Quantitative Assessment of Markers of Oxidative Stress in Mice Model of Renal Artery Stenosis
Behzad Ebrahimi1,2, Alvin Ihsani2, Arkadiusz Sitek2, Slobodan I Macura3, and Lilach O Lerman1

1Nephrology, Mayo Clinic, Rochester, MN, United States, 2Radiology, Harvard/MGH, Boston, MA, United States, 3Biochemistry and Molecular Biology, Mayo Clinic


Inability of cells to detoxify reactive oxidative species (ROS) is responsible for numerous degenerative pathological conditions. Currently there is no clinical method to assess reduction-oxidation (redox) state in vivo. In this study, using a cyclic nitroxide T1 MR probe with unique characteristics, we propose a two-tissue compartment model which provides quantitative information of markers of oxidative stress. Results demonstrated the feasibility of redox status assessment in vitro and in vivo in the stenotic mouse kidney. In the stenotic kidney, our method indicated increased renal ROS production, accompanied by preserved ability to detoxify ROS compared to the contralateral kidney.


Unperturbed biological systems have the ability to detoxify reactive oxygen species (ROS) and maintain a delicate reduction-oxidation (redox) balance. Cellular injury may disturb this balance and result in increased oxidative stress, which exerts deleterious effects on proteins, lipids, and DNA, and is responsible for numerous degenerative neurological, cardiovascular, renal, and other pathologies1-3. Currently methods for assessing oxidative stress in vivo are unsuitable for clinical translation, mainly due to their invasive nature (biopsy) or lack of clinical availability (EPR spectroscopy). In this study, we introduce a novel analysis method for quantitative MRI-based assessment of markers of oxidative stress using Tempol, a T1 MR tracer (Figure 1) and superoxide dismutase (SOD) mimetic. The absence of advanced knowledge describing the pharmacokinetics of Tempol within the tissue makes it difficult to discern dynamics of T1 contrast secondary to its reduction or re-oxidization following inflow and clearance from the organ of interest. To alleviate this limitation, we propose a novel kinetic model that accounts for the uptake and washout of Tempol, and deconvolves quantitative markers of ROS generation and cellular detoxification capability from the kinetic information.


As a proof-of-concept, using Tempol in an in vitro system, we showed the feasibility of redox state assessment in cells with oxidative stress. The experimental details are discussed in the caption of Figure 2. Imaging was performed on an Avance DRX 700WB (Bruker-BioSpin, Billerica, MA) spectrometer with a vertical 16.4 T wide bore magnet equipped with mini imaging accessories. T1-weighted MR dynamic contrast enhanced (DCE) images with TR/TE = 24/1.68ms, Matrix Size = 128x256, FOV = 2.56x2.56cm with temporal resolution of <3s were acquired over 6-7 minutes. We subsequently investigated this approach in vivo in the stenotic (STK) and contralateral (CLK) kidneys of a mouse model of unilateral renal artery stenosis (RAS) and chronic kidney disease. Five S129 mice underwent unilateral RAS surgery, as described previously4. Blood pressure was measured 3 weeks after RAS induction. Animals were scanned and kidney volumes and perfusion estimated from images collected using respiratory-gated 3D Fast Imaging with Steady Precession (3D-FISP) and arterial spin labelling (ASL), respectively. Tempol DCE imaging was then performed using the same scanning protocol as in cells. Thirty seconds through the imaging, a bolus of 350mM of Tempol was injected through a tail vein catheter over 15 seconds, and acquisition continued for 6-8 minutes (Figure 3). Pharmacokinetic and quantitative oxidative stress markers were assessed by finding the optimal parameters to the differential equations describing our two-tissue compartment model (Figure 4.B) based on our Bayesian estimation method implemented using a Metropolis-Hastings algorithm5,6 and the posterior distribution proposed by Sitek et. al.7 (Figure 4.C).


The in vitro experiment showed higher concentration of unreduced Tempol in cells exposed to X/XO compared to untreated myocytes, and indicated that the redox state of cells is detectable with Tempol DCE. In vivo, renal volume and hemodynamics confirmed RAS development in the mice. In a paired statistical analysis, the uptake, 𝑘1, in parallel to perfusion was significantly lower in all STK. While 𝑘3 was not statistically different between STK and CLK (0.46±0.2 vs. 0.69±0.6 min-1, respectively), 𝑘4 tended to be higher in the STK (0.004±0.001 vs. 1.24±0.65 min-1, respectively, p=0.06).


The in vitro study showed that Tempol DCE-MRI is sufficiently sensitive to detect subtle difference in the concentration of unreduced Tempol in the two samples, despite the significantly lower density of cells in the dialysis bags, and therefore the reduction rate, compared to tissues. The in vivo animal study demonstrated the feasibility of this method for redox state assessment in research and potentially pre-clinical applications. The similarity of 𝑘3 in both STK and CLK showed close agreement to recent studies using histological methods8, which have demonstrated preserved SOD functionality in CKD in the presence of oxidative stress. Together with higher 𝑘4, this observation may suggest that the cellular redox imbalance in the STK is determined by increased ROS production (e.g. as a result of mitochondrial injury) more than by diminished SOD activity.


Our preliminary results indicate feasibility of assessing renal redox status in vivo using Tempol DCE MRI in RAS. In contrast to previous methods, our approach has the capacity to discrete markers of oxidative stress from wash-in/wash-out driven parameters. Additional studies are needed to test the applicability and sensitivity of this method in a larger group of animals and in additional pathological conditions.


No acknowledgement found.


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Figures 1. Tempol (4-hydroxy-TEMPO), a cyclic nitroxide radical, is a T1 highly diffusive contrast agent that can cross cell membrane with ease. It is generally considered non-toxicity and is well tolerated even over prolonged courses of vascular administration 9. In cells, it is reduced by the same mechanisms which cells detoxify ROS. Once reduced, Tempol is no longer a T1 paramagnetic contrast, therefore the redox state of this probe provides a readout of the tissue redox status.

Figure 2. Two million cardiac myocytes, isolated from a pig using a previously published method10, were equally divided and suspended in two dialysis membrane bags, each filled with 1mL of saline. Oxidative stress was induced by exposing the cells in one bags to xanthine and xanthine oxidase (X/XO). Bags were immersed in a bath of Tempol dissolved in saline. As Tempol diffused into the bags, the concentration of unreduced Tempol increased in the bag with cells affected by oxidative stress (red arrow) faster than the bag with the untreated cells (black arrow).

Figure 3. CLK and STK at baseline (before injection) and during arrival of Tempol bolus. Right, representative normalized arterial input function (AIF, black) and concentration-time curve in CLK (red).

Figure 4. (A) In our two-tissue compartment model 𝐶𝑝 is the arterial input function, and 𝐶𝑜 and 𝐶𝑟 represent the concentration of Tempol (the oxide) and hydroxylamine (the reduced state, which is invisible to MR) states. The kinetic parameters 𝑘i represent the exchange rates between the compartments. 𝑘1 and 𝑘2 are the exchange rates between the intravascular and the second compartment. 𝑘2 = 𝑘1/λ where λ is the partition coefficient. 𝑘3 is the reduction rate, and 𝑘4 is the re-oxidization rate in the presence of ROS. (B) The differential equations describing our two-tissue compartment model and (C) the posterior distribution.

Table 1. Indices of RAS development in mice. Three weeks after RAS, animals tended to be hypertensive (p=0.06); (* p<0.05 vs. CLK).

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)