Differential regulation of RGS-2 by constant and oscillating PTH concentrations.

PTH has diverse effects on bone metabolism: anabolic when given intermittently, catabolic when given continuously. The cellular mechanisms underlying the varying target cell response are not clear yet. PTH induces RGS-2, a member of the Regulator of G-protein Signaling protein family, via cAMP/PKA, and inactivates PKC-mediated signaling. To investigate intracellular signaling pathways with different PTH concentration-time patterns, we treated UMR 106-01 osteoblast-like cells in a perfusion system. PTH was administered intermittently (4 min/h, 10(-7) M) or continuously at an equivalent cumulative dose (6.6 x 10(-9) M). cAMP was measured using radioimmunoassay, mRNA levels using real-time rtPCR and ribonuclease protection assay, and protein levels using Western immunoblotting. A single PTH pulse transiently increased cAMP levels by 2000% +/- 1200%. In contrast to continuous PTH exposure, cAMP induction remained unchanged with intermittent PTH, ruling out desensitization of the PTH receptor. In continuously perfused cells, RGS-2 abundance was three to five times higher than in cells intermittently exposed to PTH for up to 12 h. MKP-1 and -3 were significantly less induced with pulsatile PTH; exposure-mode-dependent differences in MMP-13 and IGFBP-5 were small. Pulsatile but not continuous PTH administration prevents PTHrP receptor desensitization and accumulation of RGS-2 in osteoblasts, which should preserve PKC-dependent signaling.

Metabolic clearance of recombinant human growth hormone in health and chronic renal failure.

Despite the increasing therapeutic use of recombinant human growth hormone (rhGH), its metabolic clearance has not been investigated in detail. To evaluate the kinetics of rhGH as a possible function of GH plasma concentration and glomerular filtration rate (GFR), we investigated the steady state metabolic clearance rate (MCR), disappearance half-life, and apparent volume of distribution of rhGH at low and high physiological as well as supraphysiological plasma GH levels during pharmacological suppression of endogenous GH secretion in human subjects with normal and reduced renal function. GH in plasma and urine was determined by an immunoradiometric assay, and GFR by inulin clearance. In all subjects MCR decreased and plasma half-life increased with increasing plasma GH concentrations (P < 0.001). MCR of rhGH was approximately half in patients with chronic renal failure at each GH level and plasma half-life was increased by 25-50%. Allowing for the linear dependence of MCR on GFR and assuming single-compartment distribution, the estimated renal fraction of total MCR was 25-53 and 4-15% in controls and patients, respectively. Saturation of extrarenal disposal of GH was suggested by an inverse hyperbolic relationship between extrarenal MCR and plasma GH concentrations in all subjects. Fractional GH excretion was up to 1,000-fold higher in patients than in controls. We conclude that MCR of hGH is a function of plasma GH concentrations and GFR. Extrarenal elimination is saturable in the upper physiological range of GH concentrations, whereas renal MCR is independent of plasma GH levels. The kidney handles GH like a microprotein involving glomerular filtration, tubular reabsorption, and urinary excretion.

No difference in intestinal strontium absorption after an oral or an intravenous 1,25(OH)2D3 bolus in normal subjects. For the European Study Group on Vitamin D in children with renal failure.

It has been suggested that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulates intestinal calcium absorption less via the intravenous (iv) than the oral route, because the first avoids direct contact of the drug with the enterocytes. However, no study has addressed the issue directly. This investigation was designed to measure the effect of a single oral or iv dose of 1,25(OH)2D3 on calcium absorption, using stable strontium (Sr) as a surrogate for calcium, and measuring the Sr fractional absorbed dose (FAD%) over 240 minutes after Sr administration. In 10 healthy volunteers, five tests were performed in a cross-over design, with a wash-out period between two consecutive tests: Sr absorption without 1,25(OH)2D3 (test A); Sr absorption immediately after either oral (test B) or iv (test C) 1,25(OH)2D3 (1.5 microg/m2 of body surface area [BSA]); Sr absorption (24 hr after either oral (test D) or iv (test E) 1, 25(OH)2D3 (1.5 microg/m2 BSA). The concurrent administration of 1, 25(OH)2D3 and Sr (tests B and C) did not significantly change the area under the Sr FAD%-time curve with respect to test A (test A: 4090 +/- 345; test B: 4510 +/- 345; test C: 4210 +/- 345), whereas Sr absorption was significantly increased (p < 0.001) when Sr was given 24 hr after either oral or iv 1,25(OH)2D3 (test D: 5710 +/- 345; test E: 5510 +/- 345). It was concluded that 1,25(OH)2D3 is likely to influence calcium absorption significantly only via its genomic effect, independent of its administration route.

Synergistic effects of parathyroid hormone and 1,25-dihydroxyvitamin D3 on proliferation and vitamin D receptor expression of rat growth cartilage cells.

We investigated possible interaction of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and PTH on: 1) proliferation (monolayer culture) and colony formation (agarose stabilized suspension cultures); 2) expression of 1,25-(OH)2D3 receptor (VDR); and 3) cAMP response to PTH, using primary cultures of chondrocytes from rat tibia proximal epiphysis. 1 alpha,25-(OH)2D3 stereospecifically stimulated DNA synthesis, cell counts, and colony formation at low concentration (10(-12) M). Within 6 h bovine PTH (bPTH)(1-34), human PTH (hPTH)(28-48) (10(-10) M), (Bu)2cAMP (1-2 mM), and 12-O-tetradecanoyl-13-acetate (10(-8) M) increased [3H]thymidine incorporation in the absence and presence of 1,25-(OH)2D3. Both PTH fragments also stimulated chondrocyte growth and colony formation in a Ca-dependent fashion. Prolonged exposure to bPTH(1-34) or hPTH(28-48) did not affect baseline DNA synthesis but increased the stimulatory effect of 1,25-(OH)2D3. This increase was inhibited in the presence of H7 (inhibition of PKC) or the monoclonal hPTH(1-38) antibody A1-70. In subconfluent chondrocyte cultures VDR was up-regulated by bPTH(1-34) and hPTH(28-48) (10(-10) M) or activators of protein kinase C (PKC), but not by (Bu)2cAMP. It was blocked by cycloheximide and actinomycin D and persisted in the presence of Ca-channel blockers. Inhibition of PKC by H7 also blocked the effect of bPTH(1-34) on VDR. The cAMP response to bPTH(1-34) was not affected by 1,25-(OH)2D3. We conclude that: 1) DNA synthesis, cell proliferation, and colony formation in chondrocyte monolayer or suspension cultures is increased by aminoterminal and midregional PTH fragments and by cAMP analogs in a Ca- dependent fashion; 2) bPTH(1-34) and hPTH(28-48) up-regulate VDR by cAMP-independent, PKC-dependent steps requiring transcriptional and translational processes; both PTH fragments also amplify the effect of 1,25-(OH)2D3 on DNA synthesis; and 3) no difference is found between the bPTH(1-34) and hPTH(28-48) fragments with respect to chondrocyte proliferation and VDR up-regulation, although the two differ with respect to stimulation of cAMP production.

Vitamin D and dexamethasone inversely regulate parathyroid hormone-induced regulator of G protein signaling-2 expression in osteoblast-like cells.

The PTH/PTHrP receptor stimulates both adenylate cyclase- and phospholipase C-dependent signaling pathways via different G proteins. The biological actions of PTH on bone are modified by steroid hormones. PTH induces expression of regulator of G protein signaling (RGS)-2, a putative preferential inhibitor of G(q)-mediated phospholipase C activation. We investigated whether steroid hormones interfere with PTH signaling by modulating PTH-induced RGS-2 expression in osteoblast-like UMR 106-01 cells. PTH (1-34) rapidly and transiently induced expression of RGS-2 mRNA and protein via the cAMP/protein kinase A pathway within 30 min, with maximal protein abundance after 2 h. PTH-induced RGS-2 preferentially bound to Galpha(q), compared with Galpha(s) protein. 1,25-(OH)(2)D(3) pretreatment enhanced PTH-induced RGS-2 mRNA and protein accumulation, whereas dexamethasone preincubation had an attenuating effect. These effects were due to modulation of the RGS-2 gene transcription rate, which increased by 35% with 1,25-(OH)(2)D(3) and decreased by 63% with dexamethasone pretreatment. RGS-2 mRNA half-life was not affected by either steroid. The transcriptional effects of dexamethasone and 1,25-(OH)(2)D(3) were independent of PTH/PTHrP receptor activation and were not explained by effects on cAMP accumulation, cAMP response element-binding protein expression or phosphorylation, or the abundance of the osteoblast-specific transcription factor core-binding factor alpha (CBFa1/Runx2), a known activator of RGS-2 expression. In conclusion, glucocorticoids and 1,25-(OH)(2)D(3) inversely modulate PTH-induced RGS-2 gene transcription. Regulation of RGS-2 may constitute a novel mechanism by which steroids modulate signaling via the PTH/PTHrP receptor and other G protein-coupled receptors in bone.

Dexamethasone impairs growth hormone (GH)-stimulated growth by suppression of local insulin-like growth factor (IGF)-I production and expression of GH- and IGF-I-receptor in cultured rat chondrocytes.

Growth depression as a side effect of glucocorticoid therapy in childhood is partially mediated by alterations of the somatotropic hormone axis. The mechanisms of interaction between glucocorticoids and somatotropic hormones on the cellular and molecular level are poorly understood. In an experimental model of primary cultured rat growth plate chondrocytes, basal as well as GH (40 ng/ml) or insulin-like growth factor (IGF)-I (60 ng/ml)-stimulated growth was suppressed dose dependently (10(-l2)-10(-7)M) by dexamethasone (Dexa). An IGF-I antibody specifically and dose dependently inhibited the GH- but not the basic fibroblast growth factor (bFGF)-stimulated cell proliferation. GH increased the IGF-I concentration in conditioned serum-free culture medium; this was reversed by concomitant Dexa. Dexa time dependently suppressed the transcription of GH receptor (GHR) messenger RNA (mRNA) and down-regulated the basal and GH-stimulated expression of GHR. Whereas no suppressive effect on basal type I IGF-receptor (IGFR) was observed, Dexa blocked the IGF-I induced increase of IGF binding. These results were confirmed by GHR and IGFR immunostaining. We conclude that Dexa impairs the GH-induced stimulation of local secretion and paracrine action of IGF-I and reduces the homologous increase of IGFR and GHR expression. The above experiments give further insight on the interaction between GH and glucocorticoids on the cellular and molecular level of growth plate chondrocytes.

Impaired postprandial regulation of insulin-like growth factor binding protein-1 in children with chronic renal failure.

Patients with chronic renal failure (CRF) have elevated plasma levels of insulin-like growth factor-1 (IGFBP-1). We sought to determine the dynamics of plasma IGFBP-1 in response to an endogenous insulin pulse during an oral glucose tolerance test (oGTT) in 12 prepubertal children with advanced CRF [glomerular filtration rate (GFR) 12.5 +/- 4 mL/min/1.73 m2] and in 9 age-, gender-, and body size-matched controls with normal renal function. Glucose and insulin responses to oGTT were significantly elevated in CRF (P < 0.01), indicating decreased sensitivity to the hypoglycemic action of insulin. Fasting plasma IGFBP-1 levels in CRF (235 +/- 40 ng/mL) were 2.5-fold increased compared with controls (94 +/- 11.6 ng/mL, P < 0.0001). In controls, plasma IGFBP-1 levels rapidly decreased with time by 52%, to a level of 45 +/- 6.7 ng/mL 180 min after the oral glucose load. In contrast, plasma IGFBP-1 levels in CRF patients slowly decreased with time by 25%, to a level of 176 +/- 28 ng/mL (P < 0.001 vs. controls) 180 min after the oral glucose load. For the group as a whole, the percent decrease in IGFBP-1 at 180 min was positively correlated with GFR (r = 0.85, P < 0.0001). Plasma GH concentrations were not statistically different at baseline, but showed a paradoxical increase in CRF patients thereafter. Plasma IGF-I concentrations at baseline were comparable in CRF patients and controls and similarly decreased by about 10% (P < 0.01) after the oral glucose load. In summary, our study shows that the decline of plasma IGFBP-1 in response to an oral glucose load is impaired in children with CRF despite increased insulin levels. This impaired postprandial decline of plasma IGFBP-1 might interfere with glucose homeostasis by blocking insulin-like activity of free IGFs in vivo and thereby contribute to glucose intolerance in uremia.

Serum insulin-like growth factors (IGFs) and IGF binding proteins 1, 2, and 3 in children with chronic renal failure: relationship to height and glomerular filtration rate. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood.

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 94 children with chronic renal failure (CRF). The results were compared with their respective age-dependent normal ranges, and the relationship with height and residual glomerular filtration rate (GFR) was examined. Each IGF and IGFBP was quantified by specific RIA. Serum IGF-I and IGF-II levels were in the normal range throughout their entire childhood in the vast majority of cases. The mean age-related IGF-I (0.07 +/- 0.14 SD score) and IGF-II levels (0.06 +/- 0.11 SD) were similar. Age-related IGF-II but not IGF-I levels showed a weak inverse linear correlation with residual GFRs (r = -0.24, P < 0.02). Mean age-related IGFBP-1 serum levels (1.04 +/- 0.09 SD) were slightly elevated, whereas mean age-related serum IGFBP-2 levels (3.25 +/- 0.20 SD) and serum IGFBP-3 levels (2.61 +/- 0.12 SD) were markedly elevated. Significant inverse correlations were found between GFRs and age-related IGFBP-1 (r = -0.42, P < 0.001), IG-FBP-2 (r = -0.56, P < 0.001), and IGFBP-3 (r = -0.28, P < 0.005), but the increase in IGFBP-2 with declining GFR was relatively more pronounced than the respective increase in IGFBP-1 and IGFBP-3. The correlation between age-related IGF-I and relative height in prepubertal children with CRF (n = 54, r = 0.43, P < 0.001) was lower than in prepubertal controls (n = 68, r = 0.67, P < 0.001), and the slope of the regression line was significantly less steep, indicating that the normal relationship between IGF-I and height is disturbed in CRF. The normal relationship between IGFBP-3 and height was disrupted in CRF. Forward stepwise regression analysis revealed that height in CRF is correlated with IGF-I and inversely correlated with IGFBP-2. We conclude that the imbalance between normal IGFs and excessive IGFBP serum levels in CRF plays a pathogenic role in the growth failure of these children.

Synchronous fluctuations of blood insulin and lactate concentrations in humans.

Oscillatory organization is a universal mode of signal transduction in living organisms. In vitro studies suggest spontaneous pulsatile fluctuations of intracellular energy metabolism. It is possible that, in vivo, some of these processes are synchronized by the pulsatile release of insulin. We assessed a potential coupling among plasma insulin, glucose, and lactate concentrations, by frequent blood sampling for 24 h in 11 healthy volunteers. Insulin sensitivity was assessed using the euglycemic hyperinsulinemic clamp technique. Lactate concentrations exhibited pulsatile fluctuations at an average interval of 84 +/- 11 min, whereas sodium and pH were nonpulsatile. The lactate concentration pulses closely corresponded to insulin oscillations, which occurred with a periodicity of 86 +/- 11 min. Blood glucose also fluctuated during daytime at an interval of 89 +/- 32 min. During nighttime, the frequency and amplitude of glucose oscillations were lower. The daytime profiles showed significant temporal coupling and pattern synchrony among insulin, lactate, and glucose. Only the close temporal relationship between insulin and lactate release persisted during nighttime. The temporal coupling and pattern synchrony between insulin and lactate were correlated inversely with insulin sensitivity, and positively with the degree of abdominal obesity. Our results suggest that: 1) the concentration of lactate, an indicator of cellular energy metabolism, fluctuates periodically in vivo; 2) the lactate concentrations fluctuate in synchrony with insulin pulses; and 3) such coupling is more pronounced in obese, insulin-resistant individuals.

Reduced concentration of serum growth hormone (GH)-binding protein in children with chronic renal failure: correlation with GH insensitivity. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood. The German Study Group for Growth Hormone Treatment in Chronic Renal Failure.

Growth retardation in children with chronic renal failure (CRF) despite normal or elevated GH levels indicates a peripheral insensitivity to the action of GH. One possible molecular mechanism is a reduced density of GH receptors in GH target organs. In humans, the circulating high affinity GH binding protein (GHBP) is thought to reflect GH receptor expression, because it is derived from the extra-cellular domain of the GH receptor by proteolytic cleavage. We, therefore, analyzed serum GHBP levels by ligand-mediated immunofunctional assay in 126 children with CRF compared to reference values obtained by analysis of 773 healthy children. In 77% of CRF patients, serum GHBP concentrations were below the mean for age- and gender-matched controls. The decrease in serum GHBP levels was related to the degree of renal dysfunction. In advanced CRF (glomerular filtration rate, < 35 mL/min.1.73 m2), mean age- and gender-adjusted GHBP levels were -1.40 +/- 0.18 SD score; 36% of patients had GHBP levels below the normal range (< -2 SD score). Children with end-stage renal disease (n = 26) had the lowest GHBP levels (-2.25 +/- 0.22 SD score). Multiple linear regression analysis revealed that body mass index, rather than glomerular filtration rate, is the prevailing determinant of serum GHBP levels in CRF. GHBP levels correlated with both the spontaneous growth rate ( r = 0.44; P < 0.0001) and the growth response to GH therapy (r = 0.48; P < 0.005), indicating decreased sensitivity to both endogenous and exogenous GH. Subcutaneous GH therapy did not consistently affect serum GHBP levels after 3 months of treatment. It is suggested that low GHBP levels in children with CRF represent a quantitative tissue GH receptor deficiency as one of the molecular mechanisms of GH insensitivity.

Control of pulsatile and tonic parathyroid hormone secretion by ionized calcium.

To investigate the effect of changes in ionized calcium on instantaneous PTH secretion, we examined seven young healthy volunteers by 1-min blood sampling under conditions of normo-, hypo-, and hypercalcemia. After a baseline period of 75 min, ambient ionized calcium was either increased or decreased by 0.2 mmol/L for 105 min by clamped infusion of calcium gluconate or sodium citrate. The characteristics of PTH secretion were analyzed by a deconvolution technique, accounting for subject-specific plasma PTH disappearance half-life, as measured during the first 15 min of calcium infusion (range, 2.04-2.93 min). The process regularity of pulsatile PTH secretion was evaluated by an approximate entropy statistic. Under baseline conditions, 32% of total PTH secretion was released in a pulsatile fashion, with a burst frequency of 6.9 +/- 0.8 h-1 and a PTH mass per burst of 2.6 +/- 0.9 pmol/L. The remaining 68% of total secretion was attributed to tonic hormone release. During the initial 30 min of induced hypocalcemia, pulsatile secretion increased by 1140%, whereas tonic secretion did not change. The preferential increase in pulsatile PTH secretion was mediated by a combined rise in burst frequency and mass released per burst. During subsequent steady state hypocalcemia, the tonic secretion rate increased (255% of baseline), whereas burst frequency and burst mass decreased (to 103% and 189% of the baseline values), restoring the baseline ratio of tonic to pulsatile PTH secretion. The regularity of PTH release increased during steady state hypocalcemia. During hypercalcemia, tonic secretion, burst mass, and burst frequency decreased by 75%, 82%, and 32%, respectively, and remained constant throughout the clamp period. We conclude that acute hypocalcemia elicits an immediate pulsatile and a delayed tonic secretory response of the parathyroid gland with increased regularity of PTH release. Acute hypercalcemia suppresses both the pulsatile and the tonic component of PTH secretion.

Multifactorial control of the elimination kinetics of unbound (free) growth hormone (GH) in the human: regulation by age, adiposity, renal function, and steady state concentrations of GH in plasma.

To evaluate the principal determinants of the MCR and plasma t1/2 of unbound (free) GH in man, we performed steady state infusions of 3 doses of recombinant human GH during pharmacological suppression (iv octreotide) of endogenous GH secretion in 24 healthy adults and 12 patients (6 adults and 6 children) with chronic renal failure (CRF). Free plasma GH was calculated from total plasma GH (measured by immunoradiometric assay) and GH-binding protein activity (radioligand assay). The MCR of free GH was determined from free plasma GH and the rate of recombinant human GH infusion. The t1/2 of free plasma GH, and the concentration and the in vivo dissociation constant (Kd) of GH-binding protein (GHBP) were estimated by dynamic modeling of the postinfusion total plasma GH concentration decay curves. The MCR of free GH decreased and the plasma GH t1/2 increased significantly with increasing plasma GH concentrations. The MCR of free GH over its physiological concentration range was positively correlated with the body mass index as a measure of relative obesity and negatively related to age, but only at supraphysiological GH concentrations. In the adult patients with CRF, the MCR of free GH was decreased at each infusion rate by 25-38%, and the t1/2 was increased by 80-170%. Children with CRF showed a significantly lower MCR and higher t1/2 of plasma free GH than adult patients. Modeling and direct measurements of the off-rate of GH from its high affinity GHBP indicated normal dissociation rate constants but decreased molar concentrations of the GHBP in uremic plasma. We conclude that the rate of elimination of free GH from plasma in man is controlled by 1) plasma total free GH concentrations, 2) relative obesity, and 3) renal function within the physiological GH concentration range, whereas 4) age is a negative predictor of MCR only at supraphysiological GH concentrations.

Insulin-like growth factor-binding protein-6 levels are elevated in serum of children with chronic renal failure: a report of the Southwest Pediatric Nephrology Study Group.

Previous studies suggest that growth retardation in children with chronic renal failure (CRF) results in part from inhibition of insulin-like growth factor (IGF) action by excess serum IGF-binding proteins (IGFBPs). Excess IGFBPs in CRF serum include IGFBP-1, -2, and -3 and a diffuse approximately 24- to 28-kDa IGFBP band identified by [125I]IGF ligand blot. The present studies characterized this diffuse approximately 24- to 28-kDa band. Initial studies identified this band as IGFBP-6, because it was immunoprecipitated by antiserum raised against a synthetic peptide of human IGFBP-6 (hIGFBP-6). Additional [125I]IGF ligand blots found that the immunoprecipitated band was 1) recognized by [125I]IGF-II but not [125I]IGF-1, 2) more abundant in CRF than in normal serum, and 3) more abundant in serum from dialyzed than nondialyzed prepubertal CRF children. Using the hIGFBP-6 antiserum in a specific and sensitive RIA, we found that serum IGFBP-6 levels were 4.7 +/- 1.7 nmol/L in 10 normal prepubertal children, 21.4 +/- 6.1 nmol/L in 44 nondialyzed prepubertal CRF children, 73.5 +/- 14.4 nmol/L in 7 dialyzed prepubertal CRF children, and 94.6 +/- 26.2 nmol/L in 14 dialyzed pubertal CRF children. IGFBP-6 levels were also elevated in 71 nondialyzed European children with CRF. In nondialyzed CRF children, serum IGFBP-6 levels 1) correlated inversely with the glomerular filtration rate, 2) did not correlate with height SD score, and 3) were not altered by 12 months of daily recombinant hGH treatment. In summary, a specific antiserum and RIA were used to demonstrate elevated levels of intact IGF-II-binding IGFBP-6 in serum of CRF children. We postulate that the excess IGFBP-6 may modulate the action of IGF-II on target tissues.

Effect of vitamin D on growth in experimental uremia.

Acceleration of growth of uremic children after administration of vitamin D has been demonstrated by various authors. This has been attributed to healing of skeletal lesions. Clinical observations suggest that vitamin D has also an effect on food intake perhaps associated with improvement of vitality. This could be confirmed in an experimental study in which uremic rats (subtotal nephrectomy) with and without vitamin D supplementation were compared with sham-operated pair-fed control rats with and without vitamin D supplementation. In uremic animals supplemented with vitamin D, weight gain and growth were significantly greater than in uremic animals on the control diet. Both with and without vitamin D supplements, weight gain and growth rate were greater in sham-operated pair-fed control than in the corresponding uremic animals. Histological abnormalities in the growth zone of uremic rats were markedly reduced by vitamin D. Since food intake was greater in vitamin D-treated uremic animals than in nonvitamin D-treated uremic animals, the increase in growth rate under vitamin D cannot be attributed exclusively to the skeletal effects of vitamin D. This study demonstrates important extraskeletal actions of vitamin D which may be associated with or causally related to the improvement of growth.

Protein restriction in the conservative management of uremia.

Protein-restricted diets are widely used in the dietary management of uremia. These diets are undoubtedly effective in ameliorating many aspects of the uremic syndrome. However, there is no consensus as to whether diets providing less than 0.6 g/kg per day of protein are nutritionally adequate and capable of preventing the wasting syndrome. Wasting is common in the adult patient with renal insufficiency as is growth failure in the uremic child. There is some evidence that wasted patients do less well on hemodialysis and are more prone to infection. Experimental studies in uremic animals point ot diminihsed efficiency of utilization of protein, increased gluconeogenesis from animo acids, and increased catabolism of protein in the fasting state; in addition, the metabolism of a number of individual amino acids is altered in uremia. In view of these multiple abnormalities, it would seem unwise to routinely provide less than the Recommended Daily Allowances of protein. More recent developments, i.e., supplementation of essential amino acids and perhaps alpha keto acids, may provide useful alternatives. One important aspect of dietary management, i.e. prevention of hyperlipidemia, has attracted surprisingly little attention so far. Therapy with protein restricted diets in nondialyzed uremic patients has to compete with other modalities of treatment currently available, i.e., hemodialysis and transplantation, in providing optimal medical rehabilitaiton of the patient.

Calbindin-D28K and -D9K and 1,25(OH)2 vitamin D3 receptor immunolocalization and mineralization induction in long-term primary cultures of rat epiphyseal chondrocytes.

Rat epiphyseal plat chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)2D3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid levels in monocytes of patients with moderate hyperlipoproteinemia.

Recent studies suggest that circulating blood monocytes may serve as a lipid clearance system in early atherosclerotic lesions. To evaluate the influence of moderate hyperlipoproteinemia on monocyte lipid concentrations, we measured fasting serum and monocyte lipid levels in 7 healthy individuals, in 7 patients with primary hypercholesterolemia and in 17 patients with secondary dyslipidemia due to chronic renal failure; 10 of these patients were treated by hemodialysis (HD) and 7 patients by continuous ambulatory peritoneal dialysis (CAPD). The hypercholesterolemic patients had elevated serum levels of total cholesterol, LDL-cholesterol and apolipoprotein (apo) B, but normal plasma triglycerides. Patients on dialysis had elevated serum levels of triglycerides, serum cholesterol (CAPD only) and VLDL- and LDL-cholesterol (CAPD only) and apo B (CAPD only), whereas HDL-cholesterol and apo A-I levels (HD only) were decreased. In monocytes, we measured the content of free cholesterol (FC), cholesteryl esters (CE) and triglycerides (TG). The normal mean intracellular concentrations of FC, CE and TG were 48.3, 1.7 and 2.4 micrograms/mg cell protein, respectively. All monocyte lipid levels were similar in patients and controls, with the exception of a decreased content of FC (30.8 micrograms/mg) in monocytes of HD patients. We conclude that moderate increases in serum lipoprotein lipid levels are not associated with lipid accumulation in monocytes.

Quantification of urinary insulin-like growth factors (IGFs) and IGF binding protein 3 in healthy volunteers before and after stimulation with recombinant human growth hormone.

We examined excretion of urinary insulin-like growth factors I and II (IGF-I and IGF-II) and their major binding protein IGFBP-3 in comparison to their respective serum concentration in nine healthy female volunteers (median age 25 years, range 22-27) under baseline conditions and after stimulation with recombinant human growth hormone (rhGH), 4.5 IU twice daily subcutaneously for a period of 3 days. The IGFs were measured in unconcentrated urine by use of recently developed, highly sensitive radioimmunoassays. The IGFBP-3 was measured by a specific radioimmunoassay. The mean (+/- SD) urinary concentrations of IGF-I (0.08 +/- 0.07 micrograms/l), IGF-II (1.02 +/- 0.47 micrograms/l) and IGFBP-3 (19.1 +/- 6.9 micrograms/l) were two to three orders of magnitude lower than in serum. The ratio of IGF-II over IGF-I concentration in urine (13:1) was five times higher than in serum (2.5:1), and the ratio of IGFBP-3 over the sum of IGF-I and IGF-II in urine (17:1) was four times higher than in serum (4:1). Urinary excretion was 63.3 +/- 46.6 ng.m-2.24h-1 for IGF-I, 1002 +/- 598 ng.m-2.24h-1 for IGF-II and 18039 +/- 4983 ng.m-2.24h-1 for IGFBP-3. Using fast protein liquid exclusion chromatography, only immunoreactive IGFBP-3 components of less than 60 kD were detected in urine, with a major peak at 20 kD. Urinary IGFBP-3 excretion correlated with serum IGFBP-3 (r = 0.61, p < 0.01) and the glomerular filtration rate (r = 0.56, p < 0.05) measured by steady-state inulin infusion clearances.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult height after GH therapy in 188 Ullrich-Turner syndrome patients: results of the German IGLU Follow-up Study 2001.

OBJECTIVES: We aimed to evaluate the factors influencing true adult height (HT) after long-term (from 1987 to 2000) GH treatment in Ullrich-Turner syndrome (UTS) based on modalities conceived in the 1980s. DESIGN: Out of 347 near-adult (>16 Years) patients from 96 German centres, whose longitudinal growth was documented within KIGS (Pharmacia International Growth Database), 188 (45, X=59%; bone age >15 Years) were available for further anthropometric measurements. RESULTS: At a median GH dose of 0.88 (10th/90th percentiles: 0.47/1.06) IU/kg per week, a gain of 6.0 (-1.3/+13) cm above the projected adult height was recorded. Variables were recorded at GH start, after 1 Year GH, puberty onset, and last visit on GH therapy. At these visits, the median ages were 11.7, 12.7, 14.2, 16.6 and 18.7 Years; and median heights, 0.4, 1.1, 1.7, 1.7 and 1.3 SDS (UTS) respectively. Height gain (DeltaHT) after GH discontinuation was 1.5 cm. Total DeltaHT correlated (P<0.001) negatively with bone age and HT SDS at GH start, but positively with DeltaHT after the first Year, DeltaHT at puberty onset, and GH duration. Final HT correlated (P<0.001) positively with HT at GH start, first-Year DeltaHT, and HT at puberty onset. Body mass index increased slightly (P<0.05), with values at start and adult follow-up correlating highly (R=0.70, P<0.001). No major side effects of GH occurred. CONCLUSIONS: GH dosages conceived in the 1980s are safe but too low for most UTS patients. HT gain and height are determined by age and HT at GH start. Height gain during the first Year on GH is indicative of overall height gain. After spontaneous or induced puberty, little gain in height occurs.

Skeletal growth in experimental uremia.

Although in recent years experimental work on growth in uremia has clarified many issues, many key questions cannot be answered with available experimental data. In our own studies on subtotally nephrectomized rats, uremic animals consumed less food and grew less. However, although low energy intake diminishes growth, it has not been established that high protein energy intake will normalize growth. We showed that uremia reduced growth (and net protein synthesis) even under conditions of controlled food intake. In renal failure the optimal dietary protein level for growth or for efficiency of utilization has not been established, particularly since protein intake has an independent injurious effect on long-term renal function. Calcium and vitamin D supplements improved growth in uremic rats, but the data cannot easily be extrapolated to humans. The growth-promoting action of 1,25(OH)2D3 was not superior to that of equipotent doses of vitamin D3. Correction of anemia and physical exercise did not improve growth. Diminished stimulation of growth cartilage cyclic AMP with PTH and augmented stimulation with calcitonin was noted in uremic animals. Growth hormone in supraphysiological doses improved growth and raised IGF carrier protein in uremic animals. Spermine, a potential uremic toxin, inhibited growth cartilage 3H-thymidine incorporation, but only in concentrations higher than that encountered in uremia.