Injury of the renal microvascular endothelium alters barrier function after ischemia
Timothy A. Sutton,1
Henry E. Mang,1
Silvia B. Campos,1
Ruben M. Sandoval,1,2
Mervin C. Yoder,3 and
Bruce A. Molitoris1,2
1Division of Nephrology, Department of Medicine,
Indiana Center for Biological Microscopy, and
3Division of Neonatology, Department of Pediatrics,
Indiana University School of Medicine, and 2Roudebush
Veterans Affairs Medical Center, Indianapolis, Indiana 46202
Submitted 3 February 2003
; accepted in final form 28 March 2003
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ABSTRACT
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The role of renal microvascular endothelial cell injury in the
pathophysiology of ischemic acute renal failure (ARF) remains largely unknown.
No consistent morphological alterations have been ascribed to the endothelium
of the renal microvasculature as a result of ischemia-reperfusion injury.
Therefore, the purpose of this study was to examine biochemical markers of
endothelial injury and morphological changes in the renal microvascular
endothelium in a rodent model of ischemic ARF. Circulating von Willebrand
factor (vWF) was measured as a marker of endothelial injury. Twenty-four hours
after ischemia, circulating vWF peaked at 124% over baseline values
(P = 0.001). The FVB-TIE2/GFP mouse was utilized to localize
morphological changes in the renal microvascular endothelium. Immediately
after ischemia, there was a marked increase in F-actin aggregates in the basal
and basolateral aspect of renal microvascular endothelial cells in the
corticomedullary junction. After 24 h of reperfusion, the pattern of F-actin
staining was more similar to that observed under physiological conditions. In
addition, alterations in the integrity of the adherens junctions of the renal
microvasculature, as demonstrated by loss of localization in vascular
endothelial cadherin immunostaining, were observed after 24 h of reperfusion.
This observation temporally correlated with the greatest extent of
permeability defect in the renal microvasculature as identified using
fluorescent dextrans and two-photon intravital imaging. Taken together, these
findings indicate that renal vascular endothelial injury occurs in ischemic
ARF and may play an important role in the pathophysiology of ischemic ARF.
von Willebrand factor; vascular endothelial cadherin; vascular permeability
BOTH SUBLETHAL AND LETHAL tubular epithelial cell injury have
been of central importance in explaining the decrement in glomerular
filtration rate that is the hallmark of acute renal failure (ARF). However,
over the last decade the paradigm of the pathophysiology of ischemic ARF has
evolved to include a complex interplay between tubular injury, inflammation,
and altered renal hemodynamics. Recent studies have provided further evidence
for the role that vascular injury, in particular endothelial cell injury,
plays in the pathophysiology of ischemic ARF
(5,
46). Endothelial cell
swelling, altered endothelial cell-cell attachment, and altered endothelial
cell-basement membrane attachment are some of the morphologic alterations that
have been observed in the renal microvasculature
(5,
12) as well as the cerebral
and coronary vasculature (40,
42) after ischemic injury.
Functional consequences of these morphological alterations include altered
vascular reactivity, increased leukocyte adherence and extravasation, altered
coagulation due to loss of normal endothelial function and/or barrier, and
increased interstitial edema that have been documented as a consequence of
ischemic ARF in animal models
(16).
Specialized cellular junctions similar to those in epithelial cells
maintain endothelial cell-cell contacts. Tight junctions are more prominent in
"tight" vascular beds, such as those between the endothelial cells
of the cerebral vasculature forming the blood-brain barrier, whereas they are
sparse and simplified in "leaky" vascular beds, such as
postcapillary venules (43).
Cadherin-containing adherens junctions are ubiquitous between endothelial
cells throughout the vasculature
(43). Recent studies
highlighting the differences in the molecular composition of junctional
complexes in various vascular beds, including those within the kidney, provide
insight into the potential functional differences in the cellular junctions in
these vascular beds (3,
11,
25,
32). Disruption of endothelial
adherens junctions in vivo by the use of an inhibitory antibody to vascular
endothelial cadherin (VE-cadherin) has been demonstrated to induce gaps
between endothelial cells, increase endothelial permeability, and promote the
accumulation of inflammatory cells in coronary and pulmonary vascular beds
(8). Furthermore, there is
evidence from in vitro studies that the interaction of endothelial cell-cell
junctions with the actin cytoskeleton plays an important role in regulating
endothelial paracellular transport
(34). Although the above
findings underscore the importance of endothelial cell-cell junctions in
maintaining the integrity of the endothelial permeability barrier, there is
essentially no in vivo information on the effect of ischemic injury on the
function and organization of these intercellular junctions in the renal
microvasculature due to previous technical difficulty in visualizing the
endothelium of the renal microvasculature in animals and human biopsy samples.
However, there are data indicating microvascular congestion and localized
interstitial edema after renal ischemia
(15,
16). Therefore, we
hypothesized that ischemic injury to the kidney results in alterations of the
endothelial actin cytoskeleton and endothelial cell-cell junctions that
contribute to increased vascular permeability and local interstitial edema. In
this study, we demonstrate that ischemic renal injury results in
disorganization of the actin cytoskeleton and loss of VE-cadherin localization
in the endothelium of the renal microvasculature and that these alterations
are accompanied temporally by increased permeability of the renal
microvasculature.
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MATERIALS AND METHODS
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Reagents and antibodies. Rhodamine-conjugated dextran (3,000 mol
wt), FITC-conjugated dextran (500,000 mol wt),
rhodamine-conjugated-phalloidin, and Hoechst 33342 were from Molecular Probes
(Eugene, OR). Rat polyclonal antibodies to mouse VE-cadherin were from BD
Pharmingen (San Diego, CA). Affinity-purified Texas red-labeled sheep anti-rat
IgG antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).
An Asserachrom vWF ELISA kit was from Diagnostica Stago (Parsippany, NJ), and
a Quantikine M rat IL-6 immunoassay kit was from R&D Systems (Minneapolis,
MN).
Animals. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) and
male FVB-TIE2/GFP mice, which express green fluorescent protein (GFP) under
the direction of the endothelial-specific receptor tyrosine kinase
(Tie2) promoter (33),
were used as described below.
Surgical procedure to induce renal ischemia. All experiments were
conducted in accordance with the Guide for the Care and Use of Laboratory
Animals (Washington, DC: National Academy Press, 1996) and approved by
the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats
weighing 200250 g were anesthetized with an intraperitoneal injection
of pentobarbital sodium (65 mg/kg) and placed on a homeothermic table to
maintain core body temperature at 37°C. For experiments involving
measurement of biochemical markers of endothelial injury, a midline incision
was made, the renal pedicles were isolated, and bilateral renal ischemia was
induced by clamping the renal pedicles for 45 min as previously described
(31). Sham surgery consisted
of an identical procedure with the exception of immediate release of the
microaneurysm clamps. Tail-vein blood samples from experimental, sham, and
nonoperative control rats were collected, stored, and processed as per
instructions of the Asserachrom vWF ELISA kit. For experiments involving live
two-photon microscopic imaging of rat kidneys, a flank incision was made over
the left kidney, the renal pedicle was isolated, and unilateral renal ischemia
was induced by clamping the left renal pedicle for 45 min as previously
described (9). For experiments
involving renal ischemia in FVB-TIE2/GFP mice, 20- to 25-g mice were
anesthetized utilizing 5% halothane for induction and 1.5% for maintenance and
placed on a homeothermic table to maintain core body temperature at 37°C.
A midline incision was made, and the left renal pedicle was isolated. Renal
ischemia was induced by clamping the renal pedicle for 32 min as described by
Kelly et al. (20).
Fluorescence microscopy. Kidney sections from anesthetized mice
were fixed ex vivo in 4% paraformaldehyde, and 50-µm vibratome sections
were obtained. Sections were stained with rhodamine-phalloidin or rat
polyclonal antimouse VE-cadherin and polyclonal Texas red-labeled sheep
anti-rat IgG secondary antibodies. Images of the microvasculature in the
corticomedullary region of the kidney were collected with an LSM-510 Zeiss
confocal microscope (Heidelberg, Germany) equipped with argon and helium/neon
lasers. For intravital fluorescence microscopy, 100 µl of
rhodamine-conjugated dextran (3,000 mol wt, 20 mg/ml in 0.9% saline), 500
µl of FITC-conjugated dextran (500,000 mol wt, 7.5 mg/ml in 0.9% saline),
and 400 µl of Hoechst 33342 (1.5 mg/ml in 0.9% saline) were injected via
the tail vein into anesthetized rats just before imaging. The left kidney of
the anesthetized rat was imaged through a retroperitoneal window via a
left-flank incision using a Bio-Rad MRC-1024MP Laser Scanning
Confocal/Multiphoton scanner (Hercules, CA) with an excitation wavelength of
800 nm attached to a Nikon Diaphot inverted microscope (Fryer, Huntley, IL) as
described by Dunn et al. (9).
Image processing was performed utilizing Metamorph software (Universal
Imaging, West Chester, PA).
Statistics. Results are expressed as means ± SE and were
analyzed for significance by paired and unpaired Student's t-tests
and ANOVA.
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RESULTS
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Biochemical evidence of renal endothelial injury during ischemia.
A variety of studies have utilized an elevated circulating von Willebrand
factor Ag (vWF) concentration as a marker of endothelial cell injury in other
organs in response to a variety of insults
(1,
6,
13,
19,
28,
35,
37) including
ischemia-reperfusion injury to the intestines
(1). Therefore, we quantified
circulating vWF in Sprageu-Dawley rats as a marker of renal endothelial injury
after renal ischemia. Twenty-four hours after a 45-min bilateral renal artery
clamp, circulating vWF reached its maximum level and was significantly
elevated (Table 1) over
baseline preclamp values (P = 0.001) and over levels in sham-operated
control animals (P = 0.029). Circulating vWF levels in sham-operated
animals were not significantly different from baseline values at 24 h.
Circulating vWF decreased by 48 h after ischemia and was not significantly
elevated over baseline values or over the level in sham-operated control
animals.
The actin cytoskeleton of renal microvascular endothelium was disrupted
after ischemia. To determine the effect of ischemia on the actin
cytoskeleton of the renal microvascular endothelium, sections of normal and
ischemic kidneys from FVB-TIE2/GFP mice were stained with rhodamine-phalloidin
and examined by confocal microscopy. Under physiological conditions, F-actin
staining was predominantly observed along the basal aspect of renal
microvascular endothelial cells (Fig.
1A). Immediately after 32 min of renal ischemia,
alterations in the actin cytoskeleton of the renal microvascular endothelium
included F-actin aggregation along the lateral aspects of the cells and
increased F-actin aggregation along the basal aspects of the cells
(Fig. 1B). After 24 h
of reperfusion subsequent to ischemia, the F-actin staining pattern more
closely resembled the pattern observed under physiological conditions
(Fig. 1C), although
there remained a more aggregated appearance of the F-actin along the basal
aspects of the renal microvascular endothelial cells than what was observed
under physiological conditions.

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Fig. 1. The actin cytoskeleton of renal endothelial cells is disrupted after renal
ischemia. The FVB-TIE2/GFP mouse, which expresses green fluorescent protein
(GFP) driven by the endothelial TIE2 promoter, was utilized to localize
changes in the actin cytoskeleton of the microvascular endothelium (green) in
the corticomedullary region of the kidney. Rhodamine-phalloidin was used to
stain F-actin (red). A: nonischemic kidney. B: kidney after
32 min of renal artery clamping. Note the increase in F-actin polymerization
and aggregation in endothelial cells of the renal microvasculature (arrow).
C: kidney after 24 h of reperfusion following 32 min of renal artery
clamping. Bar = 10 µm.
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Adherens junctions of renal microvascular endothelium were disrupted
after ischemia. To determine the effect of ischemia on the adherens
junctions of the renal microvascular endothelium, the pattern of VE-cadherin
immunostaining was examined by confocal microscopy in sections of normal and
ischemic kidneys from FVB-TIE2/GFP mice. Under physiological conditions,
VE-cadherin immunostaining was continuous along the renal microvascular
endothelium (Fig. 2). Whereas
VE-cadherin immunostaining was not limited to intracellular endothelial
contacts, it was similar to in vivo VE-cadherin immunostaining observed in
other studies (4,
8). After 32 min of ischemia,
VE-cadherin staining remained in the renal microvasculature. Twenty-four hours
after ischemia, the majority of the renal microvasculature did not stain for
VE-cadherin. The loss of VE-cadherin staining after ischemia suggested a
disruption of the normal junctional complex between endothelial cells of the
renal microvasculature. Seventy-two hours after ischemia, VE-cadherin staining
in the renal microvasculature was similar to that observed under physiological
conditions.

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Fig. 2. Adherens junctions of renal microvascular endothelium were disrupted after
ischemia. The FVB-TIE2/GFP mouse was utilized to localize changes in vascular
endothelial (VE)-cadherin (red) staining of the microvascular endothelium
(green) in the corticomedullary region of the kidney after ischemia. While
VE-cadherin staining was present under physiological conditions (arrow in
A) and immediately after 32 min of ischemia (B), loss of
VE-cadherin staining was noted after 24 h of reperfusion following 32 min of
ischemia (C). Of note, immunostaining was observed in the lumens (*)
of tubules at 24 h postischemia; however, this pattern of staining was seen in
the secondary antibody controls (data not shown), suggesting that the staining
in the tubular lumen was nonspecific secondary antibody staining and not
VE-cadherin staining. Seventy-two hours after 32 min of ischemia, VE-cadherin
staining was similar to that under physiological conditions. Bar = 10
µm.
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Alteration in renal microvascular permeability after ischemia.
Having demonstrated morphological alterations in the actin cytoskeleton and
the adherens junctions of the renal microvascular endothelium after ischemia,
we undertook the next study to examine the functional correlate of the
observed morphological alterations. Technical considerations limited the
effectiveness of two-photon intravital imaging of the corticomedullary region
in rodents, as well as intravital imaging of kidneys in FVB-TIE2/GFP mice.
Consequently, intravital imaging was performed in the cortical region of rats.
To examine alterations in vascular permeability, a rhodamine-conjugated,
low-molecular-weight (3,000) neutral dextran and a FITC-conjugated,
high-molecular-weight (500,000) neutral dextran were intravenously coinjected
into rats under physiological conditions and after ischemia. Injections of
fluorescent dextrans were undertaken at selected time points to evaluate the
microvascular permeability at that particular point in time. The renal
microcirculation was observed in vivo utilizing dual-photon confocal
microscopy. Injection of the fluorescent-labeled dextrans under physiological
conditions revealed that the high-molecular-weight dextran was maintained in
the vascular space and was not filtered by the glomerulus, whereas, the
low-molecular-weight dextran was rapidly filtered into the tubules,
accumulated in endosomes of the proximal tubule, and concentrated in the lumen
of the distal tubules as previously described
(9)
(Fig. 3A). Areas of
diminished microvascular blood flow were observed immediately after renal
ischemia; however, occasional areas of leakage of either the low- or
high-molecular-weight dextran from the microvascular space into the renal
interstitium were not observed until 2 h after renal ischemia (arrows,
Fig. 3B). Leakage of
both dextrans appeared to reach its greatest extent 24 h after ischemia
(Fig. 3C). Forty-eight
hours after ischemia, there were still some patchy areas of leakage but the
extent of the permeability defect appeared to have improved significantly
(Fig. 3D). Glomerular
filtration of the high-molecular-weight dextran into the tubular lumen was not
observed after ischemia at any of the time points studied. As might be
anticipated, leakage of the smaller dextran into the interstitium was more
diffuse than that of the larger dextran
(Fig. 4). Interestingly, areas
where leakage of both dextrans occurred were more often observed in areas of
markedly diminished microvascular flow (a supplementary video of
Fig. 4C can be viewed
at
http://ajprenal.physiology.org/cgi/content/full/00042.2003/DC1).

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Fig. 4. Extent of microvascular permeability defect is heterogeneous. A 3,000-mol
wt, rhodamine-labeled dextran (A; red in C) and a
500,000-mol wt, FITC-labeled dextran (B; green in C) were
injected via the tail vein into a rat after 24 h of reperfusion following 45
min of ischemia. AC are images obtained from the same
field. A: demonstration of the extent of microvascular extravasation
of the low-molecular-weight dextran. B: demonstration of the extent
of microvascular extravasation of the high-molecular-weight dextran.
C: color overlay of A and B, with the
low-molecular-weight dextran in red, the high-molecular-weight dextran in
green, and the arrowhead indicating an area of diminished microvascular flow.
Bar = 10 µm. A supplementary video of C is a time series taken
from the same field as shown and is composed of a 30-frame series of images
collected at 2 frames/s, projected at 10 frames/s, demonstrating the
diminished flow in the area of the greatest permeability defect.
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DISCUSSION
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Endothelial dysfunction and vasomotor alterations of the renal vasculature
have played a conceptual role in the pathophysiology of ARF for a number of
years (12). However, recent
work by Brodsky et al. (5), in
particular, has brought to light the vulnerability of the renal microvascular
endothelium to ischemic injury and has highlighted the renal vascular
endothelium as a potential target for injury in renal ischemia. To further an
understanding of the role endothelial cell injury plays in ischemic ARF, we
have utilized a renal artery clamp model of ischemic ARF in rats and mice.
While this model is imperfect, it has provided valuable insight into the
pathophysiology of ischemic injury to the kidney
(27). Our study has served to
extend confirmed previous findings and to begin to examine some of the
mechanisms by which renal microvascular endothelial injury may contribute to
altered function in one of the myriad roles to which the endothelium is
subservient.
Our finding of an elevation in circulating vWF after renal ischemic injury
contributes to the evidence that the renal vascular endothelium is injured. In
other organ systems, release of vWF from the endothelium has been demonstrated
to occur in a biphasic fashion in response to ischemia-reperfusion injury
(10,
39). These studies suggest
that hypoxia, as well as reperfusion, mediate endothelial release of vWF via
potentially different mechanisms. Furthermore, inflammatory processes can
contribute to endothelial vWF release
(41). The peak elevation of
circulating vWF in our study was monophasic and occurred 24 h after the
initial ischemic insult; thus continued hypoxia, reperfusion injury, and/or
inflammatory processes could separately or collectively play a role in
endothelial release of vWF in the renal artery-clamp model of ischemic ARF.
Whereas the peak increase in vWF occurred at the same time as the peak in
serum creatinine in our study, given the large molecular weight of the various
circulating forms of vWF (50020,000), it is doubtful that reduced renal
clearance of vWF significantly contributed to the elevation in circulating
vWF. While the intent of this portion of the investigation was to simply
provide evidence for endothelial injury, the utility of such a finding may
ultimately lie in the ability to characterize the nature and extent of the
underlying injury by circulating markers of endothelial injury and thus
provide a useful clinical correlate for disease severity, prognosis, and
therapeutic intervention. For example, endothelial release of IL-6 has been
demonstrated to be of prognostic value in sepsis where endothelial injury
plays an important role in the underlying pathophysiology
(14). Although a previous
study of intestinal ischemia-reperfusion injury did not demonstrate a
correlation between histological injury scores and the level of circulating
vWF (1), the pattern of
circulating vWF multimers or the pattern of vWF coupled with other markers of
injury may in the end prove to be a useful clinical tool in renal
ischemia-reperfusion injury.
Our examination of the morphological alterations in renal microvascular
endothelial cells revealed that the normal structure of the actin cytoskeleton
in renal microvascular endothelial cells is disrupted after ischemia.
Alteration of the normal actin cytoskeleton of endothelial cells in vitro has
been demonstrated with ATP depletion as a model of ischemic injury and with
H2O2 as a model of oxidant-mediated reperfusion injury.
ATP depletion has been demonstrated to rapidly and reversibly disrupt the
normal cortical and basal F-actin structures in endothelial cells
(17,
24,
45), resulting in F-actin
aggregation and polymerization. Oxidant-mediated endothelial cell injury also
has been demonstrated to disrupt the cortical actin band in cultured
endothelial cells (18,
29,
47). We observed that
disruption of the actin cytoskeleton in endothelial cells of the renal
microvasculature subjected to ischemic injury in vivo was most prominent
immediately after the ischemic insult. Consistent with the above-mentioned in
vitro findings, we observed an alteration of the normal cortical and basal
F-actin structures of the endothelial cell with an apparent increase in
F-actin polymerization and aggregation at the basal and basolateral aspects of
endothelial cells after ischemia. This F-actin polymerization and aggregation
was reminiscent of the alterations observed after ischemia in proximal tubular
epithelial cells (30) and
renal vascular smooth muscle cells
(26). Of note, the TIE2
promoter has been reported to be upregulated in vitro by hypoxia in human
umbilical vascular endothelial cells
(7). In our in vivo study, we
did not observe a difference in the number of vessels or a change in the
intensity of GFP signal at the selected time points.
The alterations in the actin cytoskeleton of renal microvascular
endothelial cells preceded alterations in VE-cadherin staining at endothelial
cell-cell junctions. These findings are consistent with a mechanism by which
loss of integrity of the actin cytoskeleton contributes to breakdown of the
actin-associated adherens junctions and contributes to the concomitant
permeability defect. Much of the present knowledge regarding the mechanisms
regulating endothelial cell-cell interaction during ischemic and oxidant
injury has come from in vitro models utilizing cultured endothelial cells. ATP
depletion of endothelial cell monolayers and exposure of endothelial
monolayers to oxidants such H2O2 have both been
demonstrated to increase endothelial permeability and intercellular gap
formation (22,
36,
38). Increased endothelial
permeability in these models has been associated with internalization of
VE-cadherin from endothelial adherens junctions
(2,
21). Although we did not
observe internalization of VE-cadherin after ischemia in vivo, other
mechanisms including cleavage of VE-cadherin as described in a previous in
vivo study (4) may account for
the diminished VE-cadherin staining observed in our study.
The utilization of two-photon intravital microscopy to visualize the
permeability defect in the renal microvasculature after ischemia is a
particular strength of our study. The power of this imaging technique is
demonstrated in our ability not only to simultaneously evaluate the disparity
in the permeability defect of two differently sized fluorescent probes but
also to observe a correlation, in a timed-image series, between alterations in
blood flow and severity of the permeability defect. These data imply continued
reduced blood flow results in increased permeability defects between
endothelial cells. Although these observations were limited to the cortical
area of rats due to technical considerations, presumably the permeability
defect in the corticomedullary area would be even more pronounced than what we
observed in the cortical microvasculature
(16). Implications for leakage
of plasma from the vascular space and increased interstitial edema, especially
in the corticomedullary area, include further diminishment of the compromised
medullary blood flow by extrinsic compression of peritubular capillaries
(23) and by hemoconcentration
as observed in other organs
(44).
In summary, we have demonstrated that renal microvascular injury manifested
by disruption of the actin cytoskeleton and adherens junction of endothelial
cells occurs after ischemic injury and that this injury is probably
fundamental to increased microvascular permeability and renal interstitial
edema. Further characterization of the implications of renal microvascular
injury may provide new diagnostic and therapeutic avenues in ischemic ARF.
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DISCLOSURES
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This work was supported by National Institutes of Health (NIH) Grant
DK-6062161594, the Ralph W. and Grace M. Showalter Research Trust, and
National Kidney Foundation of Indiana grants (to T. A. Sutton), NIH Grant
HL-63169 (to M. C. Yoder), and NIH Grants DK-41126, DK-53465, and DK-61594,
and Veterans Affairs Medical Research Service grants (to B. A. Molitoris).
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ACKNOWLEDGMENTS
|
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We acknowledge Simon Atkinson, Ken Dunn, and Katherine Kelly for valuable
discussions.
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FOOTNOTES
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Address for reprint requests and other correspondence: T. A. Sutton, Div. of
Nephrology/Dept. of Medicine, 1120 South Dr., Fesler Hall 115, Indianapolis,
IN 46202 (E-mail:
tsutton2{at}iupui.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
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