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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 9 2978-2984
Copyright © 1997 by The Endocrine Society

Original Studies

Insulin-Like Growth Factor-Binding Protein-6 Levels Are Elevated in Serum of Children with Chronic Renal Failure: A Report of the Southwest Pediatric Nephrology Study Group1

David R. Powell, Frances Liu, Bonita K. Baker, Raymond L. Hintz, Susan K. Durham, Eileen D. Brewer, James W. Frane, Burkhard Tonshoff, Otto Mehls, Anne-Margret Wingen, Sandra L. Watkins, Ronald J. Hogg and Phillip D. K. Lee

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 Children’s Hospital (B.T., O.M.), Heidelberg, Germany; University Children’s 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 Children’s Hospital, Clinical Care Center, MC# 3–2482, 6621 Fannin, Houston, Texas 77030. E-mail: dpowell{at}bcm.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Previous studies suggest that growth retardation in children with chronic renal failure (CRF) results in part from inhibition of insulin-like growth factor (IGF) action by excess serum IGF-binding proteins (IGFBPs). Excess IGFBPs in CRF serum include IGFBP-1, -2, and -3 and a diffuse ~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
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
MANY CHILDREN with chronic renal failure (CRF) fail to achieve an adult height consistent with their genetic potential despite aggressive supportive care (1). The GH-insulin-like growth factor (IGF)-growth plate chondrocyte axis plays an important role in linear growth. This is achieved in part because GH raises serum IGF levels and stimulates growth plate chondrocyte proliferation, with subsequent linear growth; a crucial role for IGF in this process is suggested by the observation that exogenous IGF-I stimulates linear growth in rats and humans (2, 3, 4, 5), and exogenous IGF-II stimulates linear growth in rats (5). Although growth-retarded CRF children have normal or high serum levels of GH, IGF-I, and IGF-II, serum bioactivity is decreased, a factor that may contribute to the poor linear growth of these children (1, 6, 7, 8, 9, 10).

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
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Experimental subjects and study design

Sera were obtained from 44 children with CRF who did not yet require dialysis [glomerular filtration rate (GFR) between 10–40 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 10–70 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 6.1–9.6% and 6.4–10.7%, respectively. There is no cross-reactivity with hIGFBP-1, -2, -3, -4, or -5 added at final concentrations of 1–5 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 81–118 of the hIGFBP-6 sequence [hIGFBP-6-(81–118)] 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 Student’s 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
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Immunoprecipitation of IGFBP-6 by hIGFBP-6 antiserum

Antiserum raised in goats against the synthetic hIGFBP-6-(81–118) peptide was tested for ability to immunoprecipitate hIGFBP-6 forms that bind IGFs with high affinity. As shown in Fig. 1Go, 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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  		Figure 1. IGFBP-6 immunoprecipitation. Two microliters of serum from a CRF child not yet requiring dialysis and 150 ng rhIGFBP-6 were individually immunoprecipitated with antiserum to synthetic hIGFBP-6-(81–118) peptide and then probed with a mixture of [125I]IGF-I and [125I]IGF-II. Mr markers are on the left.

 
The relative amounts of IGFBP-6 were determined in sera pooled from six dialyzed CRF children, six age-matched nondialyzed CRF children, and six age-matched normal children; all of these children were prepubertal. Each serum pool was immunoprecipitated with IGFBP-6 antiserum, separated by SDS-PAGE, and then probed with either [125I]IGF-I or [125I]IGF-II; whole serum pooled from the six dialyzed CRF children was also probed with each [125I]IGF to show the size of IGFBP-6 relative to the size of the IGFBPs present in CRF serum. As shown in Fig. 2Go, IGFBP-6 corresponded to the diffuse IGFBP band at 24–28 kDa in whole CRF sera. IGFBP-6 was more abundant in serum from dialyzed than nondialyzed CRF children, and IGFBP-6 was least abundant in normal serum; in each case, immunoprecipitated IGFBP-6 was detected by [125I]IGF-II, but not [125I]IGF-I.



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  		Figure 2. Recognition of immunoprecipitated IGFBP-6 by [125I]IGF-I and [125I]IGF-II. Sera were pooled from six CRF children treated with dialysis (DIAL), six age-matched CRF children not requiring dialysis (CRF), and six age-matched normal children (NL); all children were prepubertal. Two microliters of serum from each pool were immunoprecipitated with IGFBP-6 antiserum (IP, BP6 Ab). These immunoprecipitates and 0.8-µL aliquots of whole serum (WS) pooled from the six CRF children treated with dialysis (DIAL) were separated by SDS-PAGE, transfered to nitrocellulose, and then probed with either [125I]IGF-II (left) or [125I]IGF-I (right). The Mr values, in kilodaltons, of IGFBPs present in whole serum are shown on the left.

 
IGFBPs in serum from normal children were also compared to those in serum from age-matched nondialyzed CRF children before and after 12 months of rhGH treatment. In Fig. 3AGo, a representative [125I]IGF ligand blot compares levels of all IGFBPs in these sera as determined by their ability to bind [125I]IGFs. Relative to normal serum, CRF serum had an excess of IGFBPs in the Mr range of 24–28 kDa; the amount of these IGFBPs was not consistently affected by rhGH treatment. In Fig. 3BGo, a representative [125I]IGF ligand blot compares levels of IGFBP-6 immunoprecipitated from these sera. In general, IGFBP-6 was detected in the Mr range of 24–28 kDa, levels were higher in sera of CRF children, and levels were not consistently affected by rhGH treatment.



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  		Figure 3. Effects of CRF and rhGH on serum IGFBP-6 levels; [125I]IGF ligand blotting. A, [125I]IGF ligand blot of age-matched CRF and normal sera. Two microliters of sera taken from three CRF children (no. 5–7) both before (-) and after (+) 12 months of rhGH treatment were paired with 2 µL sera taken from three age-matched normal (NL) children (no. 1–3). Sera were separated by SDS-PAGE, transferred to nitrocellulose, and then analyzed by [125I]IGF-I and [125I]IGF-II ligand blot. B, [125I]IGF ligand blot of IGFBP-6 immunoprecipitated from age-matched CRF and normal sera. Two microliters of sera taken from four CRF children (no. 5–8) both before (-) and after (+) 12 months of rhGH treatment were paired with 2 µL sera taken from four age-matched normal (NL) children (no. 1–4). Sera were immunoprecipitated with hIGFBP-6-(81–118) antibody and then analyzed as described above. For each blot, Mr estimates are shown on the left, and the age of paired children is shown at the bottom.

 
Serum IGFBP-6 levels

As shown in Fig. 4AGo, 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. 4AGo).



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  		Figure 4. Serum IGFBP-6 levels measured by RIA. A, Effect of CRF. Sera from normal prepubertal children (NL), prepubertal CRF children not requiring dialysis (CRF), European prepubertal CRF children not requiring dialysis (E-CRF), European pubertal CRF children not requiring dialysis (E-CRF-P), prepubertal CRF children treated with dialysis (DIAL), and pubertal CRF children treated with dialysis (DIAL-P) were assayed using the IGFBP-6 RIA. The number of children in each group is in parentheses. 1, Different from all other groups (P < 0.01); 2, different from CRF and E-CRF groups (P < 0.05). B, Effect of rhGH. Prepubertal CRF children not requiring dialysis were either treated with rhGH (rhGH) or served as untreated controls (No Rx). Serum drawn at baseline (0) or after 3 or 12 months of study from each child was assayed using the IGFBP-6 RIA. Values represent the mean ± SD. The number of children in each group is in parentheses.

 
Of the 44 nondialyzed prepubertal CRF children entered into the rhGH study, 30 received daily rhGH for 1 yr and 14 were untreated controls. Figure 4BGo shows that serum IGFBP-6 levels were comparable in the two groups at baseline and that rhGH treatment had no significant effect on serum IGFBP-6 levels.

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. 5Go, 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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  		Figure 5. Size distribution of immunoassayable IGFBP-6 in peritoneal dialysate. One milliliter of peritoneal dialysate pooled from three CRF children was size-separated on a Superdex-200 column. Individual 2-mL fractions were collected and assayed for IGFBP-6 by RIA. Arrows indicate the elution positions of proteins used as Mr markers; these include aldolase (158 kDa), ovalbumin (43 kDa), myoglobin (19 kDa), and IGF-I (7 kDa). Fraction numbers are shown at the bottom.

 
Variables correlating with serum IGFBP-6 levels measured by RIA

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 0–12 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. 6Go).



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  		Figure 6. Correlation between GFR and serum IGFBP-6 levels in CRF children. The correlation between GFR and serum IGFBP-6 levels is shown for the 71 European children with CRF.

 
Serum levels of IGFs, free IGF-I, insulin, GH-binding protein, IGFBP-1, IGFBP-2, and ALS measured at 0, 3, or 12 months of the rhGH study (22) did not correlate significantly with serum IGFBP-6 levels measured in the same samples. Serum PTH levels at 0 months also did not correlate significantly with serum IGFBP-6 levels. Serum IGFBP-6 levels did correlate positively with serum IGFBP-3 levels in the 44 children at baseline (r = 0.52; P = 0.0003; Fig. 7Go) and in the 14 untreated (r = 0.56; P = 0.0385) and 30 rhGH-treated (r = 0.54; P = 0.0019) children after 12 months of study. Similarly, serum IGFBP-6 levels in the 71 European children with CRF correlated positively with serum IGFBP-3 levels (r = 0.5; P < 0.005), but did not correlate significantly with levels of IGF-I, IGF-II, insulin, IGFBP-1, or IGFBP-2.



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  		Figure 7. Correlation between serum IGFBP-3 and IGFBP-6 levels in CRF children. The correlation between serum IGFBP-3 and IGFBP-6 levels is shown for the 44 prepubertal children entered into the rhGH study.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
These studies show that CRF serum contains high levels of IGFBP-6, as determined by [125I]IGF-II ligand blot and RIA. Using both methods, serum IGFBP-6 levels were found to be higher in children with lower GFRs. In CRF children, serum IGFBP-6 levels correlated directly and significantly with serum IGFBP-3 levels measured by immunoradiometric assay, did not correlate significantly with height SD score, and were unaffected by rhGH treatment.

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 24–28 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 81–118, 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 10–70 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
 
The following centers/participants were involved with this study: Baylor College of Medicine (Houston, TX): David Powell, M.D., Eileen Brewer, M.D., Andrea Forbes, R.N., and Evelyn Janoff, R.N; Arkansas Children’s Hospital (Little Rock, AR): Eileen Ellis, M.D., Donna Floyd-Gimons, R.N., and Melissa House, R.N.; Baylor University Medical Center (Dallas, TX): Ronald Hogg, M.D., and Tammy Fisher, R.N.; California Pacific Medical Center (San Francisco, CA): Susan Conley, M.D., and Deborah Acres, R.N.; Cedars-Sinai Medical Center (Los Angeles, CA): Elaine Kamil, M.D., and Cathy Vogt, R.N.; Children’s Hospital Medical Center (Cincinnati, OH): Fred Strife, M.D.; Children’s Hospital of Buffalo (Buffalo, NY): Leonard Feld, M.D., and Cathy Sherin, R.N.; Children’s Memorial Hospital (Chicago, IL): Craig Langman, M.D., and Katy Schmeissing, R.N, M.S.; Children’s Mercy Hospital Medical Center (Kansas City, MO): Bradley Warady, M.D., and Gina Weddle, R.N.; Children’s National Medical Center (Washington DC): Mary Ellen Turner, M.D.; Cook Children’s Hospital (Fort Worth, TX): William Allen, M.D., Watson Arnold, M.D., and Peggy Brigance, R.N.; Crippled Children’s Foundation Research Center (Memphis, TN): Robert Wyatt, M.D., and Paula Miller, R.N.; Loma Linda University Medical Center (Loma Linda, CA): Shoba Sahney, M.D., and Sandi Swiridoff, R.N.; University of Oklahoma (Oklahoma City, OK): Adolfo Garnica, M.D., and James Wenzl, M.D.; Seattle Children’s Hospital Medical Center (Seattle, WA): Sandra L. Watkins, M.D., Louise Peck, R.N., and Kelly McCarthy, R.N.; Tulane University Medical Center (New Orleans, LA): Frank Boineau, M.D., Karen Welling, R.N., M.S.N., and Melissa Parenti, R.N.; University of Alabama (Birmingham, AL): Edward Kohaut, M.D., and Sandra Overstreet, R.N.; University of California (Los Angeles, CA): Robert Ettenger, M.D., Ora Yadin, M.D., and Lila Moulton, R.N.; University of Chicago Children’s Hospital (Chicago, IL): Sharon Bartosh, M.D., and Eileen Swanson, R.N.; University of Colorado Health Sciences Center (Denver, CO): Douglas Ford, M.D., Carol Salbenblatt, R.N., and Terri Bisio, R.N.; University of Texas Medical Branch (Galveston, TX): Alok Kalia, M.D., Ann Burns, R.N., and Mary Ann Armendaiz, R.N.; University of Texas Medical School (Houston, TX): Ronald Portman, M.D., and Patty Brannan, R.N.; University of Texas Southwestern Medical Center (Dallas, TX): Steven Alexander, M.D., and Nancy Simonds, R.N.; University of Utah Medical Center (Salt Lake City, UT): Miriam Turner, M.D., Richard Siegler, M.D., and Carolyn Wagner-Munford, R.N.; University of Virginia (Charlottesville, VA): Robert Chevalier, M.D., and Fern Campbell, R.N.; and SPNSG Central Office: Columbia Hospital at Medical City (Dallas, TX): Ronald J. Hogg, M.D., Director; Kaye Green, Administrative Coordinator.

The technical support of Grace Matthew and Dr. Aruna Khare is gratefully acknowledged.


   Footnotes
 
1 This work was supported by NIH Grant RO1-DK-38773 (to D.R.P.), a grant from Genentech (South San Francisco, CA), and Grant M01-RR-00069 from the General Clinical Research Centers Program, National Centers for Research Resources, NIH. Back

Received March 13, 1997.

Revised May 16, 1997.

Accepted June 2, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Powell DR. 1989 Renal disease and growth retardation. Kidney. 22:7–12.
  2. Roberts CT, Brown AL, Graham DE, et al. 1986 Growth hormone regulates the abundance of IGF-I mRNA in adult rat liver. J Biol Chem. 261:10025–10028.[Abstract/Free Full Text]
  3. Cox GN, McDermott MJ, Merkel E, et al. 1994 Recombinant human insulin-like growth factor (IGF) binding protein-1 inhibits somatic growth stimulated by IGF-I and growth hormone in hypophysectomized rats. Endocrinology. 135:1913–1920.[Abstract]
  4. Vaccarello MA, Diamond FB, Guevara-Aguirre J, et al. 1993 Hormonal, metabolic and pharmacokinetic effects of recombinant insulin-like growth factor-I in growth hormone receptor deficient (GHRD) syndrome. J Clin Endocrinol Metab. 77:273–280.[Abstract]
  5. Schoenle E, Zapf J, Hauri C, Steiner T, Froesch ER. 1985 Comparison of in vivo effects of insulin-like growth factors I and II and of growth hormone in hypophysectomized rats. Acta Endocrinol (Copenh). 108:167–174.[Medline]
  6. El-Bishti M, Counahan R, Bloom S, Chantler C. 1978 Hormonal and metabolic responses to intravenous glucose in children on regular hemodialysis. Am J Clin Nutr. 31:1865–1869.[Abstract/Free Full Text]
  7. Powell DR, Rosenfeld RG, Baker BK, Hintz RL. 1986 Serum somatomedin levels in adults with chronic renal failure: the importance of measuring insulin-like growth factor (IGF)-I and IGF-II in acid-chromatographed uremic serum. J Clin Endocrinol Metab. 63:1186–1192.[Abstract]
  8. Powell DR, Rosenfeld RG, Sperry JB, Baker BK, Hintz RL. 1987 Serum concentrations of insulin-like growth factor (IGF)-1, IGF-2 and unsaturated somatomedin carrier proteins in children with chronic renal failure. Am J Kidney Dis. 10:287–292.[Medline]
  9. Phillips LS, Fusco AC, Unterman TG, DelGreco F. 1984 Somatomedin inhibitor in uremia. J Clin Endocrinol Metab. 59:764–772.[Abstract]
  10. Blum WF, Ranke MB, Kietzmann K, Tonshoff B, Mehls O. 1991 Growth hormone resistance and inhibition of somatomedin activity by excess of insulin-like growth factor binding protein in uraemia. Pediatr Nephrol. 5:539–544.[CrossRef][Medline]
  11. Powell DR, Liu F, Baker B, Lee PDK, Belsha CW, Brewer ED, Hintz RL. 1993 Characterization of insulin-like growth factor binding protein-3 in chronic renal failure serum. Pediatr Res. 33:136–143.[Medline]
  12. Shimasaki S, Shimonaka M, Zhang H-P, Ling N. 1991 Identification of five different IGFBPs from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human. J Biol Chem. 266:10646–10653.[Abstract/Free Full Text]
  13. Shimasaki S, Gao L, Shimonaka M, Ling N. 1991 Isolation and molecular cloning of insulin-like growth factor binding protein-6. Mol Endocrinol. 5:938–948.[Abstract]
  14. Baxter RC, Martin JL. 1986 Radioimmunoassay of growth hormone-dependent insulin-like growth factor binding protein in human plasma. J Clin Invest. 78:1504–1512.
  15. Lee PDK, Hintz RL, Sperry JB, Baxter RC, Powell DR. 1990 Insulin-like growth factor binding proteins in growth retarded children with chronic renal failure. Pediatr Res. 26:308–315.[Medline]
  16. Tonshoff B, Mehls O, Heinrich U, Blum WF, Ranke MB, Schauer A. 1990 Growth-stimulating effects of recombinant human growth hormone in children with end-stage renal disease. J Pediatr. 116:561–566.[CrossRef][Medline]
  17. Liu F, Powell DR, Hintz RL. 1990 Characterization of insulin-like growth factor binding proteins in human serum from patients with chronic renal failure. J Clin Endocrinol Metab. 70:620–628.[Abstract]
  18. Hokken-Koelega ACS, Stijnen T, de Muinck Keizer-Schrama SMPF, et al. 1991 Placebo-controlled, double-blind, cross-over trial of growth hormone treatment in prepubertal children with chronic renal failure. Lancet. 338:585–590.[CrossRef][Medline]
  19. Lee PDK, Conover CA, Powell DR. 1993 Regulation and function of insulin-like growth factor binding protein-1. Proc Soc Exp Biol Med. 204:4–29.[Abstract]
  20. Blum WF, Horn N, Kratzsch J, et al. 1993 Clinical studies of IGFBP-2 by radioimmunoassay. Growth Regul. 3:100–104.[Medline]
  21. Tonshoff B, Blum WF, Wingen A-M, Mehls O. 1995 Serum insulin-like growth factors (IGFs) and IGF binding proteins 1, 2 and 3 in children with chronic renal failure: relationship to height and glomerular filtration rate. J Clin Endocrinol Metab. 80:2684–2691.[Abstract]
  22. Powell DR, Liu F, Baker BK, et al. 1997 Modulation of growth factors by growth hormone in children with chronic renal failure. Kidney Int. 51:1970–1979.[Medline]
  23. Tonshoff B, Blum WF, Mehls O. 1996 Serum insulin-like growth factors and their binding proteins in children with end- stage renal disease. Pediatr Nephrol. 10:269–274.[Medline]
  24. Burch WM, Correa J, Shively JE, Powell DR. 1990 The 25 kiloDalton insulin-like growth factor (IGF) binding protein inhibits both basal and IGF-I-mediated growth of chick embryo pelvic cartilage in vitro. J Clin Endocrinol Metab. 70:173–180.[Abstract]
  25. Daughaday WH, Rotwein P. 1989 Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum and tissue concentrations. Endocr Rev. 10:68–91.[Abstract]
  26. Bach LA, Thotakura R, Rechler MM. 1992 Human insulin-like growth factor binding protein-6 is O-glycosylated. Biochem Biophys Res Commun. 186:301–307.[CrossRef][Medline]
  27. Wingen A, Fabian-Bach C, Mehls O. 1992 Multicenter randomized study on the effect of a low-protein diet on the progression of renal failure in childhood: one-year results. Miner Electrolyte Metab. 18:303–308.[Medline]
  28. Kale AS, Liu F, Hintz RL, et al. 1996 Characterization of insulin-like growth factors (IGFs) and IGF binding proteins in peritoneal dialysate. Pediatr Nephrol. 10:467–73.[CrossRef][Medline]
  29. Baxter RC, Saunders H. 1992 Radioimmunoassay of insulin-like growth factor binding protein-6 in human serum and other body fluids. J Endocrinol. 134:133–139.[Abstract]
  30. Kiefer MC, Schmid C, Waldvogel M, et al. 1992 Characterization of recombinant human insulin-like growth factor binding proteins 4,5 and 6 produced in yeast. J Biol Chem. 267:12692–12699.[Abstract/Free Full Text]
  31. Lemozy S, Pucilowska JB, Underwood LE. 1994 Reduction of insulin-like growth factor-I (IGF-I) in protein-restricted rats is associated with differential regulation of IGF- binding protein messenger ribonucleic acids in liver and kidney, and peptides in liver and serum. Endocrinology. 135:617–623.[Abstract]
  32. Martin JL, Coverley JA, Baxter RC. 1994 Regulation of imunoreactive insulin-like growth factor binding protein-6 in normal and transformed human fibroblasts. J Biol Chem. 269:11470–11477.[Abstract/Free Full Text]
  33. Zhou Y, Mohan S, Linkhart TA, Baylink DJ, Strong DD. 1996 Retinoic acid regulates insulin-like growth factor binding protein expression in human osteoblastic cells. Endocrinology. 137:975–983.[Abstract]
  34. Gabbitas B, Canalis E. 1996 Cortisol enhances the transcription of insulin-like growth factor binding protein-6 in cultured osteoblasts. Endocrinology. 137:1687–1692.[Abstract]
  35. Okazaki R, Riggs BL, Conover CA. 1994 Glucocorticoid regulation of insulin-like growth factor binding protein expression in normal human osteoblast-like cells. Endocrinology. 134:126–132.[Abstract]
  36. Conover CA, Clarkson JT, Bale LK. 1995 Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF binding protein expression in fibroblasts. Endocrinology. 136:1403–1410.[Abstract]
  37. Tonshoff B, Eden S, Weiser E, et al. 1994 Reduced hepatic growth hormone (GH) receptor gene expression and increased plasma GH binding protein in experimental uremia. Kidney Int. 45:1085–1092.[Medline]
  38. Tonshoff B, Powell DR, Zhao D, et al. 1997 Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein (IGFBP)-1 and -2 gene expression in experimental uremia. Endocrinology. 138:938–946.[Abstract/Free Full Text]
  39. McCarthy TL, Casinghino S, Centrella M, Canalis E. 1994 Complex pattern of insulin-like growth factor binding protein expression in primary rat osteoblast enriched cultures: regulation by prostaglandin E2, growth hormone, and the insulin-like growth factors. J Cell Physiol. 160:163–175.[CrossRef][Medline]
  40. Cerro JA, Grewal A, Wood TL, Pintar JE. 1993 Tissue-specific expression of the insulin-like growth factor binding protein (IGFBP) mRNAs in mouse and rat development. Regul Pept. 48:189–198.[CrossRef][Medline]
  41. Ewton DZ, Florini JR. 1995 IGF binding proteins-4, -5 and -6 may play specialized roles during L6 myoblast proliferation and differentiation. J Endocrinol. 144:539–553.[Abstract]
  42. Wang E, Wang J, Chin E, Zhou J, Bondy CA. 1995 Cellular patterns of insulin-like growth factor system gene expression in murine chondrogenesis and osteogenesis. Endocrinology. 136:2741–2751.[Abstract]
  43. Schmid C, Schlapfer I, Keller A, Waldvogel M, Froesch ER, Zapf J. 1995 Effects of insulin-like growth factor (IGF) binding proteins (BPs)-3 and -6 on DNA synthesis of rat osteoblasts: further evidence for a role of auto-/paracrine IGF-I but not IGF-II in stimulating osteoblast growth. Biochem Biophys Res Commun. 212:242–248.[CrossRef][Medline]
  44. Bach LA, Hsieh S, Brown AL, Rechler MM. 1994 Recombinant human insulin-like growth factor (IGF) binding protein-6 inhibits IGF-II-induced differentiation of L6A1 myoblasts. Endocrinology. 135:2168–2176.[Abstract]
  45. Bach LA, Salemi R, Leeding KS. 1995 Roles of insulin-like growth factor (IGF) receptors and IGF binding proteins in IGF-II-induced proliferation and differentiation of L6A1 rat myoblasts. Endocrinology. 136:5061–5069.[Abstract]
  46. Kato M, Ishizaki A, Hellman U, et al. 1995 A human keratinocyte cell line produces two autocrine growth inhibitors, transforming growth factor-ß and insulin-like growth factor binding protein-6, in a calcium- and cell density-dependent manner. J Biol Chem. 270:12373–12379.[Abstract/Free Full Text]
  47. Shinar DM, Endo N, Halperin D, Rodan GA, Weinreb M. 1993 Differential expression of insulin-like growth factor (IGF)-I and IGF-II messenger ribonucleic acid in growing rat bone. Endocrinology. 132:1158–1167.[Abstract]
  48. Hill DJ, De Sousa D. 1990 Insulin is a mitogen for isolated epiphyseal growth plate chondrocytes from the fetal lamb. Endocrinology. 126:2661–2670.[Abstract]
  49. Froger-Gaillard B, Hossenlopp P, Adolphe M, Binoux M. 1989 Production of insulin-like growth factors and their binding proteins by rabbit articular chondrocytes: relationships with cell multiplication. Endocrinology. 124:2365–2372.[Abstract]



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