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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 5 1654-1661
Copyright © 1998 by The Endocrine Society

Original Studies

The Insulin-Like Growth Factor Axis and Growth in Children with Chronic Renal Failure: A Report of the Southwest Pediatric Nephrology Study Group1

David R. Powell, Susan K. Durham, Frances Liu, Bonita K. Baker, Phillip D. K. Lee, Sandra L. Watkins, Phil G. Campbell, Eileen D. Brewer, Raymond L. Hintz and Ronald J. Hogg

Department of Pediatrics (D.R.P., S.K.D., P.D.K.L., E.D.B.), Baylor College of Medicine, Houston, Texas 77030; Stanford University Medical School (F.L., B.K.B., R.L.H.), Stanford, California 94305; University of Washington (S.L.W.), Seattle, Washington 98105; Orthopedic Research Laboratory, Allegheny University of Health Sciences (P.G.C.), Pittsburgh, Pennsylvania 15212; and Columbia Hospital at Medical City (R.L.H.), Dallas, Texas 75230

Address all correspondence and requests for reprints to: Dr. David R. Powell, Texas Children’s Hospital, Feigin 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
 
Children with chronic renal failure (CRF) are often growth retarded despite normal serum levels of GH and insulin-like growth factors (IGFs). Recent studies suggest that excess IGF-binding proteins (IGFBPs) in the 35-kDa fractions of CRF serum contribute to CRF growth failure. This report characterizes the relationship between IGFBP-3 and IGF peptides in the serum of growth-retarded CRF children. Size-exclusion chromatography at pH 7.4 found IGFBP-3 and IGFs almost exclusively in the 150-kDa fractions of normal serum, where their molar stoichiometry was approximately 1:1. However, similar chromatography of CRF serum found a molar excess of IGFBP-3 over total IGFs in the 150-kDa fractions and large amounts of IGFs in the 35-kDa fractions. In the 150-kDa fractions of CRF serum, IGFBP-3 was present in normal amounts, but a greater than normal amount was in the form of a 29-kDa IGFBP-3 fragment. Treatment of these CRF children with recombinant human GH increased the molar excess of IGFBP-3 over total IGFs in the 150-kDa fractions, the amount of IGFBP-3 and total IGFs in the 150-kDa fractions, and the amount of IGFs, but not IGFBPs, in the 35-kDa fractions. These data suggest that in untreated CRF children, proteolysis of IGFBP-3 in the 150-kDa fractions releases IGFs to the excess IGFBPs in the 35-kDa fractions, but insufficient IGF is released to overcome the growth-inhibiting effects of these excess IGFBPs. Treatment with recombinant human GH increases levels of IGFs and IGFBP-3 in the 150-kDa fractions, and subsequent IGFBP-3 proteolysis releases sufficient IGF to overcome the growth inhibitory effects of excess IGFBPs in the 35-kDa fractions of CRF serum.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE GH-INSULIN-LIKE growth factor (IGF)-growth plate chondrocyte (GPC) axis plays a central role in linear growth. GH and IGFs act synergistically to stimulate GPC proliferation and hypertrophy (1). Beyond stimulating local IGF-I production by GPCs, GH also raises serum IGF levels, and it is now clear that circulating IGFs can stimulate linear growth (1, 2, 3, 4, 5).

IGF-I and -II are 7-kDa proteins found in serum and other body fluids at higher molecular mass, tightly bound by a family of six IGF-binding proteins (IGFBPs) (6, 7, 8). In serum from healthy individuals, most IGFs circulate in the 150-kDa serum fractions in a ternary complex of one IGF peptide, one approximately 40-kDa form of glycosylated IGFBP-3, and an approximately 86-kDa acid-labile subunit (ALS) (8, 9, 10). The remaining IGFs are found in the 35-kDa serum fractions bound to some or all of the six IGFBPs.

The profound growth failure of children with chronic renal failure (CRF) is associated with many abnormalities of the serum GH-IGF axis (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). Although IGF levels in CRF serum are in the normal range, IGF bioactivity is low (13, 14, 15, 16, 17, 18, 19, 20). This is due to excess unsaturated IGF-binding sites in CRF serum; removing these excess IGF-binding sites, by passing CRF serum through agarose beads covalently linked to IGF-II, raises serum IGF bioactivity (16). High levels of IGFBP-1, -2, -3, and -6 appear to provide the excess IGF-binding sites in CRF serum (12, 14, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 29). The ability of excess IGFBPs to inhibit IGF bioactivity is not simply an in vitro phenomenon, as IGFBP-1 injected daily for 2 weeks inhibited both GH- and IGF-I-induced growth of hypophysectomized rats (3); although IGFBP-1 was used in this study, it is likely that an excess of any unsaturated IGFBP with high affinity for IGF-I would also inhibit GH- or IGF-I-stimulated cartilage growth.

IGFBP-3 is the major serum IGFBP in postnatal life. Early work found high levels in CRF sera, suggesting that IGFBP-3 was the most likely serum IGFBP to be a CRF growth inhibitor (16, 17, 18, 30). This idea was supported by work showing that intact IGFBP-3 blocks IGF action and IGF-independent cell growth in vitro (31, 32, 33). However, IGFBP-3 also potentiates IGF-I action under some conditions; thus, IGFBP-3 is not always an IGF inhibitor (32, 33, 34, 35). Indeed, the data now suggest that IGFBP-3 is not a major inhibitor of growth or IGF action in CRF serum: 1) in children with growth failure and a glomerular filtration rate (GFR) between 10–40 mL/min·1.73 m2, serum IGFBP-3 levels are not high and do not correlate with the degree of growth failure (5); 2) the excess IGFBP-3 in CRF serum is in the form of IGFBP-3 fragments with low affinity for IGFs (16, 25, 28, 36); 3) serum levels of both IGFBP-3 and the 150-kDa complex are low in GH-deficient children and rise during the catch-up growth these children experience with recombinant human GH (rhGH) treatment (8, 37); and 4) in CRF children, the rhGH-stimulated rise in serum IGFBP-3 levels correlates directly with accelerated linear growth (5). These last two findings suggest an anabolic role for IGFBP-3, perhaps as part of the 150-kDa complex. Thus, the role of IGFBP-3 in CRF growth failure is unclear, but this IGFBP is unlikely to be a growth inhibitor.

To better understand the role of serum IGFs and IGFBP-3 in the growth failure of CRF children and in the rhGH-stimulated catch-up growth of these children, we studied the size distribution of IGF-I, IGF-II, and IGFBP-3 in serum of CRF children before and during rhGH treatment. The results suggest how IGF axis abnormalities lead to growth failure in children with CRF and how rhGH alters the IGF axis in CRF serum to allow catch-up growth.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients and study design

Sera were obtained from 44 CRF children who were part of a multicenter trial of the effects of rhGH on CRF children. Study design was approved by the institutional review board for research involving human subjects of each center; details of study design and of these 44 children were previously published (5). Inclusion criteria were 1) irreversible CRF (GFR, >10 and <40 mL/min·1.73 m2), 2) height less than the fifth percentile for chronological age, 3) age more than 2.5 yr, 4) ability to stand for height measurement, 5) bone age less than 10 yr for girls and less than 11 yr for boys, and 6) Tanner stage 1. Exclusion criteria were 1) serum albumin less than 2.5 g/dL, 2) taking medications that influence growth, 3) illnesses affecting growth, 4) diabetes mellitus, and 5) presence or history of malignancy. Children were randomized so that one third were untreated controls, and two thirds received rhGH; the groups were balanced for age, gender, height, primary renal disease, and baseline GFR. Treated children received a daily sc dose of 0.05 mg/kg rhGH (Nutropin), provided by Genentech (South San Francisco, CA). Of the 69 children initially entered into the protocol, only 44 provided fasting serum samples at baseline and 12 months, maintained their GFR above 10 and below 40 mL/min·1.73 m2 for 12 months, and remained at Tanner stage 1 for 12 months.

Sera from 10 healthy prepubertal children were used as normal control sera (5), and 1 term pregnancy serum sample was used as a positive control in the standard IGFBP-3 protease assay (38).

Serum sample preparation

All CRF patients fasted for more than 5 h before blood drawing. Blood was centrifuged, and serum was frozen (-80 C) until assay.

Most studies used pooled sera. Equal volumes of 0 and 12 month sera from the first 12 rhGH-treated children and from the first 5 untreated children to complete the study were pooled and designated pool I/rhGH/0, pool I/rhGH/12, pool I/untreated/0, or pool I/untreated/12. Equal volumes of sera from the last 18 rhGH-treated children and from the last 9 untreated children to complete 12 months of the study were pooled and designated pool II/rhGH/0, pool II/rhGH/12, pool II/untreated/0, or pool II/untreated/12. Equal volumes of sera from the 10 healthy prepubertal children were combined to make a normal serum pool.

Size-exclusion chromatography

Sephacryl S-300. One half milliliter from each of the nine serum pools was individually chromatographed at pH 7.4 on a 0.9 x 120-cm Sephacryl S-300 column, as described previously (22). Individual 2-mL column fractions were collected and frozen at -80 C until assay.

Sephadex G-50. IGF-I and IGF-II in the 2-mL fractions from the Sephacryl S-300 column were separated from IGFBPs by acid chromatography on a 0.9 x 120-cm Sephadex G-50 column (13) and then assayed by specific RIA for IGF-I and IGF-II.

Serum protein assays

IGF-I and IGF-II RIAs. IGF-containing fractions from the acid G-50 column were pooled, lyophilized, reconstituted, and assayed as described previously (22). IGF-I antiserum was provided by Drs. L. E. Underwood and J. J. Van Wyk (Chapel Hill, NC) through the National Hormone and Pituitary Program. IGF-II antibody was purchased from Amano International Enzyme Co. (Troy, VA).

IGFBP-3 immunoradiometric assay (IRMA). Fractions from the Sephacryl S-300 column were directly measured by IRMA using a commercially available kit from Diagnostic Systems Laboratories (Webster, TX), as described previously (28).

IGFBP-3 immunoblot

Aliquots of pooled CRF sera that had been size-separated by Sephacryl S-300 were separated by 12% SDS-PAGE and transfered to nitrocellulose (39). IGFBP-3 immunoblotting was performed as previously described (28, 40); {alpha}IGFBP-3g1 (28, 41), a gift from Dr. Ron Rosenfeld (Oregon Health Science Center, Portland, OR), was diluted 1:1000 and served as the IGFBP-3 antibody.

Standard IGFBP-3 protease assay

A standard IGFBP-3 protease assay was used (38); 2 µL sera from individual CRF or normal children were incubated with 30,000 cpm [125I]IGFBP-3 for 4 h at 37 C [the human IGFBP-3 used was produced in Escherichia coli by BioGrowth, Richmond, CA (currently Celtrix Pharmaceuticals, Inc.)]. Term pregnancy serum served as a positive control. After samples were separated by 12% SDS-PAGE, intact and fragmented [125I]IGFBP-3 were detected by autoradiography.

IGFBP-3 plate protease assay

[125I]IGFBP-3 (100,000 cpm) in a total volume of 25 µL 0.1 mol/L NaCO3 (pH 9.8) was placed into wells of 96-well immunological plates (Maxi-Sorb, Nunc, Fisher Scientific, Pittsburgh, PA) and air-dried in an oven overnight at 37 C. Wells were rinsed with 200 µL 10 mmol/L Na phosphate (pH 7.5) and 150 mmol/L NaCl and blocked with 200 µL Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween-80, 0.02% NaN3, and 1% BSA for 1 h at 37 C. Wells were rinsed with 200 µL 30 mmol/L Tris acetate (pH 7.4), 10 mmol/L Na phosphate, 0.1% Tween-20, and 0.02% NaN3 (assay buffer). Two microliter equivalents of sera from normal children (normal pool), CRF children before rhGH treatment (pool II/rhGH/0), or CRF children after 12 months of rhGH treatment (pool II/rhGH/12) were placed in wells in a total volume of 200 µL assay buffer. After 24 h at 37 C, 25 µL from each well were counted to measure the [125I]IGFBP-3 released. Excess trypsin (10 IU/mL; Sigma Chemical Co., St. Louis, MO) was used to determine maximal releasable [125I]IGFBP-3 after correction for nonspecific release of [125I]IGFBP-3 in assay buffer alone. Total releasable [125I]IGFBP-3 was 45,000 cpm or 45% of the counts per min plated, and nonspecific releasable [125I]IGFBP-3 was 11,500 cpm or 11.5% of the counts per min plated.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
As shown in Fig. 1Go, most immunoreactive IGFBP-3 circulated at 150 kDa in serum collected at baseline and 12 months from untreated and rhGH-treated CRF children. A smaller IGFBP-3 peak was present at 35 kDa in each sample; this peak made up about 19% of total IGFBP-3 in the baseline serum samples. After 12 months of study, IGFBP-3 was increased only in CRF children treated with rhGH, and this increase was limited to IGFBP-3 circulating at 150 kDa.



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  		Figure 1. Size distribution of immunoassayable IGFBP-3 in CRF serum before and after rhGH treatment. A, Equal volumes of 0 and 12 month sera from the first 12 rhGH-treated children and the first 5 untreated children to complete 12 months of the study were pooled and designated pool I/rhGH/0, pool I/rhGH/12, pool I/untreated/0, or pool I/untreated/12. An aliquot from each pool was size-separated on a Sephacryl S-300 column. IGFBP-3 levels were measured in each column fraction by IRMA. B, Equal volumes of 0 and 12 month sera from the last 18 rhGH-treated children and from the last 9 untreated children to complete 12 months of the study were pooled and designated pool II/rhGH/0, pool II/rhGH/12, pool II/untreated/0, or pool II/untreated/12. Aliquots from each pool were size-separated and assayed for IGFBP-3 as described in A. Arrows identify the approximate elution positions of 150- and 35-kDa serum proteins.

 
Distribution of immunoreactive IGF-I and -II in size-separated CRF sera is shown in Fig. 2Go; for ease of presentation, the sum of IGF-I and -II levels (total IGF) in each fraction is shown, but IGF-I and -II individually had the same relative distribution as total IGF in each serum pool. Significant amounts of IGF-I and -II were found in both the 150- and 35-kDa fractions of each CRF serum pool. Treating CRF children with rhGH for 12 months led to a rise in IGF-I and -II levels that was roughly equally divided between the 150- and 35-kDa CRF serum fractions. This analysis established that the observed size distribution of IGFs in CRF serum is usual for the CRF state, as 1) the same distribution of IGFs was found in two independent pools of CRF sera collected at baseline (pool II/rhGH/0 and pool II/untreated/0); and 2) a similar distribution of IGFs in CRF serum has been reported (42).



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  		Figure 2. Size distribution of immunoassayable IGFs in CRF serum before and after rhGH treatment. The Sephacryl S-300 column fractions presented in Fig. 1BGo were individually assayed for IGF-I and IGF-II by RIA as described in Materials and Methods. For each Sephacryl S-300 fraction, the sum of the IGF-I and IGF-II levels (total IGF) is presented. Arrows identify the approximate elution positions of 150- and 35-kDa serum proteins.

 
Figure 3Go shows the molar distribution of IGFBP-3 and total IGF in size-fractionated normal and CRF sera. IGFBP-3 and total IGF followed the same pattern in normal sera, with most IGFBP-3 and IGF circulating at 150 kDa in an approximately 1:1 stoichiometry. In contrast, the 150-kDa fractions of baseline CRF sera showed a molar excess of IGFBP-3, and this excess was exaggerated after 12 months of rhGH treatment.



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  		Figure 3. Comparison of the molar distribution of IGFBP-3 and total IGFs in size-fractionated normal and CRF sera. Left, Normal sera. Equal volumes of sera from the 10 normal children were pooled, and an aliquot was size-separated on the Sephacryl S-300 column. IGFBP-3, IGF-I, and IGF-II levels were assayed as described in Figs. 1Go and 2Go. Total IGF represents the sum of IGF-I and IGF-II levels for each fraction. Middle and right, CRF sera. Sera from untreated CRF children (pool II/rhGH/0) and from the same children after 12 months of rhGH treatment (pool II/rhGH/12) were size-separated by Sephacryl S-300 chromatography, and individual fractions were assayed for IGFBP-3 (see Fig. 1BGo), IGF-I, and IGF-II (see Fig. 2Go). IGFBP-3 and total IGF levels are presented together for direct comparison. Arrows identify the approximate elution positions of 150- and 35-kDa serum proteins.

 
To determine whether differences in the IGF/IGFBP-3 molar distribution between CRF and normal sera were related to different forms of IGFBP-3 circulating at 150 and 35 kDa, size-fractionated normal and baseline CRF sera were immunoblotted on the same nitrocellulose filter. In contrast to normal sera, CRF sera had lower levels of intact (41 and 38 kDa) IGFBP-3 and higher levels of a 29-kDa IGFBP-3 form (IGFBP-329) in the 150-kDa complex (Fig. 4Go). This agrees with studies showing that IGFBP-329, a glycosylated IGFBP-3 fragment, is more abundant than normal in the 150-kDa fractions of sera from CRF children (28, 36); also consistent with these studies is the excess IGFBP-329 found in the 35-kDa fractions of CRF serum.



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  		Figure 4. Immunoblot of IGFBP-3 forms in size-separated sera from normal children and children with CRF. Aliquots from the identical column fractions presented in Fig. 3Go were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using the IGFBP-3 antibody {alpha}IGFBP-3g1. A, Pooled sera from normal children. B, Pooled sera from untreated CRF children (pool II/rhGH/0). Fraction numbers are presented below the immunoblot. Arrows identify the approximate elution positions of 150- and 35-kDa serum proteins.

 
The effect of rhGH on levels of intact and fragmented IGFBP-3 in CRF sera was evaluated by immunoblot. After seven of the pool II CRF children received rhGH for 12 months, their sera showed a consistent rise in IGFBP-329 and a variable rise in intact IGFBP-3 (Fig. 5Go). In pooled sera, the rhGH-induced rise in IGFBP-329 was found primarily in the 150-kDa fractions (data not shown).



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  		Figure 5. Effect of rhGH on abundance of IGFBP-3 forms in sera of CRF children. Individual sera from seven Pool II CRF children (no. 1–7) before (0) and after (12) 12 months of rhGH treatment were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using the IGFBP-3 antibody {alpha}IGFBP-3g1. The molecular mass, in kilodaltons, of IGFBP-3 forms is shown on the left.

 
As IGFBP-329 is abundant in CRF sera, these sera were tested for increased IGFBP-3 protease activity. Individual sera from five pool II CRF children, shown in Fig. 5Go, were screened using a standard IGFBP-3 protease assay (38). As shown in Fig. 6Go, [125I]IGFBP-3 was degraded comparably by either pooled normal sera or CRF sera, whereas [125I]IGFBP-3 was extensively degraded by pregnancy serum; rhGH treatment did not affect IGFBP-3 proteolysis. The IGFBP-3 plate protease assay showed comparable results (Fig. 7Go); pooled normal sera proteolyzed [125I]IGFBP-3 to at least the same extent as pooled sera from CRF children before and after 12 months of rhGH treatment.



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  		Figure 6. IGFBP-3 protease activity in CRF serum: standard assay. Individual sera from five pool II CRF children (no. 1–5) collected before (0) and after (12) 12 months of rhGH treatment, pooled normal sera (NL), term pregnancy serum (PR), or no serum (0) were incubated with [125I]IGFBP-3 for 4 h at 37 C. Samples were separated by 12% SDS-PAGE, after which intact and fragmented [125I]IGFBP-3 were visualized by autoradiography. The molecular mass of intact [125I]IGFBP-3E. coli (29 kDa) and the major [125I]IGFBP-3E. coli fragment (16 kDa) are shown on the left.

 


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  		Figure 7. IGFBP-3 protease activity in CRF serum: plate protease assay. Two microliter equivalents of serum pooled from the 10 normal children (control), from CRF children before rhGH treatment (pool II/GH/0), or from CRF children after 12 months of rhGH treatment (pool II/GH/12) were placed in the IGFBP-3 plate protease assay. The release of [125I]IGFBP-3 was determined after a 24-h incubation at 37 C. Proteolysis is reported as a percentage of the total releasable [125I]IGFBP-3, as determined by trypsin digestion. Bars represent the mean ± SEM of triplicate determinations. Values from the GH treatment groups were compared for differences with the control value by ANOVA followed by Dunnett’s post-hoc test: control vs. pool II/rhGH/0, P = 0.088; and control vs. pool II/rhGH/12, P = 0.752.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In normal children, most serum IGFBP-3 was found in the 150-kDa serum fractions in an approximately 1:1 molar stoichiometry with total IGFs, consistent with past studies localizing most IGFBP-3 to the 150-kDa IGF/IGFBP-3/ALS ternary complex in sera from GH-replete subjects (8, 10). The present study also found most circulating IGFBP-3 in the 150-kDa fractions of CRF serum, with a smaller IGFBP-3 peak in the 35-kDa fractions. However, IGF-I and IGF-II did not follow the same distribution as IGFBP-3; a significant amount of each IGF was found in the 35-kDa fractions of CRF serum, leaving a large molar excess of IGFBP-3 over total IGFs in the 150-kDa fractions. This abnormal IGF distribution in CRF serum has been reported previously, but IGFBP-3 distribution was not examined in that study (42).

By immunoblot, IGFBP-3 was found mainly in 150-kDa fractions of both CRF and normal serum. However, these fractions of normal serum contained primarily the 41- and 38-kDa forms of glycosylated intact IGFBP-3, whereas these fractions of CRF serum contained primarily the glycosylated IGFBP-329 fragment. This is similar to our data on distribution of IGFBP-3 forms in serum of normal and dialyzed adolescents (25, 28), but differs from the data of Blum et al. (16), who found that most IGFBP-3 in CRF serum was fragmented, but in the 35-kDa fractions. In CRF serum, the finding of about 60% of IGFs in the 150-kDa fractions (Ref. 42 and present study) is consistent with the IGFBP-3 distribution reported here, but is not consistent with the IGFBP-3 distribution reported by Blum et al. (16).

The excess of IGFBP-3 over IGFs in the 150-kDa fractions of CRF serum is inconsistent with the 1:1:1 stoichiometry of IGF/IGFBP-3/ALS traditionally expected for the serum ternary complex (43), but is consistent with reports that purified IGFBP-3 and ALS form binary complexes in vitro (44) and that IGFBP-3/ALS binary complexes are abundant in normal rat serum in vivo (45, 46, 47). In rats, it is a 30-kDa IGFBP-3 fragment in the 150-kDa serum fractions that circulates free of IGFs, almost certainly due to the low affinity of this fragment for IGFs (46, 47). In the present study, most IGFBP-3 in the 150-kDa fractions of CRF serum is IGFBP-329, a fragment with low IGF affinity (25, 28, 36). Thus, IGFBP-329 in the 150-kDa fractions of CRF serum is probably the human equivalent of the 30-kDa IGFBP-3 fragment circulating free of IGFs in rat sera.

GH therapy raised IGFBP-3 levels in the 150-kDa, but not the 35-kDa, serum fractions of growth-retarded CRF children. The parallel rise in serum levels of IGFBP-3, IGFs, and ALS during rhGH therapy of these CRF children (5) suggests that rhGH stimulates formation of the IGF/IGFBP-3/ALS ternary complex. Levels of this complex rise during catch-up growth of GH-deficient children treated with GH (8, 10), suggesting the ternary complex promotes linear growth. If this complex promotes growth, it probably does so by releasing IGFs while in the circulation, as very little ternary complex crosses the vascular endothelium into interstitial fluids (28, 48).

In rat serum, IGF seems to be released from the circulating 150-kDa complex by proteolysis of IGFBP-3 to the 30-kDa fragment with low IGF affinity. The same process probably occurs in human serum; proteolysis of IGFBP-3 in ternary complexes creates IGFBP-329, which releases bound IGFs (25, 28, 36, 41). As IGFBP-3 protease activity is comparable in normal and CRF sera, the accumulation of IGFBP-329 free of IGFs in the 150-kDa fractions of CRF, but not normal sera, suggests that proteolyzed IGFBP-3 is cleared much more slowly than normal from these CRF serum fractions.

Figure 8Go summarizes the balance between IGFs and IGFBPs in the 150- and 35-kDa fractions of CRF serum before and after rhGH treatment. The major abnormality in CRF serum is an excess, in 35-kDa fractions, of IGFBPs with high IGF affinity (18, 23, 24, 26, 27, 29); these may block growth by sequestering IGFs from type I IGF receptors on target tissues. The inverse correlation between height and elevated serum levels of IGFBP-2 and -1 in CRF (5, 27) emphasizes the likely role of these two IGFBPs in growth inhibition. Untreated CRF children have normal amounts of IGFBP-3 at 150 kDa, but much of this IGFBP-3 has been proteolyzed to IGFBP-329; the low IGF affinity of this fragment probably explains the low IGF levels in the 150-kDa fractions and the high IGF levels in the 35-kDa fractions, where the IGFs are probably bound by the excess high affinity IGFBPs. GH treatment 1) increased IGFBP-3 in the 150-kDa fractions, with a fair amount of this rise in the form of IGFBP-329; 2) increased IGFs in both the 150- and 35-kDa fractions; and 3) had no major effect on levels of high affinity IGFBPs in the 35-kDa fractions. Probably, rhGH increased IGFBP-3 and IGF levels by stimulating formation of the 150-kDa IGF/IGFBP-3/ALS ternary complex; some of the IGFBP-3 in this newly formed ternary complex was proteolyzed to IGFBP-329 at a normal rate, allowing release of some of the new IGF to the high affinity IGFBPs at 35 kDa. The increase in IGF relative to high affinity IGFBPs in the 35-kDa serum fractions should make more IGF available to activate type I IGF receptors on target tissues; consistent with this hypothesis, rhGH increased free IGF-I levels in the sera of CRF children and induced catch-up growth in these children (5). The continued presence of excess IGFBP-3 relative to IGFs in the 150-kDa fractions of CRF serum is probably due to delayed clearance of the IGF-free, proteolysis-induced IGFBP-329 fragment from these CRF serum fractions. It is likely that by perturbing normal IGFBP-329 clearance, the CRF state allows the detection of excess IGFBP-3 relative to IGF at 150 kDa. This crucial finding, noted in rat serum but never before demonstrated in human serum, suggests that proteolysis of circulating, ternary-complexed IGFBP-3 plays a central role in human growth by releasing IGFs to target tissues.



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  		Figure 8. Balance between IGFBPs and IGFs in serum of CRF children before and after rhGH treatment. Levels (nanomoles per L) of IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-6 in the 150- and 35-kDa fractions of CRF serum are presented. Protein levels were measured in whole serum of 30 CRF children before (0 months) and during (12 months) rhGH treatment (for details, see reference 5). Mean IGFBP-1, IGFBP-2, and IGFBP-6 levels were assigned entirely to the 35 kDa serum fractions. The percentages of IGFBP-3 and IGFs at 150 kDa (fractions 23–27) and at 35 kDa (fractions 28–30) in sera from CRF children before and after 12 months of rhGH treatment were calculated from the data presented in Fig. 3Go; these percentages were then applied to the mean whole serum levels in Ref. 5 to calculate the amounts of each protein at 150 and 35 kDa. Both intact IGFBP-3 and IGFBP-329 were abundant in the 150-kDa fractions; IGFBP-329 was much more abundant than intact IGFBP-3 in the 35-kDa fractions.

 


   Acknowledgments
 
The following centers/participants were involved in 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 (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); and Kaye Green (Administrative Coordinator).


   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 by Grant M01-RR-00069 from the General Clinical Research Centers Program, National Centers for Research Resources, NIH. Back

Received July 30, 1997.

Revised December 3, 1997.

Accepted January 15, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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