Assessing the degree of hepatic ischemia-reperfusion injury using PBPK modeling of sodium fluorescein disposition in ex vivo machine-perfused livers.

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically-based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multi-drug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; Vmax) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a 6-parameter version of this model to control time course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only 3 unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT and found the mean estimated value for each parameter and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of Vmax for MRP2 at 30 min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP.

Effects of ROS pathway inhibitors and NADH and FADH2 linked substrates on mitochondrial bioenergetics and ROS emission in the heart and kidney cortex and outer medulla.

Mitochondria are major sources of reactive oxygen species (ROS), which play important roles in both physiological and pathological processes. However, the specific contributions of different ROS production and scavenging components in the mitochondria of metabolically active tissues such as heart and kidney cortex and outer medulla (OM) are not well understood. Therefore, the goal of this study was to determine contributions of different ROS production and scavenging components and provide detailed comparisons of mitochondrial respiration, bioenergetics, ROS emission between the heart and kidney cortex and OM using tissues obtained from the same Sprague-Dawley rat under identical conditions and perturbations. Specifically, data were obtained using both NADH-linked substrate pyruvate + malate and FADH2-linked substrate succinate followed by additions of inhibitors of different components of the electron transport chain (ETC) and oxidative phosphorylation (OxPhos) and other ROS production and scavenging systems. Currently, there is limited data available for the mitochondria of kidney cortex and OM, the two major energy-consuming tissues in the body only next to the heart, and scarce quantitative information on the interplay between mitochondrial ROS production and scavenging systems in the three tissues. The findings from this study demonstrate significant differences in mitochondrial respiratory and bioenergetic functions and ROS emission among the three tissues. The results quantify the rates of ROS production from different complexes of the ETC, identify the complexes responsible for variations in mitochondrial membrane depolarization and regulations of ROS production, and quantify the contributions of ROS scavenging enzymes towards overall mitochondrial ROS emission. These findings advance our fundamental knowledge of tissue-specific and substrate-dependent mitochondrial respiratory and bioenergetic functions and ROS emission. This is important given the critical role that excess ROS production, oxidative stress, and mitochondrial dysfunction in the heart and kidney cortex and OM play in the pathogenesis of cardiovascular and renal diseases, including salt-sensitive hypertension.

In vivo molecular imaging stratifies rats with different susceptibilities to hyperoxic acute lung injury.

99mTc-hexamethylpropyleneamine oxime (HMPAO) and 99mTc-duramycin in vivo imaging detects pulmonary oxidative stress and cell death, respectively, in rats exposed to >95% O2 (hyperoxia) as a model of acute respiratory distress syndrome (ARDS). Preexposure to hyperoxia for 48 h followed by 24 h in room air (H-T) is protective against hyperoxia-induced lung injury. This study's objective was to determine the ability of 99mTc-HMPAO and 99mTc-duramycin to track this protection and to elucidate underlying mechanisms. Rats were exposed to normoxia, hyperoxia for 60 h, H-T, or H-T followed by 60 h of hyperoxia (H-T + 60). Imaging was performed 20 min after intravenous injection of either 99mTc-HMPAO or 99mTc-duramycin. 99mTc-HMPAO and 99mTc-duramycin lung uptake was 200% and 167% greater (P < 0.01) in hyperoxia compared with normoxia rats, respectively. On the other hand, uptake of 99mTc-HMPAO in H-T + 60 was 24% greater (P < 0.01) than in H-T rats, but 99mTc-duramycin uptake was not significantly different (P = 0.09). Lung wet-to-dry weight ratio, pleural effusion, endothelial filtration coefficient, and histological indices all showed evidence of protection and paralleled imaging results. Additional results indicate higher mitochondrial complex IV activity in H-T versus normoxia rats, suggesting that mitochondria of H-T lungs may be more tolerant of oxidative stress. A pattern of increasing lung uptake of 99mTc-HMPAO and 99mTc-duramycin correlates with advancing oxidative stress and cell death and worsening injury, whereas stable or decreasing 99mTc-HMPAO and stable 99mTc-duramycin reflects hyperoxia tolerance, suggesting the potential utility of molecular imaging for identifying at-risk hosts that are more or less susceptible to progressing to ARDS.

Fluorescein clearance kinetics in blood and bile indicates hepatic ischemia-reperfusion injury in rats.

Quantitative measurement of the degree of hepatic ischemia-reperfusion injury (IRI) is crucial for developing therapeutic strategies for its treatment. We hypothesized that clearance of fluorescent dye through bile metabolism may reflect the degree of hepatic IRI. In this study, we investigated sodium fluorescein clearance kinetics in blood and bile for quantifying the degree of hepatic IRI. Warm ischemia times (WITs) of 0, 30, or 60 min followed by 1 h or 4 h of reperfusion, were applied to the median and lateral lobes of the liver in Sprague-Dawley rats. Subsequently, 2 mg/kg of sodium fluorescein was injected intravenously, and blood and bile samples were collected over 60 min to measure fluorescence intensities. The bile-to-plasma fluorescence ratios demonstrated an inverse correlation with WIT and were distinctly lower in the 60-min WIT group than in the control or 30-min WIT groups. Bile-to-plasma fluorescence ratios displayed superior discriminability for short versus long WITs when measured 1 h after reperfusion versus 4 h. We conclude that the bile-to-blood ratio of fluorescence after sodium fluorescein injection has the potential to enable the quantification of hepatic IRI severity.NEW & NOTEWORTHY Previous attempts to use fluorophore clearance to test liver function have relied on a single source of data. However, the kinetics of substrate processing via bile metabolism include decreasing levels in blood and increasing levels in bile. Thus, we analyzed data from blood and bile to better reflect fluorescein clearance kinetics.

Autophagy, TERT, and mitochondrial dysfunction in hyperoxia.

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1ß, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.

A size-modified poisson-boltzmann ion channel model in a solvent of multiple ionic species: Application to voltage-dependent anion channel.

We present a new size-modified Poisson-Boltzmann ion channel (SMPBIC) model and use it to calculate the electrostatic potential, ionic concentrations, and electrostatic solvation free energy for a voltage-dependent anion channel (VDAC) on a biological membrane in a solution mixture of multiple ionic species. In particular, the new SMPBIC model adopts a membrane surface charge density and a natural Neumann boundary condition to reflect the charge effect of the membrane on the electrostatics of VDAC. To avoid the singularity difficulties caused by the atomic charges of VDAC, the new SMPBIC model is split into three submodels such that the solution of one of the submodels is obtained analytically and contains all the singularity points of the SMPBIC model. The other two submodels are then solved numerically much more efficiently than the original SMPBIC model. As an application of this SMPBIC submodel partitioning scheme, we derive a new formula for computing the electrostatic solvation free energy. Numerical results for a human VDAC isoform 1 (hVDAC1) in three different salt solutions, each with up to five different ionic species, confirm the significant effects of membrane surface charges on both the electrostatics and ionic concentrations. The results also show that the new SMPBIC model can describe well the anion selectivity property of hVDAC1, and that the new electrostatic solvation free energy formula can significantly improve the accuracy of the currently used formula. © 2019 Wiley Periodicals, Inc.

Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.

OBJECTIVE: We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. APPROACH AND RESULTS: Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter-binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. CONCLUSIONS: Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction.

BioModME for building and simulating dynamic computational models of complex biological systems.

Summary: Molecular mechanisms of biological functions and disease processes are exceptionally complex, and our ability to interrogate and understand relationships is becoming increasingly dependent on the use of computational modeling. We have developed "BioModME," a standalone R-based web application package, providing an intuitive and comprehensive graphical user interface to help investigators build, solve, visualize, and analyze computational models of complex biological systems. Some important features of the application package include multi-region system modeling, custom reaction rate laws and equations, unit conversion, model parameter estimation utilizing experimental data, and import and export of model information in the Systems Biology Matkup Language format. The users can also export models to MATLAB, R, and Python languages and the equations to LaTeX and Mathematical Markup Language formats. Other important features include an online model development platform, multi-modality visualization tool, and efficient numerical solvers for differential-algebraic equations and optimization. Availability and implementation: All relevant software information including documentation and tutorials can be found at https://mcw.marquette.edu/biomedical-engineering/computational-systems-biology-lab/biomodme.php. Deployed software can be accessed at https://biomodme.ctsi.mcw.edu/. Source code is freely available for download at https://github.com/MCWComputationalBiologyLab/BioModME.

Mitochondrial function in lungs of rats with different susceptibilities to hyperoxia-induced Acute Lung Injury.

Adult rats exposed to hyperoxia (>95% O2) die from respiratory failure in 60-72 hours. However, rats preconditioned with >95% O2 for 48 hours followed by 24 hours in room air (H-T) acquire tolerance of hyperoxia, while rats preconditioned with 60% O2 for 7 days (H-S) become more susceptible. Our objective was to evaluate lung tissue mitochondrial bioenergetics in H-T and H-S rats. Bioenergetics were assessed in mitochondria isolated from lung tissue of H-T, H-S, and control rats. Expressions of complexes involved in oxidative phosphorylation (OxPhos) were measured in lung tissue homogenate. Pulmonary endothelial filtration coefficient (Kf) and tissue mitochondrial membrane potential (ΔΨm) were evaluated in isolated perfused lungs. Results show that ADP-induced state 3 OxPhos capacity (Vmax) decreased in H-S mitochondria but increased in H-T. ΔΨm repolarization time following ADP-stimulated depolarization increased in H-S mitochondria. Complex I expression decreased in H-T (38%) and H-S (43%) lung homogenate, whereas complex V expression increased (70%) in H-T lung homogenate. ΔΨm is unchanged in H-S and H-T lungs, but complex II has a larger contribution to ΔΨm in H-S than H-T lungs. Kf increased in H-S, but not H-T lungs. For H-T, increased complex V expression and Vmax counter the effect of the decrease in complex I expression on ΔΨm. A larger complex II contribution to ΔΨm along with decreased Vmax and increased Kf could make H-S rats more hyperoxia susceptible. Results are clinically relevant since ventilation with ≥60% O2 is often required for extended periods in Acute Respiratory Distress Syndrome patients.

Computational Modeling of Substrate-Dependent Mitochondrial Respiration and Bioenergetics in the Heart and Kidney Cortex and Outer Medulla.

Integrated computational modeling provides a mechanistic and quantitative framework to characterize alterations in mitochondrial respiration and bioenergetics in response to different metabolic substrates in-silico. These alterations play critical roles in the pathogenesis of diseases affecting metabolically active organs such as heart and kidney. Therefore, the present study aimed to develop and validate thermodynamically constrained integrated computational models of mitochondrial respiration and bioenergetics in the heart and kidney cortex and outer medulla (OM). The models incorporated the kinetics of major biochemical reactions and transport processes as well as regulatory mechanisms in the mitochondria of these tissues. Intrinsic model parameters such as Michaelis-Menten constants were fixed at previously estimated values, while extrinsic model parameters such as maximal reaction and transport velocities were estimated separately for each tissue. This was achieved by fitting the model solutions to our recently published respirometry data measured in isolated rat heart and kidney cortex and OM mitochondria utilizing various NADH- and FADH2-linked metabolic substrates. The models were validated by predicting additional respirometry and bioenergetics data, which were not used for estimating the extrinsic model parameters. The models were able to predict tissue-specific and substrate-dependent mitochondrial emergent metabolic system properties such as redox states, enzyme and transporter fluxes, metabolite concentrations, membrane potential, and respiratory control index under diverse physiological and pathological conditions. The models were also able to quantitatively characterize differential regulations of NADH- and FADH2-linked metabolic pathways, which contribute differently toward regulations of oxidative phosphorylation and ATP synthesis in the heart and kidney cortex and OM mitochondria.

Aortic Remodeling Kinetics in Response to Coarctation-Induced Mechanical Perturbations.

Background: Coarctation of the aorta (CoA; constriction of the proximal descending thoracic aorta) is among the most common congenital cardiovascular defects. Coarctation-induced mechanical perturbations trigger a cycle of mechano-transduction events leading to irreversible precursors of hypertension including arterial thickening, stiffening, and vasoactive dysfunction in proximal conduit arteries. This study sought to identify kinetics of the stress-mediated compensatory response leading to these alterations using a preclinical rabbit model of CoA. Methods: A prior growth and remodeling (G&R) framework was reformulated and fit to empirical measurements from CoA rabbits classified into one control and nine CoA groups of various severities and durations (n = 63, 5-11/group). Empirical measurements included Doppler ultrasound imaging, uniaxial extension testing, catheter-based blood pressure, and wire myography, yielding the time evolution of arterial thickening, stiffening, and vasoactive dysfunction required to fit G&R constitutive parameters. Results: Excellent agreement was observed between model predictions and observed patterns of arterial thickening, stiffening, and dysfunction among all CoA groups. For example, predicted vascular impairment was not significantly different from empirical observations via wire myography (p-value > 0.13). Specifically, 48% and 45% impairment was observed in smooth muscle contraction and endothelial-dependent relaxation, respectively, which were accurately predicted using the G&R model. Conclusions: The resulting G&R model, for the first time, allows for prediction of hypertension precursors at neonatal ages that is currently challenging to examine in preclinical models. These findings provide a validated computational tool for prediction of persistent arterial dysfunction and identification of revised severity-duration thresholds that may ultimately avoid hypertension from CoA.

Physiologically-Based Pharmacokinetic Modeling of Blood Clearance of Liver Fluorescent Markers for the Assessment of the Degree of Hepatic Ischemia-Reperfusion Injury.

During liver transplantation, ischemia-reperfusion injury (IRI) is inevitable and decreases the overall success of the surgery. While guidelines exist, there is no reliable way to quantitatively assess the degree of IRI present in the liver. Our recent study has shown a correlation between the bile-to-plasma ratio of FDA-approved sodium fluorescein (SF) and the degree of hepatic IRI, presumably due to IRI-induced decrease in the activity of the hepatic multidrug resistance-associated protein 2 (MRP2); however, the contribution of SF blood clearance via the bile is still convoluted with other factors, such as renal clearance. In this work, we sought to computationally model SF blood clearance via the bile. First, we converted extant SF fluorescence data from rat whole blood, plasma, and bile to concentrations using calibration curves. Next, based on these SF concentration data, we generated a "liver-centric", physiologically-based pharmacokinetic (PBPK) model of SF liver uptake and clearance via the bile. Model simulations show that SF bile concentration is highly sensitive to change in the activity of hepatic MPR2. These simulations suggest that SF bile clearance along with the PBPK model can be used to quantify the effect of IRI on the activity of MRP2.Clinical Relevance- This study establishes the theory necessary to generate a model for predicting the degree of IRI during liver transplantation.

Assessment of Protection Offered By the NRF2 Pathway Against Hyperoxia-Induced Acute Lung Injury in NRF2 Knockout Rats.

ABSTRACT: Nuclear factor erythroid 2-related factor (Nrf2) is a redox-sensitive transcription factor that responds to oxidative stress by activating expressions of key antioxidant and cytoprotective enzymes via the Nrf2-antioxidant response element (ARE) signaling pathway. Our objective was to characterize hyperoxia-induced acute lung injury (HALI) in Nrf2 knock-out (KO) rats to elucidate the role of this pathway in HALI. Adult Nrf2 wildtype (WT), and KO rats were exposed to room air (normoxia) or >95% O2 (hyperoxia) for 48 h, after which selected injury and functional endpoints were measured in vivo and ex vivo. Results demonstrate that the Nrf2-ARE signaling pathway provides some protection against HALI, as reflected by greater hyperoxia-induced histological injury and higher pulmonary endothelial filtration coefficient in KO versus WT rats. We observed larger hyperoxia-induced increases in lung expression of glutathione (GSH) synthetase, 3-nitrotyrosine (index of oxidative stress), and interleukin-1ß, and in vivo lung uptake of the GSH-sensitive SPECT biomarker 99mTc-HMPAO in WT compared to KO rats. Hyperoxia also induced increases in lung expression of myeloperoxidase in both WT and KO rats, but with no difference between WT and KO. Hyperoxia had no effect on expression of Bcl-2 (anti-apoptotic protein) or peroxiredoxin-1. These results suggest that the protection offered by the Nrf2-ARE pathway against HALI is in part via its regulation of the GSH redox pathway. To the best of our knowledge, this is the first study to assess the role of the Nrf2-ARE signaling pathway in protection against HALI using a rat Nrf2 knockout model.

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla.

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H2O2 emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H2O2 emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.

Depolarized mitochondrial membrane potential and protection with duroquinone in isolated perfused lungs from rats exposed to hyperoxia.

Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction. Our objective was to use a previously developed experimental-computational approach to estimate tissue Δψm in intact lungs of rats exposed to hyperoxia and to evaluate the ability of duroquinone (DQ) to reverse any hyperoxia-induced depolarization of lung Δψm. Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 h, after which lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6 G (R6G) during three single-pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. Computational modeling was used to estimate lung Δψm from R6G data. Rat exposure to hyperoxia resulted in partial depolarization (-33 mV) of lung Δψm and complex I inhibition depolarized lung Δψm by -83 mV. Results also demonstrate the efficacy of DQ to fully reverse both rotenone- and hyperoxia-induced lung Δψm depolarization. This study demonstrates hyperoxia-induced Δψm depolarization in intact lungs and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in intact lungs.NEW & NOTEWORTHY This study is the first to measure hyperoxia-induced Δψm depolarization in isolated perfused lungs. Hyperoxia resulted in a partial depolarization of Δψm, which was fully reversed with duroquinone, demonstrating the utility of this approach for assessing the impact of potential therapies that target mitochondria such as exogenous quinones.

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes.

Our goal was to quantify mitochondrial and plasma potential (Δψ(m) and Δψ(p)) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([R(e)]) were measured over time. R123 [R(e)] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R(e)] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K(+) concentration ([K(+)]), used to manipulate contributions of membrane potentials, attenuated decreases in [R(e)], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [R(e)]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [R(e)] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψ(m) (-130 ± 7 and -133 ± 4 mV), Δψ(p) (-36 ± 4 and -49 ± 4 mV), and a Pgp activity parameter (K(Pgp), 25 ± 5 and 51 ± 11 µl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψ(m) (-124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψ(m) and Δψ(p.) Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O(2) for 48 h) cells have equivalent resting Δψ(m), hyperoxic cell Δψ(m) was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction.

Substrate- and Calcium-Dependent Differential Regulation of Mitochondrial Oxidative Phosphorylation and Energy Production in the Heart and Kidney.

Mitochondrial dehydrogenases are differentially stimulated by Ca2+. Ca2+ has also diverse regulatory effects on mitochondrial transporters and other enzymes. However, the consequences of these regulatory effects on mitochondrial oxidative phosphorylation (OxPhos) and ATP production, and the dependencies of these consequences on respiratory substrates, have not been investigated between the kidney and heart despite the fact that kidney energy requirements are second only to those of the heart. Our objective was, therefore, to elucidate these relationships in isolated mitochondria from the kidney outer medulla (OM) and heart. ADP-induced mitochondrial respiration was measured at different CaCl2 concentrations in the presence of various respiratory substrates, including pyruvate + malate (PM), glutamate + malate (GM), alpha-ketoglutarate + malate (AM), palmitoyl-carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). The results showed that, in both heart and OM mitochondria, and for most complex I substrates, Ca2+ effects are biphasic: small increases in Ca2+ concentration stimulated, while large increases inhibited mitochondrial respiration. Furthermore, significant differences in substrate- and Ca2+-dependent O2 utilization towards ATP production between heart and OM mitochondria were observed. With PM and PCM substrates, Ca2+ showed more prominent stimulatory effects in OM than in heart mitochondria, while with GM and AM substrates, Ca2+ had similar biphasic regulatory effects in both OM and heart mitochondria. In contrast, with complex II substrate SUC + ROT, only inhibitory effects on mitochondrial respiration was observed in both the heart and the OM. We conclude that the regulatory effects of Ca2+ on mitochondrial OxPhos and ATP synthesis are biphasic, substrate-dependent, and tissue-specific.

Quantification of mitochondrial membrane potential in the isolated rat lung using rhodamine 6G.

Mitochondrial membrane potential (Δψm) plays a key role in vital mitochondrial functions, and its dissipation is a hallmark of mitochondrial dysfunction. The objective of this study was to develop an experimental and computational approach for estimating Δψm in intact rat lungs using the lipophilic fluorescent cationic dye rhodamine 6G (R6G). Rat lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of three single-pass phases, loading, washing, and uncoupling, in which the lungs were perfused with R6G-containing perfusate, fresh R6G-free perfusate, or R6G-free perfusate containing the mitochondrial uncoupler FCCP, respectively. This protocol was carried out with lung perfusate containing verapamil vehicle or verapamil, an inhibitor of the multidrug efflux pump P-glycoprotein (Pgp). Results show that the addition of FCCP resulted in an increase in R6G venous effluent concentration and that this increase was larger in the presence of verapamil than in its absence. A physiologically based pharmacokinetic (PBPK) model for the pulmonary disposition of R6G was developed and used for quantitative interpretation of the kinetic data, including estimating Δψm. The estimated value of Δψm [-144 ± 24 (SD) mV] was not significantly altered by inhibiting Pgp with verapamil and is comparable with that estimated previously in cultured pulmonary endothelial cells. These results demonstrate the utility of the proposed approach for quantifying Δψm in intact functioning lungs. This approach has potential to provide quantitative assessment of the effect of injurious conditions on lung mitochondrial function and to evaluate the impact of therapies that target mitochondria.NEW & NOTEWORTHY A novel experimental and computational approach for estimating mitochondrial membrane potential (Δψm) in intact functioning lungs is presented. The isolated rat lung inlet-outlet concentrations of the fluorescent cationic dye rhodamine 6G were measured and analyzed by using a computational model of its pulmonary disposition to determine Δψm. The approach has the potential to provide quantitative assessment of the effect of injurious conditions and their therapies on lung mitochondrial function.

Mechanistic computational modeling of the kinetics and regulation of NADPH oxidase 2 assembly and activation facilitating superoxide production.

Reactive oxygen species (ROS) play a crucial role in many physiological processes. However, ROS overproduction leads to oxidative stress, which plays a critical role in cell injury/death and the pathogenesis of many diseases. Members of NADPH oxidase (NOX) family, most of which are comprised of membrane and cytosolic components, are known to be the major nonmitochondrial sources of ROS in many cells. NOX2 is a widely-expressed and well-studied NOX family member, which is activated upon assembly of its membrane subunits gp91 phox and p22 phox with its cytosolic subunits p40 phox , p47 phox , p67 phox , and Rac, facilitating ROS production. NOX2 activation is also enhanced by GTP and inhibited by GDP. However, there remains a lack of a mechanistic, quantitative, and integrated understanding of the kinetics and regulation of the assembly of these subunits and their relative contributions toward NOX2 activation and ROS production. Toward this end, we have developed a mechanistic computational model, which incorporates a generalized random rapid equilibrium binding mechanism for NOX2 assembly and activation as well as regulations by GTP (activation), GDP (inhibition), and individual subunits enhancing the binding of other subunits (mutual binding enhancement). The resulting model replicates diverse published kinetic data, including subunit concentration-dependent NOX2 activation and ROS production, under different assay conditions, with appropriate estimates of the unknown model parameters. The model provides a mechanistic, quantitative, and integrated framework for investigating the critical roles of NOX2 subunits in NOX2 assembly and activation facilitating ROS production in a variety of physiological and pathophysiological conditions. However, there is also a need for better quantitative kinetic data based on current understanding of NOX2 assembly and activation in order to test and further develop this model.

Pharmacokinetics of 99mTc-HMPAO in isolated perfused rat lungs.

Lung uptake of technetium-labeled hexamethylpropyleneamine oxime (HMPAO) increases in rat models of human acute lung injury, consistent with increases in lung tissue glutathione (GSH). Since 99mTc-HMPAO uptake is the net result of multiple cellular and vascular processes, the objective was to develop an approach to investigate the pharmacokinetics of 99mTc-HMPAO uptake in isolated perfused rat lungs. Lungs of anesthetized rats were excised and connected to a ventilation-perfusion system. 99mTc-HMPAO (56 MBq) was injected into the pulmonary arterial cannula, a time sequence of images was acquired, and lung time-activity curves were constructed. Imaging was repeated with a range of pump flows and perfusate albumin concentrations and before and after depletion of GSH with diethyl maleate (DEM). A pharmacokinetic model of 99mTc-HMPAO pulmonary disposition was developed and used for quantitative interpretation of the time-activity curves. Experimental results reveal that 99mTc-HMPAO lung uptake, defined as the steady-state value of the 99mTc-HMPAO lung time-activity curve, was inversely related to pump flow. Also, 99mTc-HMPAO lung uptake decreased by ~65% after addition of DEM to the perfusate. Increased perfusate albumin concentration also resulted in decreased 99mTc-HMPAO lung uptake. Model simulations under in vivo flow conditions indicate that lung tissue GSH is the dominant factor in 99mTc-HMPAO retention in lung tissue. The approach allows for evaluation of the dominant factors that determine imaging biomarker uptake, separation of the contributions of pulmonary versus systemic processes, and application of this knowledge to in vivo studies.NEW & NOTEWORTHY We developed an approach for studying the pharmacokinetics of technetium-labeled hexamethylpropyleneamine oxime (99mTc-HMPAO) in isolated perfused lungs. A distributed-in-space-and-time computational model was fit to data and used to investigate questions that cannot readily be addressed in vivo. Experimental and modeling results indicate that tissue GSH is the dominant factor in 99mTc-HMPAO retention in lung tissue. This modeling approach can be readily extended to investigate the lung pharmacokinetics of other biomarkers and models of lung injury and treatment thereof.