Humoral factor in children with neonatal Bartter syndrome reduces bone calcium uptake in vitro

Abstract

The neonatal Bartter syndrome (NBS) is associated with a complex disorder of mineral metabolism in children, including hypercalciuria, nephrocalcinosis, and diminished bone mineral density. Although cyclooxygenase inhibition usually brings about improvement in these findings, there is a variable component which is resistant to such therapy in many children. The factor mediating this disorder has not been identified. Blood and urine from 12 children with NBS were examined. When compared with samples from normal children and adults, all (NBS) sera reduced bone calcium uptake in a bone disc bioassay. This effect persisted in the presence of parathyroid hormone (PTH) antibody and PTH receptor blockade, indicating that neither PTH nor PTH related peptide was responsible. It was eliminated by indomethacin, suggesting that prostanoid generation was essential. Protamine was also inhibitory, as was the addition of ecteola, an anion binder. Activity could be recovered from ecteola by elution with hypertonic buffer. Urine samples from children with NBS had the same calcitropic effect. The agent was removed by ecteola and recovered by hypertonic elution. Activity was eliminated by protamine and by heparinase, but not by trypsin digestion. Size exclusion centrifugation showed that the activity was associated with a material between 10 and 30 kilodaltons. Finally, urine ecteola eluates from NBS patients raised serum concentrations of calcium after intraperitoneal injection in rats. These data suggest that children with NBS have a calcitropic substance in their serum and urine which is not found in normal individuals. The substance is heparin like, and mediates its effects through prostanoid production. These studies provide additional evidence against a direct renal cause of the urinary calcium disturbance characteristic of the disorder.

Angiotensin II reduces calcium uptake into bone

“…Recently, we have shown that this bone resorption is associated with the presence of a factor that enhances bone resorption in an in vitro system and produces hypercalcemia in vivo when given intraperitoneally to rats [3]. This factor is a complex of b-FGF and a glycosaminoglycan, and increased bone resorption in vitro and in vivo with b-FGF has been shown by others [10].…”

“…A major thrust of our efforts over the past several years has been to understand the calcium disorder in NBS, characterized by massive hypercalciuria, nephrocalcinosis, and osteopenia [3]. In one of our earliest studies, we showed that untreated children with NBS had significant elevations in 1,25-dihydroxycholecalciferol [8], with corresponding evidence of enhanced enteric calcium absorption.…”

“…This experiment/control (E/C) ratio has a direct relationship to calcium uptake of bone, decreasing in the presence of factors that increase bone resorption. For example, an E/C of 0.57 has been reported with addition of 200 pg/ml parathyroid hormone to the initial incubation media [3]. Testing of 29 sera from healthy persons showed a mean E/C=1.0, and no normal individual had an E/C less than 0.90 [3].…”

“…The bone disc bioassay for calciotropic activity was performed by a described method [3]. Briefly, discs prepared from the calvaria of newborn Sprague-Dawley rats were incubated for 4 h at 38C in 0.25 ml of phosphate buffered saline (PBS, 2 mM) (KH 2 PO 4 / K 2 HPO 4 , 1:4) in 140 mM NaCl, pH 7.38, or PBS with 10 -8 M, 10 -9 M, and 10 -10 M AT II (Sigma, St. Louis, Mo., USA).…”

“…From the 1st day of life, these children have massive hypercalciuria, and quickly develop nephrocalcinosis and osteopenia [2]. Studies in our laboratory using a bone disc bioassay system have demonstrated a factor in the serum and urine of children with NBS that increases bone resorption [3,4]. Fractionation studies have identified it as a complex of basic-fibroblast growth factor (b-FGF) and a naturally occurring heparin-like glycosaminoglycan [4].…”