Renal anatomical and pathophysiological alterations are directly associated with the fluid and electrolyte balance in the kidney, which is regulated by the extracellular corticomedullary osmolality gradient[1]. A decline in corticomedullary osmolality gradient may serve as an early indicator of pathological disruption of the tubular reabsorption process. Hence, a non-invasive imaging modality for assessing this ion gradient could be a valuable tool to aid diagnosis and guide treatment of renal diseases[2]. Here, we introduce a novel magnetic resonance imaging (MRI) approach to monitor the corticomedullary osmolality gradient in vivo using hyperpolarized ¹³C-urea in a healthy porcine model with a similar kidney physiology to humans.Four healthy female Danish landrace pigs of weight 30kg were included in this study. The pigs were intubated and mechanically ventilated using a respirator system. Catheterization was performed in the left femoral vein for administration of hyperpolarized ¹³C-urea. A clinical 3T GE HDx MR scanner (GE Healthcare, Milwaukee, WI, USA) was used to acquire ¹H images with an 8-channel cardiac array receiver coil (GE Healthcare, Milwaukee, WI, USA). A ¹³C Helmholtz loop coil of 20cm diameter (PulseTeq Limited, Surrey, UK) was used for ¹³C-urea examinations. MR images of hyperpolarized ¹³C-urea in the kidney were acquired using a 3D balanced steady state free precession sequence (TE 1.2ms, TR 2.9ms, FA 30°, matrix 64x64, FOV 300x300mm², in-plane resolution 4.69mm, slice thickness 10mm) at four time points (30, 40, 50 and 75s after urea injection). To assess the feasibility of direct diuresis imaging, a furosemide bolus injection (0.5 mg/kg bodyweight) was administered intravenously to one of the pigs, and prior and (20 min) post bolus 3D MR images were acquired. Three sets of ¹H images were acquired; first, a 3D T1-weighted steady state free precession sequence (echo time (TE) 1.1ms, repetition time (TR) 2.7ms, flip angle (FA) 35°, matrix 256x256, field of view (FOV) 340x340mm², in-plane resolution 1.3mm, slice thickness 3mm); second, a T2-weighted single shot fast spin echo sequence (TE 83ms, TR 2923ms, FA 90°, matrix 512x512, FOV 380x380mm², in-plane resolution 0.7mm, slice thickness 8mm); third, a diffusion weighted echo planar spin echo sequence with additional diffusion gradients (TE 54.4ms, TR 10000ms, FA 90°, matrix 256x256, FOV 290x290mm², in-plane resolution 1.5mm, slice thickness 7mm). Raw DICOM images were transferred to OsiriX (Pixmeo, Geneva, Switzerland) for anatomical overlay and region of interest (ROI) analysis. The intra-renal ¹³C-urea gradient was investigated by projecting the image intensity along a line-profile in the cortical-pelvic axis of the axial plane. The ¹³C-urea MR signal increase after furosemide injection was assessed using histogram analysis.The obtained signal-to-noise ratio and resolution of the ¹³C-urea images was sufficient to delineate the physiological features of the kidney (Figure 1A). Furthermore, the inherent compatibility of standard structural ¹H MRI methods and hyperpolarized ¹³C imaging enabled multiple aspects of kidney structure and function to be studied in the same scan session utilizing a variety of methods (Figure 1B-C). A renal cyst was identified in one kidney in the ¹³C-urea image and confirmed as a region of abnormally high signal intensity on a T2 weighted ¹H MR image (Figure 1A-B). As seen in Figure 1, this fact was supported by an absence of urea intrusion into the cyst. The intra-renal distribution of ¹³C-urea (i.e. gradient in renal electrolyte osmolality) was visible 75s after ¹³C-urea injection, with clear signal evident in the cortex and maximum signal intensity in the medulla region (Figure 2). Prior to 75s post injection, the renal distribution of ¹³C-urea was observed to be dominated by cortical flow of urea. Figure 3 depicts low field of view ¹³C-urea images in three different orientations (axial, sagittal, coronal) of one kidney acquired 75s after urea injection. From the axial and coronal images, it is evident that there is increased signal in the medullary space compared to the cortical space, similar to previous observations in the uni-papillary rat kidney [3]. As illustrated in Figure 4, significantly increased ¹³C-urea signal intensities were observed post furosemide (diuretic) injection, and there appears to be a strong visual correlation between the diuresis action and the localization of urea in the cortical space.This study demonstrates that hyperpolarized ¹³C-urea MRI is capable of identification of intra-renal accumulation of urea and differentiation of acute renal functional states in a large animal model, with a physiological state similar to humans. The acute observed increase in ¹³C-urea MR signal following administration of the diuretic furosemide shows great promise for further investigations in patients, where this method of monitoring of response to stimuli could play a direct diagnostic role.