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. 2016 Mar 2:17:24.
doi: 10.1186/s12882-016-0235-5.

Novel methods for microCT-based analyses of vasculature in the renal cortex reveal a loss of perfusable arterioles and glomeruli in eNOS-/- mice

Daniel S Perrien  1   2   3 , Mohamed A Saleh  4   5 , Keiko Takahashi  6   7 , Meena S Madhur  8 , David G Harrison  9 , Raymond C Harris  10   11 , Takamune Takahashi  12   13
Affiliations

Novel methods for microCT-based analyses of vasculature in the renal cortex reveal a loss of perfusable arterioles and glomeruli in eNOS-/- mice

Daniel S Perrien et al. BMC Nephrol. .

Abstract

Background: Two-dimensional measures of vascular architecture provide incomplete information about vascular structure. This study applied a novel rigorous method for 3D microCT-based analysis of total and cortical renal vasculature combined with a novel method to isolate and quantify the number of perfused glomeruli to assess vascular changes in eNOS-/- mice.

Methods: Two month old male wildtype and eNOS-/- mice were perfused with heparinized saline followed by radiopaque Microfil. The Microfil-perfused vasculature of excised kidneys was imaged by μCT with an isotropic voxel-size of 5.0 μm. For analysis of renal cortical vasculature, a custom algorithm was created to define the cortical volume of interest (VOI) as the entire volume within 600 μm of the renal surface. Vessel thickness in the whole kidney or renal cortex was analyzed by plotting the distribution of vascular volume at each measured thickness and examining differences between the genotypes at individual thicknesses. A second image processing algorithm was created to isolate, identify, and extract contrast perfused glomeruli from the cortical vessels.

Results: Fractional vascular volume (vascular volume/kidney volume; VV/KV) and Vessel Number/mm (V.N) were significantly lower in eNOS-/- mice vs. WT (p < 0.05). eNOS-/- kidneys had significantly fewer perfusable vessels vs. WT in the range of 20-40 μm in thickness. The cortex of eNOS-/- kidneys had significantly lower VV, VV/cortical volume, and V.N, with an increase in the distance between vessels (all p < 0.05). The total volume of vessels in the range of 20-30 μm was significantly lower in the cortex of eNOS-/- mice compared to WT (p < 0.05). Moreover, the total number of perfused glomeruli was significantly decreased in eNOS-/- mice (p < 0.01).

Conclusions: The methods presented here demonstrate a new method to analyze contrast enhanced μCT images for vascular phenotyping of the murine kidney. These data also demonstrate that kidneys in eNOS-/- mice have severe defects in vascular perfusion/structure in the renal cortex.

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Figures

Fig. 1
Fig. 1
Workflow for defining and analyzing total and cortical renal vasculature and glomeruli by contrast enhanced microCT. a The total renal volume was defined using a single contour around outer edge of the kidney. b The resulting mask was applied in combination with a grey-scale threshold and three-dimensional Gaussian noise filter to segment perfused vasculature from the total volume and (c) the distance transformation method was used to calculate thickness. d A mask approximating the renal cortex was created using a computer generated second contour, 600 μm deeper than outer contour (a) to exclude the medullary volume from the analysis. e and f The same threshold, Gaussian noise filter, and distance transformation procedures as in b and c were then applied. g Glomerular structures were then isolated from the three-dimensional segmented cortical vascular image (e) using an additional series of transformations to yield an object from which glomerular number and density can be calculated
Fig. 2
Fig. 2
Workflow for isolation of glomeruli from other vascular structures in the renal cortex. A series of erosion, dilation, and component labeling and extraction procedures were used to separate perfused glomeruli from afferent and efferent vasculature. Representative images from each step are shown as both 2-dimensional single slide cross sections (top) and anterior views of a portion of the 3-dimensional objects (bottom). Beginning with the binarized 3-dimensional representation of the cortical vasculature (a), three voxels (15 μm) were removed from all surfaces in a 3-dimensional manner using an erosion function resulting in the complete removal of any parts of the object with thickness ≤30 μm, effectively removing any detected capillaries or arterioles connected to the glomeruli (b). c The original thickness of the now eroded vascular components (b) was restored by applying a 3-dimensional dilation of 3 voxels. d All individual, disconnected, components of the image in (c) were labeled according to the total number of voxels in each object. All objects <45 or >1000 total voxels were removed from the image. The total number of objects remaining, representing the glomeruli, was quantified
Fig. 3
Fig. 3
Three-dimensional morphometric analysis of vascular volume and structure in the whole kidney reveals specific deficiencies in the vascular structure of eNOS-/- mice. Representative images of the total perfused renal vasculature illustrate the perfusion deficiency the cortex and smaller vessels in mice eNOS-/- mice (b and d) compared to WT mice (a and c). Three-dimensional quantification revealed the deletion of eNOS did not alter (e) the total kidney volume or (f) the total vascular volume. However, (g) the vascular volume/total kidney volume and (h) the vascular number in eNOS-/- kidneys was significantly lower than in WT kidneys. i Histograms illustrating the total vascular volume at each possible thickness were created and the total volume of perfused vessels at each given thickness was compared between genotypes. This analysis demonstrated a deficit in the number of perfused vessels in the range of 20–80 μm in thickness in the eNOS-/- kidneys compared to WT. All data are mean ± SEM. *p < 0.05 vs. Wildtype by Student’s t-test
Fig. 4
Fig. 4
eNOS-/- mice have less perfusable vessels in the renal cortex. Representative images of the renal vasculature in the renal cortex illustrate the perfusion deficiency eNOS-/- mice (b and d) compared to WT mice (a and c). Three-dimensional quantification revealed the deletion of eNOS did not alter (e) the total cortical volume. However, (f) the cortical vascular volume, (g) the cortical vascular volume/cortical volume and (h) the cortical vascular number in eNOS-/- kidneys was significantly lower than in WT kidneys. i Conversely, vessel separation (the mean distance between vessels) was significantly greater in the cortex of eNOS-/- mice. j Histograms illustrating the cortical vascular volume at each possible thickness were created and the total volume of perfused vessels at each given thickness was compared between genotypes. This analysis demonstrated a deficit in the number of perfused vessels in the range of 20–40 μm in thickness in the eNOS-/- renal cortex compared to WT. All data are mean ± SEM. *p < 0.05 vs. Wildtype by Student’s t-test
Fig. 5
Fig. 5
eNOS-/- mice have dramatically fewer perfusable glomeruli. Volumetric quantification of the total number of perfused glomeruli in the renal cortical volumes of interest revealed a dramatic decrease in perfused glomeruli in eNOS-/- mice compare to Wildtype (WT) which could be appreciated both visually (a and b) and quantitatively (c). Data are mean ± SEM. *p < 0.05 vs. Wildtype by Student’s t-test
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