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Original Studies |
Baylor College of Medicine (D.R.P., S.K.D., E.D.B., P.D.K.L.), Houston, Texas 77030; Stanford University Medical School (F.L., B.K.B., R.L.H.), Stanford, California 94305; Genentech (J.W.F.), South San Francisco, California 94080; University Childrens Hospital (B.T., O.M.), Heidelberg, Germany; University Childrens Hospital (A.-M.W.), Tubingen, Germany; University of Washington (S.L.W.), Seattle, Washington 98108; and Columbia Hospital at Medical City (R.J.H.), Dallas, Texas 75230
Address all correspondence and requests for reprints to: Dr. David R. Powell, Texas Childrens Hospital, Clinical Care Center, MC# 32482, 6621 Fannin, Houston, Texas 77030. E-mail: dpowell{at}bcm.tmc.edu
| Abstract |
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24- to 28-kDa IGFBP band identified by
[125I]IGF ligand blot. The present studies characterized
this diffuse
24- to 28-kDa band. Initial studies identified this
band as IGFBP-6, because it was immunoprecipitated by antiserum raised
against a synthetic peptide of human IGFBP-6 (hIGFBP-6). Additional
[125I]IGF ligand blots found that the immunoprecipitated
band was 1) recognized by [125I]IGF-II but not
[125I]IGF-I, 2) more abundant in CRF than in normal
serum, and 3) more abundant in serum from dialyzed than nondialyzed
prepubertal CRF children. Using the hIGFBP-6 antiserum in a specific
and sensitive RIA, we found that serum IGFBP-6 levels were 4.7 ±
1.7 nmol/L in 10 normal prepubertal children, 21.4 ± 6.1 nmol/L
in 44 nondialyzed prepubertal CRF children, 73.5 ± 14.4 nmol/L in
7 dialyzed prepubertal CRF children, and 94.6 ± 26.2 nmol/L in 14
dialyzed pubertal CRF children. IGFBP-6 levels were also elevated in 71
nondialyzed European children with CRF. In nondialyzed CRF children,
serum IGFBP-6 levels 1) correlated inversely with the glomerular
filtration rate, 2) did not correlate with height SD score,
and 3) were not altered by 12 months of daily recombinant hGH
treatment. In summary, a specific antiserum and RIA were used to
demonstrate elevated levels of intact IGF-II-binding IGFBP-6 in serum
of CRF children. We postulate that the excess IGFBP-6 may modulate the
action of IGF-II on target tissues. | Introduction |
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The decrease in IGF bioactivity in CRF serum is due to an excess of proteins with high affinity for IGFs (10, 11). These IGF-binding proteins (IGFBPs) constitute a unique protein family that to date has six members numbered according to the order of their cloning (12, 13). Specific antisera have shown that levels of intact IGFBP-1, intact IGFBP-2, and a 29-kDa fragment of IGFBP-3 are elevated in CRF serum (10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Current evidence suggests that an excess of IGFBPs can impair linear growth. In children with CRF, elevated serum levels of IGFBP-2 and, to a lesser extent, IGFBP-1 correlate significantly and inversely with height SD score (21, 22). In vitro, IGFBP-1 profoundly inhibits the basal and IGF-I-stimulated growth of chick embryo pelvic cartilage (24). In vivo, IGFBP-1 administration inhibits both IGF-I- and GH-stimulated weight gain and tibial epiphyseal widening in hypophysectomized rats (3). Although interest in IGFBPs in CRF children arose because of the potential role of IGFBPs as inhibitors of IGF-stimulated linear growth, IGFs have many other biological effects (25), and these effects may also be modulated by excess IGFBPs in the CRF milieu.
A diffuse
24- to 28-kDa IGFBP band seen on [125I]IGF
ligand blot is more abundant in serum from CRF than in that from normal
children and is not precipitated by antiserum to IGFBP-1, IGFBP-2, or
IGFBP-3 (11, 17). This IGFBP has not been identified, but its molecular
mass (Mr) is consistent with that expected for IGFBP-6 (13, 26).
To determine whether IGFBP-6 was responsible for the diffuse
24- to
28-kDa band seen in CRF serum by [125I]IGF ligand
blotting, we used a specific antiserum against a synthetic human (h)
IGFBP-6 peptide. These studies showed that CRF children have elevated
serum levels of IGFBP-6, and that this IGFBP-6 is distributed in CRF
serum as a diffuse
24- to 28-kDa band.
| Subjects and Methods |
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Sera were obtained from 44 children with CRF who did not yet require dialysis [glomerular filtration rate (GFR) between 1040 mL/min·1.73 m2] and who were part of an open label, multicenter trial of the effects of recombinant hGH (rhGH) on CRF children. Of the 44 children, 30 were randomized to the rhGH treatment group, and 14 to the untreated group. The study design, approved by the institutional review board for research involving human subjects of each participating center, has been previously described (22). Characteristics of the rhGH-treated and untreated groups at baseline and during the first year of study, including GFR, anthropometrics, and serum levels of IGF-I, free IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, insulin, acid-labile subunit (ALS), and GH-binding protein (GHBP), have also been described (22).
Additional sera were obtained from 39 prepubertal and 32 pubertal European children with moderate CRF who did not yet require dialysis (GFR between 1070 mL/min·1.73 m2); samples were drawn before these children were enrolled in a series of investigations performed by the European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood (21, 27). Each child had baseline measurements of GFR, anthropometrics, and serum levels of IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, and insulin as described previously (21, 27).
Sera were also obtained from 14 pubertal and 7 prepubertal CRF children requiring dialysis; peritoneal dialysate was obtained from 3 of these children as described previously (28). A further 10 serum samples were obtained from healthy prepubertal children for use as normal control sera; these children have been described previously (22).
Assays
Serum and peritoneal dialysate samples were stored at -70 C until assay. Serum and peritoneal dialysate samples were assayed for IGFBP-6 using a RIA kit from Diagnostic Systems Laboratories (Webster, TX). The assay sensitivity is 1.1 ng/mL, with inter- and intraassay coefficients of variation ranging from 6.19.6% and 6.410.7%, respectively. There is no cross-reactivity with hIGFBP-1, -2, -3, -4, or -5 added at final concentrations of 15 µg/mL and no interference with hIGF-I or hIGF-II at a final concentration of 100 ng/mL. Goat antiserum to the synthetic peptide comprising amino acids 81118 of the hIGFBP-6 sequence [hIGFBP-6-(81118)] was obtained from Diagnostic Systems Laboratories. Some serum samples were also assayed for intact PTH by Nichols Institute (San Juan Capistrano, CA), using their PTH immunoradiometric assay.
Size-exclusion chromatography
One milliliter of peritoneal dialysate was chromatographed at 30 mL/h on a 0.9 x 120-cm column of Superdex-200 (Pharmacia, Piscataway, NJ); 0.05 mol/L Tris-HCl, pH 7.4, and 0.15 mol/L NaCl served as mobile phase. Individual 2-mL fractions were collected and frozen at -70 C until assay. The column was calibrated with aldolase (158 kDa), ovalbumin (43 kDa), myoglobin (19 kDa), and IGF-I (7 kDa).
IGFBP-6 immunoprecipitation
Staphylococcus protein-A (Pansorbin, Calbiochem, La Jolla, CA) was washed in 100 mmol/L Tris-HCl, pH 8.0, and 0.5% Nonidet P-40 (Calbiochem) and then resuspended in the original volume with this buffer. Nonspecific precipitation by protein A was eliminated by preincubating samples with 25 µL protein A for 2 h at 4 C on a rotating mixer; protein A was then removed by centrifugation. For each immunoprecipitation, 25 µL protein A were incubated with 2 µL hIGFBP-6 antiserum for 4 h at 4 C. After centrifugation, the antibody/protein A pellets were washed and resuspended in 100 mmol/L Tris-HCl, pH 8.0, and 0.5% Nonidet P-40 and then incubated at 4 C overnight on a rotating mixer with 2 µL whole serum, 10 µL whole peritoneal dialysate, 40 µL pooled column fractions from Superdex-200 chromatography of peritoneal dialysate, or 150 ng rhIGFBP-6 (Austral, San Ramon, CA). Immunoprecipitates were pelleted by centrifugation and washed three times with 100 mmol/L Tris-HCl, pH 8.0, and 0.5% Nonidet P-40. Samples were then analyzed by [125I]IGF ligand blot.
[125I]IGF ligand blot
Aliquots of serum and peritoneal dialysate immunoprecipitated with IGFBP-6 antibody, rhIGFBP-6 immunoprecipitated with IGFBP-6 antibody, and 2-µL aliquots of whole serum were individually separated by 12% SDS-PAGE under nonreducing conditions (11). Separated proteins were transfered to a nitrocellulose membrane and probed with 2 x 106 cpm [125I]IGF-I and/or [125I]IGF-II, as previously described (11, 17).
Statistics
IGFBP-6 RIA data were analyzed using a four-parameter logistic
curve fit. Data are presented as the mean ± SD.
Differences within untreated and rhGH-treated CRF groups at 3 and 12
months and between these two groups at 0, 3, and 12 months were
evaluated for significance by Students t test. Differences
among normal children and the various groups of CRF children at
baseline were evaluated for significance by ANOVA followed by
Newman-Keuls multiple range testing. Pearson correlation coefficients
were calculated to evaluate correlations between variables measured in
the CRF children. For all comparisons, differences were considered
significant when P
0.05.
| Results |
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Antiserum raised in goats against the synthetic hIGFBP-6-(81118)
peptide was tested for ability to immunoprecipitate hIGFBP-6 forms that
bind IGFs with high affinity. As shown in Fig. 1
, this antiserum immunoprecipitated both
the 23-kDa form of nonglycosylated rhIGFBP-6 and a slightly larger
IGFBP from the serum of a child with CRF. The poorly defined,
24- to
28-kDa size of this serum protein is consistent with the predicted
Mr of glycosylated hIGFBP-6 (13, 26).
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As shown in Fig. 4A
, the serum
IGFBP-6 level determined by RIA was 4.7 ± 1.7 nmol/L in 10 normal
prepubertal children (mean age, 7.4 ± 2.7 yr). In contrast, the
mean level was 21.4 ± 6.1 nmol/L in 44 nondialyzed prepubertal
CRF children (mean age, 5.6 ± 2.2 yr). In a comparable group of
39 nondialyzed prepubertal European children with CRF (mean age,
8.3 ± 2.9 yr), the serum IGFBP-6 level was 22.4 ± 13.3
nmol/L; this level was 29.5 ± 15.1 nmol/L in 32 nondialyzed
pubertal European children with CRF (mean age, 15.6 ± 2.2 yr).
Serum IGFBP-6 levels were markedly higher in CRF children requiring
chronic dialysis treatment, measuring 73.5 ± 14.4 nmol/L in 7
prepubertal children (mean age, 5.9 ± 3.9 yr) and 94.6 ±
26.2 nmol/L in 14 pubertal children (mean age, 16.7 ± 2.7 yr). Of
interest, IGFBP-6 levels were significantly higher in pubertal than in
prepubertal children with comparable severity of CRF (Fig. 4A
).
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To investigate whether the IGFBP-6 RIA is measuring intact or
fragmented IGFBP-6 in CRF fluids, this assay was used to measure
IGFBP-6 levels in size-fractionated peritoneal dialysate from CRF
children. Peritoneal dialysate was studied because it contains high
levels of IGFBP-6 by RIA (26.2 nmol/L); also, if low
Mr IGFBP-6 fragments are abundant in CRF serum,
they are likely to accumulate in peritoneal dialysate as do IGFBP-3
fragments (28). As shown in Fig. 5
, IGFBP-6 immunoactivity eluted from the Superdex-200 column as a single
sharp peak in the same fractions as ovalbumin, and no IGFBP-6 eluted in
lower Mr fractions. Consistent with this finding,
immunoprecipitable IGFBP-6 was detected only in fractions of peritoneal
dialysate in which IGFBP-6 was identified by RIA (data not shown).
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The 44 CRF children from the rhGH protocol and the 71 CRF children
from the European study have been intensively studied in terms of their
linear growth, GFR, and serum levels of a number of proteins (21, 22, 27). Baseline serum IGFBP-6 levels in the 44 CRF children from the rhGH
study did not correlate with baseline height SD score
(r = -0.04; P = 0.7978), and serum IGFBP-6 levels
measured during the first year of rhGH treatment did not correlate with
the change in height SD score between 012 months.
However, serum IGFBP-6 levels correlated strongly and inversely with
GFR in these 44 children at baseline (r = -0.52;
P = 0.0003). Consistent with these results, serum
IGFBP-6 levels did not correlate with baseline height SD
score for the 39 prepubertal European children with CRF (r = 0.09;
P = 0.59), but did correlate strongly and inversely
with GFR in the 71 European children with CRF. In this group of
European children, who had a wider GFR range than the 44 children
entered into the rhGH study, the coefficient of correlation was highest
when GFR was plotted against 1/IGFBP-6 (r = 0.65;
P < 0.0001; Fig. 6
).
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| Discussion |
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hIGFBP-6 is a 216-amino acid protein with a predicted Mr of 22.8 kDa (13), but serum IGFBP-6 migrates as a broad band at 2428 kDa as determined by [125I]IGF ligand blot analysis due to variable amounts of O-linked glycosylation (26). Quantitative estimates of IGFBP-6 levels have been limited by the lack of reliable assays. Baxter and Saunders (29) measured IGFBP-6 levels by RIA in serum and other fluids; however, their RIA is limited by a decreased ability of the antiserum to bind radiolabeled IGFBP-6 in the presence of IGF peptides. The hIGFBP-6 antiserum and RIA reported here are highly specific for hIGFBP-6 due to epitope specificity for residues 81118, a region that is not conserved among the other five IGFBPs (13) and, therefore, is probably not involved in IGF binding. Serum IGFBP-6 levels reported here for the control prepubertal population (4.7 nmol/L) are lower than levels reported by Baxter and Saunders (29) for 21 normal adults (9.6 nmol/L). This discrepancy may be due in part to differences in age and/or pubertal status, as in the present study IGFBP-6 levels were significantly higher in pubertal than in prepubertal children with comparable severity of CRF.
Compared to levels in normal prepubertal controls, serum IGFBP-6 levels were elevated 4-fold in prepubertal children with more moderate degrees of CRF (GFR between 1070 mL/min·1.73 m2) and were elevated 15-fold in prepubertal CRF children requiring dialysis. This 4- to 15-fold increase in IGFBP-6 levels is greater than that measured for serum IGFBP-1, IGFBP-2, or IGFBP-3 in CRF children (10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). The high levels of serum IGFBP-6 and their correlation with IGFBP-3, which is elevated in CRF serum due to accumulation of low Mr IGFBP-3 fragments (10, 11, 28), suggest that the RIA may be measuring an increase in IGFBP-6 fragments in CRF serum. However, immunoprecipitation followed by [125I]IGF ligand blot analysis clearly shows that CRF serum contains markedly elevated levels of intact IGFBP-6 capable of binding radiolabeled IGF-II but not IGF-I, consistent with the preferential affinity of intact IGFBP-6 for IGF-II (30). In addition, the relative abundance of this immunoprecipitated intact IGFBP-6 in serum of dialyzed CRF children >> nondialyzed CRF children >> normal children is similar to the RIA findings and suggests that the RIA is measuring primarily intact IGFBP-6.
As detecting IGFBP-6 in the immunoprecipitation studies requires
[125I]IGF ligand binding, it is possible that
non-IGF-binding IGFBP-6 fragments accumulate in CRF serum and are
detected by RIA, but not by [125I]IGF ligand blot,
similar to IGFBP-3 fragments in CRF serum (28). This issue was
evaluated by characterizing the size distribution of IGFBP-6
immunoreactivity in peritoneal dialysate after Superdex-200
chromatography. Peritoneal dialysate was studied because it contains
large amounts of IGFBP-6 and, as an ultrafiltrate of serum, it should
accumulate IGFBP-6 fragments if these fragments exist, similar to the
accumulation of IGFBP-3 fragments in this fluid (28); indeed, IGFBP-6
levels in peritoneal dialysate pooled from three CRF children were
30% of serum levels, consistent with peritoneal dialysate being an
ultrafiltrate of serum. IGFBP-6 present in peritoneal dialysate eluted
from the column as one sharp peak with a Mr
corresponding to that of intact IGFBP-6. These results suggest that
IGFBP-6 fragments do not contribute significantly to IGFBP-6 levels
measured by RIA in CRF fluids.
The etiology of increased serum levels of intact IGFBP-6 in individuals with CRF is unclear. Decreased renal clearance, increased hepatic expression, and decreased proteolysis are all possibilities that should be considered. The correlation between IGFBP-6 levels and GFR appears more hyperbolic than linear, similar to the correlation between creatinine and GFR, suggesting that IGFBP-6 accumulation in CRF serum is more likely due to decreased clearance than to increased production. Undernutrition is unlikely to be a factor leading to increased IGFBP-6 levels, because the CRF children reported here routinely receive aggressive nutritional support; moreover, other investigators have found that protein restriction does not alter hepatic or renal expression of IGFBP-6 messenger ribonucleic acid in rats (31). Glucocorticoids and retinoic acid stimulate IGFBP-6 expression in human osteoblasts in vitro (32, 33, 34), but it is unlikely that these steroid hormones are responsible for the high IGFBP-6 levels in CRF. In addition, other in vitro studies have shown no effect of glucocorticoids on IGFBP-6 expression in primary human osteoblasts (35) or fibroblasts (36).
The CRF state is associated with impaired responsiveness to GH and/or IGFs (10, 37, 38), and this could theoretically affect IGFBP-6 levels in CRF serum. Baxter and Saunders reported low serum IGFBP-6 levels in patients with acromegaly (29), suggesting that GH may suppress levels of this IGFBP. However, GH suppression does not occur in CRF; rhGH treatment did not affect serum IGFBP-6 levels in the CRF children reported here despite the fact that rhGH did stimulate linear growth and increase serum levels of IGFs, IGFBP-3, and ALS in these children (22). In addition, neither rhGH, IGF-I, nor IGF-II influenced IGFBP-6 expression in primary rat osteoblasts in vitro (39).
The physiological significance of elevated serum IGFBP-6 levels in CRF children is uncertain. Despite the ubiquitous expression of IGFBP-6 messenger ribonucleic acid in adult rat tissues (13) and the unique expression pattern during rodent myogenesis, chondrogenesis, and osteogenesis (40, 41, 42), the biological role of IGFBP-6 is not firmly established in any tissue. However, rhIGFBP-6 has been shown to inhibit IGF-II-stimulated and, to a much lesser extent, IGF-I-stimulated DNA and glycogen synthesis in rat calvarial osteoblasts, PyMS osteoblastic cells, and B-10 osteosarcoma cells in vitro (30, 43). rhIGFBP-6 also inhibited IGF-II-mediated proliferation and differentiation of L6A1 myoblasts in vitro (44, 45). In all of these studies, the higher affinity of IGFBP-6 for IGF-II relative to IGF-I (30) probably accounts for the preferential inhibition of IGF-II bioactivity by IGFBP-6. In addition, the effects of a growth inhibitor produced by human keratinocytes, later identified as IGFBP-6, was overcome by excess insulin (46); this suggests that IGFBP-6 inhibits keratinocyte DNA synthesis by sequestering IGF peptides from type I IGF receptors.
These studies suggest a possible role for IGFBP-6 as an inhibitor of IGF, and in particular IGF-II, action in a number of tissues. IGF-II is more highly expressed than IGF-I in chondrocytes of growing rodents (42, 47), and IGF-II protein levels are higher than those of IGF-I in lamb and rabbit cartilage (48, 49). Although IGFBP-6 is not expressed in murine chondrocytes after birth (42), it is possible that accumulation of IGFBP-6 in CRF serum is associated with a similar accumulation in IGF targets, such as the skeletal system. Thus, it is possible that excess IGFBP-6 in CRF children may interfere with IGF-mediated growth of cartilage and bone and could contribute to the pathogenesis of renal osteodystrophy. However, serum IGFBP-6 levels did not correlate with height SD score or PTH levels in the CRF children reported here, suggesting that interactions between serum IGFBP-6 and skeletal metabolism, if present, are probably complex.
In conclusion, a specific RIA and antiserum were used in the present study to demonstrate significantly elevated levels of intact IGF-binding IGFBP-6 in the serum of prepubertal CRF children. Further studies are needed to establish the effects of excess IGFBP-6 on skeletal growth and tissue metabolism in this population.
| Acknowledgments |
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The technical support of Grace Matthew and Dr. Aruna Khare is gratefully acknowledged.
| Footnotes |
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Received March 13, 1997.
Revised May 16, 1997.
Accepted June 2, 1997.
| References |
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