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.

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.

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.

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.

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.

A non-invasive biomarker of type III collagen degradation reflects ischaemia reperfusion injury in rats.

BACKGROUND: Maintenance of kidney function in kidney allografts remains a challenge, as the allograft often progressively develops fibrosis after kidney transplantation. Fibrosis is caused by the accumulation of extracellular matrix proteins like type I and III collagen (COL I and III) that replace the functional tissue. We assessed the concentrations of a neo-epitope fragment of COL III generated by matrix metalloproteinase-9 cleavage (C3M) in two rat models resembling the ischaemic injury taking place following kidney transplantation. METHODS: We measured C3M in urine (U-C3M) and plasma (P-C3M) samples of rats subjected to unilateral nephrectomy followed by sham operation (NTx) or ischaemia reperfusion injury (NTxIRI) as well as in rats subjected to bilateral ischaemia reperfusion injury (BiIRI). Levels of U-C3M were normalized to urinary creatinine and were correlated to plasma creatinine, blood urea nitrogen, messenger ribonucleic acid (mRNA) of markers of kidney injury, and mRNA and protein levels of markers of tissue repair and fibrosis. RESULTS: Levels of U-C3M were significantly elevated 7 days after ischaemia reperfusion in the NTxIRI. BiIRI animals showed higher levels of U-C3M after 7 and 14 days of reperfusion but not at 21 days. P-C3M did not change in any of the models. There was a significant correlation between U-C3M and mRNA levels of fibronectin, COL I alpha 1 chain (COL Ia1) and neutrophil gelatinase-associated lipocalin (NGAL), and protein levels of alpha smooth muscle actin (αSMA), fibronectin and COL III in NTxIRI but not in NTx animals. Levels of U-C3M increased significantly in the BiIRI animals subsequent to reperfusion, and mirrored the histological alterations. Furthermore, U-C3M was associated with the extent of fibrosis, and remained elevated even after plasma creatinine levels decreased. CONCLUSIONS: These results demonstrate that degradation of COL III increases after ischaemia reperfusion injury, and that U-C3M may be a non-invasive marker of tissue repair and fibrosis in the ischaemic kidney.

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.

Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping.

PURPOSE: It has been demonstrated that hyperpolarized 13 C MR is a useful tool to study cultured cells. However, cells in culture can alter phenotype, which raises concerns regarding the in vivo significance of such findings. Here we investigate if metabolic phenotyping using hyperpolarized 13 C MR is suitable for cells isolated from kidney tissue, without prior cell culture. METHODS: Isolation of tubular cells from freshly excised kidney tissue and treatment with either ouabain or antimycin A was investigated with hyperpolarized MR spectroscopy on a 9.4 Tesla preclinical imaging system. RESULTS: Isolation of tubular cells from less than 2 g of kidney tissue generally resulted in more than 10 million live tubular cells. This amount of cells was enough to yield robust signals from the conversion of 13 C-pyruvate to lactate, bicarbonate and alanine, demonstrating that metabolic flux by means of both anaerobic and aerobic pathways can be quantified using this technique. CONCLUSION: Ex vivo metabolic phenotyping using hyperpolarized 13 C MR in a preclinical system is a useful technique to study energy metabolism in freshly isolated renal tubular cells. This technique has the potential to advance our understanding of both normal cell physiology as well as pathological processes contributing to kidney disease. Magn Reson Med 78:457-461, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

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.

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.

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.

Magnetic resonance hyperpolarization imaging detects early myocardial dysfunction in a porcine model of right ventricular heart failure.

AIMS: Early detection of heart failure is important for timely treatment. During the development of heart failure, adaptive intracellular metabolic processes that evolve prior to macro-anatomic remodelling, could provide an early signal of impending failure. We hypothesized that metabolic imaging with hyperpolarized magnetic resonance would detect the early development of heart failure before conventional echocardiography could reveal cardiac dysfunction. METHODS AND RESULTS: Five 8.5 kg piglets were subjected to pulmonary banding and subsequently examined by [1-13C]pyruvate hyperpolarization, conventional magnetic resonance imaging, echocardiography, and blood testing, every 4 weeks for 16 weeks. They were compared with a weight matched, healthy control group. Conductance catheter examination at the end of the study showed impaired right ventricular systolic function along with compromised left ventricular diastolic function. After 16 weeks, we saw a significant decrease in the conversion ratio of pyruvate/bicarbonate in the left ventricle from 0.13 (0.04) in controls to 0.07 (0.02) in animals with pulmonary banding, along with a significant increase in the lactate/bicarbonate ratio to 3.47 (1.57) compared with 1.34 (0.81) in controls. N-terminal pro-hormone of brain natriuretic peptide was increased by more than 300%, while cardiac index was reduced to 2.8 (0.95) L/min/m2 compared with 3.9 (0.95) in controls. Echocardiography revealed no changes. CONCLUSION: Hyperpolarization detected a shift towards anaerobic metabolism in early stages of right ventricular dysfunction, as evident by an increased lactate/bicarbonate ratio. Dysfunction was confirmed with conductance catheter assessment, but could not be detected by echocardiography. Hyperpolarization has a promising future in clinical assessment of heart failure in both acquired and congenital heart disease.

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.

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.

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.

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.