In vivo measurement of Renal Redox Capacity in a Model of Chronic Kidney Disease using by hyperpolarized 13C dehydroascorbate (DHA) MRS
Celine A.J. Baligand1, David H. Lovett2, Lalita Uttarwar2, Jeremy Gordon1, John Kurhanewicz1, David M. Wilson1, and Zhen Jane Wang1

1Radiology and Biomedical Imaging, UCSF, San Francisco, CA, United States, 2Medicine, San Francisco Department of Veterans Affairs Medical Center/University of California San Francisco, San Francisco, CA, United States


Limited biomarkers are available for early diagnosis and monitoring of chronic kidney disease (CKD). Renal oxidative stress is a key initiator of CKD. Therefore, in vivo assessment of kidney redox capacity may provide a clinically relevant and early marker of kidney injury. The N-terminal truncated matrix metalo-protease isoform (NTT-MMP-2) transgenic mouse is a model mimicking human progressive kidney disease that is triggered by oxidative stress. Using this model, we show that hyperpolarized 13C-dehydroascobic acid MRS imaging can detect in vivo the altered redox capacity preceding any functional and histological changes, thus potentially providing an early marker of susceptibility to CKD.


Chronic kidney disease (CKD) is a major public health issue. There are currently limited biomarkers that allow early diagnosis and disease monitoring. Recently, we have shown that oxidative stress results in sustained epigenetic changes that induce the expression of a N-terminal truncated matrix metalo-protease isoform (NTT-MMP-2) [1,2,3]. Transgenic mice with NTT-MMP-2 overexpression recapitulate the hallmarks of human progressive kidney disease. They also exhibit exaggerated injury responses to ischemia/reperfusion and development of CKD, providing a mechanistic link between oxidative stress, acute kidney injury (AKI), and CKD. Therefore, the in vivo assessment of kidney redox capacity may provide a clinically relevant and early marker of progressive kidney injury. In this work, we demonstrate that hyperpolarized 13C dehydroascorbate (DHA) reduction to vitamin C (VitC) monitored by 13C MR spectroscopic imaging (MRSI), is sensitive to early changes in renal redox capacity in NTT-MMP2 mice, and can be detected before histological manifestation of CKD.


Kidney redox capacity by 13C-MRS: 5-7 month-old NTT-MMP-2 (mild kidney injury on histology, n=7) and aged-matched control mice (n=4) were fasted for 8 h and pre-treated with 250 μL of 60mM of unlabeled VitC 45 min before the experiment to minimize the possible stress induced by the DHA injection . A tail vein catheter was placed for intravascular injection of hyperpolarized DHA solution. A 2.2M solution of [1-13C] DHA in dimethyacetamide (DMA) containing 15mM OX063 trityl radical was hyperpolarized on a HyperSense DNP instrument (Oxford Instruments) [4]. The frozen sample was dissolved in distilled water containing 0.3 mM ethylenediaminetetracetic acid (EDTA). Imaging was performed on a 3T MRI scanner (GE Healthcare, Waukesha, WI) using a 13C-1H birdcage coil. 3D 13C-EPSI was acquired 30s after injection of 250μL of 15mM HP 13C DHA. Fast spin echo, T2-weighted coronal images were acquired for anatomic reference (TE=100ms, TR=4s, 6 averages). MRSI studies were then acquired 25 seconds post injection of 15mM HP 13C DHA, at 6 mm3 isotropic resolution) [5]. Spectra were analyzed in Sivic and results are reported as metabolites peak height ratios, VitC/(VitC+DHA), an index of the redox status. Reactive oxygen species detection: Fresh renal cortical frozen sections (8 µ) were incubated for 1 hour at 37 º C with the fluorescent dye, 2’-7’-DCF-diacetate (4 µM, Invitrogen/Molecular Probes). Following rinsing the sections were mounted in anti-fade/DAPI medium (Invitrogen) and examined by confocal microscopy. DCF detects several reactive oxygen species and the fluorescent signal localizes primarily to mitochondria.


2D-chemical shift 13C MRI showed the hyperpolarized DHA peak at 174 ppm and its reduced form, VitC, was detected 3.8 ppm downfield with good signal to noise in the kidney. Transgenic mice displayed significantly lower DHA reduction to VitC as shown by the kidney VitC/(VitC+DHA) ratio of 0.23 ± 0.04 compared to 0.30 ± 0.03 in WT controls (p=0.002) (Figure 1), indicating higher oxidative stress and lower redox capacity in NNT-MMP2 transgenic mice. This was in agreement with a large increase in ROS visualized on histological staining of kidney sections (Figure 2). The alteration in redox capacity preceded any histological changes of chronic kidney disease, including peri-tubular capillary rarefaction and renal tubular atrophy, which developed over time in the transgenic mice.


HP 13C DHA imaging allowed the detection of early impairment of the redox status in the NTT-MMP2 model of CKD. This was consistent with increased oxidative stress as confirmed by fluorescence microscopy of reactive oxygen species. Work is in progress to interrogate the redox capacity and perfusion alterations following an episode of AKI and during the progression of CKD. These markers have the potential to allow early diagnosis of injury and to monitor subsequent treatment response.


American Heart Association and Department of Veterans Affairs, NIH NIDDK RO1 DK097357, NIH P41EB013598


1- Cheng et al., FASEB 2006

2- Lovett D.H., et al., Plos One 2012

3- Wanga S., et al., Plos One 2015

4- Keshari K.R, et al., PNAS 2011

5- Keshari K.R, et al., Diabetes 2011


Kidney redox capacity measured by HP DHA MRS in vivo. The top panel shows typical spectra obtained from voxels placed in the kidney of wild type (A) and MMP2 (B) mice, showing conversion of DHA to Vitamin C, or from the vasculature (C), with no DHA reduction . (D) Coronal T2-weighted image showing both kidneys and typical voxels placements for 3D chemical shift acquisitions. Measures in (WT, n=4) and mmp2 transgenic mice kidneys (mmp2, n=7) are sown in (E).

Confocal microscopic images of DCF-diacetate stained kidney slices, showing mitochondrial reactive oxygen species. A large increase in signal was observed in the mmp2 transgenic slices.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)