Central and peripheral forms of C-type natriuretic peptide (CNP): evidence for differential regulation in plasma and cerebrospinal fluid

Aminoterminal proCNP (NTproCNP), a stable product of CNP gene expression and readily measured in human plasma, provides a new approach to studies of CNP which is rapidly degraded at source. CNP is detectable in human CSF but the presence and proportions of NTproCNP in CSF are unknown. Since CNP is widely expressed throughout the CNS, we hypothesized that the ratio of NTproCNP to CNP in CSF is greatly increased when compared to plasma and that CSF CNP peptides may contribute to their concentrations in the systemic circulation. Concurrent plasma and CSF concentrations of CNP forms were measured in 51 subjects undergoing spinal anesthesia for arranged orthopedic procedures. Elevated concentrations of NTproCNP (1045 ± 359 pmol/L), characterized by HPLC-RIA, were found in CSF and greatly exceeded those of CNP (7.9 ± 3.2 pmol/L). The ratio of NTproCNP to CNP in CSF (145 ± 55) was much higher than in plasma (31 ± 27). A significant inverse relation was found between plasma and CSF CNP concentrations (r = −0.29, p < 0.05). cGMP and neprilysin were unrelated to CNP levels in CSF. We conclude that CNP is differentially regulated across the brain in normal health. Despite markedly elevated levels of NTproCNP in CSF, it is unlikely that these contribute to systemic levels in healthy adults. Identifying NTproCNP as the dominant CNP form in CSF opens up the possibility of its use in future studies exploring CNP regulation within the CNS and possible applications in the diagnosis and monitoring of subjects with central neural disorders.     

Response of plasma CNP forms to acute anabolic and catabolic interventions in growing lambs

Using a novel marker of C-type natriuretic peptide (CNP) synthesis [amino-terminal pro-CNP (NT-proCNP)], we have recently shown that plasma NT-proCNP is strongly correlated with skeletal growth and markers of bone formation and is reversibly reduced by glucocorticoids. The effects on CNP of other catabolic or anabolic factors, known to affect skeletal growth, are unknown. Accordingly, we have studied the response of plasma CNP forms to acute catabolic (caloric restriction) and anabolic [growth hormone (GH) stimulation] interventions in lambs and related the findings to circulating IGF-I levels, growth velocity, and markers of bone formation. Lambs fed a reduced caloric intake (25% of normal) for 6 days exhibited reduced live weight, plasma urea, and IGF-I (P < 0.001 for all) compared with control lambs. Basal levels of NT-proCNP (40.1 +/- 0.9 pmol/l) fell promptly to a nadir (28.1 +/- 0.8 pmol/l, P < 0.001) on day 6, returning rapidly to basal levels upon refeeding. Although plasma alkaline phosphatase (ALP) fell (P < 0.001), reductions in metacarpal growth velocity were not significant within the 12-day period of study. In contrast to caloric restriction, long-acting bovine recombinant GH (2.5 mg/kg on days 0 and 6), as expected, increased plasma IGF-I more than twofold above control for 12 days (P < 0.001). Growth velocity did not differ during the 30 days of observation, and, consistent with unchanged growth velocity, plasma NT-proCNP and ALP were also unaffected. In conclusion, CNP synthesis and markers of bone formation are acutely sensitive to catabolism but unaffected by doses of GH that fail to stimulate skeletal growth.     

Growth after radiotherapy and chemotherapy in children with leukemia or lymphoma.

The effect of radio- and chemotherapy on auxological parameters was investigated in 30 children treated for acute lymphatic leukemia (ALL) or non-Hodgkins lymphoma (NHL). Growth velocity was decreased during the first year of treatment. Catch-up growth was insufficient during the following years. Thus, the whole group experienced a loss of height of 0.49 +/- 1.1 SD at 6.8 +/- 2.6 years after diagnosis. Height and growth velocity were not different between children who received 18 or 24 Gy cranial irradiation; however, growth velocity was significantly lower in children who were treated for more than 2 years or who had the more intensive chemotherapeutic protocol. Evaluation of the growth hormone (GH) response to pharmacological stimulation revealed reduced GH peaks in 47% of the patients, but there was no correlation of GH peak with growth or treatment parameters. In conclusion, the impairment of growth in children after treatment for ALL or NHL might be related to the intensity and duration of chemotherapy.     

Coenzyme Q10 concentration in the plasma of children suffering from acute lymphoblastic leukaemia before and during induction treatment

Coenzyme Q10 (CoQ10) is used by the body as an endogenous antioxidant. This property combined with its essential function in mitochondrial energy production suggests that it may have therapeutic potential in cancer treatment. As part of the body's antioxidant defence against free radical production, CoQ10 concentrations may change during anti-cancer chemotherapy. Our study measured CoQ10 concentration in the plasma of 27 children with acute lymphoblastic leukaemia (ALL) at the time of diagnosis, during induction (protocol ALL-BFM 2000), and post induction treatment. The starting values were compared to the CoQ10 concentrations in 92 healthy children. The total CoQ10 concentration and its redox status were measured by HPLC using electrochemical detection and internal standardisation. While the CoQ10 concentration in the plasma of children with ALL was within a normal range at the time of diagnosis (0.99 +/- 0.41 pmol/microl), a drastic increase was observed during induction treatment (2.19 +/- 1.01 pmol/mul on day 33). This increase was accompanied by shift in the redox status in favour of the reduced form of CoQ10. The increase in CoQ10 concentration during induction treatment may be attributed to the activation of a natural antioxidative defence mechanism, endocrine influence on CoQ10 synthesis from steroid treatment, or a shift in CoQ10 from the damaged cells to the plasma after cell lysis.     

Plasma C-Type Natriuretic Peptide as a Predictor for Therapeutic Response to Metoprolol in Children with Postural Tachycardia Syndrome

POTS is a global public-health disease, but predictor for therapeutic response to metoprolol in children with POTS is lacking. This study was designed to investigate predictive value of plasma C-type natriuretic peptide (CNP) in the therapeutic efficacy of metoprolol on postural tachycardia syndrome (POTS) in children. Totally 34 children with POTS and 27 healthy children were included in the study. The head-up test or head-up tilt test was used to check heart rate and blood pressure from supine to upright in subjects. A double antibody (competitive) sandwich immunoluminometric assay was used to detect plasma CNP. Metoprolol was used to treat children with POTS. The difference in plasma concentrations of CNP between responders and non-responders was compared. An ROC curve was used to analyze plasma CNP to predict efficacy of metoprolol on POTS in children. Plasma CNP in children with POTS was significantly higher than that of healthy children [(51.9 ± 31.4) vs. (25.1 ± 19.1) pg/ml, P <0.001]. Plasma CNP in responders to metoprolol was significantly higher than non-responders [(59.1 ± 33.5) vs. (34.8 ± 16.7) pg/ml, P = 0.037] before treatment. The ROC curve showed that area under the curve was 0.821 (95% CI 0.642–0.999). The cut-off value of plasma CNP > 32.55 pg/ml yielded a sensitivity of 95.8% and specificity of 70% in predicting therapeutic efficacy of metoprolol on POTS children. Plasma CNP might serve as a useful predictor for the therapeutic efficacy of metoprolol on POTS in children.     

Determination of selenium in blood serum of children with acute leukemia and effect of chemotherapy on serum selenium level.

The concentration of selenium in serum was measured by the neutron activation method in three groups of children: 30 healthy children, 20 children with Acute Myeloblastic Leukemia (AML) and 40 with Acute Lymphoblastic Leukemia (ALL) (L1; n = 20, L2; n = 20). The samples were taken before and after induction chemotherapy. Age, sex, FAB, initial WBC, BUN, creatinine and urinary analysis did not show a significant change in the amount of selenium in serum. Selenium concentration in serum samples of ALL children before chemotherapy showed no significant differences as compared with that of normal individuals, but there were significant differences between children with AML and normal individuals (76.46 +/- 24.59 micrograms/L vs 102.38 +/- 19.25 micrograms/L, with p < 0.02). In conclusion, the question of whether these deficiencies are responsible for the disease or are the result of a secondary effect of the cancer remain to be answered. Immediately after induction chemotherapy, the selenium concentration in the serum of ALL children decreased significantly (80.14 +/- 15.48 micrograms/L vs 110.72 +/- 28.3 micrograms/L, p < 0.001), but this was not the case for AML children. These findings may be due to the difference in the drugs administered in induction chemotherapy of ALL and AML children.     

Plasma C-type natriuretic peptide levels in healthy children

C-type natriuretic peptide (CNP) is assuming increasing importance in cardiovascular disease, and in adults its plasma levels are related to clinical and functional disease severity. Data are scarce regarding the reference values for CNP in infancy. Aim of this study was to assess the reference intervals for CNP in human healthy newborns and infants. Plasma CNP was measured in 121 healthy children divided into: 41 newborns (age 0-3 days), 24 newborns (4-30 days), 22 infants (1-12 months) and 32 children (1-12 years). A group of 32 healthy adult subjects (age 64 ± 1 years) was also studied. CNP was measured by a specific radioimmunoassay. Between- and within-assay variability resulted ≤ 30 and 20%, respectively and analytical sensitivity 0.77 ± 0.05 pg/tube. Plasma CNP resulted significantly higher in children than in adult subjects (13.6 ± 1.2 pg/ml vs. 7.4 ± 1.0 pg/ml, p=0.030). When the results were analyzed as a function of the age the reference intervals for plasma CNP resulted: 11.6 ± 2.1 pg/ml for newborns (0-3 days), 16.4 ± 3.7 pg/ml for newborns (4-30 days), 15.4 ± 2.7 pg/ml for infants (1-12 months), 13.6 ± 2.3 pg/ml for children (1-12 years) [p=0.01 newborns (4-30 days) vs. adults; p=0.03 infants (1-12 months) vs. adults]. CNP showed the highest concentrations after 12h of life with a peak between 4 and 5 days of life and with a progressive decline afterwards. According to these data at least five different reference intervals for CNP determinations should be used. These observations may be helpful for future clinical application of CNP in human children.     

Transferrin, somatomedins and growth

Plasma transferrin is significantly positively correlated to the growth velocity in normal children and children with various growth disorders. In hypopituitary dwarfs, both plasma transferrin and somatomedin are significantly lower than in controls. Acute variations of the plasma transferrin levels could be involved in the regulation of the GH secretion by a feedback mechanism. Yet, transferrin is neither a somatomedin or a somatomedin-binding protein, and the mechanisms relating growth to transferrin remain unknown.     

Treatment of juvenile rheumatoid arthritis with growth hormone

Severe growth retardation and profoundly altered body composition are observed in children with juvenile chronic arthritis receiving glucocorticoids. This study assessed the effects of growth hormone (GH) on height velocity, body composition and bone density. Fourteen patients were treated with GH (1.4 U/kg/week) for 1 year and then studied for a 2nd year off GH. The treatment increased insulin-like growth factor 1 and insulin-like growth factor binding protein 3 plasma levels. All patients showed an increase in height velocity. Lean body mass increased by 12%. After the cessation of GH therapy, height velocity fell to pretreatment values, and weight and fat mass increased markedly. Bone formation and resorption markers significantly increased during treatment and returned to pretreatment values after discontinuation of GH treatment. These results suggest that GH may partially counteract the adverse effects of glucocorticoids on growth and metabolism in patients with chronic inflammatory disease.     

Genotype-Phenotype Correlation of 2q37 Deletions Including NPPC Gene Associated with Skeletal Malformations

Coordinated bone growth is controlled by numerous mechanisms which are only partially understood because of the involvement of many hormones and local regulators. The C-type Natriuretic Peptide (CNP), encoded by NPPC gene located on chromosome 2q37.1, is a molecule that regulates endochondral ossification of the cartilaginous growth plate and influences longitudinal bone growth. Two independent studies have described three patients with a Marfan-like phenotype presenting a de novo balanced translocation involving the same chromosomal region 2q37.1 and overexpression of NPPC. We report on two partially overlapping interstitial 2q37 deletions identified by array CGH. The two patients showed opposite phenotypes characterized by short stature and skeletal overgrowth, respectively. The patient with short stature presented a 2q37 deletion causing the loss of one copy of the NPPC gene and the truncation of the DIS3L2 gene with normal CNP plasma concentration. The deletion identified in the patient with a Marfan-like phenotype interrupted the DIS3L2 gene without involving the NPPC gene. In addition, a strongly elevated CNP plasma concentration was found in this patient. A possible role of NPPC as causative of the two opposite phenotypes is discussed in this study.     

Prediction of advanced puberty by height velocity in children with acute lymphoblastic leukemia.

Pubertal development was retrospectively evaluated in 58 children with cancer, mostly acute lymphoblastic leukemia (ALL), who are in complete remission and off chemotherapy. Six girls [5 patients with ALL, and 1 with malignant lymphoma (NHL)] showed advanced puberty (25.0%, 6 of 24 female patients with ALL and NHL) through the evaluation of their growth velocity. No evidence for advanced puberty was seen in the males. All 6 girls had received cranial irradiation for central nervous system prophylaxis and systemic chemotherapy including glucocorticoid. The mean age at onset of the pubertal growth spurt in these 6 girls was significantly lower than for girls with a solid tumor [6.90 +/- 0.10 and 9.00 +/- 0.77 (mean +/- SD) years, respectively (p < 0.01)]. By simply evaluating the height velocity, we could predict advanced puberty which was ultimately associated with short stature.     

CFU-gm colony formation of peripheral blood and bone marrow in adult acute leukemia at presentation, during remission, and at relapse

Granulocyte/macrophage colony-forming unit (CFU-gm) formation was studied simultaneously in bone marrow and peripheral blood of 52 previously untreated adult patients with acute non-lymphocytic (ANLL) and 36 with acute lymphoblastic leukemia (ALL). They were followed during induction therapy at monthly intervals while in remission and in 19 ANLL and 22 ALL cases, until relapse. Patients showing a decreased colony number in the marrow but normal or increased colony numbers in the peripheral blood had a high probability of entering remission. Non-responding patients displayed an opposite pattern. The higher the degree of marrow repopulation with granulocytic progenitor cells after induction treatment, the longer remission duration and survival for ANLL patients and the longer survival for ALL patients. CFU-gm formation returned to normal in the early stages of complete remission, but then declined progressively. At ANLL and ALL relapse, colony growth was reduced markedly while cluster formation remained normal. The number of marrow colonies and clusters in ANLL were significantly higher at first and second relapse compared to the growth pattern at first presentation. A similar trend had been observed in ALL, suggesting a selection advantage.     

Selenium status of healthy immigrant parisian preschool children

Plasma selenium (Se) concentration and erythrocyte glutathione peroxidase activity (GPx) were assessed in a population of healthy preschool children two to five years old, residing in the city of Paris. In the 118 subjects, mean (±SD) plasma Se concentration was 62.10 ±13.96 μg/L, and mean GPx activity was 23.58±8.52 U/g Hb. Mean plasma Se of male children was significantly (p=0.001) higher (12%) than levels of girls. Plasma selenium levels were not correlated with erythrocyte GPx activity. Children from Mediterranean origin had a slightly lower erythrocyte GPx activity (p<0.05) than children from other regions. Mean plasma Se concentration of this group corresponded to the lower limit of intervals, which characterizes geographical regions of intermediate selenium concentrations.     

Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis

Although recent major advances have developed a much better understanding of the pathophysiological pathways, tubulointerstitial fibrosis (TIF) is still currently incurable. Therefore, early detection may mean that the condition is more manageable than it was in the past. C-type natriuretic peptide (CNP) has been found to be a potent vasodilator but a weak natriuretic factor. In addition, CNP has also been believed to be produced in tubular cells and presented as a local modulator with anti-inflammatory and anti-proliferative effects. Elimination of CNP occurs by three main mechanisms, neutral endopeptidase, natriuretic peptide receptor-C and urinary excretion. Among them, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. In the present study, subgroups of rats were subjected to unilateral ureteral obstruction (UUO) or sham operation and observed for 24 h to 3 months. Urinary CNP excretion was significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to sham-operated rats. Urinary CNP excretion was also markedly higher than CNP concentrations both in abdominal aorta and in renal vein, and almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin. Urinary CNP excretion was negatively correlated with urinary protein concentration, blood urea nitrogen and creatinine, while positively correlated with albumin. In conclusion, the increased urinary CNP excretion is strongly associated with TIF progression, and may serve as an early marker of TIF.     

Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment

Kisspeptin is a 54-amino acid peptide, encoded by the anti-metastasis gene KiSS-1, that activates G protein-coupled receptor 54 (GPR54). The kisspeptin-GPR54 system is critical to normal reproductive development. KiSS-1 gene expression is increased in the human placenta in normal and molar pregnancies. Circulating kisspeptin is dramatically increased in normal pregnancy, but levels in GTN have not previously been reported. The present study was designed to determine whether plasma kisspeptin levels are altered in patients with malignant GTN. Thirty-nine blood samples were taken from 11 patients with malignant GTN at presentation during and after chemotherapy. Blood was also sampled from nonpregnant and pregnant volunteers. Plasma kisspeptin IR and hCG concentrations were measured. Plasma kisspeptin IR concentration in nonpregnant (n = 16) females was <2 pmol/l. Plasma kisspeptin IR in females was 803 +/- 125 pmol/l in the first trimester of pregnancy (n = 13), 2,483 +/- 302 pmol/l in the third trimester of pregnancy (n = 7), and <2 pmol/l on day 15 postpartum (n = 7). Plasma kisspeptin IR and hCG concentrations in patients with malignant GTN were elevated at presentation and fell during and after treatment with chemotherapy in each patient (mean plasma kisspeptin IR: prechemotherapy 1,363 +/- 1,076 pmol/l vs. post-chemotherapy <2 pmol/l, P < 0.0001; mean plasma hCG: prechemotherapy 227,191 +/- 152,354 U/l vs. postchemotherapy 2 U/l, P < 0.0001). Plasma kisspeptin IR strongly positively correlated with plasma hCG levels (r(2) = 0.99, P < 0.0001). Our results suggest that measurement of plasma kisspeptin IR may be a novel tumor marker in patients with malignant GTN.     

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells

Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10^?8 and 10^?7 M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10^?6 M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect.     

Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice

Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs.     

Leukaemic peripheral blood plasma and bone marrow plasma: comparison of influence on lymphocyte proliferation

Abstract. Peripheral blood plasma from some children with untreated acute lymphoblastic leukaemia (ALL) exerted an inhibitory effect in vitro on phytohaemagglutinininduced lymphocyte transformation of normal peripheral blood lymphocytes. This occurred at concentrations beyond that required for optimal response as judged by reduction of blast cell formation and tritiated thymidine and tritiated uridine incorporation into DNA and RNA, respectively. In contrast, bone marrow plasma from these patients was non-inhibitory or contained significantly less inhibitory activity. Bone marrow plasma from the majority of healthy controls was superior to their peripheral blood plasma in enhancing phytohaemagglutinin-induced mitogenesis. The difference between an individual's bone marrow- and peripheral blood-derived plasma in enhancing proliferation of patient and healthy control cells was significantly greater amongst the patients than the healthy control group; this was attributed mainly to the increased inhibitory activity of ALL peripheral blood plasma compared with normal plasma. Medium conditioned by phytohaemagglutinin-stimulated normal peripheral blood lymphocytes was effective in neutralizing the inhibitory activity of ALL peripheral blood plasma. Taken together, these in vitro results are at least suggestive that in vivo , in healthy subjects, the rapidly proliferating cells in the bone marrow and the 'resting' blood cells in the circulation may be under the influence of a fine balance of different types and/or levels of humoral growth stimulatory and inhibitory factors and that in ALL an unstable balance of these factors exists. The decreased proliferation of circulating blast cells compared with bone marrow blasts in ALL may be attributed, at least in part, to exposure to the different levels of inhibitor(s) in the circulation and bone marrow as demonstrated in vitro by our results.     

Effect of human growth hormone on adrenal androgens in children with growth hormone deficiency

The effect of human growth hormone (hGH) on adrenal androgen secretion was assessed in 7 patients (5 males, 2 females) with GH deficiency but normal ACTH-cortisol function. Patients ranged in age from 9 5/12 to 14 8/12 years (median 12 years). Plasma concentrations of dehydroepiandrosterone-sulfate (DHEA-S) and urinary excretion of 17-ketosteroids (17-KS) and free cortisol were determined before, during short-term (2 U/day X 3) and after long-term (6 months) treatment with hGH. No significant change was noted in the plasma concentration or urinary excretion of steroids during the short-term administration of hGH. Despite a significant increase in growth velocity during 6 months of hGH therapy (8.2 vs. 4.5 cm/year, p less than 0.01), the plasma concentrations of DHEA-S and the urinary 17-KS and free cortisol levels were unchanged. These results fail to substantiate a role for hGH in the physiologic control of adrenal androgen secretion. Thus, the low plasma levels of adrenal androgens sometimes seen in GH-deficient patients are not due to the absence of GH per se.     

Therapeutic effect of CNP on renal osteodystrophy by antagonizing the FGF-23/MAPK pathway

Renal osteodystrophy (ROD) is highly prevalent in chronic kidney disease (CKD). Because most patients with ROD are asymptomatic in the early stage and bone biopsy remains not a routine procedure in many clinical settings; therefore, several biochemical parameters may help to identify the existence of ROD. C-type natriuretic peptide (CNP) is considered as a positive regulator of bone formation. Both urinary excretion and renal expression of CNP are markedly up-regulated in the early stages of CKD, whereas they are still progressively declined accompanied by CKD progression, which invites speculation that the progressive decline of CNP may contribute, in part, to the pathogenesis of ROD. In addition, fibroblast growth factor (FGF)-23 is a bone-derived endocrine regulator of phosphate homeostasis. The elevation of serum FGF-23 has been recognized as a common feature in CKD to maintain normophosphatemia at the expense of declining 1,25-dihydroxyvitamin D values. Since the effects of CNP and FGF-23 on bone formation appear to oppose each other, it is reasonable to propose a direct interaction of their signaling pathways during the progression of ROD. CNP and FGF-23 act through a close or reciprocal pathway and are in agreement with recent studies demonstrating a down-regulatory role of the mitogen-activated protein kinase activity by CNP. The specific node may act at the level of RAF-1 through the activation of cyclic guanosine monophosphate-dependent protein kinases II.