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INSULIN-LIKE GROWTH FACTORS (IGF) AND IGF BINDING PROTEINS IN CHILDREN WITH
CHRONIC RENAL FAILURE
Burkhard T6nshoff,* Werner F. Blum* and Otto Mehls*
*University Children's Hospital, Heidelberg, Germany ~Lilly Deutschland, Bad Homburg, Germany
The pathomechanism of growth retardation and catabolism in children with chronic renal failure (CRF) is multifactorial. Recent evidence indicates that in particular disturbances o f the somatotropic hormone axis play an important pathogenic role. In preterminal CRF serum insulin-like growth factor (IGF)-I and IGF-H levels are normal, while in end-stage renal disease (ESRD), IGF-I levels are slightly decreased and IGF-H levels slightly increased. In view of the prevailing elevated growth hormone levels in ESRD, these serum IGF-I levels appear as inadequately low. Indeed, there is both clinical and experimental evidence for a decreased hepatic IGF-I production rate in CRF. This hepatic insensitivity to the action of GH is partially owing to a reduced GH receptor expression. The action and metabolism of lGFs are modulated by specific high-affinity IGF binding proteins (IGFBPs), which bind -99% of circulating IGF. IGFBP-1, IGFBP-2, and low molecular weight IGFBP-3 fragments are increased in CRF serum in relation to the degree o f renal dysfunction. Both decreased renal filtra- tion, in particular o f low molecular weight IGFBP-3 fragments, and increased hepatic production of IGFBP-I and -2 contribute to high IGFBP serum levels. Experimental and clinical evidence suggests that these excessive high-affinity IGFBPs in CRF serum inhibit IGF action on target tissues by competition with the type 1 IGF receptor for IGF binding.
The pathomechanism of growth failure in children with chronic renal failure (CRF) is multifactorial. However, there is recent evidence that alterations of the growth hormone (GH)/insulin-like growth factor (IGF) axis play a pivotal patho-
*Correspondence to: Dr B. T0nshoff, Tel.: 49-6221-562311; Fax: 49-6221-563703, e-mail: [email protected].
Acknowledgement We gratefully acknowledge the scientific collaboration with D.R. Powell (Baylor College of Medicine, Houston, Texas, U.S.A.
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genic role [1]. The growth-stimulating effect of GH is partly mediated by circulat- ing IGF-I (endocrine action), which is mainly produced in the liver, and by locally produced IGF-I in the growth cartilage (paracrine/autocrine action). Regarding longitudinal growth, both hormones act on different cell types. GH induces differ- entiation of epiphyseal growth plate precursor cells toward chondrocytes and these GH-stimulated chondrocytes become responsive to IGF-I and concomitantly express IGF-I mRNA, whereas IGF-I stimulates the clonal expansion of differenti- ated chondrocytes, thus leading to longitudinal bone growth [2].
In CRF, diminished growth in the presence of normal or elevated GH serum levels indicates a state of GH insensitivity [1]. In addition, IGF inhibitors have been suggested to by pathogenic because of the discrepancy between normal immunore- active and decreased bioactive total IGF levels in CRF serum. Recent studies have suggested that the prevailing inhibitory effect on IGF bioactivity in CRF serum can be attributed to an excess of high affinity IGF binding proteins (IGFBPs) in CRF patients. These IGFBPs tightly bind IGFs in the circulation but also in the extravascular compartment. The biological action of IGF-I is mediated via the type I IGF receptor. Because IGFBPs bind IGFs with affinities similar or higher to those of the type I IGF receptor [3], an excess of high affinity IGFBPs has the ability to inhibit IGF action on target tissues by competing with the type I IGF receptor for IGF binding. In addition, IGFBP-1 and -3 can directly inhibit cellular proliferation in vitro independent of their ability to sequester IGF peptides [4].
Six IGFBPs have been described up to now [5]. In the circulation, three IGFBPs are well characterized. IGFBP-1 (28 kDa) is GH-independent, and insulin is the prin- cipal suppressive regulator of hepatic IGFBP-1 production [6]. IGFBP-2 (32 kDa) is the second most abundant IGFBP in the circulation. It binds IGF-II with a greater affinity than IGF-I. IGFBP-1 and IGFBP-2 inhibit IGF action under many [7, 8], but not all experimental conditions. IGFBP-I and -2 circulate in the ~35 kDa serum fraction, and they are major contributors to the unsaturated IGFBP pool found primarily in this serum fraction [9, 10]. IGFBP-3 is the predominant circulating IGFBP in postnatal life. The IGF-binding subunit is IGFBP-3/3, an acid-stable, 45 kDa glycosylated protein with a core molecular mass of 29 kDa. In contrast to the other IGFBPs, it has the unique property to associate with an acid-labile subunit (ALS) after binding either IGF-I or IGF-II. The subsequent constituted ternary complex is a high-molecular weight complex of approximately 150 kDa [11]. Its function is thought to act as a reservoir and a buffer for IGF-I and IGF-II, preventing rapid changes of free IGF levels. IGFBP-3 serum levels are positively regulated by GH secretory status [12]. It potentiates IGF action under most [13-15] but not necessarily all experimental conditions [16]. IGFBP-4, -5 and -6 are predominantly produced and located within tissues and their role in the circulation is quantitatively less important [17]. Therefore, theses IGFBPs will not be further considered here.
Since the first proposal in 1987 that unsaturated IGFBPs might participate in the growth failure of children with CRF [9], much has been learned about their regula- tion and function. Recent data about altered serum concentrations of IGFs and IGFBPs and the possible role of IGFBPs as growth inhibitors in children with CRF are reviewed below.
IGFs and IGFBPs in Chronic Renal Failure 483
I G F A N D I G F B P SERUM LEVELS IN C R F
Determination of IGFs in CRF serum by RIA is not trivial. Increased IGFBPs in CRF serum have to be removed prior to assay, since residual IGFBP in the assay mixture can interfere with antibody binding. Powell et al. [18] demonstrated first that removal of residual IGFBPs by acidic gel chromatography is necessary for reliable estimates of IGF levels in CRF serum. In our laboratory, extraction of serum samples with acid ethanol or acidic gel chromatography gave identical results provided that a high avidity anti-hlGF-I serum was used, which made the interference of residual IGFBP in the serum extract negligible [19]. Serum IGF-II levels in our studies were measured by RIA after acid-ethanol extraction and blocking residual IGFBP in the extract with an excess of IGF-I [20]. By use of this methodology we have analysed serum IGF levels in two large cohorts of children with preterminal CRF (n = 94) [21] and end-stage renal disease (ESRD) (n = 54] [22]. In preterminal CRF, IGF-I and IGF-II serum levels were in the normal range (Fig. 1). These findings confirm and extend previous reports from our group and others in smaller cohorts of children with CRF [9, 23, 24]. In ESRD however, mean age-related IGF-I levels were slightly, but significantly decreased (-1.08 + 0.17 SDS) and mean age-related IGF-II levels were slightly, but significantly elevated (1.09 +0.15 SDS) (Fig. 1).
IGF serum levels must be interpreted in relation to their respective IGFBP serum levels. Figure 1 shows the age-related IGFBP-1, -2 and -3 serum levels (measured by RIA) in the same two groups of children with preterminal CRF and ESRD. Serum levels of all three binding proteins were clearly increased in both groups, but the respective increase in children with ESRD was more pronounced than in those with preterminal CRF and residual renal function (Fig. 1). In the latter group, age- related IGFBP-1, -2 and -3 levels were inversely correlated with residual glomeru- lar filtration rate (GFR) (Fig. 2) [21]. On a molar basis, IGFBP-3 is the most
a b u n d a n t c i rcula t ing I G F B P bo th in p re te rmina l C R F (177_+4.8 nmol 1 -l) and
E S R D (215_+8.8 nmol l-l); the second mos t a b u n d a n t serum I G F B P is I G F B P - 2
(pre terminal C R F , 24.9 _+ 1.2 nmol l-I; E S R D , 48 _+4.6 nmol 1-1) (Fig. 3). The mola r
concen t ra t ion of IGFBP-1 in the c i rculat ion is relat ively low (pre terminal C R F , 2.8 -+ 0.2 nmol l-J; E S R D , 6.3 -+ 0.5 nmol l-l); hence its role as a g rowth inhib i tor in C R F
m a y be less impor tan t . Taken together , these da t a demons t r a t e a progress ive
increase o f serum I G F B P s in children with C R F in paral le l to the decline o f renal funct ion. I t is l ikely tha t the higher extent o f I G F B P excess in E S R D c o m p a r e d with
pre te rmina l C R F contr ibutes both to more severe growth re t a rda t ion and to the
lower response to exogenous G H therapy in these chi ldren [25]. This pa t t e rn o f n o r m a l serum I G F levels a n d m a r k e d l y increased I G F B P levels is
un ique for C R F . The cons te l la t ion o f increased I G F B P over I G F s also suggests
that "normal" IGF-I and IGF-II levels in CRF cannot be readily used as an indi- cation of normal production rates of these peptides. In CRF, IGF binding capacity is increased by an order of magnitude [24]. Because of the short metabolic half-life of free IGF [26] one would expect that under normal conditions increased IGF binding capacity would be immediately saturated by IGFs produced in the liver. The consequence would be a progressive increase of IGFs concomitantly with the rise in IGFBP as GFR declines. However, this tendency was only observed for IGF-II (Fig. 4) [21]. Hence the normal serum concentrations of IGF-I in pretermi- nal CRF appears to be inadequately low. This discrepancy is even more pronounced in children with ESRD, in whom slightly decreased serum IGF-I levels are found in the presence of increased IGFBPs [22] and in the presence of elevated circulating GH levels [27]. Indeed, analysis of this complex system with a mathe- matical model indicated that data from children with CRF were consistent with a markedly reduced IGF-I production rate [28]. This hypothesis is supported by recent experimental data from our group which showed a specific 50% reduction of hepatic IGF-I gene expression in uremic non-acidotic animals compared with pair- fed controls [29]. This observation adds further evidence to the concept of GH insensitivity in the uremic state [1]. There is both clinical [30, 31] and experimental evidence [32] that the molecular basis for this GH insensitivity in CRF involves decreased hepatic GH receptor expression.
MOLECULAR ALTERATIONS OF IGFBP-3 IN CRF SERUM
Serum IGFBP-3 levels in children with CRF were first reported in 1989 to be elevated by RIA [33] and later confirmed by others [34]. In normal serum, IGFBP- 3 is present as a high molecular weight complex of about 120-150 kDa. However, when sera from patients with ESRD were subjected to HPLC exclusion chro- matography, most of the immunoreactive IGFBP-3 like material eluted in the range
of 60--20 kDa showing two major peaks at about 55 and 25 kDa [24]. The molecu- lar weight of IGFBP°3 was further studied by affinity cross-linking of IGF-I and IGF-II to sera from patients with ESRD, and precipitation of the labelled complexes with specific anti-IGFBP-3 sera. Analysis of the precipitates by SDS-PAGE and autoradiography revealed radiolabelled bands at 45 and 40 kDa that represent intact IGFBP-3, and at 30, 21 and 15 kDa that represent IGFBP-3 fragments [22, 24]. These experiments demonstrated that IGFBP-3 fragments in ESRD are clearly capable of binding IGF.
Powell et al. [34] studied IGFBP-3 levels in CRF children by [125I]IGF ligand blot either with or without immunoprecipitation by IGFBP-3 antisera and found normal amounts of intact -41/38 kDa IGFBP-3 forms. Because IGFBP-3 fragments are not detected by p25I]IGF ligand blot, the discrepancy between the results obtained by RIA and ligand blot analysis was explained. It was shown that the functional 41- and 38-kDa IGFBP-3 forms in CRF serum circulate mainly in the -150 kDa frac- tions of CRF serum and that there is no deficiency of this 150 kDa complex in CRF [34]. IGF-I, IGFBP-3 and ALS from CRF sera can all reconstitute a functional -150 kDa complex in vitro [34], because intact IGFBP-3 in CRF has a normal ability to bind ALS [35]. However, the small molecular weight IGFBP-3 fragments (15-20 kDa) in CRF, which still are capable of binding IGFs [24, 34], do not allow formation of the 150 kDa complex [34], because their ability to bind ALS is greatly reduced [35]. It is suggested that the molar excess of these small molecular weight fragments of IGFBP-3 in CRF, which are small enough to enter the interstitial space, provide unsaturated sites for IGF binding and might thereby act as IGF inhibitors by competing with IGF receptors for IGF binding.
MECHANISM OF INCREASED IGFBP SERUM LEVELS IN CRF
Theoretically, increased IGFBP levels in CRF serum could result from increased production, reduced elimination by the diseased kidneys, or a combination of both factors. The inverse correlation of serum IGFBPs with residual GFR (Fig. 2) is consistent with the concept that elevated IGFBPs in CRF results from impaired renal filtration of IGFBP-1, IGFBP-2, and low-molecular weight fragments of IGFBP-3 [21, 24]. Retention of low-molecular weight proteins in the circulation as a consequence of reduced renal filtration is a well-known feature of CRF [36]. However, we observed in our analysis that the slope of the regression line between GFR and age-related IGFBP-3 levels was significantly steeper than those observed for IGFBP-1 and for IGFBP-1 (Fig. 2), indicating that the same loss of GFR resulted in a relatively higher increase of IGFBP-2 levels compared with the other two IGFBP [21]. This finding may be owing to differential renal handling of the IGFBP, although this possibility is not very likely considering the similar molecu- lar weight of IGFBP-1 (28 kDa) and IGFBP-2 (32 kDa). An alternative explana- tion is a different production rate of IGFBP-2 compared with IGFBP-1 and -3, or a combination of increased production and reduced clearance. These three possibil- ities cannot be readily distinguished in humans. However, we recently observed in a rat model of moderate CRF an increased hepatic gene expression of IGFBP-1 and -2. Hence, increased hepatic production also appears to contribute to elevated IGFBP-1 and -2 serum levels in CRF [29]. The expression of the IGFBP-1 and -2
IGFs and IGFBPs in Chronic Renal Failure 487
gene in the liver is also stimulated in other catabolic conditions, such as fasting, protein-restricted and energy-restricted rats [37].
What is the mechanism of increased IGFBP-3 fragments in CRF serum? As noted above, the inverse linear relation of age-related IGFBP-3 levels with residual GFR suggests accumulation as a consequence of reduced renal clearance. In order to test this hypothesis, we investigated a group of patients with ESRD before and after renal transplantation [22]. Indeed, the decline of immunoreactive IGFBP-3 in patients with ESRD after restoration of renal function by a functioning transplant was markedly more pronounced than in the control group, who experienced a comparable physical stress by abdominal surgery. These data are highly suggestive that accumulation of IGFBP-3 fragments in CRF results from reduced renal clear- ance. This line of reasoning is supported by our observation that urine from normal individuals contains IGFBP-3 fragments with a major peak at 20 kDa and little intact IGFBP-3 in the 40-50 kDa range [24, 38]. Since renal IGFBP-3 production under baseline conditions is low or absent [39], urinary IGFBP-3 fragments most likely represent filtered IGFBP-3 fragments from the circulation. Notably, increased proteolytic degradation of the IGFBP-3 ternary complex, as described during preg- nancy and catabolic states, appears not to be operative in patients with CRF [40, 41].
INHIBITION OF IGF BIOACTIVITY BY IGFBPS I N VITRO
IGFBPs have the potential to inhibit IGF-mediated cell proliferation in various cell culture systems. Purified IGFBP-1 even in the low concentration of 0.2 nM markedly inhibited both basal and IGF-I stimulated growth of chick embryo pelvic cartilage cells in vitro [8]. Because the IGFBP-1 concentration in CRF serum is ~5 nM and the interstitial IGFBP levels are ~1/10 of their respective serum levels [42], the IGFBP-I serum concentration in CRF appears high enough to inhibit cartilage growth in vivo. IGFBP-2 and IGFBP-3 have not been studied in cartilage models, but each can inhibit IGF-I-stimulated DNA synthesis in cultured fibroblasts [13, 14, 43].
In CRF serum, IGF bioactivity measured by sulphate incorporation into porcine costal cartilage is markedly reduced, despite normal immunoreactive serum IGF levels [24]. This discrepancy has been explained by the presence of IGF inhibitors in CRF serum. Uremic serum contains low-molecular weight (about 1 kDa) IGF inhibitors whose molecular structure, however, has not yet been defined [44]. The prevailing inhibitory effect on IGF bioactivity in CRF can rather be attributed to the IGFBPs. We have demonstrated that removal of excessive unsaturated IGFBPs from CRF patient sera by affinity chromatography with an IGF-II Sepharose column restores IGF bioactivity in the porcine growth cartilage assay [24]. This finding indicates that unsaturated IGFBPs in CRF serum inhibit the ability of IGFs to act on cartilage tissue in vitro.
It is noteworthy that unsaturated IGFBPs in CRF serum are pathophysiologically relevant only if IGFBP levels are also increased in IGF target tissues. Although this has not been shown in subjects with CRF, it is known that IGFBP-1, IGFBP-2 and low-molecular forms of IGFBP-3 migrate into lymph fluid and thereby have access to interstitial spaces [42]. An excess of IGFBPs in IGF target tissues can therefore inhibit IGF action by sequestering IGFs from the type 1 IGF receptor.
488 B. Tiinshoff et al.
INHIBITION OF IGF BIOACTIVITY BY IGFBPS I N V I V O
There is now direct evidence that circulating IGFBPs are capaole of inhibiting growth of experimental animals. The co-injection of recombinant human IGFBP-1 inhibited the GH or IGF-I stimulated weight gain and tibial epiphyseal widening of hypophysectomized rats in a dose-dependent manner [45]. Also transgenic mice overexpressing IGFBP-I are markedly growth retarded [46].
The key question is whether the imbalance between normal total IGF and the excess of unsaturated IGFBPs contributes to growth failure in the setting of clinical CRF. We have addressed this question by correlation analysis of circulating IGF-I levels with longitudinal height in children with CRF. We observed that the normal relation between circulating IGF-I and relative height is clearly disturbed in CRF (Fig. 5) [21]. The significantly less steep regression line between height and IGF-I in CRF is consistent with the presence of IGF inhibitors. Hence, the inhibition of IGF bioactivity for stimulation of longitudinal growth appears to be operative also in vivo.
We also sought to assess the relative role of IGFBP-1, -2, and -3 for growth failure in CRF [21]. There was neither a positive relation between immunoreactive IGFBP-3 (which comprises both intact IGFBP-3 and low-molecular weight frag- ments) and relative height in CRF, as observed under normal conditions [12], nor a negative correlation, which would be expected if IGFBP-3 acted merely as an IGF inhibitor in CRF. This finding may be explained by the hypothesis that intact IGFBP-3 in CRF has a growth-promoting action via formation of the 150 kDa ternary complex. However, the molar excess of small molecular weight fragments of IGFBP-3 in CRF, which are small enough to enter the interstitial space, provide unsaturated sites for IGF binding and might thereby act as IGF inhibitors.
The inverse linear relation of IGFBP-1 and IGFBP-2 with relative height in our study [21] suggests that these IGFBPs also act as growth inhibitors in CRF. The role of IGFBP-2 as an IGF inhibitor is probably more important, because IGFBP- 2 levels were higher than IGFBP-1 both in relative and in absolute terms (Fig. l and Fig. 3). Statistical analysis revealed that only IGF-I (in a positive fashion) and
IGFBP-2 (in a negative fashion) contribute independently to the prediction of rela- tive height in CRF. This finding underlines the possible role for IGFBP-2 as an IGF inhibitor in CRF.
If longitudinal growth is stimulated by circulating IGF-I and inhibited by circu- lating IGFBPs, GH therapy, an effective growth-promoting strategy in CRF, should alter this serum profile. Indeed, we previously observed that supraphysio- logical doses of rhGH induced a sharp and persistent increase of serum IGF-I levels in children with ESRD, whereas at baseline elevated serum IGFBP-3 levels increased only slightly [47]. This improved ratio of IGF-I over IGFBP-3 resulted in a three-fold increase of initially depressed IGF bioactivity, accompanied by a marked stimulation of longitudinal growth in these children. Others have observed that rhGH therapy in children with CRF induced a significant decrease of IGFBP- 1 levels [48, 49], presumably as a consequence of increased serum insulin levels, and a decrease of IGFBP-2 levels [49]. It has been argued that the decline of these low molecular weight IGFBPs is more important for the restoration of IGF bioactivity in CRF serum than the improved IGF-I/IGFBP-3 ratio, because on a molar basis, the rise in IGFBP-3 was comparable with the increase of the sum of IGF-I and IGF-II in the two controlled trials of GH therapy in CRF children [48, 49]. However, the molecular heterogeneity of IGFBP-3 in CRF serum makes a reliable estimate of molar concentrations and functional consequences difficult. Nevertheless, the observation that the growth-promoting effect of GH therapy in CRF is accompanied by a normalization of circulating IGF bioactivity [47] under- scores the relevance of an adequate ratio of circulating IGF vs. IGFBP for longi- tudinal growth in these children.
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