Prolonged exposure to high oxygen levels (hyperoxia) is unavoidable in managing severe acute respiratory distress syndrome (ARDS), but can itself worsen lung injury and increase mortality. Rats conditioned to be hyperoxia-tolerant (H-T) or hyperoxia-susceptible (H-S) provide a system for assessing the contribution of mitochondrial bioenergetics to the differential susceptibility to hyperoxia-induced ARDS and for identifying potential therapeutic targets. Due to the system's complexity, interpreting lung mitochondrial bioenergetics data from these rat models requires a computational model to define which processes are altered and how changes influence overall lung tissue bioenergetics. We developed a thermodynamically constrained computational model of lung mitochondrial bioenergetics that extends prior models by incorporating regulation by ions (Ca2+, H+, etc.) and metabolites. The model was parameterized using experimental respirometry data from isolated lung mitochondria of conditioned (H-T and H-S) and control rats with different substrates and ADP concentrations. Model parameterization showed distinct bioenergetic changes. H-S mitochondria had reduced activity in adenine nucleotide translocase (ANT), cytochrome c oxidase (CIV), complex I (CI), and glutamate-oxaloacetate transaminase (GOT). Conversely, H-T mitochondria showed increased activity of ANT and CIV. This supports greater metabolic flexibility in H-T mitochondria compared with H-S. Simulations of ARDS-related changes predicted divergent outcomes. H-S mitochondria underwent rapid failure, with redox collapse, loss of membrane potential, and ATP depletion. H-T mitochondria maintained bioenergetic homeostasis by enhancing electron supply via CI and complex II, with higher CIV activity. This comprehensive computational model provides a framework for identifying critical mitochondrial processes and therapeutic strategies to mitigate mitochondrial dysfunction in ARDS.NEW & NOTEWORTHY We developed a comprehensive computational model of lung mitochondrial bioenergetics that integrates key regulatory mechanisms. The model, parameterized using respirometry data from lung mitochondria of hyperoxia-tolerant and -susceptible rats, identified adenine nucleotide translocase, cytochrome c oxidase, and proton leak as critical determinants of lung mitochondrial bioenergetic homeostasis. Model simulations predict that deficits in these processes drive rapid bioenergetic failure in the lungs of hyperoxia-susceptible rats.

Hyperoxia is both an essential therapy and a contributor to lung injury in acute respiratory distress syndrome. We hypothesized that adult female rats are relatively protected from hyperoxia-induced acute lung injury (HALI) compared with males and that this protection is associated with sex-dependent differences in lung mitochondrial bioenergetics and H2O2 production. Adult rats were exposed to room air (normoxia) or hyperoxia (>95% O-2) for up to 60 h. Lung injury was assessed by pleural effusion, lung wet weight, pulmonary vascular filtration coefficient (K-f), histologic injury scores, and cleaved caspase-3 (CC3) staining. Expression of mitochondrial complexes I-V was quantified in lung tissue. Mitochondrial oxygen consumption rates (OCRs) and H2O2 (mtH(2)O(2)) production were measured in isolated lung mitochondria, and lung H2O2 release rate was quantified in isolated perfused lungs. Hyperoxia caused systemic and pulmonary injury in both sexes. However, compared with females, males showed greater body weight loss and larger increases in lung wet weight, pleural effusion, K-f, and CC3-positive cells. Hyperoxia decreased complex I expression in males but not females and impaired OCRs in both sexes. For both sexes, complex II was the dominant mtH(2)O(2) source. Hyperoxia nearly doubled mtH(2)O(2) production in female, but not male, mitochondria. Nevertheless, whole lung H2O2 release rate in females did not increase, consistent with enhanced tissue-level scavenging. These findings indicate attenuated severity of HALI in adult females, highlight complex II as a major mtH(2)O(2) source during hyperoxia, and support consideration of sex as a biological variable in mitochondria-targeted therapies for HALI. NEW & NOTEWORTHY We investigated sex-specific responses to hyperoxia-induced acute lung injury (HALI) in rats. Female rats exhibited attenuated severity, with less pleural effusion, pulmonary edema, and apoptosis than males. Studies in isolated mitochondria, lung tissue homogenates, and isolated lungs indicate that this attenuated severity is associated with mitochondrial adaptations and enhanced tissue H2O2 scavenging capacity. These findings underscore the importance of sex as a biological variable and identify mitochondrial complex II as a potential HALI therapeutic target.

IntroductionThe complex dynamics of chimeric antigen receptor T-cell (CAR-T cell) cytotoxicity and proliferation are potential factors that influence the clinical response to CAR-T therapy. The patient-specific functionality of CAR-T products play a role in these dynamics. CAR-T products comprise phenotypically and functionally distinct populations of cells that impact therapy response in different ways. We hypothesized that product-specific parameters exist that predict individual patient responses to therapy and that these can be elucidated by simulating the interactions of CAR-T products and tumor cells using an in vitro assay-based model.MethodsWe use an ordinary differential equation (ODE)-based pharmacokinetic (PK) and pharmacodynamic (PD) model to characterize key CAR-T cell functional parameters. Parameters for the model developed using our method are product-specific and derived from in vitro assays performed on individual patient CAR-T products from clinical trial NCT04186520.ResultsOur results demonstrate that while considerable variability is present in in vitro cytotoxicity kinetics and subsequently estimated model parameters between each product, these differences do not predict early (28 days) or late responses (90 days) after treatment across the total cohort of patients investigated. However, we show that differences in an estimated model parameter for increased CAR-T cell responsiveness to tumor cytotoxicity are correlated with durable therapy responses (no relapse through 180 days). Additionally, in a cohort of diffuse large B-cell lymphoma (DLBCL) patients, we demonstrate that a model parameter estimating cooperativity between CAR-T cells is also correlated with durable therapy responses and that may be related to differences in CD4:CD8 ratios in the CAR-T cell product.ConclusionsOverall, our work demonstrates that while pre-treatment CAR-T cell functional parameters vary on a patient and product basis, these parameters do not predict initial therapeutic responses. We find that initial therapeutic responses are possible across a range of initial product kinetic parameters. However, we observed that their potentially exist unique kinetic properties associated with the initial product that is predictive of disease relapse.

Mitochondrial Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUx), in which MICU1/2 serve as cytosolic Ca2+-sensing gatekeepers that set a Ca2+-dependent activation threshold. Coordinated control of MCUx activity is critical because mitochondrial Ca2+ uptake couples cytosolic Ca2+ signals to metabolic activation and must be regulated to prevent mitochondrial Ca2+ overload and bioenergetic dysfunction. We developed a mechanistic model that incorporates explicit MICU1/2-dependent MCUx gatekeeping and a thermodynamically constrained Ca2+ transport formulation that accounts for Mg2+ inhibition and membrane potential dependence. Cytosolic and mitochondrial Ca2+ dynamics were simulated by integrating the MCUx model into a mitochondrial cation-handling model under multiple Ca2+ stimulation protocols. Because matrix-side Ca2+ regulation of MCUx remains controversial, we also evaluated a putative matrix-side regulatory mechanism by comparing simulations with and without an added matrix-side regulatory module, rather than assuming such regulation as a required feature of the MCUx model. The model reproduces key experimental behaviors across genotypes. In wild-type mitochondria, MCUx-mediated Ca²⁺ uptake is negligible below a cytosolic Ca2+ threshold (~0.2 µM), whereas MICU1-knockout mitochondria show constitutive uptake and MICU2-knockout mitochondria exhibit an intermediate, lowered threshold. Inclusion of matrix-side MCUx regulation transiently attenuated MCUx-mediated Ca²⁺ uptake over an intermediate mitochondrial Ca2+ range, producing higher transient cytosolic Ca2+ and lower transient mitochondrial Ca2+, while both cases approached similar steady states. In addition, cytosolic Mg2+ acts as a graded inhibitor of MCUx-mediated Ca2+ uptake, limiting mitochondrial Ca2+ loading. These results provide a quantitative framework for coupled cytosolic-mitochondrial Ca²⁺ dynamics across diverse conditions.

INTRODUCTION:Transcutaneous fluorescein isothiocyanate (FITC)-sinistrin fluorescence enables convenient, minimally invasive estimation of the glomerular filtration rate (GFR) in rodents. However, the commonly used fixed-factor conversions (i.e., GFR/BW = conversion factor (21.33)/HL, where HL is sinistrin half-life and BW is body weight) assume the volume of distribution scales proportionally with BW. This assumption can bias GFR estimates when body composition shifts with growth or when extracellular fluid volume expands under high-salt intake. Here, we quantified these errors and developed improved HL-to-GFR conversion methods. METHODS:Male and female Dahl salt-sensitive rats with chronically implanted venous catheters underwent simultaneous measurements of serum and transcutaneous FITC-sinistrin fluorescence. Serum clearance provided reference GFR and serum-derived HL, and the transcutaneous signal provided transcutaneous HL. In separate cohorts, body composition and extracellular fluid were assessed by Nuclear Magnetic Resonance-Bioimpedance Spectroscopy. A regression of GFR×HL was performed using BW, age, sex, and salt intake. RESULTS:In low-salt fed rats, sex-specific BW-based models explained much of the variability in GFR×HL (females: 97.81×BW0.385; males: 203.0×BW0.855; BW in kg). Separately, using data from all rats across salt conditions, we derived a salt-dependent conversion to allow for temporal salt effects. The corrected model detected the transient increase in GFR on high-salt day 3 that was missed by a fixed factor. GFR scaled sub-linearly with BW (exponent 0.744). CONCLUSIONS:Allowing the sinistrin HL-to-GFR conversion to vary with BW and salt condition moderately improves the accuracy of HL-based FITC-sinistrin GFR estimates compared with a single fixed conversion factor and captures the transient GFR increase after salt loading. Sub-linear scaling of GFR supports allometric size adjustment (BW0.744) to reduce size-dependent bias and better reflect true GFR in growing rats.

Oxygen (O2) binding and release by hemoglobin (Hb) are governed by cooperative interactions among its four subunits. During incremental workload exercise, femoral venous oxyhemoglobin (O2Hb) saturation exhibits a reproducible, momentary increase at the gas exchange threshold-coinciding with the inflection point of the in vivo O2 nonequilibrium curve (ONC). This suggests a transient shift in Hb's binding dynamics. We hypothesized that at this threshold, Hb tetramers carrying ≤1 bound O2 become predominant. In this state, the last bound O2 promotes further cooperative binding, but its release confers no cooperative advantage for unloading, biasing toward O2 rebinding. Using the O2 equilibrium curve models of Dash et al. (Eur J Appl Physiol 116: 97-113, 2016) and Adair, we computed the distribution of Hb's O2 ligation states across 12 pooled mean femoral venous blood samples from incremental workload cardiopulmonary exercise testing of five healthy male participants. At the gas exchange threshold-where the ONC inflects and flattens-tetramers with ≤1 O2 indeed dominated. This ligation-state distribution is consistent with Perrella et al.'s (J Biol Chem 274: 2605-2608, 1999) cryogenic resolution of native human Hb, which shows that carbon monoxide-ligated Hb tetramers peak at ∼15%-20% saturation, matching femoral venous ranges at the gas exchange threshold. Our results suggest that, at sufficiently low O2Hb saturation, Hb may favor O2 rebinding over cooperative unloading. We propose that glycolytic proton production and other Bohr effectors may counter this predicted binding bias supporting continued O2 unloading. If confirmed, this mechanism unifies long-standing controversies in O2 transport physiology, framing the Hb-Bohr system as a proportional-integral controller of tissue oxygenation.NEW & NOTEWORTHY Anaerobic metabolism is usually viewed as a fallback when oxygen delivery becomes insufficient. Our analysis suggests a different role: it may preserve oxygen delivery by correcting a hemoglobin-binding bias that emerges at low oxyhemoglobin saturation. When hemoglobin tetramers carry one or no oxygen molecules, the remaining bound oxygen can promote cooperative rebinding but not cooperative release. Glycolytic proton production and other Bohr effectors may counter this bias, restoring oxygen release linking lactate, hypoxia, and cooperativity.

We examined sex-specific and substrate-dependent differences in liver mitochondrial function of adult SD rats. Liver mitochondria preferentially use GM over PM for respiration, whereas PM produces more H 2 O 2 . Female mitochondria exhibited higher respiration and H 2 O 2 production than males across all substrates, likely driven by hormonal factors and sex-specific regulatory pathways. These findings highlight the importance of both substrate and sex in shaping liver mitochondrial bioenergetics and redox function, offering insight into intrinsic metabolic differences.

Background: Kidney proximal tubules (PTs) reabsorb nearly 65% of Na + from the glomerular filtrate in the cortex (Cx). Under high salt (HS) feeding, this imposes a significant metabolic stress on the PT mitochondria to consume very high O 2 for ATP production to meet the increased energy demand. Although mitochondrial dysfunction associated with salt-sensitive (SS) hypertension in the kidney is well-recognized, there remains a poor understanding of short-term versus long-term chronic changes in the PT mitochondrial bioenergetics and the associated regulatory mechanisms required to meet the increased energy demands in SS hypertension. In this study, we hypothesized that mitochondrial bioenergetics in the kidney PTs is progressively impaired via activation of mTORC1 signaling during the development of SS hypertension. Methods: PTs were isolated from the kidney Cx of Dahl SS rats (9-10 wks old; age-matched) fed a 0.4% NaCl diet (LS) or 4.0% NaCl diet for 7, 14, and 21 days (HS7, HS14, and HS21, respectively), and permeabilized through mechanical stroking. Mitochondrial O 2 consumption rates (OCRs) were measured in the permeabilized PTs, energized with substrates for complex I (pyruvate + malate (PM)) or complex II (succinate) and stimulated with two different ADP perturbations: 1) a saturated dose of ADP and 2) sequentially increasing doses of ADP. The distinct respiratory states such as H + leak state (state 2; S2), ADP-stimulated state (state 3; S3), resting state (state 4; S4), and uncoupled state (state 5; S5) were recorded using an Oroboros Oxygraph-2k instrument to determine the efficiency and coupling of mitochondrial oxidative phosphorylation (OxPhos). To determine the regulatory role of mTORC1 signaling in the HS-induced mitochondrial impairments, the respiratory parameters of PTs were compared between HS14 SS rats chronically treated with mTORC1 inhibitor rapamycin (1.5 mg/kg i.v. infusion per day) and vehicle throughout HS feeding. Results: Permeabilized PTs exhibited significant declines in S3 and S5 OCRs in HS14 and HS21 groups as compared to the LS group when energized with PM, with the corresponding S3 OCR durations significantly increased. The S3 OCRs of permeabilized PTs energized with succinate were also significantly reduced in the late phases of SS hypertension (HS14 and HS21), indicating an overall mitochondrial inefficiency for OxPhos and coupling in the chronic phase of the HS feeding. Since the role of mTORC1 in SS hypertension is well established in previous studies from our lab, we hypothesized that this decline in the efficiency and coupling of OxPhos was associated with activation of mTORC1 signaling. Our preliminary data obtained at HS14 clearly showed that rapamycin treatment of SS rats resulted in a significant improvement of the ADP-induced S3 OCR of permeabilized PTs compared to vehicle treatment when energized with succinate under both normal (single ADP) and stressed (sequential ADP) conditions. Conclusion: In HS fed SS rats, PTs initially are able to sustain their efficiency and coupling of OxPhos for ATP production for about 10 days which is then followed by a significant decline over the late phase of SS hypertension. This ultimate reduction in the efficiency and coupling of OxPhos for ATP production is improved in PTs of SS rats treated with rapamycin, demonstrating the vital causal role of mTORC1 signaling in PT mitochondrial dysfunction during the development of SS hypertension. Funding: NIH R01 HL151587. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND:Salt-sensitive (SS) hypertension is associated with oxidative stress and impaired renal metabolism, but the mechanistic link remains unclear. We investigated the role of NOX4 (NADPH oxidase 4)-derived reactive oxygen species in shaping renal metabolic and vascular responses to high-salt intake in SS hypertension. METHODS:Male Dahl SS and SSNOX4-/- rats were maintained on a low-salt (0.4% NaCl) diet and then exposed to high salt (4.0% NaCl) for 21 days. Mean arterial pressure and renal blood flow were continuously measured, with intermittent arterial and renal venous sampling. The glomerular filtration rate was measured in separate groups of rats. Transcriptomic and metabolomic profiling of renal cortex, outer medulla, plasma, and urine was performed. RESULTS:High-salt intake led to progressive hypertension in SS rats, accompanied by transcriptomic and metabolic patterns suggestive of increased glutamate utilization, redox imbalance, and elevated reactive oxygen species. This glutamine-related profile was associated with high oxygen demand, increased proteolysis, and reduced adaptability in substrate utilization. SSNOX4-/- rats exhibited attenuated hypertension, preserved redox balance, and increased nitric oxide bioavailability. Their kidneys exhibited metabolic patterns suggestive of greater reliance on fatty acid oxidation, with indications of improved oxygen efficiency, reduced amino acid catabolism, and lactate release without upregulating glycolytic genes. The glomerular filtration rate and renal blood flow changes were similar between strains. CONCLUSIONS:NOX4-derived reactive oxygen species were linked to less favorable metabolic responses to salt loading in SS hypertension. NOX4 deletion was associated with a more protective, fatty acid oxidation-driven metabolic profile. These findings highlight redox-sensitive metabolic reprogramming as a potential therapeutic target in hypertension and chronic kidney disease.

BACKGROUND:The introduction of normothermic machine perfusion (NMP) offers new opportunities to evaluate liver graft viability before liver transplantation (LT). Under ischemic stress, multidrug resistance-associated protein 2 (MRP2) translocates from the hepatocyte membrane to the cytoplasm, resulting in loss of function. METHODS:We measured the cytoplasmic proportion of MRP2 (MRP2 internalization index, MII) by immunofluorescence colocalization analysis using CD13 as a canalicular membrane marker. RESULTS:The data showed that MII significantly correlated with ischemia time in both in situ ischemia-reperfusion injury and NMP rat models (R 2  = 0.331, P < 0.0001; R 2  = 0.632, P < 0.0001, respectively). Perfusate levels of liver injury markers at the end of NMP showed a significant positive correlation with MII for aspartate aminotransferase (R² = 0.444, P = 0.0013) and arginase 1 (R² = 0.637, P < 0.0001). Conversely, bile production exhibited a significant inverse correlation with MII (R² = 0.618, P < 0.0001). The maximum transport rate of MRP2 ( Vmax,MRP2 ), derived from kinetic modeling of sodium fluorescein biliary excretion, showed a significant inverse correlation with ischemia time (R 2  = 0.326, P = 0.0086) and MII (R 2  = 0.554, P = 0.0002). In human LT, MII values from donor liver biopsies preLT correlated significantly with peak postLT serum aminotransferase levels (R 2  = 0.398, P = 0.0007). CONCLUSIONS:MRP2 is a putative biomarker for the assessment of hepatic ischemia-reperfusion injury. The biliary excretion kinetics of sodium fluorescein reflects MRP2-mediated transport activity, providing a novel diagnostic method for predicting liver graft viability after LT.

Voltage-dependent anion channel (VDAC) is the primary conduit for regulated passage of ions and metabolites into and out of a mitochondrion. Calculating the solvation free energy for VDAC is crucial for understanding its stability, function, and interactions within the cellular environment. In this article, numerical schemes for computing the total solvation free energy for VDAC-comprising electrostatic, ideal gas, and excess free energies plus the nonpolar energy-are developed based on a nonuniform size modified Poisson-Boltzmann ion channel (nuSMPBIC) finite element solver along with tetrahedral meshes for VDAC proteins. The current mesh generation package is also updated to improve mesh quality and accelerate mesh generation. A VDAC Solvation Free Energy Calculation (VSFEC) package is then created by integrating these schemes with the updated mesh package, the nuSMPBIC finite element package, the PDB2PQR package, and the OPM database, as well as one uniform SMPBIC finite element package and one Poisson-Boltzmann ion channel (PBIC) finite element package. With the VSFEC package, many numerical experiments are made using six VDAC proteins, eight ionic solutions containing up to four ionic species, including ATP4- and Ca2+, two reference states, different boundary values, and different permittivity constants. The test results underscore the importance of considering nonuniform ionic size effects to explore the varying patterns of the total solvation free energy, and demonstrate the high performance of the VSFEC package for VDAC solvation free energy calculation.

Mitochondrial bioenergetics and hydrogen peroxide (H2O2) production play a central role in maintaining liver metabolic function and redox balance. Understanding sex dimorphism and substrate dependency in these mitochondrial processes is crucial for elucidating the regulatory mechanisms that govern male versus female differences in liver physiology in health and disease. This study aimed at investigating sex-specific and substrate-dependent alterations in liver mitochondrial respiratory rates (Jo2), membrane potential (ΔΨ), and H2O2 production and their metabolic regulation. Liver mitochondria were isolated from adult male and female Sprague-Dawley rats. Four substrate combinations-pyruvate + malate (PM), glutamate + malate (GM), succinate, and succinate with complex I inhibitor rotenone-were used to determine their impact on the activities of the electron transport chain (ETC) and TCA cycle complexes. Adenosine diphosphate (ADP) was added to determine the influence of substrates on oxidative phosphorylation (OxPhos). Jo2 and ΔΨ were measured simultaneously using an Oroboros Oxygraph-2k respirometer with the cationic rhodamine dye tetramethylrhodamine methyl ester. H2O2 production was measured spectrofluorometrically using the Amplex Red and horseradish peroxidase assay. Our results show that male and female liver mitochondria displayed distinct respiratory patterns for different substrates. GM and succinate yielded higher Jo2, whereas PM yielded the lowest Jo2. Notably, female mitochondria exhibited higher Jo2 than males across all substrates. Both ΔΨ and H2O2 production showed substrate-dependent patterns, with females exhibiting higher values than males across all substrates. These findings reveal sex-specific differences in liver mitochondrial function, driven by substrate-dependent engagement of the ETC and TCA cycle complexes toward OxPhos, with females showing higher respiratory capacity and H2O2 production.NEW & NOTEWORTHY We examined sex-specific and substrate-dependent differences in liver mitochondrial function of adult SD rats. Liver mitochondria preferentially use GM over PM for respiration, whereas PM produces more H2O2. Female mitochondria exhibited higher respiration and H2O2 production than males across all substrates, likely driven by hormonal factors and sex-specific regulatory pathways. These findings highlight the importance of both substrate and sex in shaping liver mitochondrial bioenergetics and redox function, offering insight into intrinsic metabolic differences.

Rationale: Identifying and investigating sex differences in physiological phenomena and understanding the underlying regulatory mechanisms is crucial for precision medicine. Oxidative stress is implicated in various diseases, including ischemia-reperfusion injury (IRI) with liver transplantation. Understanding sex-specific and substrate-dependent mitochondrial bioenergetics and H 2 O 2 emission in the liver is important for developing mechanism-based therapeutic strategies to mitigate IRI in liver transplantation. The aim of this study was therefore to investigate sex-specific and substrate-dependent changes in mitochondrial respiration (oxygen consumption rate; OCR), membrane potential (dPsi), and H 2 O 2 emission in the liver and their metabolic regulations. Methods: Mitochondria were freshly isolated using differential centrifugation method from adult Sprague-Dawley rat livers (n=5/group; both sexes). To assess the electron transport chain (ETC) and TCA cycle dependencies of liver mitochondria, four different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), succinate, and succinate in the presence of the complex I inhibitor rotenone (SR) were used. A single saturated dose of ADP was added to mitochondria to determine how different substrates influence mitochondrial OCR and dPsi during oxidative phosphorylation. Mitochondrial OCR and dPsi were measured simultaneously using a dual chamber Oroboros Oxygraph-2k instrument coupled to a fluorometer using the cationic rhodamine dye TMRM. The H 2 O 2 emission was measured spectrofluorometrically using the amplex red and horseradish peroxidase in the respiration buffer, resulting in resorufin fluorescence with the non-H 2 O 2 -mediated resorufin fluorescence inhibited by PMSF (phenylmethylsulfonyl fluoride), a serine protease inhibitor. Results: Male and female liver mitochondria exhibited distinct respiratory patterns in the presence of different substrates. The respiratory rates were higher when GM and succinate were utilized as substrates, whereas the use of PM showed comparatively lower respiration. Interestingly, female mitochondria exhibited significantly higher OCR than male mitochondria irrespective of the substrate utilized. Similar sex-specific and substrate-dependent responses were observed in mitochondrial dPsi and H 2 O 2 emission. PMSF effectively inhibited the non-H 2 O 2 -mediated resorufin fluorescence, implying a potential role of serine proteases in modulating oxidative processes and redox signaling in liver mitochondria. Conclusion: Sex differences were observed when mitochondria were fueled with distinct respiratory substrates that selectively activate specific complexes of the ETC and TCA cycle enzymes. Female liver mitochondria demonstrated greater capacity and efficiency in respiratory function and H 2 O 2 emission, suggesting that sex-specific differences may arise from regulatory pathways associated with respiratory substrate utilization in liver mitochondria. NIH R01 HL151587 and NSF DMS 2153387. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

We previously hypothesized that the inflection point of the oxygen dissociation curve (ODC) is linked to the gas exchange threshold (GET) during cardiopulmonary exercise testing. This hypothesis was supported by femoral venous blood gas data sampled during constant exercise below and above the GET, which showed that the ODC shifts rightward at the GET. What had gone unnoticed since these original observations in 1994 was that this rightward shift begins slightly earlier, precisely when the oxygen saturation crosses the ODC inflection point. To investigate this phenomenon, we analyzed the 1994 femoral venous blood gas data obtained during cardiopulmonary exercise testing using a modern validated mechanistic biochemical model of oxygen (O2), carbon dioxide (CO2), and proton binding to hemoglobin (Hb). We constructed the ODC for each data point, as well as the in vivo ODC-a composite curve reflecting changes in dynamic blood chemistry during exercise-to assess its alignment with the GET. The model revealed that, at the in vitro ODC inflection point (36% O2Hb saturation), the amounts of CO2 bound to Hb equalized with HbNH3+ eventually predominating. This equilibrium apparently triggered the Bohr shift, steepening the in vivo ODC to improve O2 unloading to the tissues. Shortly afterwards, the in vivo ODC reached its inflection point, matching the measured GET. Our findings support that the GET is mechanistically linked to the in vivo ODC inflection point. These results highlight the physiological relevance of determining the ODC inflection point and its alignment with HbNH3+ and CO2 binding as critical factors in understanding ODC shifts during cardiopulmonary exercise testing.

Adult rats exposed to hyperoxia (>95 % O2) die within 60-72 h from respiratory failure. However, when preconditioned with either >95 % O2 for 48 h followed by 24 h in room air (H-T) or 60 % O2 for 7 days (H-S), they acquire tolerance or susceptibility to hyperoxia, respectively. The aim was to quantify H2O2 production rate and identify sources in isolated lung mitochondria and isolated perfused lungs (IPLs) of normoxia, H-T, and H-S rats. Mitochondria were isolated from lungs, and H2O2 production rates were quantified in the presence of pyruvate-malate or succinate, with and without inhibitors of mitochondrial complex I (CI), complex II (CII), and/or H2O2 scavenging systems. Lung rate of H2O2 release was quantified in IPLs with and without CII inhibitor. Results from isolated mitochondria show that CII is the main H2O2 source, and that both H2O2 production rate and scavenging capacity were ~48 % lower in H-S mitochondria compared to normoxia. Results from IPLs show that CII is also the dominant H2O2 source from lung tissue, and that H2O2 release rate was lower in H-T lungs compared to normoxia and H-S lungs. These results suggest that for H-S rats, both mitochondrial rate of H2O2 production and scavenging capacity were significantly lower than those in normoxia mitochondria and may contribute to their increased hyperoxia susceptibility. The lower H2O2 release rate from H-T IPLs, along with no change in mitochondrial H2O2 production rate, is consistent with higher antioxidant capacity in the lungs of H-T rats, which may contribute to their hyperoxia tolerance.

Background: The kidneys have the second highest O 2 consumption in mammals next to the heart, which is closely linked to tubular Na + transport functions. Proximal tubules (PTs) are responsible for reabsorbing a majority of Na + (~65%) from the glomerular filtrate in the cortex (Cx). This is highly energy demanding and requires a huge dependence on the PT mitochondrial bioenergetics. Mitochondrial dysfunction in the kidney is implicated in the pathophysiology of various diseases, including salt-sensitive hypertension, but studies of the PT mitochondrial bioenergetics have been hampered by the limited yield of PT mitochondria during isolation. Researchers therefore utilize Cx mitochondria as surrogates of PT mitochondria given the high density of PTs in the kidney Cx. However, the Cx has different cell types besides PTs, which may influence the study. To address this limitation, we have developed methods to permeabilize isolated PTs to study its mitochondrial bioenergetics and determined its reliability by comparing against Cx mitochondrial bioenergetics. Methods: PTs were bulk isolated from one kidney of normal Sprague-Dawley rats (6-8 wks) by collagenase perfusion, digestion, and sieving, and Cx mitochondria (Cx m ) were isolated from the other kidney of the same rats using differential centrifugation method. The isolated PTs were permeabilized (PT p ) using mechanical syringe strokes at 3 mg/ml and 5 mg/ml concentrations and compared to determine optimal condition for mitochondrial bioenergetic studies. The PT p and Cx m were studied for mitochondrial respiration (JO 2 ) and membrane potential (dPsi) changes to reveal their functional differences/similarity. Dynamic changes in JO 2 and dPsi were measured in PT p and Cx m using an Oroboros Oxygraph-2k respirometer with the addition of the complex I-linked substrate pyruvate+malate (PM) and complex II-linked substrate succinate (state 2; S2) followed by ADP (state 3; S3) and the uncoupler FCCP (state 5; S5). The JO 2 during S2 and S3 were used to determine the respiratory control index (RCI = S3/S2 JO 2 ) and P/O ratio (ADP consumed/O 2 consumed). Results: The total yield of PT p was estimated to be about twice of Cx m . Hence, dynamic changes in JO 2 of 0.2 mg/ml PT p were compared against 0.1 mg/ml Cx m . Based on preliminary studies, PTs permeabilized at 3 mg/ml was chosen for further study, considering its significant higher RCI than PTs permeabilized at 5 mg/ml. The JO 2 traces of PT p closely resembled to that of Cx m , when energized with PM or succinate. Cx m and PT p did not exhibit a significant difference in states 2 and 3 JO 2 magnitudes and state 3 JO 2 duration, confirming similar mitochondrial efficiency and coupling of oxidative phosphorylation (OxPhos) in the PT p and Cx m . The RCI and P/O ratio were ~6.5 and ~2.7 with PM and ~3.5 and ~1.6 with succinate, respectively, depicting close to theoretical values, confirming the efficient coupling of PT p mitochondria similar to Cx m . In addition, dPsi of PT p and Cx m were also close, confirming the mitochondrial membrane integrity. Conclusion: Our results show that PT p have mitochondrial bioenergetics similar to that of Cx m , in terms of OxPhos and coupling efficiency and mitochondrial membrane integrity. These pivotal preliminary studies provide the foundational basis for employing isolated permeabilized PTs, as opposed to isolated Cx mitochondria, to investigate PT-specific mitochondrial bioenergetics in pathological conditions. Funding: NIH R01 HL151587. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The Goldman-Hodgkin-Katz (GHK) equations have been widely used to model ion channel kinetics in computational models of integrated cellular systems. However, they are derived based on a constant electric field, making them have low accuracy in predicting 'macroscopic' ion channel kinetics - ionic fluxes, electric currents, and membrane potentials. This paper revisits and extends the GHK equations to account for nonlinear electrostatics. A numerical scheme is then presented and analyzed to yield a computable extended GHK equation for calculating ionic fluxes in terms of a set, $ {\mathcal {S}} $ S, of 'microscopic' electrostatic potential values. Furthermore, one finite element iterative method and a Python software package are developed to solve a Poisson-Nernst-Planck ion channel model and applied to the generation of dataset $ {\mathcal {S}} $ S. Numerical results demonstrate significant differences between the extended and classical GHK equations and confirm the importance of considering charge effects in calculating ionic fluxes.