Insulin-like growth factors in normal and diseased kidney.

This article reviews the physiology of the insulin-like growth factor (IGF) system in the kidney and the changes and potential role of this system in selected renal diseases. The potential therapeutic uses of recombinant human IGF-I for the treatment of acute and chronic kidney failure are briefly discussed.

The use of bone age in clinical practice - part 1.

This review examines the role of skeletal maturity ('bone age', BA) assessment in clinical practice. BA is mainly used in children with the following conditions: short stature (addressed in part 1 of this review), tall stature, early or late puberty, and congenital adrenal hyperplasia (all addressed in part 2). Various manual and automatic methods of BA assessment have been developed. Healthy tall children tend to have advanced BA and healthy short children tend to have delayed BA in comparison to chronological age. Growth hormone (GH) treatment of children with GH deficiency leads to a catch-up in BA that is usually appropriate for the height of the child. Response to GH is dependent on BA delay in young children with idiopathic short stature, and GH dosage appears to affect BA acceleration. In chronic renal failure, BA is delayed until puberty but then increases due to increased sensitivity of the growth plate to sex steroids, thus further impairing adult height. The assessment of BA provides an important contribution to the diagnostic workup and management of children with short stature.

The IGF/IGFBP system in relation to macroscopic bone architecture in pediatric renal transplant patients.

The post-transplant bone disease of the peripheral skeleton in pediatric renal transplant recipients is characterized by an inadequately thin bone cortex in relation to muscular force. A major hormonal modulator of periosteal growth is the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system. We therefore hypothesized that the reduced cortical thickness in these patients may be due to functional IGF deficiency. To test this hypothesis, we investigated 55 patients (mean estimated glomerular filtration rate 86.3 +/- 30.0 ml/min/1.73 m(2)) in a cross-sectional study. Parameters of macroscopic bone architecture and forearm muscle size were analyzed by peripheral quantitative computed tomography (pQCT), and serum IGF/IGFBP system components were measured by specific radioimmunoassays. The mean (+/- standard deviation) standardized serum IGF-I (0.20 +/- 1.16 score) level was normal, while the mean IGF-II (1.16 +/- 0.11 score) level was significantly elevated. Serum IGFBP-1 and IGFBP-2 levels were not altered, whereas the IGFBP-3 (1.34 +/- 0.15 score) level was significantly increased. The serum IGFBP-4 level was slightly elevated (by 11%), the IGFBP-6 level was markedly (2.3-fold) elevated, while the IGFBP-5 level was comparable to that of the control. The respective age-adjusted cortical thickness at both the proximal (r = 0.407, P < 0.005) and distal (r = 0.383, P < 0.01) forearm was positively correlated with the standardized serum IGF-I level. In conclusion, the serum IGF/IGFBP system in pediatric renal transplant recipients is characterized by an increase in the levels of the inhibitory IGFBPs, IGFBP-3, -4 and -6, resulting in a functional IGF deficiency. The positive correlation of IGF-I with cortical thickness underlines the importance of this hormonal system in the modeling of bone, particularly periosteal growth.

Sexual dimorphism of body composition and insulin sensitivity across pubertal development in obese Caucasian subjects.

Background Puberty is a period of rapid growth associated with metabolic, hormonal, and body composition changes that can influence risk factors for chronic diseases such as type 2 diabetes. Objective To evaluate body composition and insulin sensitivity (IS) modifications throughout puberty in a large group of obese Caucasian subjects. Methods Five hundred and nineteen obese subjects (4-19 years), grouped according to gender and Tanner stage (T), underwent oral glucose tolerance test. Quantitative insulin check index (QUICKI) and ISI were calculated as indexes of IS. In 309 subjects, body composition by dual-energy X-ray absorptiometry, IGF1, adiponectin, and leptin were also evaluated. Results Body composition modifications were sexually dimorphic, with girls not modifying fat and lean percentage and fat distribution (P>0.15), and boys decreasing fat percentage and increasing lean percentage and central fat depot (P<0.001) across Ts. IS decreased during mid-puberty and returned to prepubertal levels by the end of puberty. Girls showed lower IS than boys (P<0.01 and =0.03 for QUICKI and ISI respectively). In multivariate analysis factors that negatively influenced IS, independently from T or age, were total fat mass and central fat depot in girls (P<0.05 and <0.01, respectively), total fat and lean mass in boys (P<0.01). IGF1, adiponectin, and leptin were not related to pubertal IS. Conclusions In obese Caucasian subjects, further decrease of IS observed during puberty is a transient phenomenon. Factors that independently from T or age influence IS are central fat depot in girls, lean amount in boys, and total fat mass in both sexes.

Growth hormone treatment started in the first year of life in infants with chronic renal failure.

Infants with chronic renal failure (CRF) are at high risk of experiencing severe growth retardation. We report a study of 12 infants with CRF who have been treated with recombinant human growth hormone (rhGH) since the age of 0.5 +/- 0.3 years. A control group comprised 15 infants with less severe CRF who were being treated during the same period, but who did not receive rhGH. Despite the infants in the rhGH group had more severe renal failure, they grew at least as well as those in the control group and experienced catch-up growth that started earlier and was more sustained; they also gained more weight. Between the age of 0.5 and 2.5 years, the height standard deviation score (HtSDS) improved from -2.0 +/- 1.2 to -0.9 +/- 0.9 in the rhGH group (p < 0.005) and from -1.6 +/- 1.6 to -1.0 +/- 1.9 in the control group (p=non significant, n.s.). The average gain in HtSDS was +1.1 +/- 0.8 in the treated group and +0.6 +/- 1.4 in the control group (p = n.s.). During the same period, the weight SDS improved from -2.2 +/- 0.9 to -0.6 +/- 1.2 (p < 0.005) and from -1.9 +/- 1.2 to -1.3 +/- 1.2 (p=n.s.) in the treatment and control groups, respectively. Nutritional intake was similar in both groups, while parathyroid hormone levels tended to increase, although not significantly, after rhGH treatment (p=n.s.). The results of this pilot study suggest that very early treatment with rhGH in patients with early-onset CRF may improve growth.

Sexual pseudo-precocity caused by a somatic activating mutation of the LH receptor preceding true sexual precocity.

AIM: We describe the clinical features of a 6-year-old boy with sexual precocity caused by a somatic activating mutation of the luteinizing hormone (LH) receptor gene preceding gonadotropin-releasing hormone (GnRH)-dependent sexual precocity. STUDY DESIGN: Genomic DNA was extracted from the right testis and from the peripheral leukocytes followed by DNA amplification and sequencing of the LH receptor gene. We described the clinical characteristics including anthropometric parameters, bone age, and endocrine evaluation when the boy presented with sexual precocity. These data were compared with the clinical and hormonal evaluation after orchiectomy preceding GnRH-dependent sexual precocity and after subsequent treatment with GnRH agonist. RESULTS: No mutation was found in the sequence of the LH receptor gene extracted from peripheral leukocytes. Interestingly, sequencing of the tumor LH receptor gene revealed a heterozygous mutation in exon 11 encoding a replacement of Asp(578)His. Despite normalization of plasma testosterone, true precocious puberty was triggered within a year. CONCLUSIONS: Inmales with GnRH-independent sexual precocity the presence of small testicular Leydig cell tumorous lesions harboring a somatic mutation of the LH receptor gene should be considered. A close follow-up of affected patients should be instigated in order to monitor recurrence or subsequent true precocity.

Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I.

The IGF/IGF binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. Evidence from in vitro studies indicates that IGFBPs may have IGF-independent effects. We analyzed the biological activity of intact IGFBP-2 and defined carboxy-terminal IGFBP-2 fragments isolated from human hemofiltrate in two cell culture systems of the growth plate: rat growth plate chondrocytes in primary culture and the mesenchymal chondrogenic cell line RCJ3.1C5.18. The IGFBP-2 fragments IGFBP-2(167-279), IGFBP-2(167-289), and IGFBP-2(104-289) exerted a strong (2- to 3-fold) mitogenic effect on growth plate chondrocytes, which was comparable with IGF-I in equimolar concentrations (7.8 nm) but was not mediated through the type 1 IGF receptor. In a dose-response experiment, the most effective concentration of IGFBP-2(104-289) for the stimulation of cell proliferation was 10 nm. This biological activity of IGFBP-2 fragments was associated with cell membrane binding, demonstrated by Western blot analysis of fractionated cell lysates and immunohistochemistry. Whereas intact IGFBP-2 did not modulate chondrocyte proliferation, partially reduced (by dithiothreitol) full-length IGFBP-2 stimulated cell proliferation to a comparable extent (3.4-fold) as carboxy-terminal IGFBP-2 fragments. The mitogenic activity of these IGFBP-2 fragments and of partially reduced full-length IGFBP-2 was mediated through the use of the MAPK/ERK 1/2. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.

Signaling mechanisms leading to regulation of proliferation and differentiation of the mesenchymal chondrogenic cell line RCJ3.1C5.18 in response to IGF-I.

Since IGF-I is an important chondrocyte growth factor, we sought to examine the intracellular mechanisms by which it exerts two of its pivotal effects, stimulation of proliferation and differentiation. We used the mesenchymal chondrogenic cell line RCJ3.1C5.18, which progresses spontaneously to differentiated growth plate chondrocytes. This differentiation process could be enhanced by exogenous IGF-I. Pharmacological inhibition of the phosphatidylinositol-3 (PI-3) kinase by LY294002, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)1/2 by U0126, the protein kinase (PK) A pathway by H-89 or KT5720, and the PKC pathway by bisindolylmaleimide suppressed IGF-I-stimulated cell proliferation. In contrast, IGF-I-enhanced early cell differentiation, as assessed by collagen type II and aggrecan gene expression, was not affected by MAPK/ERK1/2 pathway inhibition, but significantly diminished by inhibition of the PI-3 kinase, the PKC and the PKA pathway. Moreover, terminal differentiation of chondrocytes in response to IGF-I, as assessed by gene expression of alkaline phosphatase, Indian hedgehog, and collagen type X, were only interrupted by PI-3 kinase pathway inhibition. In conclusion, IGF-I exerts its differential effect on chondrocyte proliferation vs differentiation through the use of at least four partially interacting intracellular signaling pathways, whose activity is temporarily regulated. When chondrocytes progress from proliferating cells to early and terminal differentiating cells, they progressively inactivate IGF-I-related intracellular signaling pathways. This mechanism might be essential for the complex and cell stage-specific anabolic action of IGF-I in the growth plate.

Differential expression of IGF system components in proliferating vs. differentiating growth plate chondrocytes: the functional role of IGFBP-5.

The growth plate is an important target tissue for insulin-like growth factors (IGFs), but little is known about the regulation of the IGF system during the developmental sequence of chondrocytes. We therefore examined the expression profile of IGF system components in proliferating vs. differentiating growth plate chondrocytes by use of two cell culture models of the growth cartilage. In rat growth plate chondrocytes in primary culture, IGF-I expression increased twofold during the process of differentiation. IGF-binding protein-3 (IGFBP-3) expression showed a biphasic pattern of with a twofold increase at the onset of differentiation and a downregulation in late differentiating chondrocytes to 25% of baseline levels; the expression patterns of IGFBP-2, -4 and -6 were not dependent on the developmental stage. In IGF- and IGFBP-3-deficient RCJ3.1C5.18 (RCJ) mesenchymal chondrogenic cells, IGFBP-2 and -6 synthesis declined by 50% during differentiation. IGFBP-5 expression was markedly upregulated during the process of differentiation in both cell culture models. Although IGFBP-5 overexpression did not have an IGF-independent effect on RCJ cell differentiation, it promoted IGF-I-enhanced differentiation of these cells. A potential mechanism for this effect is the specific increase of Akt phosphorylation in IGFBP-5-overexpressing cells in the presence of IGF-I, indicating an increased activity of the phosphatidylinositol (PI) 3-kinase pathway. These data suggest that the developmental stage of the chondrocyte is an important determinant of IGF and IGFBP expression and imply a functional role for IGFBP-5 for upregulating IGF action during chondrocyte differentiation in vivo.

Insulin-like growth factor (IGF)-I stimulates cell proliferation and induces IGF binding protein (IGFBP)-3 and IGFBP-5 gene expression in cultured growth plate chondrocytes via distinct signaling pathways.

The bioactivity of IGF-I in the cellular microenvironment is modulated by both inhibitory and stimulatory IGF binding proteins (IGFBPs), whose production is partially under control of IGF-I. However, little is known on the IGF-mediated regulation of these IGFBPs in the growth plate. We therefore studied the effect of IGF-I on IGFBP synthesis and the involved intracellular signaling pathways in two cell culture models of rat growth plate chondrocytes. In growth plate chondrocytes in primary culture, incubation with IGF-I increased the concentrations of IGFBP-3 and IGFBP-5 in conditioned cell culture medium in a dose- and time-dependent manner. Coincubation of IGF-I with specific inhibitors of the p42/44 MAPK pathway (PD098059 or U0126) completely abolished the stimulatory effect of IGF-I on IGFBP-3 mRNA expression but did not affect increased IGFBP-5 mRNA levels. In contrast, inhibition of the phosphatidylinositol-3 kinase signaling pathway by LY294002 abrogated both IGF-I-stimulated IGFBP-3 and -5 mRNA expression. Comparable results regarding IGFBP-5 were obtained in the mesenchymal chondrogenic cell line RCJ3.1C5.18, which does not express IGFBP-3. The IGF-I-induced IGFBP-5 gene expression required de novo mRNA transcription and de novo protein synthesis. These data suggest that IGF-I modulates its activity in cultured rat growth plate chondrocytes by the synthesis of both inhibitory (IGFBP-3) and stimulatory (IGFBP-5) binding proteins. The finding that IGF-I uses different and only partially overlapping intracellular signaling pathways for the regulation of two IGFBPs with opposing biological functions might be important for the modulation of IGF bioactivity in the cellular microenvironment.

Growth hormone/insulin-like growth factor system in children with chronic renal failure.

Disturbances of the somatotropic hormone axis play an important pathogenic role in growth retardation and catabolism in children with chronic renal failure (CRF). The apparent discrepancy between normal or elevated growth hormone (GH) levels and diminished longitudinal growth in CRF has led to the concept of GH insensitivity, which is caused by multiple alterations in the distal components of the somatotropic hormone axis. Serum levels of IGF-I and IGF-II are normal in preterminal CRF, while in end-stage renal disease (ESRD) IGF-I levels are slightly decreased and IGF-II levels slightly increased. In view of the prevailing elevated GH levels in ESRD, these serum IGF-I levels appear inadequately low. Indeed, there is both clinical and experimental evidence for decreased hepatic production of IGF-I in CRF. This hepatic insensitivity to the action of GH may be partly the consequence of reduced GH receptor expression in liver tissue and partly a consequence of disturbed GH receptor signaling. The actions and metabolism of IGFs are modulated by specific high-affinity IGFBPs. CRF serum has an IGF-binding capacity that is increased by seven- to tenfold, leading to decreased IGF bioactivity of CRF serum despite normal total IGF levels. Serum levels of intact IGFBP-1, -2, -4, -6 and low molecular weight fragments of IGFBP-3 are elevated in CRF serum in relation to the degree of renal dysfunction, whereas serum levels of intact IGFBP-3 are normal. Levels of immunoreactive IGFBP-5 are not altered in CRF serum, but the majority of IGFBP-5 is fragmented. Decreased renal filtration and increased hepatic production of IGFBP-1 and -2 both contribute to high levels of serum IGFBP. Experimental and clinical evidence suggests that these excessive high-affinity IGFBPs in CRF serum inhibit IGF action in growth plate chondrocytes by competition with the type 1 IGF receptor for IGF binding. These data indicate that growth failure in CRF is mainly due to functional IGF deficiency. Combined therapy with rhGH and rhIGF-I is therefore a logical approach.

Recombinant human erythropoietin for the treatment of renal anaemia in children: no justification for bodyweight-adjusted dosage.

BACKGROUND: Drug doses for children are usually calculated by reducing adult doses in proportion to bodyweight. The clinically effective dose of recombinant human erythropoietin (epoetin) in children, however, seems to be higher than predicted by this calculation. OBJECTIVE: To determine the quantitative relationship between epoetin dose, bodyweight and response in children with end-stage renal disease. PATIENTS AND METHODS: The time-course of haemoglobin in 52 children during long-term treatment with epoetin beta was analysed by population pharmacodynamic modelling. Patients were 5-20 years old and weighed 16-53kg at the beginning of treatment. Epoetin beta was given intravenously three times per week after haemodialysis. Doses ranged from 110 to 7500IU (3-205 IU/kg). Haemoglobin versus time was described by assuming that the haemoglobin level rises after each dose due to the formation of new red blood cells, which then survive according to a logistic function. The initial rise after each dose was modelled in terms of absolute dose (not dose/kg). A parametric analysis was done with NONMEM, followed by a nonparametric analysis with NPAG. RESULTS: Dose-response was best described by a sigmoid maximum-effect (E(max)) model with median E(max) = 0.29 g/dL, median 50% effective dose (ED(50)) = 2400IU and shape parameter gamma = 2. The estimated median survival time of the epoetin-induced red blood cells, tau, was 76 days. Neither of the dose-response parameters E(max) and ED(50) showed dependence on bodyweight. The median haemoglobin response to a standard dose, 0.042 g/dL for 1000IU, was similar to that reported for adults with intravenous administration. CONCLUSIONS: Doses for children in this age range should be specified as absolute amounts rather than amounts per unit bodyweight. Initial doses can be calculated individually, based on haemoglobin level before treatment, the desired haemoglobin at steady state and the median population parameters E(max), ED(50) and tau.

Differential effects of insulin-like growth factor binding proteins-1, -2, -3, and -6 on cultured growth plate chondrocytes.

BACKGROUND: In children with chronic renal failure (CRF), impairment of longitudinal growth is in part due to excess amounts of circulating high-affinity insulin-like growth factor binding proteins (IGFBPs) that might decrease or prevent insulin-like growth factor (IGF) binding to its signaling receptor. However, it appears from the clinical studies that various IGFBPs may have contrasting effects on longitudinal growth. Because of the potential importance of the IGFBPs as modulators of longitudinal growth in pediatric CRF, the aim of the present study was to investigate the biological effects of IGFBP-1, -2, -3, and -6 on cultured growth plate chondrocytes that express the type 1 IGF receptor. METHODS: The effects of exogenous IGFBPs on IGF-independent and IGF-dependent proliferation of rat growth plate chondrocytes in primary culture were investigated. Proliferation was assessed by colony formation of agarose-stabilized long-term suspension cultures and by the [3H]thymidine assay. The effects of IGFBPs on IGF-I binding and the binding of IGFBPs to chondrocytes were assessed by binding studies with radiolabeled proteins in monolayer culture. RESULTS: Intact IGFBP-1, IGFBP-2 and IGFBP-6 inhibited in equimolar concentration the IGF-I- and IGF-II-stimulated DNA synthesis and cell proliferation, whereas the biological activity of IGFBP-3 was complex. It had an IGF-independent antiproliferative effect and also inhibited IGF-dependent chondrocyte proliferation under coincubation conditions, whereas under preincubation conditions IGFBP-3 enhanced IGF-I-responsiveness. Studies on the mechanism by which IGFBP-3 potentiated IGF activity demonstrated that under preincubation conditions IGFBP-3 is capable to associate with the cell membrane and to facilitate IGF-I cell surface binding. CONCLUSIONS: Intact IGFBP-1, IGFBP-2 and IGFBP-6 act exclusively as growth inhibitors on IGF-dependent proliferation of growth plate chondrocytes. IGFBP-3, however, can either inhibit IGF-independent and IGF-dependent cell proliferation, or enhance IGF responsiveness of chondrocytes dependent on the temporal relationship to the IGF exposure.

Intact IGF-binding protein-4 and -5 and their respective fragments isolated from chronic renal failure serum differentially modulate IGF-I actions in cultured growth plate chondrocytes.

Impairment of longitudinal growth among children with chronic renal failure (CRF) may be partly attributable to the inhibition of insulin-like growth factor (IGF) activity by an excess amount of high-affinity IGF-binding proteins (IGFBP). Elevated levels of immunoreactive IGFBP-4 in CRF serum are inversely correlated with the standardized heights of these children, whereas levels of IGFBP-5, which circulates mainly as proteolyzed fragments, are positively correlated with growth parameters. To delineate the respective effects of these IGFBP on growth cartilage, the biologic effects of intact and fragmented forms of IGFBP-4 and IGFBP-5 on rat growth plate chondrocytes in primary cultures were characterized. Intact IGFBP-4 and IGFBP-5 and the amino-terminal fragment IGFBP-5(1-169) were recombinant proteins; the carboxy-terminal fragments IGFBP-5(144-252) and IGFBP-4(136-237) and the amino-terminal fragment IGFBP-4(1-122) were purified to homogeneity from CRF hemofiltrates. Intact IGFBP-4 and, to a lesser extent, IGFBP-4(1-122) inhibited IGF-I-induced cell proliferation. In contrast, intact IGFBP-5 was stimulatory in the absence or presence of exogenous IGF-I, whereas the amino-terminal fragment IGFBP-5(1-169) was inhibitory. Studies on the mechanism by which IGFBP-4 and IGFBP-5 exert opposite effects on chondrocyte proliferation demonstrated that intact IGFBP-4 prevented the binding of (125)I-IGF-I to chondrocytes, whereas intact IGFBP-5 enhanced ligand binding and was able to bind specifically to the cell membrane. These data suggest that intact IGFBP-4 and, to a lesser extent, IGFBP-4(1-122) act exclusively as growth-inhibitory binding proteins in the growth cartilage. IGFBP-5, however, can either stimulate (if it remains intact) or inhibit (if amino-terminal forms predominate) IGF-I-stimulated chondrocyte proliferation.