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.

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.

Noninvasive Assessment of Fibrosis Following Ischemia/Reperfusion Injury in Rodents Utilizing Na Magnetic Resonance Imaging.

Fibrosis is often heterogeneously distributed, and classical biopsies do not reflect this. Noninvasive methods for renal fibrosis have been developed to follow chronic kidney diseases (CKD) and to monitor anti-fibrotic therapy. In this study, we combined two approaches to assess fibrosis regression following renal ischemia-reperfusion injury (IRI): magnetic resonance imaging (MRI) and noninvasive extracellular matrix (ECM) biomarkers. MRI was used to evaluate fibrosis in bilateral IRI in rats after reperfusion at 7, 14, and 21 days. This was performed with 1HT1 and T2* mapping, dynamic contrast-enhanced (DCE)-MRI, and chemical shift imaging (CSI)-23Na. The degradation of laminin gamma-1 chain (LG1M) and type III collagen (C3M) was measured in urine and plasma. Fibrosis was analyzed in tissue using fibronectin (FN) and alpha-smooth muscle actin (α-SMA) using quantitative polymerase chain reaction qPCR and western blotting. We found increased fibrosis 7 days after reperfusion, which dropped to sham levels after 21 days. Single kidney glomerular filtration rate (skGFR), perfusion (DCE-MRI), and total 23Na kidney content correlated positively with fibrotic markers FN and α-SMA as well as noninvasive LG1M and C3M. We showed that novel MRI protocols and ECM markers could track fibrogenic development. This could give rise to a multi-parametric practice to diagnose and assess fibrosis whilst treating kidney disease without using invasive methods.

Graft assessment of the ex vivo perfused porcine kidney using hyperpolarized [1-13 C]pyruvate.

PURPOSE: Normothermic perfusion is an emerging strategy for donor organ preservation and therapy, incited by the high worldwide demand for organs for transplantation. Hyperpolarized MRI and MRS using [1-13 C]pyruvate and other 13 C-labeled molecules pose a novel way to acquire highly detailed information about metabolism and function in a noninvasive manner. This study investigates the use of this methodology as a means to study and monitor the state of ex vivo perfused porcine kidneys, in the context of kidney graft preservation research. METHODS: Kidneys from four 40-kg Danish domestic pigs were perfused ex vivo with whole blood under normothermic conditions, using an MR-compatible perfusion system. Kidneys were investigated using 1 H MRI as well as hyperpolarized [1-13 C]pyruvate MRI and MRS. Using the acquired anatomical, functional and metabolic data, the state of the ex vivo perfused porcine kidney could be quantified. RESULTS: Four kidneys were successfully perfused for 120 minutes and verified using a DCE perfusion experiment. Renal metabolism was examined using hyperpolarized [1-13 C]pyruvate MRI and MRS, and displayed an apparent reduction in pyruvate turnover compared with the usual case in vivo. Perfusion and blood gas parameters were in the normal ex vivo range. CONCLUSION: This study demonstrates the ability to monitor ex vivo graft metabolism and function in a large animal model, resembling human renal physiology. The ability of hyperpolarized MRI and MRS to directly compare the metabolic state of an organ in vivo and ex vivo, in combination with the simple MR implementation of normothermic perfusion, renders this methodology a powerful future tool for graft preservation research.

Anatomically correct assessment of the orientation of the cardiomyocytes using diffusion tensor imaging.

Diffusion tensor imaging has been used for assessing the orientation of cardiac myocytes for decades. Striking methodological differences exist between studies when quantifying these orientations. This limits the comparability between studies, and impedes collaboration and the drawing of appropriate physiological conclusions. We have sought to elucidate these differences, permitting us to propose a standardised "tool set" that might better establish consensus in future studies. We fixed hearts from seven 25 kg pigs in formalin, and scanned them using diffusion tensor imaging. Using various angle definitions as found in literature, we assessed the orientations of cardiomyocytes, comparing them in terms of helical and intrusion angles, along with the orientation of their aggregations. The difference between assessment of the helical angle with and without relation to the epicardial curvature was 25.2° (SD: 7.9) at the base, 5.8° (1.9) at the equatorial level, and 28.0° (7.0) at the apex, ANOVA P = 0.001. In comparable fashion, the intrusion angle differed by 25.9° (12.9), 7.6° (0.98) and 17.5° (4.7), P = 0.01, and the angle of the aggregates (E3-angle) differed by 25.0° (13.5) at the base, 9.4° (1.7) at the equator, and 23.1° (6.2) apically, P = 0.003. When assessing 14 definitions used in literature to calculate the orientation of aggregates, only 4 rendered identical results. The findings show that any attempt to use projection of eigenvectors introduces considerable bias. The epicardial curvature of the ventricular cone needs to be taken into account when seeking to provide accurate quantification of the orientation of the aggregated cardiomyocytes, especially in the apical and basal regions. This means that projection of eigenvectors should be avoided prior to quantifying myocyte orientation, especially when assessing radial orientation. Based on our results, we suggest appropriate methods for valid assessment of myocyte orientation using diffusion tensor imaging.

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.

Hyperpolarized 13 C,15 N2 -Urea MRI for assessment of the urea gradient in the porcine kidney.

PURPOSE: A decline in cortico-medullary osmolality gradient of the kidney may serve as an early indicator of pathological disruption of the tubular reabsorption process. The purpose of this study was to investigate the feasibility of hyperpolarized 13 C,15 N2 -urea MRI as a biomarker of renal function in healthy porcine kidneys resembling the human physiology. METHODS: Five healthy female Danish domestic pigs (weight 30 kg) were scanned at 3 Tesla (T) using a 13 C 3D balanced steady-state MR pulse sequence following injection of hyperpolarized 13 C,15 N2 -urea via a femoral vein catheter. Images were acquired at different time points after urea injection, and following treatment with furosemide. RESULTS: A gradient in cortico-medullary urea was observed with an intramedullary accumulation 75 s after injection of hyperpolarized 13 C,15 N2 -urea, whereas images acquired at earlier time points postinjection were dominated by cortical perfusion. Furosemide treatment resulted in an increased urea accumulation in the cortical space, leading to a reduction of the medullary-to-cortical signal ratio of 49%. CONCLUSION: This study demonstrates that hyperpolarized 13 C,15 N2 -urea MRI is capable of identifying the intrarenal accumulation of urea and can differentiate acute renal functional states in multipapillary kidneys, highlighting the potential for human translation. Magn Reson Med 76:1895-1899, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Renal ischemia and reperfusion assessment with three-dimensional hyperpolarized 13 C,15 N2-urea.

PURPOSE: The aim of this work was to investigate whether hyperpolarized 13 C,15 N2 -urea can be used as an imaging marker of renal injury in renal unilateral ischemic reperfusion injury (IRI), given that urea is correlated with the renal osmotic gradient, which describes the renal function. METHODS: Hyperpolarized three-dimensional balanced steady-state 13 C magnetic resonance imaging (MRI) experiments alongside kidney function parameters and quantitative polymerase chain reaction measurements were performed in rats subjected to unilateral renal ischemia for 60-minute and 24-hour reperfusion. RESULTS: We revealed a significant reduction in the intrarenal gradient in the ischemic kidney in agreement with cortical injury markers neutrophil gelatinase-associated lipocalin and kidney injury molecule 1, as well as functional kidney parameters. CONCLUSION: Hyperpolarized functional 13 C,15 N2 urea MRI can be used to successfully detect changes in the intrarenal urea gradient post-IRI, thereby enabling in vivo monitoring of the intrarenal functional status in the rat kidney. Magn Reson Med 76:1524-1530, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Assessment of early diabetic renal changes with hyperpolarized [1-(13) C]pyruvate.

BACKGROUND: This experimental study explores a novel magnetic resonance imaging/spectroscopic (MRI/MRS) method that measures changes in renal metabolism in a diabetic rat model. This hyperpolarized metabolic MRI/MRS method allows monitoring of metabolic processes in seconds by >10 000-fold enhancement of the MR signal. The method has shown that the conversion of pyruvate to bicarbonate, i.e. pyruvate dehydrogenase (PDH) activity, is significantly altered in the myocardium already at the onset of diabetes, and the predominant Warburg effect is a valuable cancer maker via the lactate dehydrogenase (LDH) activity. We hypothesize that a similar change in PDH and LDH could be found in the early diabetic kidney. METHODS: In a streptozotocin rat model of type 1 diabetes, hyperpolarized (13) C-MRI and blood oxygenation level-dependent (1) H-MRI was employed to investigate the changes in renal metabolism in the diabetic and the control kidneys in vivo. RESULTS: The diabetic kidney showed a 149% increase in the lactate/pyruvate ratio compared with the control rat kidney, whereas the bicarbonate/pyruvate ratio was unchanged between the diabetic and the control rat kidneys, consistent with literature findings. These metabolic findings paralleled a reduced intrarenal oxygen availability as found by blood oxygenation level-dependent MRI. DISCUSSION: Hyperpolarized (13) C-MRI shows promise in the diagnosis and monitoring of early renal changes associated with diabetes, with the pyruvate/lactate ratio as an imaging biomarker for regional renal changes.