Supplementary Methods, Figure Legends 1-4 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

Supplementary Figure 2 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

Supplementary Methods, Figure Legends 1-4 from Hyperpolarized ¹³C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

Supplementary Methods, Figure Legends 1-4 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

The setup, operational procedures and performance of a cryogen-free device for producing hyperpolarized contrast agents using dissolution dynamic nuclear polarization (dDNP) in a preclinical imaging center is described. The polarization was optimized using the solid-state, DNP-enhanced NMR signal to calibrate the sample position, microwave and NMR frequency and power and flip angle. The polarization of a standard formulation to yield ~ 4 mL, 60 mM 1- 13 C-pyruvic acid in an aqueous solution was quantified in five experiments to P( 13 C) = (38 ± 6) % (19 ± 1) s after dissolution. The mono-exponential time constant of the build-up of the solid-state polarization was quantified to (1032 ± 22) s. We achieved a duty cycle of 1.5 h that includes sample loading, monitoring the polarization build-up, dissolution and preparation for the next run. After injection of the contrast agent in vivo, pyruvate, pyruvate hydrate, lactate, and alanine were observed, by measuring metabolite maps. Based on this work sequence, hyperpolarized 15 N urea was obtained (P( 15 N) = (5.6 ± 0.8) % (30 ± 3) s after dissolution).

Spin hyperpolarization enables real-time metabolic imaging of carbon-13-labeled substrates. While hyperpolarized l-(1-13C)alaninamide is a probe of the cell-surface tumor marker aminopeptidase-N (APN, CD13), its activity in vivo has not been described. Scanning the kidneys of rats infused with hyperpolarized alaninamide shows both conversion to [1-13C]alanine and several additional spectral peaks with distinct temporal dynamics. The (1-13C)alaninamide chemical shift is pH-sensitive, with a pKa of 7.9 at 37 °C, and the peaks correspond to at least three different compartments of pH 7.46 ± 0.02 (1), 7.21 ± 0.02 (2), and 6.58 ± 0.05 (3). An additional peak was assigned to the carboxyamino adduct formed by reaction with dissolved CO2. Spectroscopic imaging showed nonuniform distribution, with the low-pH signal more concentrated in the inner medulla. Treatment with the diuretic acetazolamide resulted in significant pH shifts in compartment 1 to 7.38 ± 0.03 (p = 0.0057) and compartment 3 to 6.80 ± 0.05 (p = 0.0019). While the pH of compartment 1 correlates with blood pH, the pH of compartment 3 did not correspond to the pH of urine. In vitro experiments show that alaninamide readily enters blood cells and can detect intracellular pH. While carbamate formation depends on pH and pCO2, the carbamate-to-alaninamide ratio did not correlate with either arterial blood pH or pCO2, suggesting that it may reflect variations in tissue pH and pCO2. This study demonstrates the feasibility of using hyperpolarized sensors to simultaneously image enzyme activity, pCO2, and pH in vivo.

Hyperpolarized [1- 13 C]pyruvate enables direct in vivo assessment of real-time liver enzymatic activities by 13 C magnetic resonance. However, the technique usually requires the injection of a highly supraphysiological dose of pyruvate. We herein demonstrate that liver metabolism can be measured in vivo with hyperpolarized [1- 13 C]pyruvate administered at two- to three-fold the basal plasma concentration. The flux through pyruvate dehydrogenase, assessed by 13 C-labeling of bicarbonate in the fed condition, was found to be saturated or partially inhibited by supraphysiological doses of hyperpolarized [1- 13 C]pyruvate. The [ 13 C]bicarbonate signal detected in the liver of fasted rats nearly vanished after treatment with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, indicating that the signal originates from the flux through PEPCK. In addition, the normalized [ 13 C]bicarbonate signal in fasted untreated animals is dose independent across a 10-fold range, highlighting that PEPCK and pyruvate carboxylase are not saturated and that hepatic gluconeogenesis can be directly probed in vivo with hyperpolarized [1- 13 C]pyruvate.

The circadian clock is a ubiquitous molecular time-keeping mechanism which synchronizes cellular, tissue, and systemic biological functions with 24-hour environmental cycles. Local circadian clocks drive cell type- and tissue-specific rhythms and their dysregulation has been implicated in pathogenesis and/or progression of a broad spectrum of diseases. However, the pathophysiological role of intrinsic circadian clocks in the kidney of diabetics remains unknown. To address this question, we induced type I diabetes with streptozotocin in mice devoid of the circadian transcriptional regulator BMAL1 in podocytes (cKOp mice) or in the kidney tubule (cKOt mice). There was no association between dysfunction of the circadian clock and the development of diabetic nephropathy in cKOp and cKOt mice with diabetes. However, cKOt mice with diabetes exhibited exacerbated hyperglycemia, increased fractional excretion of glucose in the urine, enhanced polyuria, and a more pronounced kidney hypertrophy compared to streptozotocin-treated control mice. mRNA and protein expression analyses revealed substantial enhancement of the gluconeogenic pathway in kidneys of cKOt mice with diabetes as compared to diabetic control mice. Transcriptomic analysis along with functional analysis of cKOt mice with diabetes identified changes in multiple mechanisms directly or indirectly affecting the gluconeogenic pathway. Thus, we demonstrate that dysfunction of the intrinsic kidney tubule circadian clock can aggravate diabetic hyperglycemia via enhancement of gluconeogenesis in the kidney proximal tubule and further highlight the importance of circadian behavior in patients with diabetes.

This document codifies the probe preparation of pyruvate and urea for dissolution dynamic nuclear polarization. Additionally, this also describes the steps recommended for estimation of T1 and polarization and quality assurance to ensure repeatability and robustness of hyperpolarized 13C probe formulation.

It was demonstrated that nonpersistent radicals can be generated in frozen solutions of metabolites such as pyruvate by irradiation with ultraviolet (UV) light, enabling radical-free dissolution DNP. Although pyruvate is endogenous, an excess of additional pyruvate may perturb metabolic processes, making it potentially unsuitable as a polarizing agent when studying fatty acids or carbohydrate metabolism. Therefore, the aim of the study was to characterize solutions containing endogenously-occurring alternatives to pyruvate as UV-induced nonpersistent radical precursors for in vivo hyperpolarized MRI. The metabolites alpha-ketovalerate (AKV) and alpha-ketobutyrate (AKB) are analogues of pyruvate and were chosen as potential radical precursors. Sample formulations containing AKV and AKB were studied with UV-visible spectroscopy, irradiated with UV light, and their nonpersistent radical yields were quantified with ESR and compared to pyruvate. The addition of 13C labeled substrates to the sample matrix altered the radical yield of the precursors. Using AKB increased the 13C-labeled glucose liquid state polarization to 16.3 +/- 1.3% compared with 13.3 +/- 1.5% obtained with pyruvate, and 8.9 +/- 2.1% with AKV. For [1-13C]butyric acid, polarization levels of 12.1 +/- 1.1% for AKV and 12.9 +/- 1.7% for AKB were achieved. Hyperpolarized [1-13C]butyrate metabolism in the heart revealed label incorporation into [1-13C]acetylcarnitine, [1-13C]acetoacetate, [1-13C]butyrylcarnitine, [5-13C]glutamate and [5-13C]citrate. This study demonstrates the potential of AKV and AKB as endogenous polarizing agents for in vivo radical-free hyperpolarized MRI. UV-induced, nonpersistent radicals generated in endogenous metabolites enable high polarization without requiring radical filtration, thus simplifying the quality-control tests in clinical applications.

As both a consumer and producer of glucose, the kidney plays a significant role in glucose homeostasis. Measuring renal gluconeogenesis requires invasive techniques, and less invasive methods would allow renal gluconeogenesis to be measured more routinely. Magnetic resonance spectroscopy and imaging of infused substrates bearing hyperpolarized carbon-13 spin labels allows metabolism to be detected within the body with excellent sensitivity. Conversion of hyperpolarized 1-13C pyruvate in the fasted rat liver is associated with gluconeogenic flux through phosphoenolpyruvate carboxykinase (PEPCK) rather than pyruvate dehydrogenase (PDH), and this study tested whether this was also the case in the kidney. The left kidney was scanned in fed and overnight-fasted rats either with or without prior treatment by the PEPCK inhibitor 3-mercaptopicolinic acid (3-MPA) following infusion of hyperpolarized 1-13C pyruvate. The 13C-bicarbonate signal normalized to the total metabolite signal was 3.2-fold lower in fasted rats (p = 0.00073) and was not significantly affected by 3-MPA treatment in either nutritional state. By contrast, the normalized [1-13C]aspartate signal was on average 2.2-fold higher in the fasted state (p = 0.038), and following 3-MPA treatment it was 2.8-fold lower in fed rats and 15-fold lower in fasted rats (p = 0.001). These results confirm that, unlike in the liver, most of the pyruvate-to-bicarbonate conversion in the fasted kidney results from PDH flux. The higher conversion to aspartate in fasted kidney and the marked drop following PEPCK inhibition demonstrate the potential of this metabolite as a marker of renal gluconeogenesis.

The intense but transient increase in nuclear magnetic resonance signal provided by dynamic nuclear polarization (DNP) has enabled a range of new applications, including rapid, minimally invasive metabolic imaging at the second timescale. This chapter provides an overview of the hyperpolarized carbon-13 labeled agents prepared by dissolution DNP used in vivo and their biochemical interactions, categorized by metabolic pathway. The required characteristics of a suitable probe and an efficiently polarizing formulation are described, as are practical considerations for formulation development and metabolite identification. Relevant biochemical principles to aid in the interpretation of the apparent metabolism of the probes are introduced with illustrative examples.

d ‐amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d ‐amino acids to their corresponding α ‐keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d ‐amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N ‐methyl‐ d ‐aspartate (NMDA) receptor hypo/hyper‐function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d ‐[1‐ 13 C]alanine. Following a bolus of hyperpolarized d ‐alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate‐to‐ d ‐alanine ratio was comparable to the values measured when the l ‐enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d ‐[1‐ 13 C]alanine to lactate and pyruvate was detected in blood ex vivo , and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo ; however, the bicarbonate‐to‐ d ‐alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1‐ 13 C]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d ‐[1‐ 13 C]alanine as a probe of d ‐amino acid metabolism.

AIM:Arginase 2 (ARG2) is a mitochondrial enzyme that catalyses hydrolysis of l-arginine into urea and l-ornithine. In the kidney, ARG2 is localized to the S3 segment of the proximal tubule. It has been shown that expression and activity of this enzyme are upregulated in a variety of renal pathologies, including ischemia-reperfusion (IR) injury. However, the (patho)physiological role of ARG2 in the renal tubule remains largely unknown.METHODS:We addressed this question in mice with conditional knockout of Arg2 in renal tubular cells (Arg2lox/lox /Pax8-rtTA/LC1 or, cKO mice).RESULTS:We demonstrate that cKO mice exhibit impaired urea concentration and osmolality gradients along the corticomedullary axis. In a model of unilateral ischemia-reperfusion injury (UIRI) with an intact contralateral kidney, ischemia followed by 24 hours of reperfusion resulted in significantly more pronounced histological damage in ischemic kidneys from cKO mice compared to control and sham-operated mice. In parallel, UIRI-subjected cKO mice exhibited a broad range of renal functional abnormalities, including albuminuria and aminoaciduria. Fourteen days after UIRI, the cKO mice exhibited complex phenotype characterized by significantly lower body weight, increased plasma levels of early predictive markers of kidney disease progression (asymmetric dimethylarginine and symmetric dimethylarginine), impaired mitochondrial function in the ischemic kidney but no difference in kidney fibrosis as compared to control mice.CONCLUSION:Collectively, these results establish the role of ARG2 in the formation of corticomedullary urea and osmolality gradients and suggest that this enzyme attenuates kidney damage in ischemia-reperfusion injury.

SA-BDPA is a water-soluble, narrow-line width radical previously used for dynamic nuclear polarization (DNP) signal enhancement in solid-state magic angle spinning NMR spectroscopy. Here, we report the first study using SA-BDPA under dissolution DNP conditions (6.7 T and 1.15 K). Longitudinal-detected (LOD)-electron spin resonance (ESR) and 13C DNP measurements were performed on samples containing 8.4 M [13C]urea dissolved in 50:50 water:glycerol (v/v) doped with either 60 or 120 mM SA-BDPA. Two distinct DNP mechanisms, both "pure" thermal mixing and a well-resolved solid effect could clearly be identified. The radical's ESR line width (30-40 MHz), broadened predominantly by dipolar coupling, excluded any contribution from the cross effect. Microwave frequency modulation increased the enhancement by DNP at the lower radical concentration but not at the higher radical concentration. These results are compared to data acquired with trityl radical AH111501, highlighting the unusual 13C DNP properties of SA-BDPA.

Under normal conditions, the heart mainly relies on fatty acid oxidation to meet its energy needs. Changes in myocardial fuel preference are noted in the diseased and failing heart. The magnetic resonance signal enhancement provided by spin hyperpolarization allows the metabolism of substrates labeled with carbon-13 to be followed in real time in vivo. Although the low water solubility of long-chain fatty acids abrogates their hyperpolarization by dissolution dynamic nuclear polarization, medium-chain fatty acids have sufficient solubility to be efficiently polarized and dissolved. In this study, we investigated the applicability of hyperpolarized [1-C-13]octanoate to measure myocardial medium-chain fatty acid metabolism in vivo. Scanning rats infused with a bolus of hyperpolarized [1-C-13]octanoate, the primary metabolite observed in the heart was identified as [1-C-13]acetylcarnitine. Additionally, [5-C-13]glutamate and [5-C-13]citrate could be respectively resolved in seven and five of 31 experiments, demonstrating the incorporation of oxidation products of octanoate into the tricarboxylic acid cycle. A variable drop in blood pressure was observed immediately following the bolus injection, and this drop correlated with a decrease in normalized acetylcarnitine signal (acetylcarnitine/octanoate). Increasing the delay before infusion moderated the decrease in blood pressure, which was attributed to the presence of residual gas bubbles in the octanoate solution. No significant difference in normalized acetylcarnitine signal was apparent between fed and 12-hour fasted rats. Compared with a solution in buffer, the longitudinal relaxation of [1-C-13]octanoate was accelerated similar to 3-fold in blood and by the addition of serum albumin. These results demonstrate the potential of hyperpolarized [1-C-13]octanoate to probe myocardial medium-chain fatty acid metabolism as well as some of the limitations that may accompany its use.

Under normal conditions, the heart mainly relies on fatty acid oxidation to meet its energy needs. Changes in myocardial fuel preference are noted in the diseased and failing heart. The magnetic resonance signal enhancement provided by spin hyperpolarization allows the metabolism of substrates labeled with carbon‐13 to be followed in real time in vivo. Although the low water solubility of long‐chain fatty acids abrogates their hyperpolarization by dissolution dynamic nuclear polarization, medium‐chain fatty acids have sufficient solubility to be efficiently polarized and dissolved. In this study, we investigated the applicability of hyperpolarized [1– 13 C]octanoate to measure myocardial medium‐chain fatty acid metabolism in vivo. Scanning rats infused with a bolus of hyperpolarized [1– 13 C]octanoate, the primary metabolite observed in the heart was identified as [1– 13 C]acetylcarnitine. Additionally, [5‐ 13 C]glutamate and [5‐ 13 C]citrate could be respectively resolved in seven and five of 31 experiments, demonstrating the incorporation of oxidation products of octanoate into the tricarboxylic acid cycle. A variable drop in blood pressure was observed immediately following the bolus injection, and this drop correlated with a decrease in normalized acetylcarnitine signal (acetylcarnitine/octanoate). Increasing the delay before infusion moderated the decrease in blood pressure, which was attributed to the presence of residual gas bubbles in the octanoate solution. No significant difference in normalized acetylcarnitine signal was apparent between fed and 12‐hour fasted rats. Compared with a solution in buffer, the longitudinal relaxation of [1– 13 C]octanoate was accelerated ~3‐fold in blood and by the addition of serum albumin. These results demonstrate the potential of hyperpolarized [1– 13 C]octanoate to probe myocardial medium‐chain fatty acid metabolism as well as some of the limitations that may accompany its use.

Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1-13C]glycine and acquiring dynamic 13C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1-13C]glycine and its product [1-13C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1-13C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-13C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-13C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1-13C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.

Under normal conditions, the heart mainly relies on fatty acid oxidation to meet its energy needs. Changes in myocardial fuel preference are noted in the diseased and failing heart. The magnetic resonance signal enhancement provided by spin hyperpolarization allows the metabolism of substrates labeled with carbon-13 to be followed in real time in vivo. Although the low water solubility of long-chain fatty acids abrogates their hyperpolarization by dissolution dynamic nuclear polarization, medium-chain fatty acids have sufficient solubility to be efficiently polarized and dissolved. In this study, we investigated the applicability of hyperpolarized [1-C-13]octanoate to measure myocardial medium-chain fatty acid metabolism in vivo. Scanning rats infused with a bolus of hyperpolarized [1-C-13]octanoate, the primary metabolite observed in the heart was identified as [1-C-13]acetylcarnitine. Additionally, [5-C-13]glutamate and [5-C-13]citrate could be respectively resolved in seven and five of 31 experiments, demonstrating the incorporation of oxidation products of octanoate into the tricarboxylic acid cycle. A variable drop in blood pressure was observed immediately following the bolus injection, and this drop correlated with a decrease in normalized acetylcarnitine signal (acetylcarnitine/octanoate). Increasing the delay before infusion moderated the decrease in blood pressure, which was attributed to the presence of residual gas bubbles in the octanoate solution. No significant difference in normalized acetylcarnitine signal was apparent between fed and 12-hour fasted rats. Compared with a solution in buffer, the longitudinal relaxation of [1-C-13]octanoate was accelerated similar to 3-fold in blood and by the addition of serum albumin. These results demonstrate the potential of hyperpolarized [1-C-13]octanoate to probe myocardial medium-chain fatty acid metabolism as well as some of the limitations that may accompany its use.