Ultrasound super-resolution imaging provides a noninvasive assessment of renal microvasculature changes during mouse acute kidney injury

Abstract

Acute kidney injury (AKI) is a risk factor for the development of chronic kidney disease (CKD). One mechanism for this phenomenon is renal microvascular rarefaction and subsequent chronic impairment in perfusion. However, diagnostic tools to monitor the renal microvasculature in a noninvasive and quantitative manner are still lacking. Ultrasound super-resolution imaging is an emerging technology that can identify microvessels with unprecedented resolution. Here, we applied this imaging technique to identify microvessels in the unilateral ischemia-reperfusion injury mouse model of AKI-to-CKD progression in vivo. Kidneys from 21 and 42 day post- ischemia-reperfusion injury, the contralateral uninjured kidneys, and kidneys from sham-operated mice were examined by ultrasound super-resolution and histology. Renal microvessels were successfully identified by this imaging modality with a resolution down to 32 μm. Renal fibrosis was observed in all kidneys with ischemia-reperfusion injury and was associated with a significant reduction in kidney size, cortical thickness, relative blood volume, and microvascular density as assessed by this imaging. Tortuosity of the cortical microvasculature was also significantly increased at 42 days compared to sham. These vessel density measurements correlated significantly with CD31 immunohistochemistry (R2=0.77). Thus, ultrasound super-resolution imaging provides unprecedented resolution and is capable of noninvasive quantification of renal vasculature changes associated with AKI-to-CKD progression in mice. Hence, this technique could be a promising diagnostic tool for monitoring progressive kidney disease.

Keywords: acute kidney injury; chronic kidney disease; diagnostic imaging; fibrosis; microvascular rarefaction; ultrasound super-resolution.

Conflict of interest statement

Disclosure

The authors have declared that no conflict of interest exists.

Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Experimental design for in vivo ultrasound super-resolution imaging on mouse acute kidney injury model.

(A) Timeline of the experiment (B) Ischemia-reperfusion injury (IRI) is performed on the right kidney to induce the acute kidney injury (AKI). The sham, contralateral, and injured kidneys at 21-days and injured kidneys at 42-days post injury (n=5 for each group) were scanned by ultrasound in long axis for ultrasound super-resolution (USR) images. The kidneys were excised immediately after the scan for fibrosis analysis and immunohistochemistry with CD31 staining focusing on renal vasculature around the corticomedullary junction. (C) From the reconstructed USR images of the kidneys, kidney changes including area, vessel density, and microvasculature tortuosity were assessed.

Figure 2.. IRI leads to renal fibrosis.

Mice were subjected to unilateral IRI and both the affected and contralateral kidneys were recovered at either 21 or 42 days after injury. (A) Fibrosis was detected with Masson’s Trichrome stain (blue staining) as well as picrosirius red stain (dark red). When the picrosirius slides were viewed under polarized light, birefringence denotes specific staining for collagens. (B) Fibrosis scoring of kidneys. (C-D) mRNA levels of collagen (Col3) are dramatically elevated in all injured kidneys compared to the contralateral kidneys. mRNA levels of VEGF are dramatically decreased in all injured kidneys. Data are expressed as mean ± standard error. * P < 0.0001 compared to contralateral kidneys, no differences were found between the 21 and 42 day injured kidneys.

Figure 3.. Overlaid B-mode and Ultrasound super-resolution (B-USR) images for sham kidneys, contralateral kidneys, injured kidneys at 21-days post injury, and injured kidneys at 42-days post injury.

Column A shows the USR images of the five sham kidneys. Column B shows the contralateral kidneys. Column C and column D show the USR images of injured kidney scanned at 21 days and 42 days after injury, respectively. To provide an anatomical landmark, the major renal vessel branches, aorta, cortex, medulla, and dorsal skin were marked by white arrows in the image of the injured kidney from the mouse number 6. Overall decrease in size and increase in vasculature rarefaction were observed over time.

Figure 4.. Quantitative assessment of the changes in overall morphology and renal blood volume of sham, contralateral, and IRI kidneys.

(A) Kidney cross-sectional area measured from the long-axis US B-mode images. Cross-sectional areas of the sham, contralateral, 21 days post injury and 42 days post injury kidneys were 49.20 ± 1.58 mm2, 51.61 ± 3.57 mm2, 37.54 ± 1.55 mm2, 35.67 ± 2.414 mm2. A significant decrease in kidney area after IRI was observed by ultrasound measurement. (B) Weight of the sham (160.50 ± 4.83 g), contralateral (197.70 ± 8.08 g), and injured kidneys (21 days: 96.94 ± 6.22 g, 42days: 80.70 ± 6.14 g). Significant reduction in weight was found, which supports the size decrease measured by US. (C) Cortex thickness of the kidneys measured from the US images. Significant decrease of the cortex thickness of the injured kidneys (21days: 1.23 ±0.04 mm, 42 days: 1.10 ±0.08 mm) compared to sham (1.76 ±0.03 mm) and contralateral (1.88 ±0.08 mm) was found. (D) US estimation of relative blood volume (rBV). The average rBV of the sham kidneys, contralateral kidneys, and IRI kidneys at 21 days and 42 days were 34.66% ± 1.99%, 35.85% ± 1.88%, 22.35% ± 1.38%, and 26.30% ± 1.92%, respectively. Significant decrease of rBV on IRI kidney was observed. (E) Vessel density in the cortex measured by US. A significant reduction in 21 days (39.77 ± 2.69%) and 42 days (46.47 ± 2.47%) post-injury kidneys compared to sham (64.44 ± 1.80%) and contralateral (66.96 ± 2.66%) kidneys was found. (F) Vessel density in the corticomedullary junction measured by US. A significant decrease in 21 days (26.17 ± 1.28%) and 42 days (27.60 ± 1.37%) compared to sham (47.14 ± 2.41%) and contralateral (49.59 ± 2.42%) was found. (n=5, ANOVA with post-hoc Tukey HSD test, *P<0.05; **P<0.01; ***P<0.001.)

Figure 5.. Vessel density in the corticomedullary junction by histology and correlation with US imaging.

(A) Representative CD31 staining of the sham, contralateral, and IRI kidneys. (B) Vessel density measured as positively stained area fraction of the vessels in corticomedullary junction. (Sham: 12.14 ± 0.75%, contralateral: 15.02 ± 0.47%, 21 days: 7.56 ± 0.22%, 42 days: 8.82 ± 0.73%) Injured kidneys exhibited a decrease of vessel density in the ROI compared either sham or contralateral kidney. (n=5, ANOVA with post-hoc Tukey HSD test, *P<0.05; **P<0.01; ***P<0.001.) (C) Significant correlation between the histology and USR measurement of the vessel density in the corticomedullary junction was found (P value < 0.001, correlation coefficient: 0.77). (n=20, Pearson’s correlation analysis)

Figure 6.. Tortuosity of the sham, contralateral and IRI kidneys.

(A) Representative USR images of the cortical vessels from the sham and 42-days post-injury kidneys are shown. White arrows indicate the curved and aggregated vessels from the 42-days post-injury kidney. (B) Significant increase of cortical microvasculature tortuosity is shown in the kidneys at 42-days post injury compared to sham. (n=5, ANOVA with post-hoc Tukey HSD test, *P<0.05.)