Magnetic resonance imaging as a noninvasive adjunct to conventional assessment of functional differences between kidneys in vivo and during ex vivo normothermic machine perfusion.

Normothermic machine perfusion (NMP) is increasingly considered for pretransplant kidney quality assessment. However, fundamental questions about differences between in vivo and ex vivo renal function, as well as the impact of ischemic injury on ex vivo physiology, remain unanswered. This study utilized magnetic resonance imaging (MRI), alongside conventional parameters to explore differences between in vivo and ex vivo renal function and the impact of warm ischemia on a kidney's behavior ex vivo. Renal MRI scans and samples were obtained from living pigs (n = 30) in vivo. Next, kidney pairs were procured and exposed to minimal, or 75 minutes of warm ischemia, followed by 6 hours of hypothermic machine perfusion. Both kidneys simultaneously underwent 6-hour ex vivo perfusion in MRI-compatible NMP circuits to obtain multiparametric MRI data. Ischemically injured ex vivo kidneys showed a significantly altered regional blood flow distribution compared to in vivo and minimally damaged organs. Both ex vivo groups showed diffusion restriction relative to in vivo. Our findings underscore the differences between in vivo and ex vivo MRI-based renal characteristics. Therefore, when assessing organ viability during NMP, it should be considered to incorporate parameters beyond the conventional functional markers that are common in vivo.

The number of glomeruli and pyruvate metabolism is not strongly coupled in the healthy rat kidney.

PURPOSE: The number of glomeruli is different in men and women, as they also present different prevalence and progression of chronic kidney disease. A recent study has demonstrated a potential difference in renal metabolism between sexes, and a potential explanation could be the differences in glomeruli number. This study investigates the potential correlation between glomerular number and pyruvate metabolism in healthy kidneys. METHODS: This study is an experimental study with rats (N = 12). We used cationized-ferritin MRI to visualize and count glomeruli and hyperpolarized [1-13 C]pyruvate to map the metabolism. Dynamic contrast-enhanced MRI was used to analyze kidney hemodynamics using gadolinium tracer. RESULTS: Data showed no or subtle correlation between the number of glomeruli and the pyruvate metabolism. Minor differences were observed in the number of glomeruli (female = 24,509 vs. male = 26 350; p = .16), renal plasma flow (female = 606.6 vs. male= 455.7 ml/min/100 g; p = .18), and volume of distribution (female = 87.44 vs. male = 76.61 ml/100 ml; p = .54) between sexes. Mean transit time was significantly prolonged in males compared with females (female = 8.868 s vs. male = 10.63 s; p = .04). CONCLUSION: No strong statistically significant correlation between the number of glomeruli and the pyruvate metabolism was found in healthy rat kidneys.

Remodeling after myocardial infarction and effects of heart failure treatment investigated by hyperpolarized [1-13 C]pyruvate magnetic resonance spectroscopy.

PURPOSE: Hyperpolarized [1-13 C]pyruvate MRS can measure cardiac metabolism in vivo. We investigated whether [1-13 C]pyruvate MRS could predict left ventricular remodeling following myocardial infarction (MI), long-term left ventricular effects of heart failure medication, and could identify responders to treatment. METHODS: Thirty-five rats were scanned with hyperpolarized [1-13 C]pyruvate MRS 3 days after MI or sham surgery. The animals were re-examined after 30 days of therapy with ß-blockers and ACE-inhibitors (active group, n = 12), placebo treatment (placebo group, n = 13) or no treatment (sham group, n = 10). Furthermore, heart tissue mitochondrial respiratory capacity was assessed by high-resolution respirometry. Metabolic results were compared between groups, over time and correlated to functional MR data at each time point. RESULTS: At 30 ± 0.5 days post MI, left ventricular ejection fraction (LVEF) differed between groups (sham, 77% ± 1%; placebo, 52% ± 3%; active, 63% ± 2%, P < .001). Cardiac metabolism, measured by both hyperpolarized [1-13 C]pyruvate MRS and respirometry, neither differed between groups nor between baseline and follow-up. Three days post MI, low bicarbonate + CO2 /pyruvate ratio was associated with low LVEF. At follow-up, in the active group, a poor recovery of LVEF was associated with high bicarbonate + CO2 /pyruvate ratio, as measured by hyperpolarized MRS. CONCLUSION: In a rat model of moderate heart failure, medical treatment improved function, but did not on average influence [1-13 C]pyruvate flux as measured by MRS; however, responders to heart failure medication had reduced capacity for carbohydrate metabolism.

Organ-specific metabolic profiles of the liver and kidney during brain death and afterwards during normothermic machine perfusion of the kidney.

We investigated metabolic changes during brain death (BD) using hyperpolarized magnetic resonance (MR) spectroscopy and ex vivo graft glucose metabolism during normothermic isolated perfused kidney (IPK) machine perfusion. BD was induced in mechanically ventilated rats by inflation of an epidurally placed catheter; sham-operated rats served as controls. Hyperpolarized [1-13 C]pyruvate MR spectroscopy was performed to quantify pyruvate metabolism in the liver and kidneys at 3 time points during BD, preceded by injecting hyperpolarized[1-13 C]pyruvate. Following BD, glucose oxidation was measured using tritium-labeled glucose (d-6-3H-glucose) during IPK reperfusion. Quantitative polymerase chain reaction and biochemistry were performed on tissue/plasma. Immediately following BD induction, lactate increased in both organs (liver: eµd 0.21, 95% confidence interval [CI] [-0.27, -0.15]; kidney: eµd 0.26, 95% CI [-0.40, -0.12]. After 4 hours of BD, alanine production decreased in the kidney (eµd 0.14, 95% CI [0.03, 0.25], P < .05). Hepatic lactate and alanine profiles were significantly different throughout the experiment between groups (P < .01). During IPK perfusion, renal glucose oxidation was reduced following BD vs sham animals (eµd 0.012, 95% CI [0.004, 0.03], P < .001). No differences in enzyme activities were found. Renal gene expression of lactate-transporter MCT4 increased following BD (P < .01). In conclusion, metabolic processes during BD can be visualized in vivo using hyperpolarized magnetic resonance imaging and with glucose oxidation during ex vivo renal machine perfusion. These techniques can detect differences in the metabolic profiles of the liver and kidney following BD.

Detection of acute kidney injury with hyperpolarized [13 C, 15 N]Urea and multiexponential relaxation modeling.

PURPOSE: To assess the utility of Laplacian fitting to describe the differences in hyperpolarized [13 C, 15 N]urea T2 relaxation in ischemic and healthy rodent kidneys. METHODS: Six rats with unilateral renal ischemia were investigated. [13 C, 15 N]Urea T2 mapping was undertaken with a radial fast spin echo method, with subsequent postprocessing performed with regularized Laplacian fitting. RESULTS: Simulations showed that Laplacian fitting was stable down to a signal-to-noise ratio of 20. In vivo results showed a significant increase in the mono- and decrease in biexponential pools in ischemia reperfusion injury kidneys, in comparison to healthy (14 ± 10% versus 4 ± 2%, 85 ± 10% versus 95 ± 3%; P < .05). CONCLUSION: We demonstrate, for the first time, the differences in multiexponential behavior of [13 C, 15 N]urea between the healthy and ischemic rodent kidney. The distribution of relaxation pools were found to be both visually and numerically significantly different. The ability to improve the information level in hyperpolarized MR, by using the relaxation contrast mechanisms is an appealing option, that can easily be adopted in large animals and even in clinical studies 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.

Glucagon infusion alters the hyperpolarized 13 C-urea renal hemodynamic signature.

Renal urea handling is central to the urine concentrating mechanism, and as such the ability to image urea transport in the kidney is an important potential imaging biomarker for renal functional assessment. Glucagon levels associated with changes in dietary protein intake have been shown to influence renal urea handling; however, the exact mechanism has still to be fully understood. Here we investigate renal function and osmolite distribution using [13 C,15 N] urea dynamics and 23 Na distribution before and 60 min after glucagon infusion in six female rats. Glucagon infusion increased the renal [13 C,15 N] urea mean transit time by 14%, while no change was seen in the sodium distribution, glomerular filtration rate or oxygen consumption. This change is related to the well-known effect of increased urea excretion associated with glucagon infusion, independent of renal functional effects. This study demonstrates for the first time that hyperpolarized 13 C-urea enables monitoring of renal urinary excretion effects in vivo.

Acute renal metabolic effect of metformin assessed with hyperpolarised MRI in rats.

AIMS/HYPOTHESIS: Metformin inhibits hepatic mitochondrial glycerol phosphate dehydrogenase, thereby increasing cytosolic lactate and suppressing gluconeogenesis flux in the liver. This inhibition alters cytosolic and mitochondrial reduction-oxidation (redox) potential, which has been reported to protect organ function in several disease states including diabetes. In this study, we investigated the acute metabolic and functional changes induced by metformin in the kidneys of both healthy and insulinopenic Wistar rats used as a model of diabetes. METHODS: Diabetes was induced by intravenous injection of streptozotocin, and kidney metabolism in healthy and diabetic animals was investigated 4 weeks thereafter using hyperpolarised 13C-MRI, Clark-type electrodes and biochemical analysis. RESULTS: Metformin increased renal blood flow, but did not change total kidney oxygen consumption. In healthy rat kidneys, metformin increased [1-13C]lactate production and reduced mitochondrial [1-13C]pyruvate oxidation (decreased the 13C-bicarbonate/[1-13C]pyruvate ratio) within 30 min of administration. Corresponding alterations to indices of mitochondrial, cytosolic and whole-cell redox potential were observed. Pyruvate oxidation was maintained in the diabetic rats, suggesting that the diabetic state abrogates metabolic reprogramming caused by metformin. CONCLUSIONS/INTERPRETATION: This study demonstrates that metformin-induced acute metabolic alterations in healthy kidneys favoured anaerobic metabolism at the expense of aerobic metabolism. The results suggest that metformin directly alters the renal redox state, with elevated renal cytosolic redox states as well as decreased mitochondrial redox state. These findings suggest redox biology as a novel target to eliminate the renal complications associated with metformin treatment in individuals with impaired renal function.

Hyperpolarized 13 C,15 N2 -urea T2 relaxation changes in acute kidney injury.

PURPOSE: To investigate the correlation between renal ischemia and 13 C-urea T2 relaxation rate in an acute kidney injury (AKI) rat model. METHODS: Six rats subjected to unilateral renal ischemia were investigated. Creatinine clearance, urine output, plasma creatinine as well as blood-urea nitrogen (BUN) values were acquired before and after the procedure. 1 H T2* mapping was acquired using blood oxygenation level dependent (BOLD) MRI and hyperpolarized 13 C-urea T2 mapping was acquired using a 2D golden-angle radial approach. Kidney perfusion was estimated using noncontrast flow alternating inversion recovery arterial spin labeling. RESULTS: All rats showed clinical signs of AKI with increased plasma creatinine and increased BUN. Whole kidney 13 C-urea T2 significantly decreased 26% (P = 0.001) 24 h after reperfusion. A significantly different (3.7 times steeper; P = 0.008) osmolality gradient was observed in the contralateral kidney (P = 0.008; R2 = 0.86) compared with the postischemic kidney (P = 0.0004, R2 =0.97). Whole kidney T2* signal (P = 0.14) and T2* gradient (P = 0.26) was similar between the two kidneys. Oxygen availability dependency on 13 C-urea T2 was investigated by means of the correlation between the BOLD and T2 signals; a statistically significant difference (P = 0.03) was found in the contralateral kidney (P = 0.0001; R2 = 0.95), but not in the postischemic kidney (P = 0.31; R2 = 0.25). CONCLUSION: We demonstrate that hyperpolarized [13 C,15 N2 ]urea T2 relaxation correlates with renal oxygen tension ( T2*) in the healthy contralateral kidney, but not in the postischemic kidney. The whole kidney T2 relaxation difference between the postischemic and contralateral kidney may originate from altered blood volume in the postischemic kidney. Magn Reson Med 80:696-702, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Effects of anesthesia on renal function and metabolism in rats assessed by hyperpolarized MRI.

PURPOSE: Anesthesia is necessary for most animal studies requiring invasive procedures. It is well documented that various types of anesthesia modulate a wide variety of important metabolic and functional processes in the body, and as such, represent a potential limitation in the study design. In the present study, we aimed to investigate the renal functional and metabolic consequences of 3 typical rodent anesthetics used in preclinical MRI: sevoflurane, inaction, and a mixture of fentanyl, fluanisone, and midazolam (FFM). METHODS: The renal effects of 3 different classes of anesthetics (inactin, servoflurane, and FFM) were investigated using functional and metabolic MRI. The renal glucose metabolism and hemodynamics was characterized with hyperpolarized [1-13 C]pyruvate MRI and by DCE imaging. RESULTS: Rats receiving sevoflurane or FFM had blood glucose levels that were 1.3-fold to 1.4-fold higher than rats receiving inactin. A 2.9-fold and 4.8-fold increased 13 C-lactate/13 C-pyruvate ratio was found in the FFM mixture anesthetized group compared with the sevoflurane and the inactin anesthetized groups. The FFM anesthesia resulted in a 50% lower renal plasma flow compared with the sevoflurane and the inactin anesthetized groups. CONCLUSION: This study demonstrates different renal metabolic and hemodynamic changes under 3 different anesthetics, using hyperpolarized MR in rats. Inactin and sevoflurane were found to affect the renal hemodynamic and metabolic status to a lesser degree than FFM. Sevoflurane anesthesia is particularly easy to induce and maintain during the whole anesthesia procedure, and as such, represents a good alternative to inaction, although it alters the blood glucose level.

In situ lactate dehydrogenase activity: a novel renal cortical imaging biomarker of tubular injury?

Renal ischemia-reperfusion injury is the state of which a tissue experiences injury after a phase of restrictive blood supply and recirculation. Ischemia-reperfusion injury (I/R-I) is a leading cause of acute kidney injury (AKI) in several disease states, including kidney transplantation, sepsis, and hypovolemic shock. The most common methods to evaluate AKI are creatinine clearance, plasma creatinine, blood urea nitrogen, or renal histology. However, currently, there are no precise methods to directly assess renal injury state noninvasively. Hyperpolarized 13C-pyruvate MRI enables noninvasive accurate quantification of the in vivo conversion of pyruvate to lactate, alanine, and bicarbonate. In the present study, we investigated the in situ alterations of metabolic conversion of pyruvate to lactate, alanine, and bicarbonate in a unilateral I/R-I rat model with 30 min and 60 min of ischemia followed by 24 h of reperfusion. The pyruvate conversion was unaltered compared with sham in the 30 min I/R-I group, while a significant reduced metabolic conversion was found in the postischemic kidney after 60 min of ischemia. This indicates that after 30 min of ischemia, the kidney maintains normal metabolic function in spite of decreased kidney function, whereas the postischemic kidney after 60 min of ischemia show a generally reduced metabolic enzyme activity concomitant with a reduced kidney function. We have confidence that these findings can have a high prognostic value in prediction of kidney injury and the outcome of renal injury.

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.

Diabetes induced renal urea transport alterations assessed with 3D hyperpolarized 13 C,15 N-Urea.

PURPOSE: In the current study, we investigated hyperpolarized urea as a possible imaging biomarker of the renal function by means of the intrarenal osmolality gradient. METHODS: Hyperpolarized three-dimensional balanced steady state 13 C MRI experiments alongside kidney function parameters and quantitative polymerase chain reaction measurements was performed on two groups of rats, a streptozotocin type 1 diabetic group and a healthy control group. RESULTS: A significant decline in intrarenal steepness of the urea gradient was found after 4 weeks of untreated insulinopenic diabetes in agreement with an increased urea transport transcription. CONCLUSION: MRI and hyperpolarized [13 C,15 N]urea can monitor the changes in the corticomedullary urea concentration gradients in diabetic and healthy control rats. Magn Reson Med 77:1650-1655, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Hyperpolarized 13C urea relaxation mechanism reveals renal changes in diabetic nephropathy.

PURPOSE: Our aim was to assess a novel (13) C radial fast spin echo golden ratio single shot method for interrogating early renal changes in the diabetic kidney, using hyperpolarized (HP) [(13) C,(15) N2 ]urea as a T2 relaxation based contrast bio-probe. METHODS: A novel HP (13) C MR contrast experiment was conducted in a group of streptozotocin type-1 diabetic rat model and age matched controls. RESULTS: A significantly different relaxation time (P = 0.004) was found in the diabetic kidney (0.49 ± 0.03 s) compared with the controls (0.64 ± 0.02 s) and secondly, a strong correlation between the blood oxygen saturation level and the relaxation times were observed in the healthy controls. CONCLUSION: HP [(13) C,(15) N2 ]urea apparent T2 mapping may be a useful for interrogating local renal pO2 status and renal tissue alterations. Magn Reson Med, 2015. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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.

Sex Differences in Kidney Function and Metabolism Assessed Using Hyperpolarized [1-13C]Pyruvate Interleaved Spectroscopy and Nonspecific Imaging.

Metabolic sex differences have recently been shown to be particularly important in tailoring treatment strategies. Sex has a major effect on fat turnover rates and plasma lipid delivery in the body. Differences in kidney structure and transporters between male and female animals have been found. Here we investigated sex-specific renal pyruvate metabolic flux and whole-kidney functional status in age-matched healthy Wistar rats. Blood oxygenation level-dependent and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) were used to assess functional status. Hyperpolarized [1-13C]pyruvate was used to assess the metabolic differences between male and female rats. Female rats had a 41% ± 3% and 41% ± 5% lower absolute body and kidney weight, respectively, than age-matched male rats. No difference was seen between age-matched male and female rats in the kidney-to-body weight ratio. A 56% ± 11% lower lactate production per mL/100 mL/min was found in female rats than in age-matched male rats measured by hyperpolarized magnetic resonance and DCE MRI. Female rats had a 33% ± 11% higher glomerular filtration rate than age-matched male rats measured by DCE MRI. A similar renal oxygen tension (T2*) was found between age-matched male and female rats as shown by blood oxygenation level-dependent MRI. The results were largely independent of the pyruvate volume and the difference in body weight. This study shows an existing metabolic difference between kidneys in age-matched male and female rats, which indicates that sex differences need to be considered when performing animal experiments.

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.

Assessment of mouse liver [1-13C]pyruvate metabolism by dynamic hyperpolarized MRS.

Hyperpolarized [1-13C]pyruvate magnetic resonance (MR) spectroscopy has the unique ability to detect real-time metabolic changes in vivo owing to its high sensitivity compared with thermal MR and high specificity compared with other metabolic imaging methods. The aim of this study was to explore the potential of hyperpolarized MR spectroscopy for quantification of liver pyruvate metabolism during a hyperinsulinemic-isoglycemic clamp in mice. Hyperpolarized [1-13C]pyruvate was used for in vivo MR spectroscopy of liver pyruvate metabolism in mice. Mice were divided into two groups: (i) non-stimulated 5-h fasted mice and (ii) hyperinsulinemic-isoglycemic clamped mice. During clamp conditions, insulin and donor blood were administered at a constant rate, whereas glucose was infused to maintain isoglycemia. When steady state was reached, insulin-stimulated mice were rapidly infused with hyperpolarized [1-13C]pyruvate for real-time tracking of the dynamic distribution of metabolic derivatives from pyruvate, such as [1-13C]lactate, [1-13C]alanine and [13C]bicarbonate. Isotopomer analysis of plasma glucose confirmed 13C-incorporation from [1-13C]pyruvate into glucose was increased in fasted mice compared with insulin-stimulated mice, demonstrating an increased gluconeogenesis in fasted mice. The AUC ratios for [1-13C]alanine/[1-13C]pyruvate (38.2%), [1-13C]lactate/[1-13C]pyruvate (41.8%) and [13C]bicarbonate/[1-13C]pyruvate (169%) all increased significantly during insulin stimulation. Hyperpolarized [1-13C]pyruvate can be used for in vivo MR spectroscopy of liver pyruvate metabolism during hyperinsulinemic-isoglycemic clamp conditions. Under these conditions, insulin decreased gluconeogenesis and increased [1-13C]alanine, [1-13C]lactate and [13C]bicarbonate after a [1-13C]pyruvate bolus. This application of in vivo spectroscopy has the potential to identify impairments in specific metabolic pathways in the liver associated with obesity, insulin resistance and nonalcoholic fatty liver disease.

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.

Hyperpolarized [1-13 C] pyruvate as a possible diagnostic tool in liver disease.

Introduction of hyperpolarized magnetic resonance in preclinical studies and lately translation to patients provides new detailed in vivo information of metabolic flux in organs. Hyperpolarized magnetic resonance based on 13 C enriched pyruvate is performed without ionizing radiation and allows quantification of the pyruvate conversion products: alanine, lactate and bicarbonate in real time. Thus, this methodology has a promising potential for in vivo monitoring of energetic alterations in hepatic diseases. Using 13 C pyruvate, we investigated the metabolism in the porcine liver before and after intravenous injection of glucose. The overall mean lactate to pyruvate ratio increased significantly after the injection of glucose whereas the bicarbonate to pyruvate ratio was unaffected, representative of the levels of pyruvate entering the tricarboxylic acid cycle. Similarly, alanine to pyruvate ratio did not change. The increased lactate to pyruvate ratio over time showed an exponential correlation with insulin, glucagon and free fatty acids. Together, these data, obtained by hyperpolarized 13 C magnetic resonance spectroscopy and by blood sampling, indicate a hepatic metabolic shift in glucose utilization following a glucose challenge. Our findings demonstrate the capacity of hyperpolarized 13 C magnetic resonance spectroscopy for quantifying hepatic substrate metabolism in accordance with well-known physiological processes. When combined with concentration of blood insulin, glucagon and free fatty acids in the blood, the results indicate the potential of hyperpolarized magnetic resonance spectroscopy as a future clinical method for quantification of hepatic substrate metabolism.