Am J Physiol Renal Physiol 288: F198 –F206, 2005.
First published September 21, 2004; doi:10.1152/ajprenal.00244.2003.
HIF-1␣ expression follows microvascular loss in advanced
murine adriamycin nephrosis
Lukas Karolis Kairaitis,1 Yiping Wang,1 Max Gassmann,2
Yuet-Ching Tay,1 and David Charles Hamlyn Harris1
1
The University of Sydney at Westmead Millennium Institute, Sydney, New South Wales, Australia;
and 2Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland
Submitted 14 July 2003; accepted in final form 16 September 2004
capillary; chronic; tubular
has been shown to have effects
potentially relevant to the pathogenesis of chronic renal injury.
Hypoxia has profibrotic effects in vitro, invoking the transcription of matrix proteins (28, 29, 35), profibrotic growth factors
(15, 28, 35), and endothelin-1 (ET-1) (23, 34). In addition,
distinct hypoxia-dependent pathways may exist for the induction of apoptosis (6, 43). Since renal fibrosis and tubular
atrophy are inevitable components of the tubulointerstitial
disease that accompanies all progressive renal diseases, it has
been suggested that cellular responses to hypoxia may represent a “final common pathway” for the development of these
pathological changes (9, 10).
CELLULAR HYPOXIA IN VITRO
Address for reprint requests and other correspondence: L. Kairaitis, Dept. of
Renal Medicine, Westmead Hospital, Westmead, NSW 2145, Australia (Email: [email protected]).
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To date, supportive evidence for this theory has been indirect. Progressive renal diseases are associated with both a
progressive loss of vascular structures (4, 21, 22, 26, 30) and an
excess of vasoconstrictor substances such as ANG II and ET-1
(33, 52), both of which may decrease oxygen supply to the
renal parenchyma. In conjunction with these changes, there are
alterations in renal oxygen demand due to the processes of
hypertrophy, atrophy, and loss of tissue components, all of
which may be present in variable degrees. The net effect in
terms of the timing and severity of tubular hypoxia, therefore,
remains to be elucidated.
We previously reported a progressive reduction in binding of
the hypoxia marker EF5 in murine adriamycin nephrosis, a
model of human chronic renal disease (20a). Although this
result could be interpreted as indicating less hypoxia in this
model, we were unable to correct for alterations in delivery or
binding of the EF5 marker occurring as a consequence of renal
injury. To further assess hypoxia in this model, we examined a
potentially more reliable marker of cellular hypoxia [nuclear
localization of the ␣-subunit of hypoxia-inducible factor-1
(HIF-1␣)]. HIF-1 is the prototypical factor involved in transcriptional responses to cellular hypoxia (46). While the ␤-subunit of HIF-1 is hypoxia independent, the ␣-subunit is stabilized in an oxygen-dependent manner (reviewed in Ref. 18).
Stabilization of HIF-1␣ allows it to bind to the ␤-subunit and
translocate to the nucleus, where it induces the transcription of
multiple genes involved in homeostasis and disease pathogenesis. Detection of nuclear HIF-1␣ is therefore recognized as a
marker of cellular hypoxia (20, 44, 50) that reflects local
oxygen supply/demand ratios within the tissue.
Nuclear HIF-1␣ localization was examined at different time
points in the course of murine adriamycin nephrosis by quantitative immunohistochemistry and Western blotting. The degree of nuclear HIF-1␣ accumulation was interpreted in the
context of changes to the cortical microcirculation and measurement of the angiogenic growth factor VEGF.
MATERIALS AND METHODS
Murine Adriamycin Nephrosis
BALB/c mice were cared for in the Department of Animal Care,
Westmead Hospital, under the ethical guidelines outlined in the Code
of Practice for the Care and Use of Animals for scientific purposes
established by the Australian National Health and Medical Research
Council (NHMRC). Six-week-old male inbred BALB/c mice, weighing 22–25 g, were kept under standard conditions with unrestricted
The costs of publication of this article were defrayed in part by the payment
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0363-6127/05 $8.00 Copyright © 2005 the American Physiological Society
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Kairaitis, Lukas Karolis, Yiping Wang, Max Gassmann,YuetChing Tay, and David Charles Hamlyn Harris. HIF-1␣ expression
follows microvascular loss in advanced murine adriamycin nephrosis.
Am J Physiol Renal Physiol 288: F198 –F206, 2005. First published
September 21, 2004; doi:10.1152/ajprenal.00244.2003.—Cellular
hypoxia has been proposed as a major factor in the pathogenesis of
chronic renal injury, yet to date there has been no direct evidence to
support its importance. Therefore, we examined cortical hypoxia in an
animal model of chronic renal injury (murine adriamycin nephrosis;
AN) by assessing nuclear localization of the oxygen-dependent ␣-subunit of hypoxia-inducible factor-1 (HIF-1␣) in animals 7, 14, and 28
days after adriamycin. Results were assessed in conjunction with
quantitation of the cortical microvasculature (by CD34 immunostaining) and cortical expression of VEGF. Cortical apoptosis was also
examined by terminal deoxynucleotidyl transferase dUTP nick-end
labeling staining. A dramatic and significant increase in nuclear
localization of HIF-1␣ was seen 28 days after adriamycin in the
context of severe glomerular and tubulointerstitial damage. Areas of
nuclear HIF-1␣ staining did not colocalize with areas of cellular
apoptosis. AN was also associated with a significant attenuation of the
peritubular capillaries that was significant at 14 and 28 days after
adriamycin. Cortical VEGF expression fell in a stepwise manner from
day 7 until day 28 after adriamycin. In conclusion, these data are
consistent with a significant increase in cellular hypoxia occurring in
the advanced stages of murine AN. Increased cortical hypoxia was
preceded by significant reductions in both the number of peritubular
capillaries (i.e., oxygen supply) and the angiogenic cytokine VEGF.
Apart from providing the first direct evidence for cellular hypoxia in
a model of chronic renal disease, these results suggest that a primary
dysregulation of angiogenesis may be the cause of increased hypoxia
in this model.
CORTICAL HYPOXIA IN MURINE ADRIAMYCIN NEPHROSIS
Comparative Histology
Quantitative histological measurements were obtained from the
outer cortex using a modification of previously published methods
(40, 47). Five-micrometer paraffin-embedded sections were stained
with periodic acid-Schiff (PAS). Thirty random cortical images were
taken using an SV-microdigital camera (Sound Vision, Framingham,
MA) fitted to an Axioskop microscope (Zeiss, Oberkochen, Germany). Blinded analysis was then made using image-analysis software
(Optimas version 6.5, Media Cybernetics, Seattle, WA).
Twenty outer cortical glomeruli sectioned through the hilum were
analyzed. The outline of the glomerular tuft was traced with a mouse
for an estimate of tuft volume. As an index of glomerulosclerosis
(GS%), a 24-bit color threshold was used to detect PAS-positive
material within the glomerular tuft, and the proportion of PAS staining
within the tuft was expressed as a percentage.
The volume fraction of the interstitium was estimated in 10 nonoverlapping fields using an 8 ⫻ 11 counting grid. The proportion of
interstitial grid intersections was calculated and expressed as a percentage.
Tubular measurements were obtained from 50 randomly selected
cortical tubules/animal. Using line morphometry, the tubular diameter
was estimated as the shortest axis through the center of the imaged
tubule. Tubular cell height was assessed using line morphometry by
measuring the radial distance between the basement membrane and
the lumen of a line drawn through the center of the largest nucleus
visible in the tubular cross section of interest.
Immunostaining for HIF-1␣
A well-characterized chicken polyclonal anti-HIF-1␣ antibody (5)
was used as a primary antibody. Five-micrometer cryosections were
fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min and
incubated with a 1:50 concentration of primary antibody overnight at
4°C. A peroxidase-conjugated rabbit anti-chicken IgY antibody (1:
100, Pierce) was used as a secondary antibody and peroxidaseconjugated goat anti-rabbit IgG (Dako, Carpinteria, CA) as a tertiary
antibody. Antibody binding was localized using nickel-enhanced
diaminobenzidine (Pierce). Kidney and liver tissue from mice exposed
to in vivo hypoxia (10% O2, 6 h) was used as a positive control.
Double immunostaining was performed to look for evidence of
HIF-1␣ nuclear staining within interstitial inflammatory cells. HIF-1␣
staining was performed as above with the substitution of a Texas
red-conjugated goat anti-rabbit antibody (Molecular Probes) as the
tertiary antibody. Following HIF-1␣ staining, sections were incubated
with rat monoclonal antibodies against murine macrophages (clone
F4/80, 1:100 dilution, BD Biosciences, San Diego, CA), murine CD4
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cells (clone RM4 –5, 1:100, BD Biosciences), and murine CD8 cells
(clone 53– 6.7, 1:100, BD Biosciences) for 1 h at room temperature. A
fluorescein-conjugated goat Fab⬘ fragment to rat immunoglobulins
(1:50, Serotec, Oxford, UK) was used as a secondary fluorescent
reagent for the inflammatory cells.
Cortical nuclear staining of HIF-1␣ was analyzed semiquantitatively using image-analysis software (Optimas version 6.5). Ten
random cortical images (each measuring 257 ⫻ 214 ␮m) were taken
from each animal. Using the software, positively stained nuclei within
the positive control tissue (hypoxic mouse liver) were used to set a
24-bit color “threshold” that recognized positively stained nuclei
within the positive control image. When a suitable threshold was
selected, it was then applied to the experimental slides to enable an
unbiased comparison of the number of positive nuclei within the
samples.
Western Blotting for HIF-1␣
Nuclear proteins were extracted from snap-frozen samples of renal
cortex as previously described (40). Aliquots (25 ␮g) of nuclear
proteins were resolved on an 8% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The chicken IgY anti-HIF-1␣
(1:100) was used as a primary antibody and peroxidase-conjugated
rabbit anti-chicken IgY antibody (1:1,000, Pierce) as the secondary
antibody. Sites of antibody binding were revealed by immersion in an
enhanced chemiluminescent substrate (ECL, Amersham) and exposure to autoradiographic film (Amersham). Comparative densitometry
was performed using a Molecular Dynamics personal densitometer SI
(Amersham) and ImageQuant image-analysis software (Amersham).
Colocalization of HIF-1␣ and Terminal Deoxynucleotidyl
Transferase dUTP Nick-End Labeling Staining
Apoptosis within tissue samples was examined by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining.
HIF-1␣ staining was performed as above using a Texas red-conjugated anti-rabbit antibody (Molecular Probes) as the tertiary antibody.
TUNEL staining was then performed using a commercially available
kit (in situ cell death detection kit, Roche Diagnostics, Mannheim,
Germany). Fluorescent microscopy was performed using a Leica
DM-LB fluorescent microscope (Wetzlar, Germany) fitted with a
digital camera (Spot RT, Diagnostic Instruments, Sterling
Heights, MI).
Assessment of Microvasculature
Immunostaining for murine CD34. The distribution of the endothelial cell marker CD34 (7, 12, 39) within the cortex was examined
by immunohistochemistry and image analysis. Five-micrometer paraffin-embedded sections were dewaxed. Antigen retrieval was performed for 20 min at 95–99°C using a commercial target retrieval
buffer (Dako). A rat monoclonal antibody to mouse CD34 (1:400,
MEC 14.7, Cedarlane Laboratories) was used for 1 h at room temperature. Biotin-conjugated rabbit anti-rat immunoglobulin (Dako)
was used as a secondary antibody. Sites of secondary antibody
binding were demonstrated using a streptavidin-peroxidase conjugate
(Dako) with diaminobenzidine as the enzyme substrate.
Peritubular capillary density was estimated using an adaptation of
previously published methods (21). Ten random outer cortical fields
(measuring 257 ⫻ 214 ␮m) were obtained from each animal. The
capillary rarefaction index (CRI) was then measured using imageanalysis software (Optimas version 6.5). Using a 10 ⫻ 12 counting
grid, the proportion of grid squares that did not contain CD34-staining
capillaries was calculated and expressed as a percentage.
Western blotting for CD34. Aliquots (50 ␮g) of cytoplasmic
proteins were resolved on an 8% SDS-polyacrylamide gel and transferred to a nitrocellulose membranes. The MEC14.7 antibody was
used at a concentration of 1:2,000, and biotinylated rabbit anti-rat IgG
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access to food and water in a 12:12-h light-dark cycle. Murine
adriamycin nephrosis (AN) (47) was established by a single injection
of adriamycin (doxorubicin hydrochloride, Pharmacia and Upjohn,
Perth, Australia) at a dose of 11 ␮g/g into the tail vein of 6-wk-old
male BALB/c mice. Animals (6 – 8/time point) were killed at 7, 14,
and 28 days after adriamycin injection. At death, samples of serum
and bladder urine were collected. The kidneys were rapidly removed
and transferred to a metal plate cooled on ice for sectioning. The
kidney poles were snap frozen in liquid nitrogen and stored at ⫺70°C.
The remaining kidney was sectioned coronally. One-half of the
kidneys were placed in cryomoulds, embedded in optimum cooling
temperature compound (Tissue-Tek, Sakura Finetechnicals, Torrance,
CA) by immersion in liquid nitrogen-cooled isopentane, and stored at
⫺70°C. The other half was immersed in 10% neutral-buffered formalin for 24 h, dehydrated in graded alcohols, and embedded in
paraffin. Creatinine was measured in serum and urine samples by the
Jaffé reaction using a Hitachi 747 multianalyzer (Tokyo, Japan). The
same machine was used to measure serum albumin by the bromocresyl green method and urine protein using the benzethonium chloride
turbidimetric method.
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CORTICAL HYPOXIA IN MURINE ADRIAMYCIN NEPHROSIS
Table 1. Time course of renal functional and histological changes in male BALB/c mice after injection with 11␮g/g
adriamycin
Animal Group
Parameter
Control
(n ⫽ 6)
Day 7 Postadriamycin
(n ⫽ 8)
Day 14 Postadriamycin
(n ⫽ 7)
Day 28 Postadriamycin
(n ⫽ 6)
Serum creatinine, ␮mol/l
Serum albumin, g/l
Urine protein/creatinine ratio, g/mmol
Glomerular tuft area, ␮m2
Glomerulosclerosis index (%PAS material in tuft)
Cortical tubular cell height, ␮m
Interstitial area, % of cortex
20.8⫾5.3
25.0⫾1
1.2⫾0.9
2,272⫾165
32.5⫾4.2
13.1⫾1.5
11.3⫾3.5
16.8⫾1.5†
18.6⫾9.7*
2,221⫾137
35.0⫾7.6
12.0⫾0.8
16.8⫾6.3
32.6⫾14.0
15.8⫾0.3†
23.8⫾14.7†
2,842⫾285†
42.3⫾8.7†
7.9⫾1.7†
28.0⫾7.2†
63.7⫾29.1†
16.3⫾2.2†
29.7⫾16.6†
3,258⫾484†
46.4⫾6.0†
5.6⫾0.4†
32.7⫾6.0†
Values are means ⫾ SD. n, No. of mice; PAS, periodic acid-Schiff. *P ⱕ 0.05 compared with control group. †P ⱕ 0.01 compared with control group.
Assessment of Cortical VEGF
Immunostaining for VEGF. Four-micrometer paraffin-embedded
sections were incubated with a 1:50 dilution of goat polyclonal
antibody against VEGF (P20, Santa Cruz Biotechnology, Santa Cruz,
CA). Preliminary staining of acetone-fixed cryosections showed
VEGF staining in the glomerular mesangium, cortical tubules, and
vascular smooth muscle. The degree of glomerular VEGF staining
was less prominent in paraffin-embedded sections but was restored if
antigen retrieval was performed. Antigen retrieval was not performed
for routine staining due to the minimal contribution of glomerular
VEGF to the total.
Peroxidase-conjugated rabbit anti-goat antibody (1:200, Dako) was
used as the secondary antibody and peroxidase-conjugated goat antirabbit antibody (1:100, Dako) as the tertiary antibody. Cortical VEGF
staining was analyzed semiquantitatively using image-analysis software (Optimas version 6.5) using an adaptation of published methods
(21). Ten random images (measuring 257 ⫻ 214 ␮m) were obtained
from the outer cortex of each animal. Using the software, a 24-bit
color threshold was used to identify positively stained areas in each
image. The area of each image represented by positively stained areas
was then calculated by the software and expressed as a percentage.
Western blotting for VEGF. Aliquots (50 ␮g) of cytoplasmic
proteins were resolved on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Staining was performed using a
1:100 dilution of the primary antibody and peroxidase-conjugated
rabbit anti-goat Ig (Dako, 1:2,000) as the secondary antibody. Comparative densitometry was performed as described above.
Statistical Comparisons
Statistical comparisons between animal groups were performed
using a computer-based statistical package (SPSS version 8, SPSS,
Chicago, IL). Statistical significance between groups was tested using
one-way ANOVA and the least squares method of post hoc analysis.
A P value of 0.05 was considered to represent statistical significance.
RESULTS
Functional and Pathological Effects of Murine AN
As previously described (47), these animals developed progressive renal disease characterized by proteinuria, hypoalbuminemia, and impaired renal function (Table 1). Three mice
died after adriamycin treatment (2 in the 28-day group and 1 in
the 14-day group). Measured values for serum creatinine at 7
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days after adriamycin (5.8 ⫾ 5.5 ␮M) were outside the linear
range of the analyzer used (stated as 9 –2,000 ␮M) and were
excluded from analysis. Serum creatinine levels were statistically greater than control at 28 days after adriamycin. AN
animals developed proteinuria, as evidenced by a stepwise
increase in the urine protein/creatinine ratio that was significantly greater than control after 7 days.
Pathologically, this model was associated with progressive
glomerular, tubular, and interstitial injury, all of which were
severe at 28 days after adriamycin (Table 1). Glomerular
changes observed included progressive glomerular enlargement and an increase in glomerulosclerosis. Progressive tubular atrophy was reflected by loss of the luminal brush border
and a stepwise reduction in tubular cell height. The interstitium
was expanded with a mononuclear cell infiltrate and increased
PAS-positive matrix.
Immunostaining and Western Blotting for HIF-␣
Strong nuclear staining of HIF-1␣ was seen in the kidney
and liver tissue from animals exposed to 10% oxygen (Fig.
1A). In the normal kidney, there was faint staining in cortical
nuclei, predominantly in distal tubules (Fig. 1B). Glomeruli
demonstrated no nuclear staining, and proximal tubules
showed faint nuclear staining only. No nuclear staining was
seen in the medulla or papilla.
The pattern of nuclear staining of HIF-1␣ was significantly
altered in AN. Although HIF-1␣ staining was not obviously
different from control at days 7 and 14 after adriamycin, a
dramatic increase in nuclear HIF-1␣ staining was seen in the
renal cortex at 28 days after adriamycin (Fig. 1C). Nuclear
HIF-1␣ staining was most apparent in areas of extensive
tubular dilatation and interstitial inflammation. In these areas,
nuclear HIF-1␣ staining was seen in both tubular and interstitial cells. Double fluorescent staining for HIF-1␣ and inflammatory cell-surface markers showed nuclear HIF-1␣ staining
within macrophages, CD4 lymphocytes, and CD8 lymphocytes
in cortical samples from animals obtained 28 days after adriamycin (Fig. 2).
Semiquantitative analysis of the number of positive nuclei
per unit of cortical area reflected these observations (Fig. 1D).
There was a dramatic increase in the number of positively
stained nuclei at the 28-day time point (191 ⫾ 115 vs. 9 ⫾ 17
nuclei/unit area, P ⬍ 0.01). An identical pattern was seen when
nuclear protein extracts were probed for HIF-1␣ (Fig. 1E).
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(1:2,000, Dako) was used as the secondary antibody. Sites of antibody
binding were revealed by immersion in streptavidin-HRP (1:3,000,
Dako) before chemiluminescent development. Comparative densitometry was performed as described above.
CORTICAL HYPOXIA IN MURINE ADRIAMYCIN NEPHROSIS
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Colocalization of HIF-1␣ and TUNEL Staining
TUNEL staining revealed apoptotic nuclei within the middle
epithelial layers of the positive control tissue (murine esophagus) consistent with the normal process of keratinocyte matu-
ration (Fig. 3A). Double staining revealed occasional apoptotic
nuclei within the epithelial layers of the esophagus that also
stained positive for HIF-1␣, although the majority of apoptotic
nuclei were negative (Fig. 3A). No apoptosis was visible in the
normal kidney (not shown). Apoptotic nuclei were detectable
Fig. 2. Double staining for HIF-1␣ (red) and mononuclear inflammatory cells (green) in samples from animals at 28 days after adriamycin. Positive staining
within the nuclei of dilated cortical tubules is seen in all sections. Nuclear HIF-1␣ staining was also seen within macrophages (left), CD4 lymphocytes (middle),
and CD8 lymphocytes (right) in the adjacent interstitium (arrows).
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Fig. 1. Hypoxia-inducible factor (HIF)-1␣ staining in tissue samples. In the positive control tissue (hypoxic mouse liver), there was strong nuclear staining of hepatocytes
(A). Weak nuclear staining of occasional distal tubular nuclei was seen in the cortex of the control kidney (B, arrowheads) with minimal staining of proximal tubular
nuclei and no glomerular nuclear staining. Although the number of HIF-1␣-positive nuclei was not obviously increased at days 7 and 14 after adriamycin,
extensive cortical nuclear staining was seen 28 days after adriamycin (C), occurring particularly in atrophic tubules (arrowheads). In addition, there was
strong nuclear staining of cells within the expanded tubulointerstitium (arrows). Semiquantitative analysis of nuclear HIF-1␣ staining in the cortex (D) showed
a significant increase in the number of positive nuclei in the cortex 28 days after adriamycin. Western blotting of nuclear protein extracts for HIF-1␣ revealed
a protein band of ⬃100 kDa (E). Comparative densitometry showed a significant increase in band density at 28 days after adriamycin. Values are means ⫾ SD.
**P ⬍ 0.01 (ANOVA).
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CORTICAL HYPOXIA IN MURINE ADRIAMYCIN NEPHROSIS
after adriamycin. There were scanty apoptotic nuclei at day 7
after adriamycin, with apoptotic nuclei at days 14 and 28 after
adriamycin largely confined to occasional epithelial cells lining
atrophic tubules and sloughed nuclei present within the tubular
lumen (Fig. 3, B and C). Double staining of the AN samples
did not demonstrate areas of overlap of the HIF-1␣ or apoptotic
stains (Fig. 3, B and C).
Assessment of Cortical Microvasculature
CD34 immunostaining of the normal renal cortex revealed a dense capillary network that in some areas appeared to completely encircle individual tubules (Fig. 4A).
The glomerular capillary network also exhibited positive
staining. A dense intertubular capillary network was also
noted in the medulla and papilla (not shown). AN was
associated with significant changes in the density of the
cortical microvasculature. There was a significant reduction
in visible capillaries from day 14 that was most prominent
28 days after adriamycin (Fig. 4B). The reduction in capillaries was particularly prominent in areas of interstitial
expansion and tubular atrophy. In keeping with these observations, there was a significant increase in the capillary
rarefaction index after adriamycin (Fig. 4C). Microvascular
density was reduced threefold at 14 days after adriamycin
(P ⬍ 0.01) and fivefold at 28 days (P ⬍ 0.01). Western
blotting of protein extracts (Fig. 4D) showed a similar
Fig. 4. Assessment of the cortical microvasculature in murine adriamycin nephrosis.
CD34 immunohistochemistry of normal cortex revealed an extensive peritubular capillary plexus (A). Glomerular (gl) capillaries
were also demonstrated. Adriamycin nephrosis was associated with attenuation of the
peritubular capillary plexus, with a dramatic
reduction evident at 28 days after adriamycin
(B). These changes were particularly prominent in areas of interstitial expansion and
tubular atrophy. Semiquantitative analysis of
CD34 immunostaining (C) revealed a progressive loss of staining (an increase in the
rarefaction index) that was significant by 14
days after adriamycin. Western blotting of
cortical protein extracts (D) showed similar
results, with a progressive reduction in the
80-kDa CD34 protein detectable at 14 and 28
days after adriamycin. Values are means ⫾
SD. *P ⬍ 0.05, **P ⬍ 0.01 (ANOVA).
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Fig. 3. Double staining for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and HIF-1␣ in murine tissues. TUNEL-positive nuclei are
stained green, and HIF-1␣-positive nuclei are stained red. Areas of colocalization are orange or yellow. There was extensive apoptosis in the epithelium of the
murine esophagus (A; L, lumen). Although occasional esophageal nuclei showed double staining (arrowheads), the majority of apoptotic nuclei did not
demonstrate HIF-1␣ staining. Apoptotic nuclei were also detected in the adriamycin nephrosis samples. Fourteen days after adriamycin (B), there was evidence
of tubular cell apoptosis with occasional apoptotic nuclei present within luminal cellular casts (not shown). Apoptotic tubular epithelial cells in did not
demonstrate HIF-1␣ positivity. Cortical samples from 28 days after adriamycin (C) showed extensive HIF-1␣ staining. Apoptotic nuclei were confined to
intraluminal cell casts with no clear evidence of colocalization of the 2 stains.
CORTICAL HYPOXIA IN MURINE ADRIAMYCIN NEPHROSIS
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stepwise reduction in cortical CD34 expression that was
significant at 14 and 28 days after adriamycin.
Immunostaining and Western Blotting for VEGF
Extensive tubular staining for VEGF was seen in the normal
kidney, with accentuation of distal tubules (Fig. 5A). There was
a reduction in tubular VEGF staining following adriamycin,
particularly in areas of tubular damage or atrophy (Fig. 5B).
Semiquantitative analysis of cortical VEGF expression reflected these changes. There was a significant reduction in
cortical VEGF detectable at 7 days after adriamycin (Fig. 5C),
with further stepwise reductions at later time points. Western
blotting of cortical proteins also showed a similar decremental
pattern of VEGF expression (Fig. 5D).
DISCUSSION
The results presented in this paper describe in detail the time
course of cortical HIF-1␣ expression in murine AN, a robust
model of human focal glomerulosclerosis. Increased expression and nuclear localization of HIF-1␣ were seen in the late
stages of this model and were localized to tubular and interstitial cells in areas of extensive parenchymal injury. This
finding represents the first direct evidence of hypoxia in an
animal model of chronic renal disease. Increased HIF-1␣
expression in these experiments followed significant reductions
in both peritubular capillary density (a surrogate measure of
oxygen supply) and reduced cortical expression of the angiogenic growth factor VEGF. Areas of cellular hypoxia did not
colocalize with areas of tubular apoptosis in this model.
Two separate methods were used to compare nuclear localization of HIF-1␣ in the current experiments (immunostaining
and Western blotting). Consistent with previous reports, we
found minimal nuclear staining of HIF-1␣ in the normal kidney
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(44, 45, 51). Cortical nuclear HIF-1␣ was significantly increased by whole animal hypoxia as well as in the later stages
of AN. This pattern of nuclear localization is consistent with
increased stabilization of HIF-1␣ in the advanced stages of this
model. The cellular fate of HIF-1␣ is determined by the
activity of a redox-sensitive pathway (reviewed in Ref. 18). In
the presence of oxygen, the ␣-subunit undergoes prolyl hydroxylation and is targeted for proteasomic degradation. In the
absence of oxygen, this hydroxylation does not occur, and after
translocation to the nucleus, HIF-1␣ is free to bind to the
␤-subunit (the aryl hydrocarbon nuclear receptor translocator)
to form the HIF-1 complex that, in turn, interacts with hypoxiaresponsive elements in the promoter region of multiple genes.
Other than hypoxia, a number of other stimuli may potentially
affect the stabilization of HIF-1␣ in this model, including the
proinflammatory molecules TNF-␣, IL-1␤ (2, 17), and nitric
oxide (NO) (19, 37). It is noteworthy, however, that the effects
of TNF-␣, IL-1␤, and NO on HIF-1␣ stabilization are based on
in vitro experiments. A consistent difficulty with the interpretation of these experimental systems is that these molecules
may well exert their in vitro effects by influencing oxygen
consumption, weakening the case for a direct effect of these
stimuli on HIF-1␣ stabilization. Furthermore, extrapolation of
the results obtained with the use of NO donors in vitro is made
more complex by the fact that the production of NO in vivo is
an oxygen-dependent process (16), and exogenous NO donors
may themselves disrupt the redox-dependent process of
HIF-1␣ stabilization in a way that is not relevant to NO
produced in vivo (19, 37). The effects of endogenous TNF-␣,
IL-1␤, and NO on HIF-1␣ stabilization in the current experiments are therefore uncertain.
The presence of increased nuclear HIF-1␣ in the later stages
of this model is strongly suggestive of a significant increase in
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Fig. 5. Assessment of cortical VEGF expression. VEGF immunostaining of the normal kidney (A) showed prominent tubular
staining with enhancement of distal tubules
(d). Tubular staining was reduced 28 days
after adriamycin (B), particularly in dilated
atrophic tubules (a). Sections at 7 and 14
days showed intermediate changes (not
shown). Semiquantitative analysis of cortical
VEGF expression (C) showed a significant
reduction of VEGF immunostaining 7 days
after adriamycin (P ⬍ 0.01). Further stepwise reductions were seen at 14 and 28 days.
Western blotting of cortical protein extracts
(D) showed a similar stepwise reduction of
the 26-kDa VEGF protein. Values are
means ⫾ SD. **P ⬍ 0.01 (ANOVA).
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AJP-Renal Physiol • VOL
tion of the capillary rarefaction index assumes that the volume
of tissue assessed in the normal kidney is unchanged in AN.
This model is associated with tubular dilatation, tubular loss,
and interstitial expansion, all of which affect the volume of
tissue to a variable amount. The reduction of CD34 protein in
AN by Western blotting (which is not subject to the same
limitations) was further evidence that the cortical microvasculature is reduced in this model.
A further result from these experiments is that cortical
expression of the angiogenic cytokine VEGF was significantly
reduced as the disease progressed, consistent with results
obtained in other models of chronic renal injury (8, 21, 31).
This result may seem contradictory to the finding of increased
hypoxia in this model, particularly since VEGF is both induced
by hypoxia and transcriptionally activated by HIF-1 (11, 25,
27). There are several explanations for this apparent disparity.
First, HIF-1 is not the only known transcriptional activator of
VEGF, with experimental evidence suggesting a role for SP-1
(1, 41) as well as the oncogene ras (3, 32). VEGF transcription
is also inhibited by the anti-oncogenes p53 (36) and p73 (42).
Also relevant are previous experiments showing that VEGF
expression is reduced in cutaneous models of inflammation at
a time when HIF-1␣ expression is increased (2) and the finding
that VEGF production in response to hypoxia was reduced in
the presence of macrophage-derived cytokines in vitro (21).
These latter findings are potentially relevant to the reduction of
VEGF observed in the current experiments, since AN is associated with a prominent mononuclear infiltrate (47). It is
therefore likely that the finding of reduced VEGF in AN (as
well as in other renal disease models) is multifactorial in
origin.
Whatever the cause, the finding that VEGF is reduced in AN
has potential relevance to the increased HIF-1␣ expression that
occurs in the late stages of this model. If cellular hypoxia is
caused by a reduction in oxygen supply engendered by microvascular loss in this model, it is also possible, in turn, that the
reduction in peritubular capillaries may in fact be secondary to
reduced local production of VEGF, since VEGF is an important survival factor for endothelial cells (13, 14). In support of
this, the reductions in cortical VEGF seen in AN were observed before a detectable reduction in peritubular capillaries,
which, in turn, preceded a detectable increase in cellular
hypoxia.
In summary, we showed a significant increase in nuclear
HIF-1␣ localization in murine AN, consistent with the presence of increased cellular hypoxia in the later stages of this
model. HIF-1-dependent mechanisms do not appear to be the
predominant cause of apoptosis in this model, and the timing of
the increase in nuclear HIF-1␣ would argue against a significant role for hypoxia in either parenchymal injury or interstitial
fibrosis in this model. The observed reductions in both the
density of peritubular capillaries and expression of the principal angiogenic growth factor VEGF suggest that the increase in
nuclear HIF-1␣ in AN is secondary to a disruption of mechanisms involved in the maintenance of the normally extensive
capillary network within the kidney.
ACKNOWLEDGMENTS
In vivo hypoxia experiments to provide positive controls for HIF-1␣
staining were performed in collaboration with Dr. Scott Geller and Prof.
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cellular hypoxia in advanced disease. Before these data were
gathered, experimental evidence to support the presence of
“chronic renal hypoxia” has been based largely on indirect
findings, including the observed reduction in peritubular capillaries (4, 21, 22, 26, 30) and extrapolation of the profibrotic
effects of hypoxia in vitro (28, 29, 35). The principal advantage
of a HIF-1␣-based method of hypoxia detection is that it gives
direct evidence of transcriptionally relevant hypoxia at a cellular level. The HIF-1 pathway is activated in vivo when
oxygen demand outweighs supply (18). This delicate balance
cannot be inferred from microelectrode measurements that
have previously been regarded as the “gold standard” for direct
hypoxia measurement.
The finding of increased HIF-1␣ stabilization in AN warrants a consideration of whether HIF-1-dependent stimuli may
contribute to the pathogenesis of renal injury in this model.
Although hypoxia in vitro induces a wide range of profibrotic
effects, including the stimulation of profibrotic growth factors
(15, 28, 35), matrix proteins (28, 29, 35), and matrix stabilizers, it was notable in the current experiments that nuclear
HIF-1␣ was increased only in the later stages of this model, at
a time when interstitial fibrosis was already present. This
temporal association makes it unlikely that hypoxia contributes
significantly to the development of interstitial fibrosis in this
model. However, hypoxia could contribute to pathological
changes occurring in the late stages of AN. Our finding that
apoptosis was more extensive in the later stages of this model
in conjunction with the possible existence of HIF-1-dependent
pathways for apoptosis (6) prompted us to examine for evidence of colocalization of HIF-1␣ and a nuclear marker of
apoptosis (TUNEL staining). Using a previously described
fluorescent technique (49), we found evidence of colocalization of HIF-1␣ and TUNEL staining in nuclei of the esophageal epithelium, a tissue in which both cellular hypoxia (24,
38) and apoptosis (48) occur under normoxic conditions. In the
kidney sections, apoptotic nuclei were most prominent in the
later stages of AN, with TUNEL labeling of occasional tubular
epithelial cells and intraluminal cellular casts. Although
HIF-1␣ staining was detectable in the same sections, there was
no colocalization of HIF-1␣ or apoptosis in the kidney samples. This result suggests that HIF-1-dependent pathways are
unlikely to contribute significantly to tubular apoptosis in this
model but may instead represent an attempted homeostatic
response to relative oxygen deficiency within the tubular cells
in advanced AN.
To investigate the potential causes of hypoxia in this model,
we measured peritubular capillary density as a surrogate measure of oxygen supply. AN was associated with a significant
reduction in CD34-labeled peritubular capillaries, consistent
with similar findings in other chronic renal diseases (4, 21, 22,
26, 30). This reduction in capillary density preceded the detection of increased cortical hypoxia and would suggest that a
reduction in oxygen supply plays a role in the development of
hypoxia. Consistent with this hypothesis was the finding that
the areas of greatest capillary rarefaction at day 28 after
adriamycin were those with the greatest degree of tubulointerstitial injury and correlated morphologically with the areas
within which HIF-1␣ nuclear staining was seen. Two methods
(CD34 immunostaining and Western blotting) were used to
assess the cortical microvasculature in this model. Western
blotting was performed because the method used for calcula-
CORTICAL HYPOXIA IN MURINE ADRIAMYCIN NEPHROSIS
Jonathon Stone from the Department of Retinal Biology, the University of
Sydney.
GRANTS
This work was supported by grants from the National Health and Medical
Research Council of Australia and the Swiss National Science Foundation.
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