title: Novel methods for microCT-based analyses of vasculature in the renal cortex reveal a loss of perfusable arterioles and glomeruli in eNOS-/- mice.
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.
authors.affiliation: Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN, 37212, USA. daniel.s.perrien@vanderbilt.edu.|Department of Orthopaedic Surgery and Rehabilitation, Medical Center East, South Tower, Suite 4200, Nashville, TN, 37232, USA. daniel.s.perrien@vanderbilt.edu.|Vanderbilt University Institute of Imaging Sciences, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. daniel.s.perrien@vanderbilt.edu.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Perrien
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, 33516, Egypt. mohamed.a.saleh@Vanderbilt.Edu.|Division of Clinical Pharmacology in the Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. mohamed.a.saleh@Vanderbilt.Edu.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Saleh
authors.affiliation: Department of Nephrology, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. keiko.takahashi@Vanderbilt.Edu.|Vanderbilt O'Brien Mouse Kidney Physiology and Disease Center, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. keiko.takahashi@Vanderbilt.Edu.
authors.forename: Keiko
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Division of Clinical Pharmacology in the Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. meenakshi.s.madhur@Vanderbilt.Edu.
authors.forename: Meena S
authors.initials: MS
authors.lastname: Madhur
authors.affiliation: Division of Clinical Pharmacology in the Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. david.g.harrison@Vanderbilt.Edu.
authors.forename: David G
authors.initials: DG
authors.lastname: Harrison
authors.affiliation: Department of Nephrology, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. raymond.harris@Vanderbilt.Edu.|Vanderbilt O'Brien Mouse Kidney Physiology and Disease Center, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. raymond.harris@Vanderbilt.Edu.
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Harris
authors.affiliation: Department of Nephrology, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. takamune.takahashi@Vanderbilt.Edu.|Vanderbilt O'Brien Mouse Kidney Physiology and Disease Center, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. takamune.takahashi@Vanderbilt.Edu.
authors.forename: Takamune
authors.initials: T
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D001160:Arterioles / Q000000981:diagnostic imaging*; D007672:Kidney Cortex / Q000098:blood supply / Q000000981:diagnostic imaging*; D007678:Kidney Glomerulus / Q000098:blood supply / Q000000981:diagnostic imaging*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D009929:Organ Size; D012077:Renal Artery / Q000000981:diagnostic imaging*; D055114:X-Ray Microtomography / Q000379:methods
chemical_list: D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse