Hyperpolarized 13 C magnetic resonance evaluation of renal ischemia reperfusion injury in a murine model

Abstract

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) ¹³ C dehydroascorbate (DHA) and ¹³ C pyruvate magnetic resonance spectroscopy (MRS) to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline and 7 days after unilateral ischemia reperfusion injury (IRI). Compared with the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP ¹³ C vitamin C/(vitamin C + DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP ¹³ C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP ¹³ C lactate/pyruvate ratio at day 7 compared with baseline, although the ¹³ C lactate/pyruvate ratio was not significantly different between the IRI and contralateral sham-operated kidneys at day 7. Arterial spin labeling magnetic resonance imaging (MRI) demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS) and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP ¹³ C MRS for the non-invasive assessment of oxidative stress and mitochondrial PDH activity following renal IRI.

Keywords: Acute kidney injury; [1−13C]dehydroascorbic acid; [1−13C]pyruvate; hyperpolarization; magnetic resonance spectroscopy; mitochondrial dysfunction; oxidative stress.

Fig.1

A: Representative voxel placement and HP DHA ¹³C spectra in the kidney subjected to IRI and the contralateral sham-operated kidney at day 7. B: HP ¹³C VitC/(VitC+DHA) ratio at baseline and day 7. The kidneys subjected to IRI showed significantly lower VitC/(VitC+DHA) ratio at day 7 when compared to baseline, and when compared to the contralateral sham-operated kidneys at day 7.

Fig. 2

A: Overlay of the voxel placement from the 2D chemical shift acquisition on the T2 weighted anatomic image. B: Representative spectrum through the kidney voxel after the injection of HP ¹³C pyruvate in the IRI kidney at day 7. HP ¹³C metabolites visualized included lactate (Lac), alanine (Ala), and bicarbonate (Bic). Hydrate=¹³C pyruvate hydrate. C: The kidneys subjected to IRI showed significantly lower ¹³C Bic/Pyr ratio at day 7 when compared to the baseline, and when compared to the contralateral sham-operated kidneys at day 7. D: The HP ¹³C Lac/Pyr ratio increased significantly from baseline to day 7 in the IRI kidneys. However, the ¹³C Lac/Pyr ratio was not significantly different between the IRI and the contralateral sham-operated kidneys at day 7. E: The HP ¹³C Ala/Pyr ratio remained the same following IRI. F: The HP ¹³C Bic/Lac ratio decreased significantly from baseline to day 7 in the IRI kidneys. The ¹³C Bic/Lac ratio also decreased slightly from baseline to day 7 in the contralateral sham-operated kidneys. The ¹³C Bic/Lac ratio was significantly lower in the IRI than the contralateral sham-operated kidneys at day 7.

Fig. 3

A: Example of a renal perfusion map as measured by ¹H ASL MRI (Kidney subjected to IRI: arrows; contralateral sham-operated kidney: arrowheads). B: The kidneys subjected to IRI showed a significant decrease in perfusion, while the perfusion remained virtually unchanged in the contralateral sham-operated kidneys.

Fig. 4

Histologic features of unilateral IRI model on PAS stained renal sections. A: Kidneys from control mice not subjected to IRI showed normal tubular structures. B: Kidneys at 48 hours after being subjected to unilateral IRI showed moderate injury with tubular dilatation and cast formation (arrows). C: Kidneys at 7 days after being subjected to unilateral IRI showed decreased tubular dilatation and cast formation, associated with regenerating tubular epithelial cells (arrow). D: The contralateral sham-operated kidneys at day 7 showed scattered foci of tubular epithelial cell necrosis associated with cellular cast formation (arrow). A, B, C: × 200 magnification. D: × 400 magnification.

Fig. 5

Example of DCF staining of the renal sections. The kidneys subjected to IRI (B) showed markedly higher DCF staining (green color) consistent with increased level of reactive oxygen species (ROS), when compared to the contralateral sham-operated kidneys (A) at day 7.