Infused IGF-I/IGFBP-3 complex causes glomerular localization
of IGF-I in the rat kidney
Alexander
Sandra,
Mary
Boes,
Brian L.
Dake,
John B.
Stokes, and
Robert S.
Bar
Departments of Internal Medicine and Anatomy, Diabetes and
Endocrinology Research Center, Veterans Administration Medical Center,
The University of Iowa, Iowa City, Iowa 52246
 |
ABSTRACT |
Insulin-like growth
factor I (IGF-I) increases renal blood flow, glomerular filtration rate
(GFR), and proximal tubule reabsorption of phosphate in humans and
rodents. The biological effects of IGF-I are likely to be influenced by
cellular localization of IGF-I within the kidney. We therefore tested
whether the renal localization of infused IGF-I could be altered if
given with selected IGF-binding proteins (IGFBPs). Rats were treated
with intravenous injections of
125I-labeled IGF-I,
125I-IGFBP-3, or
125I-IGFBP-4 alone or with
complexes of 125I-IGF-I and
IGFBP-3 or IGFBP-4. The cellular localization of IGF and the IGFBP
within the kidney was then determined.
125I-IGF-I,
125I-IGFBP-4, and
125I-IGF-I/IGFBP-4 complexes were
found almost exclusively in vacuolar structures (endosomes) of proximal
renal tubules. In contrast, about one-third of renal
125I-IGFBP-3 and
125I-IGF-I/IGFBP-3 was localized
to glomeruli. When 125I-IGF-I was
given alone, 3% was found in glomeruli and 89% in proximal tubules.
When given as 125I-IGF-I/IGFBP-3,
29% was in glomeruli and 65% in proximal tubules. We conclude that
the cellular localization of IGF-I within the kidney can be directed to
glomerular elements if the IGF-I is given with IGFBP-3.
renal redistribution of insulin-like growth factors
 |
INTRODUCTION |
THE INSULIN-LIKE GROWTH FACTORS (IGFs) IGF-I and IGF-II
exert both metabolic and mitogenic effects in several tissues
throughout the body (18, 20, 28). The IGFs are present in the highest concentration in the circulation, where >90% of IGF-I and/or
-II circulates as part of a 150-kDa trimeric complex composed of IGF, IGF-binding protein (IGFBP)-3, and an acid labile subunit (5, 6).
When in the 150-kDa complex, IGF is thought to be
biologically inert, and the complex is largely confined to the
circulation (7). At the tissue level, biological effects of IGF,
whether derived from the circulation or produced by the tissue itself, are dependent on a complex interplay involving IGF, ambient
IGF-binding proteins (IGFBP-1 through IGFBP-6), IGFBP protease(s),
and specific IGF receptors of the target cell (4, 8, 25). Of the six high-affinity IGF-binding proteins, IGFBP-3 and specific
IGFBP-3 fragments have the most diverse biological effects, causing
both inhibition and potentiation of specific IGF functions while also having intrinsic biological activities independent of IGF (10, 11,
24, 25, 27).
In the intact animal, perhaps the most IGF-sensitive tissue/organ is
the kidney. In both rodents and humans, treatment with relatively low
doses of IGF-I causes a rapid increase in renal plasma flow and
glomerular filtration rate (14-16). To gain insight into the
potential influence of IGFBP-3 on IGF-mediated renal function, in the
present study rats were infused with IGF-I alone, IGFBP-3 alone, or
IGFBP-3 plus IGF-I, and the tissue localization within the kidney was
determined by autoradiographic methods. Localization data for infused
IGFBP-3 were then compared with the tissue localization data in the
kidney of a second group of animals perfused with IGFBP-4 or
IGFBP-4/IGF-I complexes. As previously reported by Arany et al. (2),
IGFBP-3 was found throughout the kidney, with particular localization
in cellular elements of the glomerulus. In contrast, IGF-I, as well as
IGFBP-4, was found almost exclusively in proximal renal tubules, with
striking enrichment in vacuolar structures of the tubules. When IGF-I
was infused with IGFBP-3, the localization of IGF-I more closely
approximated that of IGFBP-3, with an increase of IGF-I in the
glomerulus and a decrease in localization within the proximal tubules.
These data indicate that the kidney localization, and perhaps renal bioactivity, of infused IGF-I can be altered if the IGF-I is infused in
the presence of IGFBP-3.
 |
MATERIALS AND METHODS |
IGF-I (Intergen, Purchase, NY), IGFBP-3 (a gift from Celtrix, Santa
Clara, CA), and IGFBP-4 [made in Baculovirus as per protocol of
Invitrogen (San Diego, CA)] were iodinated with
Na125I by the lactoperoxidase
method as previously described (13). Specific activities of
125I-labeled IGF-I, IGFBP-3,
and IGFBP-4 ranged from 40 to 100 µCi/µg protein. Heart perfusion
buffer (HPB) consisted of Hanks' balanced salt solution,
pH 7.4, with glucose (1 g/l), fatty acid-poor albumin (1 mg/ml;
Intergen), and 1 mM HEPES (Sigma Chemical, St. Louis, MO) in place of
sodium bicarbonate.
Perfusion Studies
Sprague-Dawley rats, 250-274 g (Harlan Laboratories, Indianapolis,
IN), were anesthetized with methoxyflurane (Pitman-Moore, Mundelein,
IL) before tail vein injections with IGF-I and/or IGFBPs. 125I-IGF-I,
125I-IGFBP-3, and
125I-IGFBP-4 [2 × 107 counts/min (cpm) in 300 µl
HPB] were each injected into the tail vein and allowed to
circulate for 1 or 10 min. The chest cavity of the animal was then
opened, a 19-gauge needle was inserted into the left ventricle, and 200 µl of blood were removed for analysis of degradation of the
125I-labeled protein. To clear
blood from the circulation, the rat was next perfused through the
ventricular needle with 100 ml of PBS, 37°C, at 130 mmHg by gravity
flow. To fix tissues, the perfusate was then changed to a solution of
2.5% glutaraldehyde in 67 mM sodium phosphate, and 100 ml of the
fixing solution were infused.
Rats were also treated with either IGFBP-3 and
125I-IGF-I or IGFBP-4 and IGF-I.
Treatment of an animal with both IGFBP and IGF-I was done in two ways.
1) Rats were preperfused with
unlabeled IGFBP by injecting 100 µg of unlabeled IGFBP (IGFBP-3 or
IGFBP-4) in 300 µl HPB, a mixture that was allowed to circulate for
10 min. The rat was then given 2 × 107 cpm
125I-IGF-I in 300 µl HPB; this
was allowed to circulate for 1 min, after which the kidney tissues were
fixed and analyzed. 2) Rats were
infused with a mixture of
125I-IGF-I (2 × 107 cpm) and unlabeled IGFBP-3 or
IGFBP-4 in a 1:10 IGF-I/IGFBP molar ratio. After 1 min, the tissues
were fixed and prepared for analysis.
Tissue Fixation
Light-microscopic level autoradiography.
Kidneys were rapidly dissected and cleaned of perirenal fat and
connective tissue. The kidneys were then cut coronally, processed by
standard methods, and embedded in paraffin. Sections (8 µm) were cut,
mounted on slides, deparaffinized, and coated with Kodak NTB2 nuclear
track emulsion. After 1-2 wk of development, the slides were
processed with D-19 developer and hypofix. The slides were stained with
hematoxylin and eosin and were analyzed (as described in
Analysis) for silver grain
distribution with a light microscope equipped with a ×60 and a
×100 objective lens.
Electron-microscopic level autoradiography.
Rats were injected as described for light-microscopic level
autoradiography except that the counts per minute of the injected IGF-I
or binding protein were increased 10-fold. After removal of the
kidneys, representative cortical and medullary areas were dissected out
and processed for electron microscopy by postfixing in osmium
tetroxide, dehydration in graded ethanol, and embedding in Epoxy resin.
Thin (50 nm) sections of the tissue in the areas of the glomerulus and
tubules were cut, mounted on nickel grids, and covered with a monolayer
of Ilford L-5 emulsion. After 2-4 wk of development, the grids
were processed using Microdol-x developer. Grids were stained with lead
citrate and uranyl acetate before being viewed in a Hitachi E600
electron microscope.
Analysis
Silver grains at both the light- and electron-microscopic levels were
counted and analyzed for localization. For
125I-IGF-I alone,
125I-IGFBP-3 alone, and IGFBP-3
plus 125I-IGF-I, the data
represent the distribution of ~60,000 silver grains counted at the
light level in at least three independent experiments. An equal number
of microscopic fields were randomly chosen and analyzed in each
treatment group, and individual silver grains were assigned to a
cellular compartment. The cellular compartment assigned was based on
the localization of the grains to a distance within 0.2 µm. The
density of background silver grains on the periphery of each slide was
subtracted from the density over tissue sections. This background
averaged 5% of the grain density associated with cells. Values are
means ± SE of at least five rat kidneys. For studies with IGFBP-4,
both kidneys from a single rat were analyzed.
 |
RESULTS |
Rats were infused with 125I-IGF-I,
125I-IGFBP-3, or
125I-IGFBP-4. The relative
distribution of radioactivity in several organs/tissue is given in
Table 1. After 1-min circulation of the
125I-labeled protein, the kidneys
were removed and analyzed for 125I
grains at the light- and electron-microscopic levels.
125I-IGF-I was also infused
1) with IGFBP-3 or IGFBP-4 and
2) after animals had been pretreated
with intravenous IGFBP-3 or IGFBP-4. Results were similar whether
125I-labeled compounds were
allowed to circulate for 1 or 10 min. Data reported in Tables 1 and
2 and in Figs. 1-5 reflect 1-min circulation times for 125I-labeled
compounds.
IGFBP-4 and IGF-I Perfusions
At the light-microscopic level, autoradiographic grain counts
demonstrated that infused
125I-IGF-I and
125I-IGFBP-4 were each localized
to the proximal tubule, with a glomerulus-to-proximal tubule ratio of
~1:30 (Fig. 1, Table 2). When
125I-IGF-I was coinfused with
unlabeled IGFBP-4 after IGFBP-4 pretreatment, or chemically
cross-linked to IGFBP-4 (data not shown), the distribution of
125I-IGF-I within the kidney was
unchanged (Table 2). With higher magnification at the light level, the
majority of the silver grains for both
125I-IGF-I and
125I-IGFBP-4 appeared localized
over vacuoles near the apical surface of the tubule epithelium (Fig.
2). Electron microscopy revealed that
intracellular silver grains representing IGF-I or IGFBP-4 were
predominantly localized to vacuolar structures, most likely early
endosomes resulting from endocytotic activity (Fig.
3). After 1 and 10 min of circulation,
125I-IGF-I and
125I-IGFBP-4 in the blood were
>90% intact, as judged by TCA precipitation or SDS-PAGE analysis.

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Fig. 1.
Light-microscopic level autoradiograph showing localization of
125I-labeled insulin-like growth
factor-binding protein (IGFBP)-4 in rat kidney. Label is primarily
found in proximal tubule epithelium (arrows). * Tubule joining a
glomerulus. Bar, 10 µm.
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Fig. 2.
Light-microscopic level micrograph (with higher magnification than that
shown in Fig. 1) of 125I-IGFBP-4
in rat proximal tubule. Silver grains are mostly localized in apical
portion of epithelial cells. Bar, 10 µm.
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Fig. 3.
Electron-microscopic autoradiograph of
125I-IGFBP-4 in rat proximal
tubule. Label is found in apical brush border (BB) and is associated
with vacuolar system of kidney epithelial cell (arrows). Bar, 1 µm.
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IGFBP-3
In contrast to IGFBP-4, the renal distribution of infused
125I-IGFBP-3 was shifted to the
glomerulus with a glomerulus-to-proximal tubule ratio of ~2:3, i.e.,
39% of the IGFBP-3 grains were in glomeruli, 57% in proximal tubules,
and 3 and 1% in distal tubules and collecting ducts, respectively
(Fig. 4, Table 2). Electron microscopy of
the glomerulus revealed that 55% of the silver grains were associated
with podocytes or podocyte processes. The remaining grains within the
glomeruli were distributed between the capillary endothelium/basement
membrane (30%) and mesangium (15%) (Fig. 5).

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Fig. 4.
Light-microscopic level micrograph of
125I-IGFBP-3 in rat kidney. Silver
grains are localized in both proximal tubule cells (arrows) and
glomerulus (arrowheads). Bar, 10 µm.
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Fig. 5.
Electron-microscopic autoradiograph of
125I-IGFBP-3 localized in
glomerulus of rat kidney. Silver grains are distributed among podocytes
(arrows), basement membrane (arrowheads), and mesangium cells (*). Bar,
1 µm.
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125I-IGF-I was also infused in
IGFBP-3-treated rats. This was done in two ways.
1) Rats were first preperfused with
IGFBP-3 (100 µg injected in tail vein). The unlabeled IGFBP-3 was
allowed to circulate for 10 min, and then
125I-IGF-I (2 × 107 cpm in 300 µl buffer) was
injected in the tail vein and allowed to circulate for 1 min.
2)
125I-IGF-I (2 × 107 cpm) and unlabeled IGFBP in a
1:10 ratio were coinfused and allowed to circulate for 1 min. Both
treatments resulted in a changed renal distribution of
125I-IGF-I (Table 1). In rats
preperfused with IGFBP-3, 8% of grains were in the glomeruli and 77%
were in the proximal tubule. In rats in which
125I-IGF-I was coinfused with
IGFBP-3, the redistribution of
125I-IGF-I to glomerular
structures was more striking, with 29% of grains localized to the
glomerulus and 65% to the proximal tubule, a distribution similar to
that of rats treated with
125I-IGFBP-3 alone (39%
glomeruli, 57% proximal tubule) and clearly different from renal
localization of 125I-IGF-I in rats
treated solely with 125I-IGF-I
(3% glomeruli, 89% proximal tubule). After 1 min of circulation, >90% of 125I-IGF-I and
125I-IGFBP-3 in the blood
represented intact protein, as judged by TCA precipitation or SDS-PAGE
analysis.
 |
DISCUSSION |
In the present study, 125I-IGF-I,
125I-IGFBP-4, and
125I-IGFBP-3 were infused into
rats, and the distribution of the labeled proteins within the kidney
was determined. In addition, the effect of each binding protein on the
renal localization of infused
125I-IGF-I was also determined.
Infused IGF-I, IGFBP-4, and IGFBP-4 with IGF-I were localized in
endocytotic vacuoles of the proximal tubules, the major degradative
pathway in the kidney. In contrast, for infused IGFBP-3,
nearly 40% of the renal IGFBP-3 was retained in glomeruli, associated
with podocytes, endothelium/basement membrane, and mesangial cells. Of
perhaps greatest interest, the localization of infused IGF-I within the
kidney was substantially altered when IGF-I was introduced after
IGFBP-3 pretreatment or, more strikingly, if IGF-I and IGFBP-3 were
infused together. When given as an IGF-I/IGFBP-3 coinfusion, 29% of
renal IGF-I was in glomeruli vs. only 3% of IGF-I in the glomeruli
when IGF-I was injected alone.
In both humans and rats, treatment with IGF-I results in a rapid
increase in glomerular filtration rate (GFR), renal blood flow, and
proximal tubular reabsorption of phosphate. The precise cellular
mechanisms by which IGF-I mediates these effects are not entirely
clear. Type 1 IGF (IGF-I) receptors, as well as the ability to
synthesize IGF-I, are both properties of the three cell types of the
glomerulus (endothelial, mesangial, and epithelial cells) (3, 12). In
contrast, the proximal tubule has a high density of IGF-I receptors but
little ability to synthesize IGF-I, whereas, in humans, the cells of
the distal tubule and collecting ducts synthesize substantial IGF-I but
lack specific IGF-I receptors. The mismatch of IGF-I receptors and
endogenous IGF-I synthesis along the nephron has suggested the
potential for autocrine, paracrine, and endocrine functions of IGF-I
within the kidney (17). Furthermore, it may be reasonable to speculate
that the IGF-I-induced increase in renal plasma flow and perhaps in GFR
is likely mediated through the endothelial and mesangial cells of the
glomerulus, whereas the increase in phosphate reabsorption is initiated
through IGF-I interaction with the dense concentration of type 1 IGF
receptors in the proximal tubule, the major site of phosphate
reabsorption in the kidney (9).
Further impacting on the results of the present study is the finding
that, in rats and perhaps humans, the kidney is the major site of
degradation of injected or infused IGF-I. This occurs despite the
presence of increased receptors for IGF-I in other tissues, e.g.,
skeletal muscle and heart, as well as the presence of enzymes capable
of degrading IGF-I in most tissues of the rat. In a recent report,
Tanaka et al. (30) injected rats with recombinant human IGF-I and
determined localization and degradation of IGF-I in tissues 15 and 60 min after injection. The kidney was the only tissue having a higher
concentration of IGF-I than plasma and accounted for 69% of the
elimination of the injected IGF-I from the circulation. Therefore, it
is not unreasonable to speculate that altering the localization of
injected IGF-I within the kidney by giving it with IGFBP-3
could potentially alter the effects of exogenously administered IGF-I.
In recent years, several clinical trials of IGF-I therapy have
suggested beneficial effects of IGF-I in disorders as diverse as growth
hormone insensitivity (Laron dwarfism) (22), renal failure (26),
insulin resistance syndromes and diabetes mellitus (21, 29), AIDS (23),
and osteoporosis (19). In these clinical studies it was determined that
the frequency and severity of side effects progressively worsened as
the dose of IGF-I was increased, often limiting the amount of IGF-I
that could be given. Thus, at subcutaneous IGF-I doses >160
µg · kg
1 · day
1,
chronic IGF-I therapy became difficult because of side effects. In
attempts to diminish IGF-I side effects while maintaining its beneficial actions, a limited number of studies have reported successfully giving IGF-I to humans as IGF-I/IGFBP-3 complexes (1). An
additional rationale for giving IGF-I as a complex with IGFBP-3 is
based on the finding that when IGF-I and IGFBP-3 in the circulation are
present as part of the 150-kDa complex (IGFBP-3/IGF-I/acid labile
subunit), the IGF-I and IGFBP-3 are largely limited to the vascular
compartment, having plasma half-lives of ~16 h (2, 3, 17). However,
when IGF-I is in the ~50-kDa IGF-I/IGFBP-3 complex, the complex
readily traverses the capillary endothelium, having a plasma half-life
of ~25 min. The findings of the present study suggest an additional
potential renal benefit of combined IGF-I/IGFBP-3 therapy. When IGF-I
is given in this complex, not only could higher doses of IGF-I be given
to humans, but the potential diversion of IGF-I away from degradative
pathways in the kidney could potentiate the renal effects of IGF-I.
Studies in humans comparing the renal effects of therapy with IGF-I
alone or IGF-I given as IGF-I/IGFBP-3 complex are required to
corroborate or refute this speculation.
 |
ACKNOWLEDGEMENTS |
This work was supported by research funds from the Veterans
Administration and National Institute of Diabetes and Digestive and
Kidney Diseases Grants DK-25421, DK-25295, and DK-52617.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: R. S. Bar, The Univ. of Iowa, Dept. of
Internal Medicine, ENDO-3E19 VA Medical Center, Iowa City, IA 52246.
Received 23 January 1998; accepted in final form 3 April 1998.
 |
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