Susceptibility-weighted Imaging For Renal Iron Overload Assessment: A Pilot Study

Purpose: To explore the feasibility of susceptibility-weighted imaging (SWI) for evaluating renal iron overload. Methods: Twenty-eight rabbits were randomly assigned into the control (n = 14) and iron (n = 14) groups. In the 0th week, the study group was injected with iron dextran. Both groups underwent SWI examination at the 0th, 8th, and 12th week. The signal intensity (SI) of the cortex and medulla was assessed. Angle radian value (ARV) calculated with phase image was taken as the quantitative value for cortical and medullary iron deposition. After the 12th week, the left kidneys of rabbits were removed for pathology. The difference in the ARV among the three groups was analyzed using the Kruskal–Wallis test. The difference of iron content between the two groups was analyzed through an independent sample t-test. Results: In the iron group: at the 12th week, eight rabbits were found to have decreased SI of only the cortex, and the other six rabbits had decreased SI of the cortex and medulla by the same degree; the ARV of the cortex at the 8th and 12th week was significantly higher than that of the 0th week (P < 0.05); the ARV of the six rabbits' medulla at the 12th week was significantly higher than that of the 0th week, 8th week, and the other eight rabbits at the 12th week (P < 0.05); at the 12th week, eight rabbits (iron group) were found to have many irons only deposit in the cortex, and the others were found to have many irons deposit in both cortex and medulla; the iron content of cortex and six rabbits' medulla in the iron group was significantly higher than that of the control (P < 0.05). Conclusion: The ARV of SWI can be used to assess the excess iron deposition in the kidneys quantitatively. Excessive iron deposition mainly occurs in the cortex or medulla and causes their SWI SI to decrease.

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Keywords: iron deposition, kidney, susceptibility-weighted imaging

Introduction

Iron is one of the essential microelements for organisms.1,2 Under normal conditions, humans maintain the balance between absorption, use, and loss.3 However, once the iron is excessive, the human body has no mechanisms to clear the excess iron.4 Excess iron will deposit in some of the organs and is detrimental.4 The kidney is one such organ that gets affected commonly. Iron overload has been affirmed as a dangerous factor for kidney dysfunction, which is associated with chronic kidney disease (CKD) caused by conditions like diabetic nephropathy, hypertensive kidney injury, and renal fibrosis.3–6 It was reported that iron deposition was observed in the proximal and distal tubules of the kidney in human CKD.5,7 Excessive iron promotes increased free radical generation and oxidative stress, which causes renal cellular and tissue damage.5,7,8 On the other hand, dietary iron restriction or the treatment through chelating agents can alleviate renal iron overload, thereby inhibiting the progression of the pre-existing renal injury.5,8,9 It follows that the degree of renal iron deposition is related to its injury. Accurate and effective evaluation of excessive iron deposition in the kidney is of great value in monitoring renal injury in CKD patients.10

At present, Prussian blue staining9 can be used to analyze the distribution of excess iron deposition in the kidney, and an atomic absorption spectrophotometer can be used to measure renal iron content. However, both methods are invasive and require a tissue sample, which is not suitable for clinical follow-up monitoring of excessive iron deposition in the kidney for CKD patients. To address these requirements of safe, non-invasive, and ability to offer repeated evaluations for monitoring, MRI offers the possibility of a viable alternative through susceptibility-weighted imaging (SWI), an emerging functional MRI technique. It uses tissue magnetic susceptibility difference to generate a unique contrast that differs from that obtained with conventional MRI.11,12 Combining the phase and magnitude images, SWI offers a good demonstration of paramagnetic signals.13 Iron is a paramagnetic substance, as evidenced by its short T2 relaxation time.10 It has been demonstrated that SWI can measure the tissue iron concentration reliably, which was consistent with the autopsy examination results.14 Hence, SWI can be regarded as a reliable marker through tracking iron overload in various associated diseases, thereby analyzing progression.10

So far, SWI has been used to detect and quantify iron deposition in the liver11 and brain13,14 tissue. Even after an extensive literature search, the papers evaluating renal iron deposition were next to none. In this study, we used animal experiments to explore the value of SWI in the qualitative and quantitative detection of excessive iron deposition in the kidney, considering the results of Prussian blue staining and atomic absorption spectrophotometer as the reference standard.

Materials and Methods

This study was approved by the ethics committee of The Third Affiliated Hospital of Soochow University (Approval number: 2019026).

Animal modeling and grouping

We used Twenty-eight purebred healthy New Zealand white rabbits (provided by Suzhou Huqiao Biotechnology Limited Company, Suzhou, China), each weighing 2.0–2.5 kg, 2–3 months old, 16 males and 12 females, grown at a room temperature of 22°C, clean environment, fed with complete formula fodder, and purified water. All rabbits were randomly divided into the following two groups:

1. Iron group: 14 rabbits (7 males and 7 females). On the first day of the 0th week, after documenting the body weight, a suspension of iron dextran containing 20 mg/ml of iron was injected into the gluteal muscles at a dose of 3 ml/kg.

2. Control group: 14 rabbits (9 males and 5 females). No iron was injected.

MR examination

The schedule of MRI examination in the iron and control group is as follows: on the first day of the 0th, 8th, and 12th week, respectively.

To reduce the intestinal peristalsis artifacts, food intake was restricted for a period of 8 hours before the examination. Anesthesia was achieved by injecting 3% pentobarbital sodium solution into the hind leg muscles at a dose of 1 ml/kg before the scanning. During the examination, the head was entered first, and the left kidney was scanned while in the left lateral position. The scanning range was from the upper pole to the lower pole of the kidney. All MRI images were acquired on a 3.0 T MRI system (Magnetom Verio; Siemens Healthcare, Erlangen, Germany) with a standard eight-channel phased array body matrix coil. The MRI protocols are shown in Table 1. The sequence of SWI produced the final magnitude image, maximum intensity projection image, phase image, and SWI image.

Image analysis

All images were analyzed by two physicians with more than five years of work experience in the interpretation of abdominal MRI. At the yoga.via post-processing work-station (Siemens), they entered the viewing interface and simultaneously opened the T2-weighted images (T2WI), SWI, and phase sequences and selected the largest central-level image of the kidney. The analysis was done as follows: (1) Qualitative analysis: On the T2WI and SWI, the signal intensity (SI) of the renal cortex and medulla was observed. (2) Quantitative analysis: According to the T2WI and SWI, the cortical region was manually delineated on the SWI at the central level of the kidney, avoiding the boundary area that may affect the signal value, so did the medulla. By applying the copy and paste function of the workstation, the cortical and medullary region in the SWI was, respectively, copied to the phase image. Then, the cortical and medullary regions in the phase image were, respectively, divided into three sub-regions of roughly equal area, including the front, middle, and back (Fig. 1). The phase value was obtained by, respectively, drawing the region of interest in the three sub-regions manually. The average value of the three sub-regions phase value was taken for the phase value (X) of the entire renal cortex and medulla, respectively. The angle radian value (ARV) was calculated by the following formula: ARV = (–X × π)/4096, and was utilized for the quantification of iron deposition, where X had a range from –4096 to 4095.15 The unit of ARV is radian.

Pathological examination

After the 12th week MRI scan, all rabbits were still under anesthesia. At this time, all rabbits were sacrificed by air embolization, and the left kidneys were removed. Enough tissues of the renal central level were sampled and fixed in 10% neutral buffered formalin. As per the routine protocol, tissues were dehydrated, made transparent, wax-impregnated, paraffin-embedded, sectioned, stained with hematoxylin-eosin and Prussian blue, and evaluated for renal iron deposition under brightfield microscopy.

The remaining renal cortical and medullary tissues were sent to the Guangdong Medical Laboratory Animal Center, and measurement of the cortical and medullary iron content was done by atomic absorption spectrophotometer.

Statistical analysis

For statistical analysis, SPSS 22.0 software (IBM, Armonk, NY, USA) was used. The data were expressed as the median (interquartile range) (M [Q1 and Q3]) and the Mann–Whitney U test was used to compare the difference in the ARV between the two groups. Kruskal–Wallis test was used to compare the differences in the ARV among multiple groups. An independent sample t-test was used to compare the difference of the renal iron content measured by the atomic absorption spectrophotometer between the two groups. Analysis of variance was used to compare the difference of the renal iron content measured by the atomic absorption spectrophotometer among multiple groups. Spearman rank correlation analysis was used to analyze the correlation between the angle radian values and the renal iron content measured by the atomic absorption spectrophotometer. The symbol r was used to represent the correlation coefficient.16 The correlation was interpreted as follows: r > 0 was considered a positive correlation; r < 0 was considered a negative correlation; |r| = 1 was considered a perfect correlation; 0.7 ≤ |r| < 1 was considered a high correlation; 0.4 ≤ |r| < 0.7 was considered a moderate correlation; 0 ≤ |r| < 0.4 was considered a low correlation; and r = 0 was considered zero correlation.16 P < 0.05 was considered statistically significant.

Fig. 2 The flowchart shows the change in SI for the renal cortex and medulla on T2WI and SWI images in the control and iron agent group at different time points. SI, signal intensity; SWI, susceptibility-weighted imaging; T2W, T2-weighted; T2WI, T2-weighted image.