Katherine Wesseling-Perry

PATTERNS OF FGF-23, DMP1 AND MEPE EXPRESSION IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Fibroblast growth factor 23 (FGF-23), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) are skeletal proteins involved in the regulation of phosphate homeostasis and bone metabolism. Circulating FGF-23 levels are increased in patients with chronic kidney disease (CKD); however, FGF-23 skeletal expression and its regulation by DMP1 and MEPE have yet to be evaluated. Thus, expression of these three proteins was characterized by immunohistochemistry in 32 pediatric and young adult patients with CKD stages 2-5. When compared to normal controls, bone FGF-23 and DMP1 expression were increased in all stages of CKD; significant differences in bone FGF-23 and DMP1 expression were not detected between pre-dialysis CKD and dialysis patients. Bone MEPE expression in CKD did not differ from controls. FGF-23 was expressed in osteocyte cell bodies located at the trabecular periphery. DMP1 was widely expressed in osteocyte cell bodies and dendrites throughout bone. MEPE was also expressed throughout bone, but only in osteocyte cell bodies. Bone FGF-23 expression correlated directly with plasma levels of the protein (r=0.43, p<0.01) and with bone DMP1 expression (r=0.54, p<0.01) and expression of both proteins were inversely related to osteoid accumulation. Bone MEPE expression was inversely related to bone volume. In conclusion, skeletal FGF-23 and DMP1 expression are increased in CKD and are related to skeletal mineralization. The patterns of expression of FGF-23, MEPE, and DMP1 differ markedly in trabecular bone, suggesting that individual osteocytes may have specialized functions. Increases in bone FGF-23 and DMP1 expression suggest that osteocyte function is altered early in the course of CKD.

Circulating Fibroblast Growth Factor 23 in Patients with End-Stage Renal Disease Treated by Peritoneal Dialysis Is Intact and Biologically Active

CONTEXT Fibroblast growth factor 23 (FGF23) regulates phosphorus homeostasis and vitamin D metabolism. Circulating FGF23 levels are elevated in inherited and acquired hypophosphatemic disorders that can cause rickets or osteomalacia. Particularly increased concentrations of FGF23 are observed in patients with chronic kidney disease (CKD), in which increased FGF23 is associated with more rapid disease progression, improved bone mineralization, the development of left ventricular hypertrophy, and increased mortality. OBJECTIVE Our objective was to determine whether the markedly elevated levels of immunoreactive FGF23 in CKD represent accumulation of intact, biologically active hormone, C-terminal cleavage fragments, or both. DESIGN Biologically active FGF23 in plasma from CKD patients treated by peritoneal dialysis was quantified using a cell-based Egr-1 reporter assay; bioactive FGF23 levels were compared with those measured with immunometric FGF23 assays detecting either intact hormone alone or intact hormone and C-terminal fragments. SETTING AND PATIENTS Adult and pediatric patients with end-stage renal disease treated with peritoneal dialysis participated in the study at a tertiary referral center. RESULTS Serially diluted patient samples revealed levels of bioactive FGF23 that ran in parallel to CHO cell-derived recombinant human FGF23. FGF23 bioactivity was inhibited by an anti-FGF23 antibody. Levels of bioactive and immunoreactive FGF23 were tightly correlated, and Western blot analysis of FGF23 immunoprecipitated with anti-FGF23 antibodies from plasma of dialysis patients revealed only a single prominent protein band, which was indistinguishable from recombinant intact FGF23, without clear evidence for FGF23 fragments. CONCLUSIONS Our results provide strong evidence for the conclusion that virtually all circulating FGF23 in dialysis patients is intact and biologically active.

Relationship between Plasma Fibroblast Growth Factor-23 Concentration and Bone Mineralization in Children with Renal Failure on Peritoneal Dialysis

CONTEXT Fibroblast growth factor (FGF)-23 is produced in bone, and circulating levels are markedly elevated in patients with end-stage kidney disease, but the relationship between plasma levels of FGF-23 and bone histology in dialysis patients with secondary hyperparathyroidism is unknown. OBJECTIVE The aim of the study was to evaluate the correlation between plasma levels of FGF-23 and bone histology in pediatric patients with end-stage kidney disease who display biochemical evidence of secondary hyperparathyroidism. DESIGN We performed a cross-sectional analysis of the relationship between plasma FGF-23 levels and bone histomorphometry. SETTING The study was conducted in a referral center. STUDY PARTICIPANTS Participants consisted of forty-nine pediatric patients who were treated with maintenance peritoneal dialysis and who had serum PTH levels (1st generation Nichols assay) greater than 400 pg/ml. INTERVENTION There were no interventions. MAIN OUTCOME MEASURE Plasma FGF-23 levels and bone histomorphometry were measured. RESULTS No correlation existed between values of PTH and FGF-23. Bone formation rates correlated with PTH (r = 0.44; P < 0.01), but not with FGF-23. Higher FGF-23 concentrations were associated with decreased osteoid thickness (r = -0.49; P < 0.01) and shorter osteoid maturation time (r = -0.48; P < 0.01). CONCLUSIONS High levels of FGF-23 are associated with improved indices of skeletal mineralization in dialyzed pediatric patients with high turnover renal osteodystrophy. Together with other biomarkers, FGF-23 measurements may indicate skeletal mineralization status in this patient population.

Calcitriol and doxercalciferol are equivalent in controlling bone turnover, suppressing parathyroid hormone, and increasing fibroblast growth factor-23 in secondary hyperparathyroidism.

We compared the effects of calcitriol and doxercalciferol, in combination with either calcium carbonate or sevelamer, on bone, mineral, and fibroblast growth factor-23 (FGF-23) metabolism in patients with secondary hyperparathyroidism. A total of 60 pediatric patients treated with peritoneal dialysis were randomized to 8 months of therapy with either oral calcitriol or doxercalciferol, combined with either calcium carbonate or sevelamer. Bone formation rates decreased during therapy and final values were within the normal range in 72% of patients. A greater improvement in eroded surface was found in patients treated with doxercalciferol than in those given calcitriol. On initial bone biopsy, a mineralization defect was identified in the majority of patients which did not normalize with therapy. Serum phosphate concentrations were controlled equally well by both binders, but serum calcium levels increased during treatment with calcium carbonate, and serum parathyroid hormone levels were decreased by 35% in all groups. Baseline plasma FGF-23 values were significantly elevated and rose over fourfold with calcitriol and doxercalciferol, irrespective of phosphate binder. Thus, doxercalciferol is as effective as calcitriol in controlling serum parathyroid hormone levels and suppressing the bone formation rate. Sevelamer allows the use of higher doses of vitamin D. Implications of these changes on bone and cardiovascular biology remain to be established.

Suppression of Iron-Regulatory Hepcidin by Vitamin D

The antibacterial protein hepcidin regulates the absorption, tissue distribution, and extracellular concentration of iron by suppressing ferroportin-mediated export of cellular iron. In CKD, elevated hepcidin and vitamin D deficiency are associated with anemia. Therefore, we explored a possible role for vitamin D in iron homeostasis. Treatment of cultured hepatocytes or monocytes with prohormone 25-hydroxyvitamin D or active 1,25-dihydroxyvitamin D decreased expression of hepcidin mRNA by 0.5-fold, contrasting the stimulatory effect of 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D on related antibacterial proteins such as cathelicidin. Promoter-reporter and chromatin immunoprecipitation analyses indicated that direct transcriptional suppression of hepcidin gene (HAMP) expression mediated by 1,25-dihydroxyvitamin D binding to the vitamin D receptor caused the decrease in hepcidin mRNA levels. Suppression of HAMP expression was associated with a concomitant increase in expression of the cellular target for hepcidin, ferroportin protein, and decreased expression of the intracellular iron marker ferritin. In a pilot study with healthy volunteers, supplementation with a single oral dose of vitamin D (100,000 IU vitamin D2) increased serum levels of 25D-hydroxyvitamin D from 27±2 ng/ml before supplementation to 44±3 ng/ml after supplementation (P<0.001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation (P<0.05). These data show that vitamin D is a potent regulator of the hepcidin-ferroportin axis in humans and highlight a potential new strategy for the management of anemia in patients with low vitamin D and/or CKD.

Fibroblast growth factor 23 inhibits extrarenal synthesis of 1,25-dihydroxyvitamin D in human monocytes

Vitamin D is a potent stimulator of monocyte innate immunity, and this effect is mediated via intracrine conversion of 25‐hydroxyvitamin D (25OHD) to 1,25‐dihydroxyvitamin D (1,25(OH)2D). In the kidney, synthesis of 1,25(OH)2D is suppressed by fibroblast growth factor 23 (FGF23), via transcriptional suppression of the vitamin D‐activating enzyme 1α‐hydroxylase (CYP27B1). We hypothesized that FGF23 also suppresses CYP27B1 in monocytes, with concomitant effects on intracrine responses to 1,25(OH)2D. Healthy donor peripheral blood mononuclear cell monocytes (PBMCm) and peritoneal dialysate monocyte (PDm) effluent from kidney disease patients were assessed at baseline to confirm the presence of mRNA for FGF23 receptors (FGFRs), with Klotho and FGFR1 being more strongly expressed than FGFR2/3/4 in both cell types. Immunohistochemistry showed coexpression of Klotho and FGFR1 in PBMCm and PDm, with this effect being enhanced following treatment with FGF23 in PBMCm but not PDm. Treatment with FGF23 activated mitogen‐activated protein kinase (MAPK) and protein kinase B (Akt) pathways in PBMCm, demonstrating functional FGFR signaling in these cells. FGF23 treatment of PBMCm and PDm decreased expression of mRNA for CYP27B1. In PBMCm this was associated with downregulation of 25OHD to 1,25(OH)2D metabolism, and concomitant suppression of intracrine induced 24‐hydroxylase (CYP24A1) and antibacterial cathelicidin (LL37). FGF23 suppression of CYP27B1 was particularly pronounced in PBMCm treated with interleukin‐15 to stimulate synthesis of 1,25(OH)2D. These data indicate that FGF23 can inhibit extra‐renal expression of CYP27B1 and subsequent intracrine responses to 1,25(OH)2D in two different human monocyte models. Elevated expression of FGF23 may therefore play a crucial role in defining immune responses to vitamin D and this, in turn, may be a key determinant of infection in patients with chronic kidney disease (CKD).

Early Skeletal and Biochemical Alterations in Pediatric Chronic Kidney Disease

BACKGROUND AND OBJECTIVES The relationship between parathyroid hormone, fibroblast growth factor 23 (FGF-23), and indices of bone turnover and mineralization in children with early CKD is unknown; thus, this study characterizes the features of renal osteodystrophy and their relationship to biochemical markers of mineral metabolism. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Fifty-two patients 2-21 years of age with predialysis CKD underwent tetracycline-labeled bone biopsy. Anthropomorphic measurements and biochemical values were obtained at the time of biopsy. RESULTS Serum phosphorus levels were increased in 4% of patients with stage 3 CKD and 43% of those with stage 4/5 CKD. Parathyroid hormone concentrations were elevated in 36% of patients with stage 2, 71% with stage 3, and 93% with stage 4/5 CKD, whereas FGF-23 values were elevated in 81% of all patients, regardless of CKD stage. Bone turnover was normal in all patients with stage 2, but was increased in 13% with stage 3 and 29% with stage 4/5 CKD. Defective mineralization was present in 29% of patients with stage 2, 42% with stage 3, and 79% with stage 4/5 CKD. Defective skeletal mineralization was associated with lower serum calcium levels and increased parathyroid hormone concentrations. CONCLUSIONS Elevated circulating FGF-23 levels and defects in skeletal mineralization early in the course of CKD suggest that factors other than the traditional markers of mineral deficiency play a crucial role in the development of renal bone disease.

Value of the New Bone Classification System in Pediatric Renal Osteodystrophy

BACKGROUND AND OBJECTIVES Although lesions of renal osteodystrophy have traditionally been defined by bone turnover, alterations in skeletal mineralization and volume are also prevalent and may contribute to significant morbidity in patients with chronic kidney disease (CKD). The study presented here was undertaken to compare the traditional spectrum of renal osteodystrophy defined by bone turnover to a new classification system that includes T (turnover), M (mineralization), and V (volume) and to determine the value of biochemical parameters as predictors of specific TMV lesions. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Pediatric patients (n = 161) treated with peritoneal dialysis were enrolled into the study. RESULTS Increased bone turnover and abnormal mineralization were prevalent (57% and 48%, respectively); bone volume was normal or increased in all subjects. Predictive algorithms for different skeletal diagnoses were established by Classification and regression tree analysis. Serum parathyroid hormone (PTH) less than 400 pg/ml in combination with alkaline phosphatase values less than 400 IU/L provided the highest correct prediction rate for patients with both normal bone turnover and normal mineralization. Levels of PTH were higher and serum calcium levels were lower in patients with defective mineralization, irrespective of bone turnover. CONCLUSIONS Although no single biochemical marker is able to provide a complete assessment of renal osteodystrophy, a combination of serum calcium, alkaline phosphatase, and PTH levels may lead to a more precise noninvasive assessment of turnover and mineralization abnormalities in this population.

Disordered FGF23 and mineral metabolism in children with CKD.

BACKGROUND AND OBJECTIVES In children with CKD, information is limited regarding the prevalence and determinants of fibroblast growth factor 23 excess and 1,25-dihyroxyvitamin D deficiency across the spectrum of predialysis CKD. This study characterized circulating concentrations of fibroblast growth factor 23 and 1,25-dihyroxyvitamin D, and investigated their interrelationships and associations with GFR and secondary hyperparathyroidism in children with CKD who were enrolled in the Chronic Kidney Disease in Children observational cohort study. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Plasma fibroblast growth factor 23 concentrations and determinants of mineral metabolism were measured in 464 children ages 1-16 years with predialysis CKD. GFR was measured by plasma disappearance of iohexol in 70% of participants and estimated by the Chronic Kidney Disease in Children estimating equation using serum creatinine and cystatin C concentrations in the remainder of the participants. Participants were grouped according to CKD stage and by 10-ml/min categories of GFR. RESULTS Median GFR for the cohort was 45 ml/min per 1.73 m(2) (interquartile range=33-57; range=15-109). Plasma fibroblast growth factor 23 concentration was above the normal range in 67% of participants (with higher levels observed among participants with lower GFR) before higher levels of serum parathyroid hormone and phosphorus were observed. Plasma fibroblast growth factor 23 levels were 34% higher in participants with glomerular disease than in participants with nonglomerular disease, despite similar GFR. Serum phosphorus levels, adjusted for age, were significantly lower at GFR of 60-69 ml/min per 1.73 m(2) than higher GFR, but thereafter they became higher in parallel with fibroblast growth factor 23 as GFR declined. Serum 1,25-dihyroxyvitamin D concentrations were lower in those participants with low GFR values, high fibroblast growth factor 23 levels, 25-hydroxyvitamin D deficiency, and proteinuria. Secondary hyperparathyroidism was present in 55% of participants with GFR<50 ml/min per 1.73 m(2). CONCLUSION In children with predialysis CKD, high plasma fibroblast growth factor 23 is the earliest detectable abnormality in mineral metabolism, and levels are highest in glomerular diseases.

The consequences of chronic kidney disease on bone metabolism and growth in children

Growth retardation, decreased final height and renal osteodystrophy (ROD) are common complications of childhood chronic kidney disease (CKD), resulting from a combination of abnormalities in the growth hormone (GH) axis, vitamin D deficiency, hyperparathyroidism, hypogonadism, inadequate nutrition, cachexia and drug toxicity. The impact of CKD-associated bone and mineral disorders (CKD-MBD) may be immediate (serum phosphate/calcium disequilibrium) or delayed (poor growth, ROD, fractures, vascular calcifications, increased morbidity and mortality). In 2012, the clinical management of CKD-MBD in children needs to focus on three main objectives: (i) to provide an optimal growth in order to maximize the final height with an early management with recombinant GH therapy when required, (ii) to equilibrate calcium/phosphate metabolism so as to obtain acceptable bone quality and cardiovascular status and (iii) to correct all metabolic and clinical abnormalities that can worsen bone disease, growth and cardiovascular disease, i.e. metabolic acidosis, anaemia, malnutrition and 25(OH)vitamin D deficiency. The aim of this review is to provide an overview of the mineral, bone and vascular abnormalities associated with CKD in children in terms of pathophysiology, diagnosis and clinical management.