggie Kirby ◽  
Theodore S. Jennaro ◽  
Alan E. Jones ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Regression Models ◽  
Consumption Rate ◽  
P Value ◽  
Linear Regression Models ◽  
Mitochondrial Oxygen Consumption ◽  
Non Invasive ◽  
Consumption Rates ◽  
Multiple Linear Regression Models

Background: Sepsis-induced alterations in mitochondrial function contribute to organ dysfunction and mortality. Measuring mitochondrial function in vital organs is neither feasible nor practical, highlighting the need for non-invasive approaches. Mitochondrial function may be reflected in the concentrations of metabolites found in platelets and whole blood (WB) samples. We proposed to use these as alternates to indirectly estimate platelet mitochondrial oxygen consumption rate (mOCR) in sepsis patients. Methods: We determined the relationships between platelet mOCR and metabolites in both platelets and WB, as measured by quantitative 1H-NMR metabolomics. The associations were identified by building multiple linear regression models with stepwise forward-backward variable selection. We considered the models to be significant with an ANOVA test (p-value ≤ 0.05) and a positive predicted-R2. Results: The differences in adjusted-R2 and ANOVA p-values (platelet adj-R2: 0.836 (0.0003), 0.711 (0.0004) vs. WB adj-R2: 0.428 (0.0079)) from the significant models indicate the platelet models were more associated with platelet mOCR. Conclusions: Our data suggest there are groups of metabolites in WB (leucine, acetylcarnitine) and platelets (creatine, ADP, glucose, taurine) that are associated with platelet mOCR. Thus, WB and platelet metabolites could be used to estimate platelet mOCR.

Effect of hyperthermia and acidosis on equine skeletal muscle mitochondrial oxygen consumption

2020 ◽  
pp. 1-10
Author(s):  
M.S. Davis ◽  
M.R. Fulton ◽  
A. Popken
Keyword(s):  
Skeletal Muscle ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Muscle Fatigue ◽  
Mitochondrial Function ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Mitochondrial Oxygen Consumption ◽  
Biochemical Basis ◽  
Complex Ii

The skeletal muscle of exercising horses develops pronounced hyperthermia and acidosis during strenuous or prolonged exercise, with very high tissue temperature and low pH associated with muscle fatigue or damage. The purpose of this study was to evaluate the individual effects of physiologically relevant hyperthermia and acidosis on equine skeletal muscle mitochondrial function, using ex vivo measurement of oxygen consumption to assess the function of different mitochondrial elements. Fresh triceps muscle biopsies from 6 healthy unfit Thoroughbred geldings were permeabilised to permit diffusion of small molecular weight substrates through the sarcolemma and analysed in a high resolution respirometer at 38, 40, 42, and 44 °C, and pH=7.1, 6.5, and 6.1. Oxygen consumption was measured under conditions of non-phosphorylating (leak) respiration and phosphorylating respiration through Complex I and Complex II. Data were analysed using a one-way repeated measures ANOVA and data are expressed as mean ± standard deviation. Leak respiration was ~3-fold higher at 44 °C compared to 38 °C regardless of electron source (Complex I: 22.88±3.05 vs 8.08±1.92 pmol O2/mg/s), P=0.002; Complex II: 79.14±23.72 vs 21.43±11.08 pmol O2/mg/s, P=0.022), resulting in a decrease in efficiency of oxidative phosphorylation. Acidosis had minimal effect on mitochondrial respiration at pH=6.5, but pH=6.1 resulted in a 50% decrease in mitochondrial oxygen consumption. These results suggest that skeletal muscle hyperthermia decreases the efficiency of oxidative phosphorylation through increased leak respiration, thus providing a specific biochemical basis for hyperthermia-induced muscle fatigue. The effect of myocellular acidosis on mitochondrial respiration was minimal under typical levels of acidosis, but atypically severe acidosis can lead to impairment of mitochondrial function.

Abstract 15731: Sildenafil Improves Mitochondrial Function in Failing Single Ventricles

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Anastacia M Garcia ◽  
Carissa A Miyano ◽  
Raleigh Joschner ◽  
Matthew Stone ◽  
Brian L Stauffer ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Complex I ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Mitochondrial Oxygen Consumption ◽  
Ventricular Tissue ◽  
Substrate Inhibitor ◽  
Phosphodiesterase 5 ◽  
One Way Anova

Introduction: Heart failure (HF) remains a leading cause of death and indication for transplant in single ventricle congenital heart disease (SV). However, little is known regarding the molecular mechanisms leading to HF in SV. The purpose of this study was to characterize mitochondrial function in the myocardium of failing (SVHF) and non-failing (SVNF) SV patients compared to biventricular NF controls (BVNF). Furthermore, we investigated the effect of ex vivo treatment with the phosphodiesterase-5 inhibitor (PDE5i) sildenafil on mitochondrial function. Methods: Freshly explanted ventricular tissue was saponin permeabilized and mitochondrial oxygen consumption was measured sequentially throughout the electron transport system using SUbstrate-Inhibitor-Titration (“SUIT”) protocols and an Oroboros O2k high resolution respirometer. Permeabilized ventricular tissue was treated for 40 min with sildenafil [1μM] prior to measurement of oxygen consumption. A Western blot for PDE5 was performed in isolated mitochondrial proteins from SVHF subjects ± PDE5i. Results: Compared to BVNF (n=15) and SVNF (n=6), SVHF (n=8) hearts have decreased function of Complex I and Complex I and II (A, B), and decreased maximal respiration (C), all of which improve with acute ex vivo treatment with sildenafil in SVHF (SVHF+PDE5i, n=6). Importantly, mitochondrial function is impaired in BVNF+PDE5i (n=5) and SVNF+PDE5i hearts (n=5) (A-C, one-way Anova p<0.05). PDE5 protein is expressed in SVHF mitochondria, but expression is not affected by ex vivo PDE5i treatment (D). Conclusions: Our results indicate that mitochondrial function is impaired in SVHF, PDE5 protein is expressed in SVHF mitochondria, and PDE5i improves mitochondrial function in SVHF, but may be detrimental to mitochondrial function in SVNF and BVNF. Together these data suggest that mitochondrial PDE5 is a potential therapeutic target, but that indiscriminate use of PDE5i in SV patients may not be advisable.

scholarly journals Mechanisms of the interaction of nitroxyl with mitochondria

2004 ◽  
Vol 379 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Sruti SHIVA ◽  
Jack H. CRAWFORD ◽  
Anup RAMACHANDRAN ◽  
Erin K. CEASER ◽  
Tess HILLSON ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Protein ◽  
Present Evidence ◽  
Physiological Regulation ◽  
Isolated Mitochondria ◽  
Angeli's Salt ◽  
Biological Responses ◽  
Protein Thiols ◽  
Proteomics Approach

It is now thought that NO• (nitric oxide) and its redox congeners may play a role in the physiological regulation of mitochondrial function. The inhibition of cytochrome c oxidase by NO• is characterized as being reversible and oxygen dependent. In contrast, peroxynitrite, the product of the reaction of NO• with superoxide, irreversibly inhibits several of the respiratory complexes. However, little is known about the effects of HNO (nitroxyl) on mitochondrial function. This is especially important, since HNO has been shown to be more cytotoxic than NO•, may potentially be generated in vivo, and elicits biological responses with some of the characteristics of NO and peroxynitrite. In the present study, we present evidence that isolated mitochondria, in the absence or presence of substrate, convert HNO into NO• by a process that is dependent on mitochondrial concentration as well as the concentration of the HNO donor Angeli's salt. In addition, HNO is able to inhibit mitochondrial respiration through the inhibition of complexes I and II, most probably via modification of specific cysteine residues in the proteins. Using a proteomics approach, extensive modification of mitochondrial protein thiols was demonstrated. From these data it is evident that HNO interacts with mitochondria through mechanisms distinct from those of either NO• or peroxynitrite, including the generation of NO•, the modification of thiols and the inhibition of complexes I and II.

scholarly journals Sirtuin 5 depletion impairs mitochondrial function in human proximal tubular epithelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Timo N. Haschler ◽  
Harry Horsley ◽  
Monika Balys ◽  
Glenn Anderson ◽  
Jan-Willem Taanman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mitochondrial Function ◽  
Kidney Injury ◽  
Tubular Epithelial Cells ◽  
Atp Production ◽  
Form And Function ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular

AbstractIschemia is a major cause of kidney damage. Proximal tubular epithelial cells (PTECs) are highly susceptible to ischemic insults that frequently cause acute kidney injury (AKI), a potentially life-threatening condition with high mortality. Accumulating evidence has identified altered mitochondrial function as a central pathologic feature of AKI. The mitochondrial NAD+-dependent enzyme sirtuin 5 (SIRT5) is a key regulator of mitochondrial form and function, but its role in ischemic renal injury (IRI) is unknown. SIRT5 expression was increased in murine PTECs after IRI in vivo and in human PTECs (hPTECs) exposed to an oxygen/nutrient deprivation (OND) model of IRI in vitro. SIRT5-depletion impaired ATP production, reduced mitochondrial membrane potential, and provoked mitochondrial fragmentation in hPTECs. Moreover, SIRT5 RNAi exacerbated OND-induced mitochondrial bioenergetic dysfunction and swelling, and increased degradation by mitophagy. These findings suggest SIRT5 is required for normal mitochondrial function in hPTECs and indicate a potentially important role for the enzyme in the regulation of mitochondrial biology in ischemia.

Tafazzin Deficiency Reduces Basal Insulin Secretion and Mitochondrial Function in Pancreatic Islets from Male Mice

Endocrinology ◽  
2021 ◽  
Author(s):  
Laura K Cole ◽  
Prasoon Agarwal ◽  
Christine A Doucette ◽  
Mario Fonseca ◽  
Bo Xiang ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Mitochondrial Function ◽  
Ex Vivo ◽  
Cellular Respiration ◽  
Rna Seq ◽  
Β Cell ◽  
Mitochondrial Oxygen Consumption ◽  
Low Glucose

Abstract Tafazzin (TAZ) is a cardiolipin (CL) biosynthetic enzyme important for maintaining mitochondrial function. TAZ impacts both the species and content of CL in the inner mitochondrial membrane which are essential for normal cellular respiration. In pancreatic β-cells, mitochondrial function is closely associated with insulin secretion. However, the role of TAZ and CL in the secretion of insulin from pancreatic islets remains unknown. Male 4-month-old doxycycline-inducible TAZ knock-down (TAZ KD) mice and wild-type littermate controls were utilized. Immunohistochemistry was used to assess β-cell morphology in whole pancreas sections, while ex vivo insulin secretion, CL content, RNA-Seq analysis and mitochondrial oxygen consumption were measured from isolated islet preparations. Ex vivo insulin secretion under non-stimulatory low-glucose concentrations was reduced ~52% from islets isolated from TAZ KD mice. Mitochondrial oxygen consumption under low-glucose conditions was also reduced ~58% in islets from TAZ KD animals. TAZ-deficiency in pancreatic islets was associated with significant alteration in CL molecular species and elevated polyunsaturated fatty acid CL content. In addition, RNA-Seq of isolated islets showed that TAZ KD increased expression of extracellular matrix genes which are linked to pancreatic fibrosis, activated stellate cells and impaired β-cell function. These data indicate a novel role for TAZ in regulating pancreatic islet function, particularly under low-glucose conditions.

Calcium transport and inner mitochondrial membrane damage in renal cortical mitochondria

1985 ◽  
Vol 248 (6) ◽  
pp. F876-F889 ◽  
Author(s):  
J. M. Weinberg ◽  
H. D. Humes
Keyword(s):  
Fatty Acid ◽  
Membrane Permeability ◽  
Respiratory Function ◽  
Mitochondrial Membrane ◽  
Membrane Damage ◽  
Ruthenium Red ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Membrane Permeability ◽  
Isolated Mitochondria

Ca2+ uptake and efflux processes, as they are manifested during procedures used for isolation of renal cortical mitochondria, were characterized in order to provide a better basis for making inferences from isolated mitochondria about the in vivo state of mitochondrial Ca2+ homeostasis in both normal and injured tissues and to better define the mechanisms by which Ca2+ mediates injury to renal cortical mitochondria. Mitochondrial Ca2+ uptake predictably occurred when the capacity of the Ca2+ chelator added to the isolating medium to maintain free Ca2+ in the submicromolar range was exhausted unless ruthenium red was present to specifically inhibit the Ca2+ uniport. Ca2+ uptake during isolation ultimately led to loss of accumulated Ca2+ and intramitochondrial K+ as well as to deterioration of respiratory function. Extramitochondrial Ca2+ also evoked the latter two events in the absence of Ca2+ uptake but only at much higher medium Ca2+ levels than were required when Ca2+ uptake was allowed to occur. Studies using mitochondria loaded with known amounts of Ca2+ at 4 degrees C and then subjected to a reisolation procedure including all the steps of normal isolation demonstrated that phosphate markedly potentiated Ca2+-induced alterations of mitochondrial membrane permeability properties. Of several agents studied singly, fatty acid-free albumin was most effective in blocking Ca2+ + phosphate-induced alterations of mitochondrial membrane permeability. The protective effect of fatty acid-free albumin was further enhanced by combining it with Mg2+, dibucaine, or oligomycin + ADP. This study thus quantitatively defined conditions under which Ca2+ uptake can be expected to occur during mitochondrial isolation, demonstrated that the effects of this Ca2+ uptake on mitochondrial properties are similar to those previously elucidated in mitochondria studied at warmer temperatures, and defined methods suitable for blocking such Ca2+ movements and their deleterious effects.

scholarly journals Sodium Thiosulfate Improves Intestinal and Hepatic Microcirculation Without Affecting Mitochondrial Function in Experimental Sepsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Jan Schulz ◽  
Sandra Kramer ◽  
Yasin Kanatli ◽  
Anne Kuebart ◽  
Inge Bauer ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Sodium Thiosulfate ◽  
Experimental Sepsis ◽  
Hepatic Microcirculation ◽  
Mitochondrial Oxygen Consumption ◽  
Intestinal Microcirculation ◽  
Male Wistar Rats ◽  
Tissue Homogenates ◽  
Channel Dependent

IntroductionIn the immunology of sepsis microcirculatory and mitochondrial dysfunction in the gastrointestinal system are important contributors to mortality. Hydrogen sulfide (H2S) optimizes gastrointestinal oxygen supply and mitochondrial respiration predominantly via K(ATP)-channels. Therefore, we tested the hypothesis that sodium thiosulfate (STS), an inducer of endogenous H2S, improves intestinal and hepatic microcirculation and mitochondrial function via K(ATP)-channels in sepsis.MethodsIn 40 male Wistar rats colon ascendens stent peritonitis (CASP) surgery was performed to establish sepsis. Animals were randomized into 4 groups (1: STS 1 g • kg-1 i.p., 2: glibenclamide (GL) 5 mg • kg-1 i.p., 3: STS + GL, 4: vehicle (VE) i.p.). Treatment was given directly after CASP-surgery and 24 hours later. Microcirculatory oxygenation (µHBO2) and flow (µflow) of the colon and the liver were continuously recorded over 90 min using tissue reflectance spectrophotometry. Mitochondrial oxygen consumption in tissue homogenates was determined with respirometry. Statistic: two-way ANOVA + Dunnett´s and Tukey post - hoc test (microcirculation) and Kruskal-Wallis test + Dunn’s multiple comparison test (mitochondria). p &lt; 0.05 was considered significant.ResultsSTS increased µHbO2 (colon: 90 min: + 10.4 ± 18.3%; liver: 90 min: + 5.8 ± 9.1%; p &lt; 0.05 vs. baseline). Furthermore, STS ameliorated µflow (colon: 60 min: + 51.9 ± 71.1 aU; liver: 90 min: + 22.5 ± 20.0 aU; p &lt; 0.05 vs. baseline). In both organs, µHbO2 and µflow were significantly higher after STS compared to VE. The combination of STS and GL increased colonic µHbO2 and µflow (µHbO2 90 min: + 8.7 ± 11.5%; µflow: 90 min: + 41.8 ± 63.3 aU; p &lt; 0.05 vs. baseline), with significantly higher values compared to VE. Liver µHbO2 and µflow did not change after STS and GL. GL alone did not change colonic or hepatic µHbO2 or µflow. Mitochondrial oxygen consumption and macrohemodynamic remained unaltered.ConclusionThe beneficial effect of STS on intestinal and hepatic microcirculatory oxygenation in sepsis seems to be mediated by an increased microcirculatory perfusion and not by mitochondrial respiratory or macrohemodynamic changes. Furthermore, the effect of STS on hepatic but not on intestinal microcirculation seems to be K(ATP)-channel-dependent.

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