Super-Resolution Ultrasound Imaging of Renal Vascular Alterations in Zucker Diabetic Fatty Rats during the Development of Diabetic Kidney Disease

Abstract

Individuals with diabetes at risk of developing diabetic kidney disease (DKD) are challenging to identify using currently available clinical methods. Prognostic accuracy and initiation of treatment could be improved by a quantification of the renal microvascular rarefaction and the increased vascular tortuosity during the development of DKD. Super-resolution ultrasound (SRUS) imaging is an in vivo technique capable of visualizing blood vessels at sizes below 75 µm. This preclinical study aimed to investigate the alterations in renal blood vessels' density and tortuosity in a type 2 diabetes rat model, Zucker diabetic fatty (ZDF) rats, as a prediction of DKD. Lean age-matched Zucker rats were used as controls. A total of 36 rats were studied, subdivided into ages of 12, 22, and 40 weeks. Measured albuminuria indicated the early stage of DKD, and the SRUS was compared with the ex vivo micro-computed tomography (µCT) of the same kidneys. Assessed using the SRUS imaging, a significantly decreased cortical vascular density was detected in the ZDF rats from 22 weeks of age compared to the healthy controls, concomitant with a significantly increased albuminuria. Already by week 12, a trend towards a decreased cortical vascular density was found prior to the increased albuminuria. The quantified vascular density in µCT corresponded with the in vivo SRUS imaging, presenting a consistently lower vascular density in the ZDF rats. Regarding vessel tortuosity, an overall trend towards an increased tortuosity was present in the ZDF rats. SRUS shows promise for becoming an additional tool for monitoring and prognosing DKD. In the future, large-scale animal studies and human trials are needed for confirmation.

Keywords: chronic kidney disease; diagnostic imaging; renal injury; type 2 diabetes; ultrasound localization microscopy.

Figure 1

Overview of the experimental sequence. Before the rat was anesthetized, blood glucose from the tail vein was measured. After anesthesia, a blood sample via a catheter in the carotid artery as well as urine collection via a catheter in the ureter were acquired. The 10 min SRUS imaging was performed on the left kidney. Just before the animal was euthanized, the right kidney was excised for histology. Finally, the left kidney was perfused with CT contrast and excised to be scanned. Figure created with BioRender.com, accessed February 2023.

Figure 2

Segmentation of a rat kidney separated into cortex, outer medulla, and inner medulla in a SRUS image. (a) The red represents the cortex, the dark blue the outer medulla, and the turquoise area represents the inner medulla. (b) The vessel tracks in the cortex after regional separation. The microbubble flow directions are illustrated by the color wheel in the upper right corner, e.g., green trajectories in the cortex representing cortical radial veins. Scale bar on (a): 2 mm.

Figure 3

µCT processing and density analysis. The coronal slice of a µCT scan is portrayed from a 22-week-old lean Zucker rat with the following: (a) the cortex defined by red borders and the mask of small vessels included in the cortical vascular density; (b) the medulla defined by the green border and the mask of small vessels included in the density analysis for the medulla region (note the exclusion of the large segmental and arcuate vessels); and (c) colored voxels representing the full vessel segmentation and the subsequent vessel radius estimation. The color indicates the local vessel radius in accordance with the colormap to the right. Size bar: 2 mm.

Figure 4

Bodyweight, blood glucose, albumin-to-creatinine ratio (ACR), and creatinine clearance (eGFR). The displayed parameters measured at the study end in the lean Zucker rats (LZRs) and the Zucker diabetic fatty rats (ZDF) at an age of 12, 22, and 40 weeks. The orange triangles represent the lean rats and the blue circles the rats with diabetes. A significant difference is indicated by p < 0.05, * 0.01–0.05, ** 0.001–0.01, and **** <0.0001. The lines indicate mean ± SD.

Figure 5

Histological evaluation of glomeruli. Qualitative grading of glomerulopathy in PAS-stained kidney tissue from the lean Zucker rats (LZRs) and the Zucker diabetic fatty rats (ZDF) by week 12, 22, and 40. Grade 2 is displayed in light colors with respect to the specific pathology evaluated and is defined as the specimens with moderate involvement. Grade 3 is displayed in dark colors and is defined as the number of specimens with severe involvement.

Figure 6

PAS-stained kidney tissue with a section of the cortex. Each age group is represented in the lean Zucker rats (LZRs) and the Zucker diabetic fatty rats (ZDF). (a–d) shows the glomeruli without any pathology. (e) displays microaneurysms (black arrow) and glomerular atrophy (green arrow). (f) presents microaneurysms (black arrow), glomerular atrophy (green arrow), sclerosis (orange arrow), and dilated atrophic tubuli containing proteins (blue arrow). Scale bar in (a): 100µm.

Figure 7

SRUS images of kidneys from the Zucker diabetic fatty (ZDF) rats and the lean Zucker rats (LZRs) by weeks 12, 22, and 40. (b,d,f) illustrate a less dense renal vasculature in the ZDF rats compared with their age-matched LZR (a,c,e). Hydronephrosis (arrows) was found in all the kidneys examined. The direction of the MBs, hence the blood flow’s direction, is illustrated by the color wheel in the upper right corner, e.g., green color represents tracked MBs moving downwards, in the renal vein it correlates with blood flow out of the kidney. Scale bar in (a): 2 mm.

Figure 8

Vascular density measurements from the SRUS images. The vascular density is shown for the cortex, outer medulla, and inner medulla from the lean Zucker rats (LZRs) and the Zucker diabetic fatty rats (ZDF) by weeks 12, 22, and 40. A significant difference is indicated by p < 0.05, * 0.01–0.05, ** 0.001–0.01, and **** <0.0001. The lines indicate mean ± SD.

Figure 9

Track tortuosity measurements from the SRUS images. This Figure illustrates the track tortuosity in the cortex, outer medulla, and inner medulla measured in the lean Zucker rats (LZRs) and the Zucker diabetic fatty rats (ZDF) at weeks 12, 22, and 40. The lines indicate mean ± SD.

Figure 10

Vascular density measured with the µCT. This Figure shows the vascular density measured in the cortex and medulla of six rats: a lean Zucker rat (LZR) and a Zucker diabetic fatty (ZDF) rat from the 12, 22, and 40 weeks age groups.

Figure 11

Visual comparison between the µCT and SRUS images. (a–c) displays the kidney from a lean Zucker rat and (d–f) the kidney from a Zucker diabetic fatty rat, both at 22 weeks of age. (a,d) shows the SRUS images, (b,e) the µCT images (maximum intensity projection of 33 slices), and (c,f) displays the overlay of the two different modalities. The image slices from the µCT volume were found using Tomviz (version 1.9.0). Due to the transformation of the structure from in vivo to ex vivo, the SRUS images were scaled to match the µCT image in the following way: 50% transparency was used on the intensity of the SRUS image, image (c)’s image was rotated 3.45 degrees to the right, increased 5.7% in height, and decreased 3.6% in length. (f) was increased 2% in height and decreased 11.5% in length.