Aminoguanidine ameliorates changes in the IGF system in experimental diabetic nephropathy.

BACKGROUND: Formation of advanced glycation end-products (AGEs) has been implicated in the development of diabetic complications. As well as causing changes in structural proteins, AGEs may also alter gene expression of growth factors in vitro. The insulin-like growth factor (IGF) system, including IGF-I and modulatory IGF binding proteins (IGFBPs), is dysregulated during the development of diabetic nephropathy. METHODS: Quantitative in situ hybridization histochemistry and immunohistochemistry were used to determine the effects of aminoguanidine, an inhibitor of AGE formation, on gene expression of IGF-I and IGFBPs in kidneys of long-term (8 months duration) streptozotocin-diabetic rats. RESULTS: Diabetes was associated with increased renal expression of IGFBP-1 mRNA (diabetes 824+/-236 vs control 264+/-76 arbitrary units, P<0.01) and decreased expression of mRNAs for IGF-I (diabetes 39+/-7 vs control 185+/-23 arbitrary units, P<0.001) and IGFBP-4 (diabetes 139+/-25 vs control 383+/-54 arbitrary units, P<0.001). Aminoguanidine treatment inhibited the effects of diabetes on renal expression of mRNA for IGF-I, IGFBP-1 and IGFBP-4. The changes in IGF-I and IGFBP-1 mRNA levels were reflected in altered peptide levels. In diabetic kidneys, IGFBP-5 mRNA levels were slightly decreased to 75% of control levels (P<0.01); aminoguanidine had no effect on IGFBP-5 mRNA levels. CONCLUSIONS: These results suggest that amelioration of changes in the renal IGF system by aminoguanidine may contribute to the renoprotective effects of the latter, which have been previously shown to inhibit structural and functional aspects of diabetic nephropathy in the rat.     

Differential distribution of insulin-like growth factor-1 and insulin-like growth factor binding proteins in experimental diabetic rat kidney

Insulin-like growth factor-1 (IGF-1) is a peptide growth factor, and its activity is modulated by interaction with the family of IGF binding proteins (IGFBP-1 to 6). IGF-1 is detected in rat kidney and has metabolic and growth effects. To explore the possible involvement of IGFBPs in glomerular hypertrophy in streptozotocin (STZ)-induced diabetic rat, the immunolocalization of IGF-1 and IGFBPs were investigated. IGF-1 was gradually increased in the glomeruli of diabetic rats and correlated with glomerular hypertrophy. IGFBP-1 was transiently increased at 1 week after the STZ injection and declined to control level during the following period. In contrast, IGFBP-4 was increased in the diabetic glomeruli throughout the observation period. With insulin treatment, the levels of IGF-1, IGFBP-1 and 4 were normalized and glomerular hypertrophy was prevented. Initial glomerular hypertrophy of diabetic nephropathy is a related IGF-1 action, which may be modulated by IGFBP-1 and 4.     

Inhibitory effect of a growth hormone receptor antagonist (G120K-PEG) on renal enlargement, glomerular hypertrophy, and urinary albumin excretion in experimental diabetes in mice.

Growth hormone (GH) and IGFs have a long and distinguished history in diabetes, with possible participation in the development of renal complications. To investigate the effect of a newly developed GH receptor (GHR) antagonist (G120K-PEG) on renal/glomerular hypertrophy and urinary albumin excretion (UAE), streptozotocin-induced diabetic and nondiabetic mice were injected with G120K-PEG every 2nd day for 28 days. Placebo-treated diabetic and nondiabetic animals were used as reference groups. Placebo-treated diabetic animals were characterized by growth retardation, hyperphagia, hyperglycemia, increased serum GH levels, reduced serum IGF-I, IGF-binding protein (IGFBP)-3, and liver IGF-I levels, increased kidney IGF-I, renal/glomerular hypertrophy, and increased UAE when compared with nondiabetic animals. No differences were seen between the two diabetic groups with respect to body weight, food intake, blood glucose, serum GH, IGF-I, and IGFBP-3 levels or hepatic IGF-I levels. Kidney IGF-I, kidney weight, and glomerular volume were normalized, while the rise in UAE was partially attenuated in the G120K-PEG-treated diabetic animals. No effect of G120K-PEG treatment on any of the parameters mentioned above was seen in nondiabetic animals. In conclusion, administration of a GHR antagonist in diabetic mice has renal effects without affecting metabolic control and circulating levels of GH, IGF-I, or IGFBP-3, thus indicating that the effect of G120K-PEG may be mediated through a direct inhibitory effect on renal IGF-I through the renal GHR. The present study suggests that specific GHR blockade may present a new concept in the treatment of diabetic kidney disease.     

Expression of insulin-like growth factor binding proteins in healing tendon lesions.

he treatment of overuse tendon injuries with exogenous growth factors such as insulin-like growth factor-I (IGF-I) may facilitate an improved return to sustained athletic function. The biological effects of IGF-I are exerted under the control of a complex of IGF receptors, binding proteins, and proteases. This IGF system includes a family of six structurally related high-affinity IGF binding proteins (IGFBPs) that protect IGF-I from local proteases and restrict access of IGF-I to its receptor. This study describes the expression of the IGFBPs in flexor tendon after acute injury and during healing over time. Collagenase-induced lesions were created in the tensile region of the flexor digitorum superficialis tendon of both forelimbs of 14 horses. Tendons were harvested from euthanatized horses 1, 2, 4, 8, or 24 weeks following injury. Gene expression was quantitated by fluorescent real-time PCR, and protein expression was evaluated by Western ligand blot (WLB). Message for IGFBPs 2 to 6 was expressed in both normal and healing tendon. No IGFBP-1 mRNA was detected in equine tendon. Message expression for IGFBP-2, -3, and -4 increased following injury, whereas message expression for IGFBP-5 and -6 decreased. Protein expression for IGFBP-2, -3, and -4 was detected by WLB in normal tendon and showed a marked increase following injury. Protein for IGFBP-5 and -6 was not detectable by WLB in normal or healing tendon. The results of this study document the IGFBP response of flexor tendons to injury and healing, which provides information necessary for the design of protocols that may enhance tendon healing through manipulation of IGF-I ligand and binding protein levels.     

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.     

Focal induction of IGF binding proteins in proximal tubules of diabetic rat kidney.

Diabetes-associated kidney enlargement is associated with increased kidney insulinlike growth factor I (IGF-I) binding. IGF-I binds to the type I IGF receptor, which mediates most of its actions, and to specific binding proteins (IGFBPs), which modulate its actions. To explore the nature and extent of IGF-I binding in the kidney, in vitro autoradiography was used to map the distribution of IGF binding in control and diabetic rat kidney. Specificity studies were performed with increasing concentrations of unlabeled IGF-I, IGF-II, des(1-3)IGF-I (an IGF-I derivative that binds to receptors normally but with decreased affinity to binding proteins), and insulin. In control rats, diffuse binding was found throughout the kidney with increased density in the papilla. Binding specificity in the cortex and outer medulla was typical of the type I IGF receptor (IGF-I = des[1-3]IGF-I greater than IGF-II much greater than insulin). Binding in the outer medulla of diabetic kidney was typical of the type I IGF receptor. A marked focal increase in proximal tubular binding occurred in 13 of 22 postpubertal diabetic rats. Binding specificity of the proximal tubular binding was consistent with the predominance of an IGF binding protein (IGF-I = IGF-II greater than des[1-3]IGF-I with minimal displacement by insulin). Northern-blot analysis revealed increased IGFBP-1 and IGFBP-3 mRNA in cortical tissue from diabetic rats displaying increased proximal tubular binding but not from diabetic rats not displaying this phenomenon. As cell surface association of IGFBPs is linked to potentiation of IGF activity, a possible mechanism for potentiation of local IGF-I action may be provided.     

The Insulin-Like Growth Factors (IGFs) and IGF Binding Proteins in Postnatal Development of Murine Mammary Glands

The insulin-like growth factors are mitogens and survival factors for normal mammary epithelialcells in vitro. Data reviewed here demonstrate that mRNAs for IGF-I and IGF-II, the IGFtype I receptor and the IGFBPs are expressed locally in mammary tissue during pubertal andpregnancy-induced growth and differentiation of murine mammary glands. IGF-I, IGF-II andthe IGF-IR were expressed in terminal end buds (TEBs) in virgin glands during ductal growth.In addition, IGF-II and IGF-IR mRNAs were expressed in ductal and alveolar epithelium inglands throughout postnatal development. Consistent with these results, IGF-I promoted ductalgrowth and proliferation in mouse mammary glands in organ culture. In addition to endogenousexpression of the IGFs and IGF-IR, the IGFBPs showed a varied pattern of expression inmammary tissue during postnatal development. For example, IGFBP-3 and -5 mRNAs wereexpressed in TEBs and ducts while IGFBP-2 and -4 mRNAs were expressed in stromal cellsimmediately surrounding the epithelium. These results support a role for the IGFs and IGFBPsas local mediators of postnatal mammary gland growth and differentiation.     

Prostatic involution in men taking finasteride is associated with elevated levels of insulin-like growth factor-binding proteins (IGFBPs)-2, -4, and -5

BACKGROUND: Insulin-like growth factor-binding proteins (IGFBPs)-2, -4, and -5 are associated with upregulation of apoptosis in the ovary. The purpose of this study was to assess the roles of IGF-I and IGFBPs during involution of the prostate. Frozen and fixed tissue was collected by transurethral prostatectomy from Caucasian men, aged 52-82 years, scheduled for prostatectomy for benign prostatic hyperplasia, who took either placebo (n = 7) or the 5alpha-reductase inhibitor finasteride for 6 days to 6 years (n = 15) prior to surgery. METHODS: Intraprostatic androgen levels were measured by radioimmunoassay. Tissues were immunostained for IGF-I and IGFBP-2, -3, -4, and -5, and staining was quantitated by computerized image analysis. Serial sections were stained for markers of apoptosis (TUNEL and tissue transglutaminase) and IGFBP-2, -4, or -5. RESULTS: IGF-I staining was significantly decreased in the medium-term (18-43 days) treatment group and remained so for the duration of the study (P = 0.026). IGFBP-3 staining was unchanged in the early and medium-term treatment groups; however, a transient earlier rise in the level of this proapoptotic protein cannot be ruled out. The percentage of epithelial cell area staining positively for IGFBP-2 increased significantly, from 1.6 +/- 0.5 in the placebo group to 12.0 +/- 2.0 (P < 0.0001), and 7.6 +/- 1.9 (P = 0.003) in the short (6-13 days) and medium-term treatment groups, respectively. IGFBP-4 staining increased from 2.2 +/- 0.6 to 9.8 +/- 1.9 (P < 0.0001) and 7.4 +/- 1.2 (P = 0.004) in the short and medium-term groups, respectively, and IGFBP-5 staining increased from 0.2 +/- 0.1 to 3.8 +/- 2.0 (P = 0.004) in the medium-term group. The results from serial sections showed that IGFBP-2 and -4 costained with markers of apoptosis, while IGFBP-5 did not. CONCLUSIONS: These results indicate that IGFBP-2, -4, and -5 are associated with prostatic involution. Because of the timing and distribution of expression, we hypothesize that IGFBP-2 and -4 have a role as signals for apoptosis, but that IGFBP-5 likely does not.     

Paradoxical body and kidney growth in potassium deficiency

In the growing animal, K deficiency (KD) retards body growth, but paradoxically stimulates renal growth. If KD persists, interstitial infiltrates appear and eventually tubulointerstitial fibrosis develops. In patients with chronic KD, renal cysts may form and with time tubulointerstitial disease with renal failure develops. Since early in KD, kidney IGF-I levels increase and may be a cause of the renal hypertrophy, and as TGF-beta promotes hypertrophy and fibrosis, we examined the expression of these growth factors in chronic KD. Rats were given a KD diet or pair or ad-lib fed a normal K diet. After 21 days, KD rats weighed less than pair fed controls, while the kidneys were 49% larger Serum IGF-I and kidney IGF-I protein levels were depressed, as were IGF-I mRNA levels, and is largely attributable to decreased food intake. Kidney IGFBP-1 and TGF-beta mRNA levels were increased (p < 0.05). There was marked hypertrophy and adenomatous hyperplasia of outer medullary collecting ducts, hypertrophy of thick ascending limbs of Henle (TALH) and interstitial infiltrates. Both nephron segments stained strongly for IGF-I and IGFBP-1. Only the non-hyperplastic TALH was strongly TGF-beta positive. Interstitial infiltrates containing monocytes/macrophages were prominent. These findings are consistent with a sustained role for IGF-I in promoting the renal hypertrophy of KD and appear to be caused by local trapping of IGF-I by the over-expressed IGFBP-1. Localization of TGF-beta to the hypertrophied non-hypoplastic tubules containing IGF-I, suggests that TGF-beta may be acting to convert the proliferative action of IGF-I into a hypertrophic response. TGF-beta may also contribute to the genesis of the tubulointerstitial infiltrate. Finally, the reduced levels of serum IGF-1 levels may be a cause of the blunted body growth.     

Insulin-like growth factor binding proteins as growth inhibitors in children with chronic renal failure

Children with chronic renal failure (CRF) often fail to attain an adult height consistent with their genetic potential. The growth hormone (GH)/insulin-like growth factor (IGF)/growth plate chondrocyte axis has been intensively studied in these children to determine the basis for this growth failure. Evidence suggests that hepatic GH resistance results in deficient expression of IGF-I. However, serum IGF-I levels are usually normal and it is IGF-I action on target tissues which is inhibited, possibly by the presence of excess high-affinity IGF binding proteins (IGFBPs) in CRF serum. In this paper we evaluate the roles of IGFBP-1, -2, and -3 as growth inhibitors in CRF children. The data support a role for each of these IGFBPs as growth inhibitors. Currently, IGFBP-1 meets most criteria expected of a growth inhibitor, but IGFBP-2 and -3 will likely also meet these criteria and may well be important contributors to the growth failure of CRF. Ultimately, many or all of the six IGFBPs may be found to contribute to the excess high-affinity IGF binding sites which are a hallmark of CRF serum and are possible contributors to the growth failure of CRF children.     

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.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

The roles of IGF-I and IGFBP-3 in the regulation of proximal tubule, and renal cell carcinoma cell proliferation

BACKGROUND: Insulin-like growth factor I (IGF-I), a potent proximal tubule cell (PTC) mitogen, has been implicated in the progression of many human cancers. Our previous work on human renal tissues has suggested that IGF-I and several of its binding proteins (IGFBP-3 and -6) are up-regulated in clear cell renal cell carcinoma (RCC). METHODS: To further elucidate the role of IGF-I and IGFBPs in RCC growth, immunohistochemistry, thymidine incorporation, and Western analysis were performed in primary cultures of normal PTC (priPTC) and clear-cell RCC (priRCC), as well as in SN12K1 cells (a cell line derived from metastatic RCC). RESULTS: By immunohistochemistry, IGFBP-3 and IGF-I were prominently expressed in SN12K1 cells, and weakly expressed in priPTC and priRCC. Incubation with 100 ng/mL IGF-I significantly augmented DNA synthesis by priPTC (mean +/- SD 120.7%+/- 19.7% of controls, P < 0.05), priRCC (238.7%+/- 279.9% of controls, P < 0.01), and SN12K1(120.0%+/- 22.9% of controls, P < 0.05). Neutralizing antibodies to IGF-I and IGF-I receptor significantly suppressed SN12K1 growth (81.9%+/- 13.5% of control, P < 0.01 and 87.4%+/- 16.2% of control, P < 0.05, respectively). Removal of endogenous IGFBP-3 by an anti-IGFBP-3 increased SN12K1 DNA synthesis (243.9%+/- 35.3% of control, P < 0.001), which was partially abrogated by coincubation with exogenous IGFBP-3 (135.97%+/- 5.9% of controls, P < 0.001). Using Western analysis, IGFBP-3 expression was enhanced in IGF-I-stimulated SN12K1 cells exposed to exogenous IGF-I. Coincubation with anti-IGFBP-3 further enhanced IGF-I-induced DNA synthesis. CONCLUSION: RCC cells express IGF-I and IGFBP-3, and are responsive to exogenous IGF-I stimulation. Moreover, in SN12K1 cells (derived from metastatic RCC), autocrine IGF-I and IGFBP-3 actions, respectively, stimulated and inhibited growth. These results suggest that IGF-I and IGFBP-3 may be potential candidates for therapeutic manipulation in patients with advanced RCC.     

Insulin-like growth factor system components in hyperparathyroidism and renal osteodystrophy

BACKGROUND: The insulin-like growth factor (IGF) system plays a key role in regulation of bone formation. In patients with renal osteodystrophy, an elevation of some IGF binding proteins (IGFBPs) has been described, but there is no study measuring serum levels of both IGF-I and IGF-II as well as IGFBP-1 to -6 in different forms of renal osteodystrophy and hyperparathyroidism. METHODS: In a cross-sectional study, we investigated 319 patients with mild (N = 29), moderate (N = 48), preuremic (N = 37), and end-stage renal failure (ESRF; N = 205). The ESRF group was treated by hemodialysis (HD; N = 148), peritoneal dialysis (PD; N = 27), or renal transplantation (RTX; N = 30). As controls without renal failure, we recruited age-matched healthy subjects (N = 87) and patients with primary hyperparathyroidism (pHPT; N = 25). Serum levels of total and free IGF-I, IGF-II, IGFBP-1 to -6, and biochemical bone markers including intact parathyroid hormone (PTH), bone alkaline phosphatase (B-ALP), and osteocalcin (OSC) were measured by specific immunometric assays. IGF system components and bone markers were correlated with clinical and bone histologic findings. Mean values +/- SEM are given. RESULTS: With declining renal function a significant increase was measured for IGFBP-1 (range 7- to 14-fold), IGFBP-2 (3- to 8-fold), IGFBP-3 (1.5- to 3-fold), IGFBP-4 (3- to 19-fold), and IGFBP-6 (8- to 25-fold), whereas IGFBP-5 levels tended to decrease (1.3- to 1. 6-fold). In contrast, serum levels of IGF-I, free IGF-I, and IGF-II remained constant in most patients. Compared with renal failure patients, pHPT patients showed a similar decline in IGFBP-5 levels and less elevated levels of IGFBP-1 (3.5-fold), IGFBP-2 (2-fold), IGFBP-3 (1.2-fold), and IGFBP-6 (4-fold) but no elevation of IGFBP-4 levels. In all subjects, free and total IGF-I levels showed significant negative correlations with IGFBP-1, IGFBP-2, and IGFBP-4 (that is, inhibitory IGF system components) and significant positive correlations with IGFBP-3 and IGFBP-5 (that is, stimulatory IGF system components). A positive correlation was observed between IGF-II and IGFBP-6. ESRF patients with mixed uremic bone disease and histologic evidence for osteopenia revealed significantly (P < 0.05) higher levels of IGFBP-2 and IGFBP-4 but lower IGFBP-5 levels. Histologic parameters of bone formation showed significant positive correlations with serum levels of IGF-I, IGF-II, and IGFBP-5. In contrast, IGFBP-2 and IGFBP-4 correlated positively with indices of bone loss. Moreover, dialysis patients with low bone turnover (N = 24) showed significantly (P < 0.05) lower levels of IGFBP-5, PTH, B-ALP, and OSC than patients with high bone turnover. CONCLUSION: Patients with primary and secondary hyperparathyroidism showed lower levels of the putative stimulatory IGFBP-5 but higher levels of IGFBP-1, -2, -3, and -6, whereas total IGF-I and IGF-II levels were not or only moderately increased. The marked increase in serum levels of IGFBP-4 appeared to be characteristic for chronic renal failure. IGFBP-5 correlated with biochemical markers and histologic indices of bone formation in renal osteodystrophy patients and was not influenced by renal function. Therefore, IGFBP-5 may gain significance as a serological marker for osteopenia and low bone turnover in long-term dialysis patients.     

Serum Levels of Insulin-Like Growth Factor (IGF); IGF-Binding Proteins-3, -4, and -5; and Their Relationships to Bone Mineral Density and the Risk of Vertebral Fractures in Postmenopausal Women

We previously found that serum levels of insulin-like growth factor I (IGF-I) and IGF-binding protein (IGFBP)-3, but not IFGBP-2, were associated with bone mineral density (BMD) and the risk of vertebral fractures. The aim of the present study was to investigate the roles of IGFBP-4 and -5 in age-dependent bone loss and vertebral fracture risk in postmenopausal Japanese women and to compare them with those of IGF-I and IGFBP-3. One hundred and ninety-three Japanese women aged 46–88 years (mean 62.5) were enrolled in the cross-sectional study. BMD was measured at the lumbar spine, femoral neck, ultradistal radius (UDR), and total body by dual-energy X-ray absorptiometry. Serum levels of IGFBP-4 and -5 as well as IGF-I and IGFBP-3 were measured by radioimmunoassay. Serum levels of IGF-I, IGFBP-3, and IGFBP-5 declined with age, while serum IGFBP-4 increased with age. Multiple regression analysis was performed between BMD at each skeletal site and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, and serum creatinine. BMD at the UDR was significantly and positively correlated with all serum levels of IGF-I and IGFBPs measured (P < 0.01), while BMD at the femoral neck was correlated with none of them. Serum IGF-I level was significantly and positively correlated with BMD at all sites except the femoral neck (P < 0.01), while serum IGFBP-3 and -4 levels were significantly and positively correlated with only radial BMD (P < 0.01). Serum IGFBP-5 level was positively correlated with UDR BMD (P < 0.001) and negatively correlated with total BMD (P < 0.05). Serum IGF-I, IGFBP-3, and IFGBP-5 levels were significantly lower in women with vertebral fractures than in those without fractures (mean ± SD: 97.1 ± 32.1 vs. 143.9 ± 40.9 ng/dl, P < 0.0001; 2.18 ± 1.02 vs. 3.23 ± 1.07 μg/ml, P < 0.0001; 223.6 ± 63.3 vs. 246.5 ± 71.5 ng/ml, P = 0.0330, respectively). When multivariate logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, serum creatinine, and serum alubumin as independent variables, IGF-I and IGFBP-3 were selected as indices affecting the presence of vertebral fractures [odds ratio (OR) = 0.29, 95% confidential interval (CI) 0.15–0.57 per SD increase, P = 0.0003 and OR = 0.31, 95% CI 0.16–0.61 per SD increase, P = 0.0007, respectively]. To compare the significance values, IGF-I, IGFBP-3, and age were simultaneously added as independent variables in the analysis. IGFBP-3 was more strongly associated with the presence of vertebral fractures than IGF-I and age (P = 0.0006, P = 0.0148, and P = 0.0013, respectively). Thus, after comprehensive measurements of serum levels of IGF-I and IGFBPs, it seems that serum IGF-I level is most efficiently associated with bone mass and that serum IGFBP-3 level is most strongly associated with the presence of vertebral fractures in postmenopausal women among the IGF system components examined.     

PARADOXICAL BODY AND KIDNEY GROWTH IN POTASSIUM DEFICIENCY

In the growing animal, K deficiency (KD) retards body growth, but paradoxically stimulates renal growth. If KD persists, interstitial infiltrates appear and eventually tubulointerstitial fibrosis develops. In patients with chronic KD, renal cysts may form and with time tubulointerstitial disease with renal failure develops. Since early in KD, kidney IGF-I levels increase and may be a cause of the renal hypertrophy, and as TGF-β promotes hypertrophy and fibrosis, we examined the expression of these growth factors in chronic KD. Rats were given a KD diet or pair or ad-lib fed a normal K diet. After 21 days, KD rats weighed less than pair fed controls, while the kidneys were 49 % larger Serum IGF-I and kidney IGF-I protein levels were depressed, as were IGF-I mRNA levels, and is largely attributable to decreased food intake. Kidney IGFBP-1 and TGF-β mRNA levels were increased (p < 0.05). There was marked hypertrophy and adenomatous hyperplasia of outer medullary collecting ducts, hypertrophy of thick ascending limbs of Henle (TALH) and interstitial infiltrates. Both nephron segments stained strongly for IGF-I and IGFBP-l. Only the non-hyperplastic TALH was strongly TGF-β positive. Interstitial infiltrates containing monocytes/macrophages were prominent. These findings are consistent with a sustained role for IGF-I in promoting the renal hypertrophy of KD and appear to be caused by local trapping of IGF-I by the over-expressed IGFBP-1. Localization of TGF-β to the hypertrophied non-hypoplastic tubules containing IGF-I, suggests that TGF-β may be acting to convert the proliferative action of IGF-I into a hypertrophic response. TGF-β may also contribute to the genesis of the tubulointerstitial infiltrate. Finally, the reduced levels of serum IGF-1 levels may be a cause of the blunted body growth     

Expression of insulin-like growth factor-I and transforming growth factor-beta in hypokalemic nephropathy in the rat

BACKGROUND: Potassium deficiency (KD) in the rat retards body growth but stimulates renal enlargement caused by cellular hypertrophy and hyperplasia, which is most marked in the outer medulla. If hypokalemia persists, interstitial infiltrates appear and eventually fibrosis. Since early in KD insulin-like growth factor-I (IGF-I) levels in the kidney are elevated, suggesting that it may be an early mediator of the exaggerated renal growth, and as transforming growth factor-beta (TGF-beta) promotes cellular hypertrophy and fibrosis, we examined the renal expression of these growth factors in prolonged KD. METHODS: Rats were given a K-deficient diet or were pair fed or ad libitum fed a K-replete diet for 21 days. Growth factor mRNA levels were measured in whole kidney and protein expression localized by immunohistochemistry. RESULTS: KD rats weighed less than pair-fed controls, while the kidneys were 49% larger. Their serum IGF-I and kidney IGF-I protein levels were depressed, as were their IGF-I mRNA levels in liver, kidney, and muscle. These changes can largely be attributed to decreased food intake. In contrast, kidney IGF binding protein-1 (IGFBP-1) mRNA and TGF-beta mRNA levels were increased significantly. Histology of outer medulla revealed marked hypertrophy and adenomatous hyperplasia of the collecting ducts and hypertrophy of the thick ascending limbs of Henle with cellular infiltrates in the interstitium. Both nephron segments immunostained strongly for IGF-I and IGFBP-1, but only the nonhyperplastic enlarged thick ascending Henle limb cells immunostained for TGF-beta, which was strongly positive. Prominent interstitial infiltrates with ED1 immunostained monocytes/macrophages were present. CONCLUSIONS: These findings are consistent with a sustained role for IGF-I in promoting the exaggerated renal growth of KD and appear to be mediated through local trapping of IGF-I by the overexpressed IGFBP-1, which together with IGF-I can promote renal growth. The selective localization of TGF-beta to hypertrophied nonhyperplastic nephron segments containing IGF-I raises the possibility that TGF-beta may be serving to convert the mitogenic action of IGF-I into a hypertrophic response in these segments. It is also conceivable that TGF-beta may be a cause of the tubulointerstitial infiltrate. Finally, the low circulating IGF-I levels likely contribute to the impaired body growth.     

Prostaglandin E2 stimulates synthesis of insulin-like growth factor binding protein-3 in rat bone cells in vitro.

Prostaglandin E2 is produced by bone cells and increases cyclic AMP in these cells. Like PTH and dibutyryl cyclic AMP, PGE2 is a potent stimulator of IGF-I synthesis in cultured rat osteoblasts and inhibits DNA synthesis and type I procollagen gene expression. In addition, PGE2 inhibits the response of the cells toward IGF-I after 1 day but not after 4 days of incubation. Rat calvaria osteoblasts constitutively release IGFBPs into the culture medium, in particular IGFBP-2 and IGFBP-3. Like growth hormone, PGE2 stimulates the accumulation of IGFBP-3. PGE2 rapidly increases IGF-I and IGFBP-3 mRNA expression in calvaria cells, with a time course clearly different from that observed in response to growth hormone. Thus, PGE2 modifies not only the synthesis of IGF-I but also that of IGFBP-3 in skeletal tissue.     

Effects of growth hormone on insulin-like growth factors and binding proteins before and after renal transplantation

A bstract. Three short children with severe chronic renal failure were treated with recombinant human growth hormone (rhGH) for 2 years. Each received a transplant in the 2nd year. Serum collected before and during rhGH therapy was analysed retrospectively by Western ligand blot and immunoblotting techniques. In addition, radioimmunoassays for insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), acid-labile subunit (ALS) and IGFBP-1 were performed. IGFBPs in serum, detected by Western ligand blot, were identified as IGFBP-3, -2, -1 and -4 by immunoblot. The serum concentration of IGF-I in each child rose approximately fourfold with rhGH before transplantation and subsequently remained elevated. IGFBP-3 levels rose to double the pretreatment value, but dropped to normal levels following transplantation, while ALS rose with rhGH treatment and remained increased after transplantation. IGFBP-1 levels changed little with rhGH but fell following transplantation. A low molecular weight form of IGFBP-3 was noted at 30 kilodaltons on immunoblot which was not clearly seen on the ligand blot. IGFBP-2 was present as a distinct band on Western ligand blot before transplantation and appeared decreased in intensity subsequently. IGFBP-1, seen on immunoblot clearly before transplant, disappeared after the transplant. rhGH successfully improved growth in these children, in association with a fourfold increase in IGF-I levels, which was maintained following transplantation. The reduction in IGFBPs following transplantation suggests correction of impaired clearance by the diseased kidney. Received September 5, 1994; received in revised form November 8, 1995; accepted November 21, 1995     

Serum insulin-like growth factors and their binding proteins in children with end-stage renal disease

Serum levels of insulin-like growth factor-I (IGF-I), IGF-II, and IGF binding protein-1 (IGFBP-1), IGFBP-2, and IGFBP-3 were measured in 54 children with end-stage renal disease (ESRD). The results were compared with their respective age-dependent normal ranges. IGFs and IGFBPs were quantified by specific radioimmunoassay. Serum IGF-I in children with ESRD tended to cluster in the low-normal range. Mean age-related serum IGF-I levels were slightly, but significantly decreased (–1.08±0.17 SDS). In view of the prevailing elevated growth hormone levels in ESRD, these serum, IGF-I levels must be interpreted as inadequately low. In contrast to IGF-I, individual serum IGF-II levels were either in the uppernormal range or clearly elevated. Mean age-related IGF-II (1.09±0.15 SDS) was lightly, but significantly elevated. Mean age-related IGFBP-1 serum levels (2.20±0.10 SDS) were moderately increased, while mean age-related serum IGFBP-2 (5.65±0.36) and IGFBP-3 levels (3.60±0.19) were markedly elevated. Affinity cross-linking of¹²⁵iodine-IGF-II to sera from patients with ESRD and immunoprecipitation with a specific antiserum showed that low molecular weight IGFBP-3 fragments in ESRD serum are capable of binding IGF. In patients with ESRD, a rapid and persistent decline of immunoreactive IGFBP-3 in response to restoration of renal function by renal transplantation was observed. This finding indicates that renal dysfunction contributes to high immunoreactive, IGFBP-3 levels in ESRD. In conclusion, the imbalance between normal total IGF levels and the excess of IGFBPs in ESRD is likely to play a role in growth failure in these children.