A continuous flow bioreactor system for high-throughput hyperpolarized metabolic flux analysis.

Hyperpolarized carbon-13 labeled compounds are increasingly being used in medical MR imaging (MRI) and MR imaging (MRI) and spectroscopy (MRS) research, due to its ability to monitor tissue and cell metabolism in real-time. Although radiological biomarkers are increasingly being considered as clinical indicators, biopsies are still considered the gold standard for a large variety of indications. Bioreactor systems can play an important role in biopsy examinations because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo. We demonstrate here a proof-of-concept bioreactor and microcoil receive array setup that allows for ex vivo preservation and metabolic NMR spectroscopy on up to three biopsy samples simultaneously, creating an easy-to-use and robust way to simultaneously run multisample carbon-13 hyperpolarization experiments. Experiments using hyperpolarized [1-13C]pyruvate on ML-1 leukemic cells in the bioreactor setup were performed and the kinetic pyruvate-to-lactate rate constants ( k PL ) extracted. The coefficient of variation of the experimentally found k PL s for five repeated experiments was C V = 35 % . With this statistical power, treatment effects of 30%-40% change in lactate production could be easily differentiable with only a few hyperpolarization dissolutions on this setup. Furthermore, longitudinal experiments showed preservation of ML-1 cells in the bioreactor setup for at least 6 h. Rat brain tissue slices were also seen to be preserved within the bioreactor for at least 1 h. This validation serves as the basis for further optimization and upscaling of the setup, which undoubtedly has huge potential in high-throughput studies with various biomarkers and tissue types.

Assessment of focal renal ischemia-reperfusion injury in a porcine model using hyperpolarized [1-13 C]pyruvate MRI.

PURPOSE: Ischemic injury in the kidney is a common pathophysiological event associated with both acute kidney injury and chronic kidney disease; however, regional ischemia-reperfusion as seen in thromboembolic renal disease is often undetectable and thus subclinical. Here, we assessed the metabolic alterations following subclinical focal ischemia-reperfusion injury with hyperpolarized [1-13 C]pyruvate MRI in a porcine model. METHODS: Five pigs were subjected to 60 min of focal kidney ischemia. After 90 min of reperfusion, a multiparametric proton MRI protocol was performed on a clinical 3T scanner system. Metabolism was evaluated using 13 C MRI following infusion of hyperpolarized [1-13 C]pyruvate. Ratios of pyruvate to its detectable metabolites (lactate, bicarbonate, and alanine) were used to quantify metabolism. RESULTS: The focal ischemia-reperfusion injury resulted in injured areas with a mean size of 0.971 cm3 (±1.019). Compared with the contralateral kidney, the injured areas demonstrated restricted diffusion (1269 ± 83.59 × 10-6 mm2 /s vs. 1530 ± 52.73 × 10-6 mm2 /s; p = 0.006) and decreased perfusion (158.8 ± 29.4 mL/100 mL/min vs. 274 ± 63.1 mL/100 mL/min; p = 0.014). In the metabolic assessment, the injured areas displayed increased lactate/pyruvate ratios compared with the entire ipsilateral and the contralateral kidney (0.35 ± 0.13 vs. 0.27 ± 0.1 vs. 0.25 ± 0.1; p = 0.0086). Alanine/pyruvate ratio was unaltered, and we were unable to quantify bicarbonate due to low signal. CONCLUSION: MRI with hyperpolarized [1-13 C]pyruvate in a clinical setup is capable of detecting the acute, subtle, focal metabolic changes following ischemia. This may prove to be a valuable future addition to the renal MRI suite.

Gadolinium-enhanced MRI visualizing backflow at increasing intra-renal pressure in a porcine model.

INTRODUCTION: Intrarenal backflow (IRB) is known to occur at increased intrarenal pressure (IRP). Irrigation during ureteroscopy increases IRP. Complications such as sepsis is more frequent after prolonged high-pressure ureteroscopy. We evaluated a new method to document and visualize intrarenal backflow as a function of IRP and time in a pig model. METHODS: Studies were performed on five female pigs. A ureteral catheter was placed in the renal pelvis and connected to a Gadolinium/ saline solution 3 ml/L for irrigation. An occlusion balloon-catheter was left inflated at the uretero-pelvic junction and connected to a pressure monitor. Irrigation was successively regulated to maintain steady IRP levels at 10, 20, 30, 40 and 50 mmHg. MRI of the kidneys was performed at 5-minute intervals. PCR and immunoassay analyses were executed on the harvested kidneys to detect potential changes in inflammatory markers. RESULTS: MRI showed backflow of Gadolinium into the kidney cortex in all cases. The mean time to first visual damage was 15 minutes and the mean registered pressure at first visual damage was 21 mmHg. On the final MRI the mean percentage of IRB affected kidney was 66% after irrigation with a mean maximum pressure of 43 mmHg for a mean duration of 70 minutes. Immunoassay analyses showed increased MCP-1 mRNA expression in the treated kidneys compared to contralateral control kidneys. CONCLUSIONS: Gadolinium enhanced MRI provided detailed information about IRB that has not previously been documented. IRB occurs at even very low pressures, and these findings are in conflict with the general consensus that keeping IRP below 30-35 mmHg eliminates the risk of post-operative infection and sepsis. Moreover, the level of IRB was documented to be a function of both IRP and time. The results of this study emphasize the importance of keeping IRP and OR time low during ureteroscopy.

Detection of increased pyruvate dehydrogenase flux in the human heart during adenosine stress test using hyperpolarized [1-13C]pyruvate cardiovascular magnetic resonance imaging.

BACKGROUND: Hyperpolarized (HP) [1-13C]pyruvate cardiovascular magnetic resonance (CMR) imaging can visualize the uptake and intracellular conversion of [1-13C]pyruvate to either [1-13C]lactate or 13C-bicarbonate depending on the prevailing metabolic state. The aim of the present study was to combine an adenosine stress test with HP [1-13C]pyruvate CMR to detect cardiac metabolism in the healthy human heart at rest and during moderate stress. METHODS: A prospective descriptive study was performed between October 2019 and August 2020. Healthy human subjects underwent cine CMR and HP [1-13C]pyruvate CMR at rest and during adenosine stress. HP [1-13C]pyruvate CMR images were acquired at the mid-left-ventricle (LV) level. Semi-quantitative assessment of first-pass myocardial [1-13C]pyruvate perfusion and metabolism were assessed. Paired t-tests were used to compare mean values at rest and during stress. RESULTS: Six healthy subjects (two female), age 29 ± 7 years were studied and no adverse reactions occurred. Myocardial [1-13C]pyruvate perfusion was significantly increased during stress with a reduction in time-to-peak from 6.2 ± 2.8 to 2.7 ± 1.3 s, p = 0.02. This higher perfusion was accompanied by an overall increased myocardial uptake and metabolism. The conversion rate constant (kPL) for lactate increased from 11 ± 9 *10-3 to 20 ± 10 * 10-3 s-1, p = 0.04. The pyruvate oxidation rate (kPB) increased from 4 ± 4 *10-3 to 12 ± 7 *10-3 s-1, p = 0.008. This increase in carbohydrate metabolism was positively correlated with heart rate (R2 = 0.44, p = 0.02). CONCLUSIONS: Adenosine stress testing combined with HP [1-13C]pyruvate CMR is feasible and well-tolerated in healthy subjects. We observed an increased pyruvate oxidation during cardiac stress. The present study is an important step in the translation of HP [1-13C]pyruvate CMR into clinical cardiac imaging. Trial registration EUDRACT, 2018-003533-15. Registered 4th of December 2018, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2018-003533-15.

Concentration-dependent effects of dichloroacetate in type 2 diabetic hearts assessed by hyperpolarized [1-13 C]-pyruvate magnetic resonance imaging.

Personalized medicine or individualized therapy promises a paradigm shift in healthcare. This is particularly true in complex and multifactorial diseases such as diabetes and the multitude of related pathophysiological complications. Diabetic cardiomyopathy represents an emerging condition that could be effectively treated if better diagnostic and, in particular, better therapeutic monitoring tools were available. In this study, we investigate the ability to differentiate low and high doses of metabolically targeted therapy in an obese type 2 diabetic rat model. Low-dose dichloroacetate (DCA) treatment was associated with increased lactate production, while no or little change was seen in bicarbonate production. High-dose DCA treatment was associated with a significant metabolic switch towards increased bicarbonate production. These findings support further studies using hyperpolarized [1-13 C]-pyruvate magnetic resonance imaging to differentiate treatment effects and thus allow for personalized titration of therapeutics.

Hyperpolarized pyruvate to measure the influence of PKM2 activation on glucose metabolism in the healthy kidney.

The purpose of the current study was to investigate if hyperpolarized [1-13 C]pyruvate can inform us on the metabolic consequences for the kidney glucose metabolism upon treatment with the pyruvate kinase M2 (PKM2) activator TEPP-46, which has shown promise as a novel therapeutic target for diabetic nephropathy. A healthy male Wistar rat model was employed to study the conversion of [1-13 C]pyruvate to [1-13 C]lactate in the kidney 2 and 4 h after treatment with TEPP-46. All rats were scanned with hyperpolarized [1-13 C]pyruvate kidney MR and vital parameters and blood samples were taken after scanning. The PKM2 activator TEPP-46 increases the glycolytic activity in the kidneys, leading to an increased lactate production, as seen by hyperpolarized pyruvate-to-lactate conversion. The results are supported by an increase in blood lactate, a decreased blood glucose level and an increased pyruvate kinase (PK) activity. The metabolic changes observed in both kidneys following treatment with TEPP-46 are largely independent of renal function and could as such represent a new and extremely sensitive metabolic readout for future drugs targeting PKM2. These results warrant further studies in disease models to evaluate if [1-13 C]pyruvate-to-[1-13 C]lactate conversion can predict treatment outcome.

Cardiac pH-Imaging With Hyperpolarized MRI.

Regardless of the importance of acid-base disturbances in cardiac disease, there are currently no methods for clinical detection of pH in the heart. Several magnetic resonance imaging techniques hold translational promise and may enable in-vivo mapping of pH. We provide a brief overview of these emerging techniques. A particular focus is on the promising advance of magnetic resonance spectroscopy and imaging with hyperpolarized 13C-subtrates as biomarkers of myocardial pH. Hyperpolarization allows quantification of key metabolic substrates and their metabolites. Hereby, pH-sensitive reactions can be probed to provide a measure of acid-base alterations. To date, the most used substrates are [1-13C]pyruvate and 13C-labeled bicarbonate; however, others have been suggested. In cardiovascular medicine, hyperpolarized magnetic resonance spectroscopy has been used to probe acid-base disturbances following pharmacological stress, ischemia and heart failure in animals. In addition to pH-estimation, the technique can quantify fluxes such as the pivotal conversion of pyruvate to lactate via lactate dehydrogenase. This capability, a good safety profile and the fact that the technique is employable in clinical scanners have led to recent translation in early clinical trials. Thus, magnetic resonance spectroscopy and imaging may provide clinical pH-imaging in the near future.

The hemodynamic and metabolic effects of spironolactone treatment in acute kidney injury assessed by hyperpolarized MRI.

Renal ischemia-reperfusion injury (IRI) is one of the most common types of acute kidney injury. Spironolactone has shown promising kidney protective effects in renal IRI in rats. We investigated the hemodynamic and metabolic effects of spironolactone (100 mg/kg) administered immediately after 40 min unilateral kidney ischemia in rats. Hyperpolarized MRI using co-polarized [1-13 C]pyruvate and [13 C,15 N2 ]urea as well as 1 H dynamic contrast-enhanced (DCE) MRI was performed 24 h after induction of ischemia. We found a significant decrease in renal blood flow (RBF) in the ischemic kidney compared with the contralateral one measured using DCE and [13 C,15 N2 ]urea. The RBF measured using [1-13 C]pyruvate and [13 C,15 N2 ]urea was significantly altered by spironolactone. The RBFs in the ischemic kidney compared with the contralateral kidney were decreased similarly as measured using both [13 C,15 N2 ]urea and [1-13 C]pyruvate in the spironolactone-treated group. Spironolactone treatment increased the perfusion-corrected pyruvate metabolism by 54% in both the ischemic and contralateral kidney. Furthermore, we showed a correlation between vascular permeability using a histological Evans blue analysis and the ratio of the volumes of distribution (VoDs), ie VoD-[13 C,15 N2 ]urea/VoD-[1-13 C]pyruvate. This suggests that [13 C,15 N2 ]urea/[1-13 C]pyruvate VoD ratio may be a novel indicator of renal vascular permeability associated with renal damage in rodents.

Hyperpolarized [1,4-13C]fumarate imaging detects microvascular complications and hypoxia mediated cell death in diabetic nephropathy.

Today, there is a general lack of prognostic biomarkers for development of renal disease and in particular diabetic nephropathy. Increased glycolytic activity, lactate accumulation and altered mitochondrial oxygen utilization are hallmarks of diabetic kidney disease. Fumarate hydratase activity has been linked to mitochondrial dysfunction as well as activation of the hypoxia inducible factor, induction of apoptosis and necrosis. Here, we investigate fumarate hydratase activity in biofluids in combination with the molecular imaging probe, hyperpolarized [1,4-13C2]fumarate, to identify the early changes associated with hemodynamics and cell death in a streptozotocin rat model of type 1 diabetes. We found a significantly altered hemodynamic signature of [1,4-13C2]fumarate in the diabetic kidneys as well as an systemic increased metabolic conversion of fumarate-to-malate, indicative of increased cell death associated with progression of diabetes, while little to no renal specific conversion was observed. This suggest apoptosis as the main cause of cell death in the diabetic kidney. This is likely resulting from an increased reactive oxygen species production following uncoupling of the electron transport chain at complex II. The mechanism coupling the enzyme leakage and apoptotic phenotype is hypoxia inducible factor independent and seemingly functions as a protective mechanism in the kidney cells.

Metabolic reprogramming associated with progression of renal ischemia reperfusion injury assessed with hyperpolarized [1-13C]pyruvate.

Acute kidney injury is a major clinical challenge affecting as many as 1 percent of all hospitalized patients. Currently it is not possible to accurately stratify and predict the outcome of the individual patient. Increasing evidence supports metabolic reprogramming as a potential target for new biomarkers. Hyperpolarized [1-13C]pyruvate imaging is a promising new tool for evaluating the metabolic status directly in the kidneys. We here investigate the prognostic potential of hyperpolarized [1-13C]pyruvate in the setting of acute kidney injury in a rodent model of ischemia reperfusion. A significant correlation was found between the intra-renal metabolic profile 24 hours after reperfusion and 7 days after injury induction, as well as a correlation with the conventional plasma creatinine biomarker of renal function and markers of renal injury. This leads to a possible outcome prediction of renal function and injury development from a metabolic profile measured in vivo. The results support human translation of this new technology to renal patients as all experiements have been performed using clinical MRI equipment.

Hyperpolarized [1-13 C] alanine production: A novel imaging biomarker of renal fibrosis.

PURPOSE: Renal tubulointerstitial fibrosis is strongly linked to the progressive decline of renal function seen in chronic kidney disease. State-of-the-art noninvasive diagnostic modalities are currently unable to detect the earliest changes associated with the onset of fibrosis. This study was undertaken to evaluate the potential for detecting the earliest alterations in fibrogenesis using a biofluid-based method and metabolic hyperpolarized [1-13 C]pyruvate imaging. METHODS: We evaluated renal fibrosis in a combined ischemia reperfusion-induced and streptozotocin-induced diabetic nephropathy rodent model by hyperpolarized [1-13 C]pyruvate MRI and correlated the metabolic MRI parameters with biomarkers of fibrosis measured on renal tissue and plasma/urine. RESULTS: The hyperglycemic rats experienced maladaptive injury repair after the ischemic insults, as shown by the elevation in the injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Renal function was significantly impaired in the ischemic hyperglycemic kidney, as seen in the reduced perfusion and single-kidney glomerular filtration rate. A deranged energy metabolism was detected in the ischemic hyperglycemic kidney, as seen in the reduced fractional perfusion of lactate. Renal fibrosis biomarkers correlated significantly with the alanine production. CONCLUSION: Hyperpolarized carbon-13 MRI provides a promising approach to assess renal fibrosis in an animal model of fibrotic chronic kidney disease. In particular, the metabolic supply of amino acids for fibrogenesis (alanine production) correlates well with biomarkers of fibrosis. Thus, [1-13 C]pyruvate-to-[1-13 C]alanine conversion might be a candidate for noninvasive assessment of renal fibrogenesis.

High Intrarenal Lactate Production Inhibits the Renal Pseudohypoxic Response to Acutely Induced Hypoxia in Diabetes.

Intrarenal hypoxia develops within a few days after the onset of insulinopenic diabetes in an experimental animal model (ie, a model of type-1 diabetes). Although diabetes-induced hypoxia results in increased renal lactate formation, mitochondrial function is well maintained, a condition commonly referred to as pseudohypoxia. However, the metabolic effects of significantly elevated lactate levels remain unclear. We therefore investigated in diabetic animals the response to acute intrarenal hypoxia in the presence of high renal lactate formation to delineate mechanistic pathways and compare these findings to healthy control animals. Hyperpolarized 13C-MRI and blood oxygenation level-dependent 1H-MRI was used to investigate the renal metabolism of [1-13C]pyruvate and oxygenation following acutely altered oxygen content in the breathing gas in a streptozotocin rat model of type-1 diabetes with and without insulin treatment and compared with healthy control rats. The lactate signal in the diabetic kidney was reduced by 12%-16% during hypoxia in diabetic rats irrespective of insulin supplementation. In contrast, healthy controls displayed the well-known Pasteur effect manifested as a 10% increased lactate signal following reduction of oxygen in the inspired air. Reduced expression of the monocarboxyl transporter-4 may account for altered response to hypoxia in diabetes with a high intrarenal pyruvate-to-lactate conversion. Reduced intrarenal lactate formation in response to hypoxia in diabetes shows the existence of a different metabolic phenotype, which is independent of insulin, as insulin supplementation was unable to affect the pyruvate-to-lactate conversion in the diabetic kidney.

Metabolic consequences of lactate dehydrogenase inhibition by oxamate in hyperglycemic proximal tubular cells.

Diabetic kidney disease (DKD) is associated with altered metabolic patterns, leading to increased lactate production even in the presence of sufficient oxygen supply. Studies have shown hyperglycemia to be an important factor in determining development of DKD. Here we explore the metabolic consequences of lactate dehydrogenase (LDH) inhibition exerted by the LDH inhibitor, oxamate, in the isolated rat renal proximal tubular cells (NRK-52E) under hyperglycemic conditions. Cells treated with oxamate (100 mM) for 24 h, with or without high D-glucose (25 mM) load, were investigated with hyperpolarized [1-13C]pyruvate in a 1T NMR system. Respiratory measurements using an oxygen microsensor system was conducted. Oxamate treatment of cells with or without the presences of high D-glucose, reduced the lactate production/accumulation with 36.5% or 22.5% respectively. Reduced proliferation, hypertrophic effects, as well as elevated vascular endothelial growth factor (VEGF) expression in the NRK-52E cells were found. The increased glycolytic flux in high D-glucose cultured NRK-52E cells resulted in an upregulation of the cellular oxygen consumption rate upon treatment with oxamate. Our findings suggested that in vitro cultured NRK-52E cells exposed to hyperglycemic conditions, could redirect the glycolytic flux towards oxidative phosphorylation by LDH inhibition. This link between aerobic and anaerobic metabolism may be determined by the redox balance (NAD+/NADH ratio). In conclusion, hyperglycemic conditions and oxamate treatment alters the metabolic phenotype of NRK-52E cells towards increased oxygen utilization mediated by a decreased NAD+/NADH ratio, which in turn decreases cell proliferation/survival.

Hyperpolarized [1-13 C]pyruvate MRI can image the metabolic shift in cardiac metabolism between the fasted and fed state in a porcine model.

PURPOSE: Owing to its noninvasive nature, hyperpolarized MRI may improve delineation of myocardial metabolic derangement in heart disease. However, consistency may depend on the changeable nature of cardiac metabolism in relation to whole-body metabolic state. This study investigates the impact of feeding status on cardiac hyperpolarized MRI in a large animal model resembling human physiology. METHODS: Thirteen 30-kg pigs were subjected to an overnight fast, and 5 pigs were fed a carbohydrate-rich meal on the morning of the experiments. Vital parameters and blood samples were registered. All pigs were then scanned by hyperpolarized [1-13 C]pyruvate cardiac MRI, and results were compared between the 2 groups and correlated with circulating substrates and hormones. RESULTS: The fed group had higher blood glucose concentration and mean arterial pressure than the fasted group. Plasma concentrations of free fatty acids (FFAs) were decreased in the fed group, whereas plasma insulin concentrations were similar between groups. Hyperpolarized MRI showed that fed animals had increased lactate/pyruvate, alanine/pyruvate, and bicarbonate/pyruvate ratios. Metabolic ratios correlated negatively with FFA levels. CONCLUSION: Hyperpolarized MR can identify the effects of different metabolic states on cardiac metabolism in a large animal model. Unlike previous rodent studies, all metabolic derivatives of pyruvate increased in the myocardium of fed pigs. Carbohydrate-rich feeding seems to be a feasible model for standardized, large animal hyperpolarized MRI studies of myocardial carbohydrate metabolism.

Ex Vivo Human Placenta Perfusion, Metabolic and Functional Imaging for Obstetric Research-A Feasibility Study.

Placenta metabolism is closely linked to pregnancy outcome, and few modalities are currently available for studying the human placenta. Here, we aimed to investigate a novel ex vivo human placenta perfusion system for metabolic imaging using hyperpolarized [1-13C]pyruvate. The metabolic effects of 3 different human placentas were investigated using functional and metabolic magnetic resonance imaging. The placenta glucose metabolism and hemodynamics were characterized with hyperpolarized [1-13C]pyruvate magnetic resonance imaging and by dynamic contrast-enhanced (DCE) imaging. Hyperpolarized [1-13C]pyruvate showed a decrease in the 13C-lactate/13C-pyruvate ratio from the highest to the lowest metabolic active placenta. The metabolic profile was complemented by a more homogenous distributed hemodynamic response, with a longer mean transit time and higher blood volume. This study shows different placenta metabolic and hemodynamic features associated with the placenta functional status using hyperpolarized magnetic resonance ex vivo. This study supports further studies using ex vivo metabolic imaging of the placenta alterations associated with pregnancy complications.

Renal Energy Metabolism Following Acute Dichloroacetate and 2,4-Dinitrophenol Administration: Assessing the Cumulative Action with Hyperpolarized [1-13C]Pyruvate MRI.

Numerous patient groups receive >1 medication and as such represent a potential point of improvement in today's healthcare setup, as the combined or cumulative effects are difficult to monitor in an individual patient. Here we show the ability to monitor the pharmacological effect of 2 classes of medications sequentially, namely, 2,4-dinitrophenol, a mitochondrial uncoupler, and dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, both targeting the oxygen-dependent energy metabolism. We show that although the 2 drugs target 2 different metabolic pathways connected ultimately to oxygen metabolism, we could distinguish the 2 in vivo by using hyperpolarized [1-13C]pyruvate magnetic resonance imaging. A statistically significantly different pyruvate dehydrogenase flux was observed by reversing the treatment order of 2,4-dinitrophenol and dichloroacetate. The significance of this study is the demonstration of the ability to monitor the metabolic cumulative effects of 2 distinct therapeutics on an in vivo organ level using hyperpolarized magnetic resonance imaging.

Imaging porcine cardiac substrate selection modulations by glucose, insulin and potassium intervention: A hyperpolarized [1-13 C]pyruvate study.

Cardiac metabolism has received considerable attention in terms of both diagnostics and prognostics, as well as a novel target for treatment. As human trials involving hyperpolarized magnetic resonance in the heart are imminent, we sought to evaluate the general feasibility of detection of an imposed shift in metabolic substrate utilization during metabolic modulation with glucose-insulin-potassium (GIK) infusion, and thus the limitations associated with this strategy, in a large animal model resembling human physiology. Four [1-13 C]pyruvate injections did not alter the blood pressure or ejection fraction over 180 min. Hyperpolarized [1-13 C]pyruvate conversion showed a generally high reproducibility, with intraclass correlation coefficients between the baseline measurements at 0 and 30 min as follows: lactate to pyruvate, 0.85; alanine to pyruvate, 1.00; bicarbonate to pyruvate, 0.83. This study demonstrates that hyperpolarized [1-13 C]pyruvate imaging is a feasible technique for cardiac studies and shows a generally high reproducibility in fasted large animals. GIK infusion increases the metabolic conversion of pyruvate to its metabolic derivatives lactate, alanine and bicarbonate, but with increased variability.

Fumarase activity: an in vivo and in vitro biomarker for acute kidney injury.

Renal ischemia/reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI), and at present, there is a lack of reliable biomarkers that can diagnose AKI and measure early progression because the commonly used methods cannot evaluate single-kidney IRI. Hyperpolarized [1,4-13C2]fumarate conversion to [1,4-13C2]malate by fumarase has been proposed as a measure of necrosis in rat tumor models and in chemically induced AKI rats. Here we show that the degradation of cell membranes in connection with necrosis leads to elevated fumarase activity in plasma and urine and secondly that hyperpolarized [1,4-13C2]malate production 24 h after reperfusion correlates with renal necrosis in a 40-min unilateral ischemic rat model. Fumarase activity screening on bio-fluids can detect injury severity, in bilateral as well as unilateral AKI models, differentiating moderate and severe AKI as well as short- and long-term AKI. Furthermore after verification of renal injury by bio-fluid analysis the precise injury location can be monitored by in vivo measurements of the fumarase activity non-invasively by hyperpolarized [1,4-13C]fumarate MR imaging. The combined in vitro and in vivo biomarker of AKI responds to the essential requirements for a new reliable biomarker of AKI.

Antioxidant treatment attenuates lactate production in diabetic nephropathy.

The early progression of diabetic nephropathy is notoriously difficult to detect and quantify before the occurrence of substantial histological damage. Recently, hyperpolarized [1-13C]pyruvate has demonstrated increased lactate production in the kidney early after the onset of diabetes, implying increased lactate dehydrogenase activity as a consequence of increased nicotinamide adenine dinucleotide substrate availability due to upregulation of the polyol pathway, i.e., pseudohypoxia. In this study, we investigated the role of oxidative stress in mediating these metabolic alterations using state-of-the-art hyperpolarized magnetic resonance (MR) imaging. Ten-week-old female Wistar rats were randomly divided into three groups: healthy controls, untreated diabetic (streptozotocin treatment to induce insulinopenic diabetes), and diabetic, receiving chronic antioxidant treatment with TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) via the drinking water. Examinations were performed 2, 3, and 4 wk after the induction of diabetes by using a 3T Clinical MR system equipped with a dual tuned 13C/1H-volume rat coil. The rats received intravenous hyperpolarized [1-13C]pyruvate and were imaged using a slice-selective 13C-IDEAL spiral sequence. Untreated diabetic rats showed increased renal lactate production compared with that shown by the controls. However, chronic TEMPOL treatment significantly attenuated diabetes-induced lactate production. No significant effects of diabetes or TEMPOL were observed on [13C]alanine levels, indicating an intact glucose-alanine cycle, or [13C]bicarbonate, indicating normal flux through the Krebs cycle. In conclusion, this study demonstrates that diabetes-induced pseudohypoxia, as indicated by an increased lactate-to-pyruvate ratio, is significantly attenuated by antioxidant treatment. This demonstrates a pivotal role of oxidative stress in renal metabolic alterations occurring in early diabetes.