Kidney International, Vol. 61 (2002), pp.
1056–1063
Enhanced renal cortical vascularization in
experimental hypercholesterolemia
MICHAEL D. BENTLEY, MARTIN RODRIGUEZ-PORCEL, AMIR LERMAN,
MIRIT HERSHMAN SARAFOV, J. CARLOS ROMERO, LAURA I. PELAEZ,
JOSEPH P. GRANDE, ERIK L. RITMAN, and LILACH O. LERMAN
Department of Internal Medicine, Divisions of Hypertension and Cardiovascular Diseases,
Department of Physiology and Biophysics, and the Department of Laboratory Medicine and Pathology,
Mayo Clinic, Rochester, and Minnesota State University, Mankato, Minnesota
Enhanced renal cortical vascularization in experimental hyper-
cholesterolemia.
Background. Experimental hypercholesterolemia is associ-
ated with pro-inflammatory changes and impaired regulation
of tissue perfusion, which may lead to neovascularization. How-
ever, it is yet unknown whether such changes take place in the
kidney. In this study, using a novel three-dimensional (3-D)
micro computed-tomography technique we tested the hypothe-
sis that hypercholesterolemia was associated with increased
microvascular density in the renal cortex.
Methods. Kidneys were excised from pigs after 12 weeks of
either a normal (N ⫽ 6) or high cholesterol (HC; N ⫽ 5) diet,
histology slides processed, and a segmental renal artery injected
with a radio-opaque intravascular silicone polymer. Renal sam-
ples were scanned with micro computed-tomography, trans-
verse and three-dimensional images were reconstructed, and
microvessels (80 to 360 ␮m in diameter) counted in situ.
Results. Serum cholesterol levels were significantly higher
in hypercholesterolemic compared to normal pigs (383 ⫾ 76 vs.
81 ⫾ 7 mg/dL, P ⬍ 0.01), and microvascular spatial density
was significantly higher in their inner and middle renal cortex
(189 ⫾ 7 vs. 126 ⫾ 6 microvessels/cm2, P ⬍ 0.0001). Hypercho-
lesterolemic kidneys also showed mild interstitial mononuclear
infiltration and heavier immunostaining of vascular endothelial
growth factor, but no other signs of morphological damage.
Conclusions. These results demonstrate that early diet-induced
hypercholesterolemia is associated with increased microvascu-
lar density in the renal cortex, which precedes signs of overt
renal morphological damage. These alterations may potentially
affect regulation and/or spatial distribution of intrarenal blood
flow in hypercholesterolemia, and may participate in renal dis-
ease progression.
Key words: kidney, microcirculation, hypercholesterolemia, micro com-
puted tomography, neovascularization.
Received for publication July 20, 2001
and in revised form October 19, 2001
Accepted for publication October 22, 2001
 2002 by the International Society of Nephrology
1056
Hypercholesterolemia (HC) is a common risk factor
for early atherosclerosis. Prior to appearance of overt
atherosclerotic changes in the vascular wall, HC induces
vascular functional changes that may lead to local isch-
emia [1] and vascular remodeling [2, 3]. We have recently
demonstrated that diet-induced HC in pigs was charac-
terized by an increase in the density of coronary adventi-
tial vasa vasorum [2] and intramyocardial microvessels
[4]. The mechanisms underlying new vessel formation
may include local tissue hypoxia and/or up-regulation of
growth factors, such as vascular endothelial growth fac-
tor (VEGF) [5–7].
In the kidney, abnormal lipid metabolism can modify
and accelerate glomerular and vascular damage [8]. Mal-
adjusted renal blood flow responses may involve repeti-
tive renal insults [9], and activation of local intrarenal
growth factors [10] may be associated with fibrosis and
new blood vessel growth [11, 12]. Similar to the heart [13],
limbs [14], and other tissues exposed to ischemia [15],
the deleterious effects of HC in the kidney also may
lead to subtle structural alterations such as new vessel
formation. However, to date it is unknown whether HC
alters intrarenal microvascular structure, partly because
of technical limitations in the analysis of microvascular
architecture.
The renal microvasculature has been studied by a vari-
ety of two-dimensional light microscopic techniques, in
which quantitation is limited by the preparation and ori-
entation of tissue sections, and by three-dimensional
scanning electron microscopic techniques, in which deep
vessels may be hidden or obscured by superimposed ves-
sels at the prepared surface. Micro-computed tomogra-
phy (␮CT) is a novel and powerful imaging technique
that permits the assessment of the three-dimensional
pattern of renal microvascular structure in situ [16], and
provides a useful means for the study of the spatial distri-
bution and connectivity of vessels within an organ [17].
 Bentley et al: Renal vasculature in hypercholesterolemia
This method provides a multidimensional means to study
large representative samples of renal vasculature without
limitations related to plane of section or superimposition.
Therefore, this study was designed to test the hypothe-
sis that experimental HC in pigs is associated with in-
creased intra-renal microvascular density. For this pur-
pose, kidney samples were removed from pigs following
a 12-week high cholesterol diet, and studied in vitro with
␮CT. Renal tissue also was examined histologically for
signs of parenchymal damage, inflammation, and immu-
nostaining for VEGF.
METHODS
This study was approved by the Institutional Animal
Care and Use Committee. Eleven domestic, crossbred
female pigs (weighing 55 to 65 kg) were studied after 12
weeks of either a normal chow (N ⫽ 6) or a 2% HC
diet (N ⫽ 5) (Harlan Teklad, Madison, WI, USA) [9].
After completion of diet, the pigs were anesthetized with
a combination of ketamine (12.5 mg/kg IM) and xylazine
(1 mg/kg IM). A blood sample was drawn from an ear
vein cannula for determination of serum lipid levels.
Heparin (10,000 units) was then infused into the ear vein
cannula, and the pigs euthanized with a lethal dose of
pentobarbital sodium (Sleepaway威, Fort Dodge Labora-
tories, IA, USA).
Preparation of tissue
Immediately following euthanasia, the left kidney was
removed by a flank incision and immersed in Kreb’s
solution containing heparin (10 U/mL) to prevent drying.
A lobe of tissue was removed from one end of the kidney
and immersed in 10% buffered formalin (Sigma, St.
Louis, MO, USA). This tissue was subsequently embed-
ded in paraffin, and slides were stained with hematoxylin
and eosin (H&E), periodic acid-Schiff (PAS), elastin-
van Gieson’s, and VEGF immunohistochemistry.
For VEGF immunohistochemistry, the tissue sections
were deparaffinzed, rehydrated, treated with rabbit anti-
VEGF (A-20; Santa Cruz Biotechnology, Inc, Santa
Cruz, CA, USA), biotinylated secondary antibody (goat
anti-rabbit). Additional slides were incubated without
primary antibody to determine the background level of
nonspecific binding. After antibody treatment, the tissue
sections were incubated with streptavidin horseradish
peroxidase, treated with 3-amino-9-ethylcarbazole for
color development, and counter-stained with hematoxy-
lin. The intensity of immunostaining was evaluated by
two observers blinded in regards to experimental treat-
ment, and graded in each kidney according to the follow-
ing scale: none ⫽ 0, low ⫽ 1, moderate ⫽ 2, and heavy ⫽
3, as we previously described [18]. The grading of the
two investigators were averaged for each kidney, and
compared between the groups.
1057
Micro-CT procedure
A saline-filled cannula (PE 190) was advanced into a
segmental artery perfusing the intact end of the kidney,
tied in place, and an infusion of 0.9% saline (containing
10 units/mL heparin) was initiated at 10 mL/min (Syringe
Infusion Pump 22; Harvard Apparatus, Holliston, MA,
USA) under physiological perfusion pressure (100
mm Hg). After 10 to 15 minutes the saline infusion was
replaced with infusion (0.8 mL/min) of freshly mixed
radio-opaque silicone polymer, containing lead chro-
mate (Microfil MV122; Flow Tech, Inc., Carver, MA,
USA). This infusion was continued until the polymer
drained freely from the segmental vein. After complete
polymerization, a lobe of the polymer-filled tissue was
trimmed from the kidney and placed in 10% buffered
formalin. This lobe was glycerinated, encased in paraffin,
and scanned using a ␮CT scanner as previously described
[17]. The paraffin encasement served to physically stabi-
lize the lobe for scanning and prevented exposure to air
during the scan. Following the scan, the three-dimen-
sional volume images were reconstructed. The recon-
structed images consisted of cubic voxels of 20 ␮m on
a side and were displayed at 40 ␮m cubic voxels for
subsequent analysis.
Analysis of images
Image analysis was carried out using the ANALYZE娃
software package (Biomedical Imaging Resource, Mayo
Foundation, Rochester, MN, USA), which provides tools
to compute, display and analyze reconstructed volume
images [16]. For analysis of the cortex, the three-dimen-
sional tomographic images were oriented so that the z-axis
was parallel to the radial vessels. To calculate cortical
thickness, the number of sections perpendicular to the
z-axis was counted between the cortico-medullary junc-
tion and the outermost surface, and multiplied by the
section thickness. Based on the number of cortical sec-
tions, the cortex was then divided into 10 equidistant
zones, starting at the juxtamedullary cortex. For analy-
sis, zones 1 to 3 were considered to belong in the inner
third, zones 4 to 7 in the middle third, and zones 8 to
10 in the outer third of the cortex. A 40-␮m thick section
with an area of 16 mm2 (100 ⫻ 100 voxels) was subse-
quently analyzed at each zone perpendicular to the z-axis.
The number of blood vessels in the sample region was
determined by interactively matching, placing and count-
ing 80 to 360 ␮m circular spots, sized at 1-voxel (40 ␮m)
diameter increments, over images of the vessels. Because
of the parallel arrangement of the vessels, results are
expressed as number of vessels per cm2.
In addition, radial vessels and their branches were
three-dimensionally isolated using a “connectivity” soft-
ware application, and the vascular tree ramifications ex-
amined in each group.
1058 Bentley et al: Renal vasculature in hypercholesterolemia
Table 1. Plasma lipid profiles (mg/dL) in pigs fed a 12-week
normal (N ⫽ 6) or high-cholesterol (N ⫽ 5) diet
Total
cholesterol HDL TG
Normal 81 ⫾ 7 37 ⫾ 3 41 ⫾ 8
Hypercholesterolemia 383 ⫾ 76a 81 ⫾ 11a 45 ⫾ 6
Abbreviations are: HDL, high-density lipoprotein; LDL, low-density lipo-
protein; TG, triglycerides.
a
P ⱕ 0.001 compared to normal
Statistical analysis
Results are expressed as mean ⫾ SEM. The Student
unpaired t test was used to detect differences in serum
cholesterol values and differences in cortical thickness.
Analysis of variance (ANOVA) was used to detect dif-
ferences in vessel number between the control and HC
groups among vessel diameter categories and among the
zones of the cortex. If significant differences were found,
the unpaired Student t test was used to detect specific
differences. Differences were considered significant if
P ⬍ 0.05.
RESULTS
Serum levels of total, high-density lipoprotein (HDL)
and low-density lipoprotein (LDL) cholesterol were sig-
nificantly elevated in the HC compared to the control
group (Table 1), confirming HC in this group.
Cortical microvascular architecture
The cortical thickness (that is, the distance from the
cortico-medullary junction to the outer surface) was sim-
ilar in the control and HC kidneys (8.5 ⫾ 1.1 and 9.8 ⫾
1.0 mm, respectively, P ⫽ NS). Cortical vessels that were
clearly distinguishable and could be counted ranged from
large (diameters greater than 360 ␮m) to small radial
vessels (diameters 80 ␮m). Several branching orders of
radial vessels extended from the arcuate vessels periph-
erally (Fig. 1). The vessels appeared more densely packed
in the HC kidneys than in the control kidneys (Figs. 1
and 2), and when the connectivity of the vascular trees
of radial vessels was examined, the HC kidneys appeared
to have more ramifications (sprouting) than did the con-
trol kidneys (Fig. 3).
Cortical microvascular number and size
The overall number of vessels ranging in size between
80 and 360 ␮m was significantly greater in the HC than
in the control kidneys (189 ⫾ 7 vs. 126 ⫾ 6 vessels/cm2,
P ⬍ 0.0001, Fig. 4), which showed significant differences
in the distribution pattern among the size categories of
the vessels (Fig. 5). These differences were most pro-
nounced in the inner two thirds of the cortex (Figs. 4
and 5). Most notably, in the inner and middle cortex of
HC kidneys there were significantly more vessels with
diameters of 80 ␮m compared to control kidneys (P ⫽
0.003 and P ⫽ 0.03, respectively, Fig. 5). In the inner
LDL
cortex of HC kidneys there were also significantly more
36 ⫾ 7 vessels with diameters greater than 360 ␮m (P ⫽ 0.018),
225 ⫾ 35a
while in the middle cortex of these kidneys there were
significantly more 240 ␮m (P ⫽ 0.035) and 320 ␮m (P ⫽
0.006) vessels compared to the control (Fig. 5). On the
other hand, in the outer cortex the only microvessels
whose number was significantly greater in the HC com-
pared to the control kidneys were those with diameters
of 280 ␮m (P ⫽ 0.04).
Renal histology
There were no obvious differences between the groups
in glomerular morphology (for example, segmental or
global glomerulosclerosis) as assessed by evaluation of
PAS-stained slides, and no appreciable differences in
vascular medial thickness as assessed by elastin-van Gie-
son’s staining. The elastin-van Gieson’s staining also re-
vealed an extensive peri-arterial loose connective sheath
around the arcuate and large radial arteries. This sheath
attenuated as the arterial diameter diminished, and there
was no appreciable difference between control and HC
kidneys. There were also no obvious differences between
control and HC kidneys in the H&E stained slides in
regards to glomerular, tubular, or vascular structure.
However, in HC kidneys H&E staining disclosed mild
focal chronic inflammatory infiltrates of mononuclear
cells, consisting of lymphocytes admixed with occasional
macrophages and plasma cells, which were most evident
in the deep cortical and outer medullary interstitium.
Positive immunostaining for VEGF was apparent, in
both control and HC kidneys, in the thick ascending
limbs and collecting ducts of the medullary rays. How-
ever, in HC kidneys the intensity of the staining was
heavier, consistently ranging from moderate to heavy,
whereas in the control kidneys the intensity ranged from
mild to moderate (2.2 ⫾ 0.1 vs. 1.6 ⫾ 0.2, P ⫽ 0.03,
respectively, Fig. 6). The immunostaining was especially
obvious in the tubules that were closely associated with
the radial vessels. In addition, intensely stained cells were
observed among the inflammatory infiltrates of the HC
kidneys (Fig. 6).
DISCUSSION
This study demonstrates that experimental diet-induced
HC is associated with increased spatial density of the intra-
renal cortical microvasculature, particularly in its inner
zone. These alterations may involve increased sprouting
from intracortical vessels, and potentially may play a
role in regulation and/or spatial distribution of intrarenal
blood flow.
Experimental and clinical data indicate a potential dam-
Bentley et al: Renal vasculature in hypercholesterolemia 1059

Fig. 1. Micro-computed tomography three-

dimensional (␮CT) images of cortex and me-
dulla in kidney lobes removed from control
and high cholesterol pigs, showing a visually
appreciable increase in spatial density of inner
cortical microvessels in the latter. The dis-
played voxel size is 40 ␮m and the imaged
tissue is 30 sections (1200 ␮m) thick.

Fig. 2. Sample cross-sections (40 ␮m thick)

of inner, middle, and outer cortical tissue of
control and high cholesterol kidneys, showing
increased number of inner cortical microves-
sels in high cholesterol kidneys. The displayed
voxel size is 40 ␮m, and the imaged area is
16 mm2 (Scale bar is 500 ␮m).
1060 Bentley et al: Renal vasculature in hypercholesterolemia
Fig. 3. Comparison of radial vessels rendered from ␮-CT volume im-
ages of control and high cholesterol kidneys. In each kidney the vessels
extend radially from the arcuate origin at the bottom of the images to
the outermost cortex at the top. The vascular tree of the high cholesterol
kidney shows a sizable increase in ramifications compared to the control
kidney. The displayed voxel size is 40 ␮m (Scale bar is 500 ␮m).
Fig. 4. Number of vessels ranging between 80 and 360 ␮m in diameter
in the cortical zones of control (䊏) and high cholesterol (䊉) kidneys.
*Significant difference (P ⬍ 0.05) between control and high cholesterol
groups.
Fig. 5. Size distribution pattern of microvessels in the (A) outer, (B)
middle and (C ) inner cortical regions of control ( ) and high cholesterol
(䊏) kidneys. *Significant difference (P ⬍ 0.05) between control and
high cholesterol groups.
aging effect of abnormal lipid metabolism on the kidney.
Direct cellular effects of oxidized LDL cholesterol and
free oxygen radicals generated in HC may underlie dam-
age to the renal parenchyma and vasculature [19], inter-
stitial injury, and glomerular disease [20]. Furthermore,
we have previously shown that early diet-induced HC
was associated with impaired renal vascular responses
both in vitro [21] and in vivo [9]. However, it remained
unknown whether these functional abnormalities were
associated with microvascular structural alterations.
 Bentley et al: Renal vasculature in hypercholesterolemia
Fig. 6. Light microscope sections of vascular endothelial growth factor
(VEGF) immunohistochemistry in cortical tissue from control (A) and
high cholesterol (B) kidneys. Note that VEGF immunostaining (arrows)
in the thick ascending limbs and collecting ducts in the medullary rays
and around the blood vessels is heavier in the high cholesterol kidney
compared to the control kidney. (C ) Focal mononuclear infiltration in
a high cholesterol kidney. Positive immunostaining for VEGF is ob-
served in many of the infiltrating cells (inset, ⫻4 enlargement).
In the current study we observed in the diet-induced
HC pig model a marked increase in the spatial density
of intra-renal microvessels in the renal cortex. These
changes in microvascular density were most notable in
the inner cortex, while the outer cortex did not show
marked differences between HC and normal pig kidneys.
The location of these structural alterations may be partly
1061
related to the unique anatomy and regulation of the
juxtamedullary vasculature. Lemly and Kriz noted that
cortical arteries were surrounded by a sheath of loose
connective tissue containing lymphatic vessels, which
was most extensively developed around the juxtamedul-
lary arcuate and radial vessels [22]. They speculated that
these sheaths might serve as pathways for interstitial
distribution of lymphocytes and macrophages, as well as
kidney-borne regulatory substances [22]. Similar peri-
arterial sheaths were indeed observed in histologic sec-
tions in the current study. Furthermore, the kidney is
often exposed to systemic challenges (such as dietary,
drugs, toxins, and circulating humoral factors), and im-
paired renal perfusion responses could be accompanied
by episodes of renal insults. We have previously shown
that the juxtamedullary cortex has a lower perfusion
profile relative to other cortical zones [23]. Although
global renal ischemia occurs only during extreme reduc-
tions in renal blood flow, repetitive insults in HC could
conceivably be associated with regional hypoxia, espe-
cially in sensitive intrarenal regions like the outer me-
dulla or the adjacent inner cortex [24], and thereby lead
to new vessel formation [25]. Interestingly, in choles-
terol-fed rabbits with advanced atherosclerosis, lipid de-
position and foam cell formation were observed in a
similar anatomic region [26], which also may support
differential regional propensity and/or sensitivity to HC.
Nonetheless, our findings in early diet-induced HC were
not associated with other signs of morphological damage,
and may represent one of the earliest structural effects
of high-cholesterol diet on the kidney.
Various mechanisms may be responsible for the in-
creased microvascular density in the kidney. HC can
stimulate release of various cytokines and growth factors
[27, 28], including VEGF [29]. Although HC does not
induce new capillary formation in ischemic limb muscle
[30] or in the rabbit aorta [31], neovascularization has
been shown around very early atherosclerotic carotid
lesions [32] and in the coronary adventitia [2] and myo-
cardium [4] of HC pigs, prior to any morphological ab-
normalities of the vascular wall. Our study demonstrates
that similar enhancement of microvascular spatial den-
sity in HC occurs in the renal parenchyma. Notably, this
enhancement was most evident in the innermost cortex,
where mild inflammatory infiltrates of mononuclear cells
were observed in histological preparations. Indeed, mac-
rophages and T lymphocytes are commonly observed in
the vascular wall at every stage of evolution of athero-
sclerosis [28], and this study shows that they can also be
found in the renal cortex in an early stage of HC. These
cells mediate inflammatory responses including release
of cytokines and growth factors [28], and may thereby
induce new vessel growth [33].
In particular, in the present study the thick ascending
limbs and collecting ducts of the medullary rays, espe-
1062 Bentley et al: Renal vasculature in hypercholesterolemia
cially those in close association with the radial vessels,
showed positive immunostaining for VEGF. In adult tis-
sues, blood vessel growth and formation is under tight
control by a variety of growth factors, including VEGF
[5], and constitutive VEGF production has been demon-
strated in adult kidneys in a variety of cell types [34–36].
However, its production can be up-regulated by factors
such as ischemia, nitric oxide, and oxidized LDL [5,
37–39], often within or in the vicinity of infiltrating in-
flammatory cells [40, 41], and in turn increases vascular
permeability and promotes angiogenesis [5] in nearby
tissue. In the current study, the intensity of tubular immu-
nostaining for VEGF in the medullary rays was greater in
the HC kidneys than in the control kidneys, and intense
staining also was observed in many of the infiltrating
cells. Speculatively, VEGF could have been involved in
the increased cortical microvascular density.
The increased vascular density was mostly due to the
microvessels under 360 ␮m in diameter, which are the
major determinants of vascular resistance [42, 43]. Al-
though the increased vascular density in HC categorized
according to vessel size showed asymmetric distribution
throughout the cortex, the functional significance of this
pattern remains to be defined. Furthermore, it is fairly
well established that nephron number does not apprecia-
bly increase after birth [44, 45]. Therefore, the observed
microvessels were likely involved in sustaining parenchy-
mal perfusion, and could have reflected a compensatory
response to inadequate renal blood supply.
On the other hand, it is possible that the increased
vessel number in the HC kidneys may not have resulted
from new vessel formation, but rather from native vessels
that dilated in response to inadequate renal perfusion, or
as a result of other stimuli. Decrements of renal perfusion
may be associated with the visual appearance of new
vessels [46] and collateral arterial circulation [47], and
native coronary microvascular collaterals progressively
dilate during acute myocardial ischemia [48]. Alterna-
tively, extravascular space contraction in HC could have
artifactually increased regional vascular density, but this
is improbable because cortical thickness in the HC pigs
was similar to normal, and renal cortical volume in this
model is indistinguishable from control [9]. In addition,
the spatial differences in vessel numbers in the HC kid-
neys were increased in the inner two thirds but not outer
third of the cortex, arguing against systematic artifacts
such as changes in extravascular space. Finally, the en-
hanced immunostaining for VEGF may support the no-
tion of new vessel formation in the HC kidneys.
Use of the ␮CT, a novel and powerful imaging tech-
nique, permits visualization and analysis of the three-
dimensional spatial distribution pattern and geometry of
the intra-renal microvasculature in situ [16]. In the pres-
ent study, the vascular ramification pattern in ␮CT im-
ages of the control kidneys was similar to that described
by Vodenicharov and Simoens [49]. Moreover, the den-
sity and distribution of cortical microvessels was compa-
rable to that described by Xu et al using histological
methods [50]. In the present study, we counted all vessels
observed in the imaged sections, and hence it is possible
that some of the observed alterations were partly in the
venous as well as arterial circulation.
In summary, we observed an increase in spatial micro-
vascular density in early experimental HC within the inner
renal cortex. This finding may reflect alterations in renal
perfusion or cytokine activation, and may potentially
interfere with regulation and/or spatial distribution of
intrarenal blood flow, since newly generated vessels may
show abnormal functional or structural characteristics
[48, 51]. Hence, subtle microvascular structural alter-
ations may affect renal disease progression in HC.
ACKNOWLEDGMENTS
This study was supported in part by grants HL-03621, HL-63282, and
RR-11800 from the NIH, and by the Mayo Foundation. The authors
are grateful to the following individuals for their skillful technical
assistance: Mr. James D. Krier, Ms. Denise A. Reyes, Mr. Steven M.
Jorgensen, Ms. Patricia E. Lund, Ms. Paula Carlson, and Ariel Feld-
stein, MD. The authors would also like to thank Ms. Julie M. Patterson
for her assistance with the preparation of illustrations.
Reprint requests to Lilach O. Lerman, M.D., Ph.D., Division of
Hypertension, Mayo Clinic, 200 First Street SW, Rochester, Minnesota
55905, USA.
E-mail: Lerman.Lilach@Mayo.Edu
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