Phosphate transport by fibroblasts from patients with hypophosphataemic vitamin D-resistant rickets B.

Escoubet, C. Silve, S. Balsan and C. Amiel

INSERM U251 and Département de Physiologie, Faculté de Médecine X Bichat, Université Paris 7, France *CNRS URA 583, Faculté de Médecine Necker-Enfant-Malades, Université Paris 5, Paris, France revised manuscript received

Paris,

15 November 1991

ABSTRACT

It is accepted that renal phosphate wasting is the basis of hypophosphataemia in vitamin D-resistant hypophosphataemic rickets (VDRR). Abnormal renal adaptation to phosphate deprivation has also been reported in these patients. We studied sodium\x=req-\ dependent phosphate transport and its modulation by phosphate deprivation in skin fibroblasts cultured from healthy subjects and patients with VDRR. Control fibroblasts exhibited high-affinity sodium\x=req-\ dependent phosphate transport (77 \m=+-\12 \g=m\mol/l)which resembled the ubiquitous transport of renal and non\x=req-\ renal cells. Phosphate deprivation (incubation in low phosphate medium) increased the maximal velocity (Vmax) of the transport by 2\m=.\7-foldafter 24 h, with no change in the affinity. The increase in Vmax was dependent on gene transcription and protein synthesis. The sodium-dependent phosphate transport exhibited in fibroblasts from VDRR patients did not significantly differ from that of control subjects, except that the Vmax of the phosphate transport was higher in

INTRODUCTION

Vitamin D-resistant hypophosphataemic rickets (VDRR), first described by Albright, Butler & Bloomberg (1937), is the most common form of vitamin D-resistant rickets (Rasmussen & Tenenhouse, 1989). The primary defect responsible for the hypophosphataemia of affected subjects is unknown. It is accepted, however, that decreased renal reabsorption of phosphate is the basis of the low plasma phosphate concentration observed in these patients (Glorieux & Scriver, 1972; Scriver, 1974; Thakker & O'Riordan, 1988; Rasmussen & Tenenhouse, 1989). I. deed, studies conducted in a murine homologue of the human disease, the Hyp mouse (Eicher, Southard, Scriver & Glorieux, 1976), have localized the renal phosphate leak to the proximal tubular cells of the kidney

cells from

patients with VDRR under normal and phosphate-deprivation conditions, although the difference was significant only after 24 h of phosphate deprivation (Vmax: 22\m=.\6\m=+-\2\m=.\4pmol/mg protein per s in VDRR vs 16 \m=+-\3\pmol/mg m=.\6 protein per s in controls, P < 0\m=.\05). These data demonstrate that sodium-coupled phosphate transport in human skin fibroblasts has

the

properties

of

ubiquitous sodium-phosphate

co\x=req-\

transport and show that this transport is not deficient

in

patients with VDRR. Indeed paradoxically the Vmax was 40% higher in VDRR than in control subjects after 24 h of phosphate deprivation. The transport must be either different from that of kidney cells responsible for the phosphate leak, or differently

modulated. Therefore, skin fibroblasts cannot be used to determine the molecular defect responsible for the renal phosphate leak in VDRR patients. Journal of Endocrinology (1992) 133, 301\p=n-\309

with

a decreased maximal velocity (Vmax) of the apical sodium-phosphate co-transport (Tenenhouse, Klugerman & Neal, 1989). More recently an abnor¬ mal protein has been identified by two-dimensional gel electrophoresis of brush border membrane vesicle proteins from the Hyp mouse (Ford & Molitoris, 1991) and by injection of mRNA prepared from Hyp mouse kidney in occytes which led to an abnormal phosphate uptake (Nagawaka, Arab & Ghishan, 1991). Although these studies support the hypothe¬ sis that the abnormality involved in the phosphate leak is localized to the proximal tubular cells, they do not allow a characterization of the primary de¬ fect. In particular, it is still not known if the defect

resides in the transporter itself or in involved in its regulation by hormonal factors.

a or

factor local

An alteration of the cellular mechanisms of to phosphate deprivation could be involved in this disease, since a defect in the renal response to phosphate deprivation has been described in patients with VDRR (Insogna, Broadus & Gertner, 1983), and in the Hyp mouse (Muhlbauer, Bonjour & Fleisch, 1982), with a reduced stimulation of phos¬

adaptation

phate reabsorption. In addition, sodium-dependent phosphate uptake was not stimulated after phosphate deprivation in primary cultures of Hyp mouse kidney cells (Kinoshita, Fukase, Nakada & Fujita, 1987). Studies in VDRR patients are hampered by the relative inaccessibility of the cells likely to express the defect. Indeed, although the existence of an associated defect in the activity of other cells, in particular intestinal (Short, Binder & Rosenberg, 1973; Meyer, Meyer, Gray & Bruns, 1987; Brault, Meyer & Meyer, 1988), bone (Ecarot-Charrier, Glorieux, Travers et al. 1988) and tooth cells (Shields, Scriver, Reade et al. 1990) has been suggested, these cells are not more readily accessible for study in patients than proximal tubular cells. These observations raise the possibility, however, that the abnormality of phosphate transport responsible for the disease is expressed in cell types other than renal.

Sodium-dependent phosphate transport has been found in most cell types (Caverzasio, Brown, Biber et al. 1985; Escoubet, Djabali & Amiel, 1989) including fibroblasts (Hilborn, 1975; Greenberg, Barsh, Ho & Cunningham, 1977; Lever, 1978; Kemp, Khouja, Bevington et al. 1989). In addition, adaptation of sodium-phosphate co-transport

to

phosphate depri¬

vation has been demonstrated in cultured renal (Caverzasio et al. 1985; Escoubet et al. 1989) and non-renal (Escoubet et al. 1989) cells, suggesting that human fibroblasts could provide a model for the study of sodium-phosphate co-transport including its

adaptation to phosphate deprivation. Since fibroblasts are relatively easily obtained in human subjects, the present study was undertaken to test the hypothesis that abnormal sodium-phosphate table

co-transport is exhibited in skin fibroblasts from

VDRR patients, compared with control subjects, thus providing a tool to characterize the defect present in VDRR patients. Part of this study was presented by the authors at the Meeting of the American Society of Nephrology, Washington, 1990. PATIENTS AND METHODS

Patients were personally evaluated and treated of us (S.B.), and two (patients 3 and 4) were referred for medical counselling. All showed clinical, biochemical and radiological features of VDRR. X-linked dominant transmission was demonstrated in three cases (Table 1). Patient 4 was the son of patient 3. All patients had hypophosphataemia caused by an isolated renal tubular defect in the reabsorption of phosphate. No associated tubular dysfunction was present, as judged by the absence of glycosuria, aminoaciduria and hyperchloraemic acidosis. All patients were normocalcaemic and hypophosphataemic, and simultaneous measurement of urinary calcium demonstrated that none had hypercalciuria. At diagnosis of the disease, all patients had radio¬ logical lesions of rickets and/or osteomalacia with femoral bowing. The age, sex, age at diagnosis and biochemical data at the time of the skin biopsy are listed in Table 1. All patients were treated with la-hydroxyvitamin D (Leo Laboratories, Montigny Le Bretonneux, France), given as a single morning dose

Four

by

patients

one

before breakfast, and phosphate supplement (Phos¬ phate Sandoz, Sandoz Laboratories, Switzerland) in four divided dosages. Seven healthy subjects (four males aged 1, 2, 29 and 34 years and three females aged 1, 2-5 and 25 years) who had to undergo surgery served as controls. None had abnormal calcium or phosphate metabolism and none were immobilized before the time of surgery.

1. Clinical and metabolic features of the patients Patients

Normal range

1

Age (years) Sex

F

Diagnosis age

2y7m

sP(mmol/l)

sp 2-37 0-75

Transmission sCa (mmol/1)

11 F

39 F

10 M

16 M

34 M

17m sp 2-37 0-75

26y

8y

2y3m

3y

X

2-35 0-8

X 2-26 0-8

sp

2-32 0-6

X 2-28 0 75

2-2-2-6 a: 0-8-1-5 c: 112

Abbreviations: F, female; M, male; y, year; m, month; sp, sporadic; X, X-linked; a, adults; c, children; s, serum; Ca, calcium;

P, phosphate.

Skin

biopsies

Informed consent was obtained from all patients and/or their parents. Skin biopsies were obtained at the time of necessary surgery for control subjects and by punch biopsy from the upper arm for VDRR patients. Biopsies were placed directly in the complete culture medium: Dulbecco's minimum essential medium (DMEM) supplemented with 100 U penicil¬ lin G/ml, 50 µg streptomycin/ml, 1 mmol glutamine/1 and 10% (v/v) fetal calf serum. Fibroblast cell lines were established as previously described (Silve, Santora, Breslau et al. 1986). Briefly, biopsies were minced and incubated overnight at room temperature in medium supplemented with 2 mg collagenase/ml (Sigma Chemical Co., St Louis, MO, U.S.A.). The medium containing collagenase was removed on the next day and the fragments were cultured in complete medium in 25 cm2 Costar culture flasks. Fibroblasts migrated from the skin expiants and were allowed to grow for 2-3 weeks. Cells were then passaged at a dilution of 1 to 4 on a weekly basis. Experiments were performed between passages four and ten. For the experiments, monolayers were trypsinized and the cells counted and replated on 24-well dishes (15 000 cells/cm2) containing 0-5 ml complete medium. Cells were grown at 37 °C in an atmosphere of 5% C02. Medium was changed twice a week. Cultures reached confluence within 6-7 days. Experiments were per¬ formed on day 8 after plating. The confluence of control and VDRR cells was similar as evaluated by the protein content per well (44-9 + 5 pg in controls vs 40-6 ± 7 pg in VDRR, 25) and by the number of cells per cm2 (36 407 + 28720, « 20 in controls and 37 870 + 2929, n= 18 in VDRR, means ± s.e.m.).

uptake in choline chloride medium did not vary sig¬ nificantly with the different experimental conditions and was always below 5% of that in the NaCl medium. Unless otherwise stated, uptake in choline chloride medium was subtracted from the uptake in NaCl medium.

Kinetics of sodium-dependent phosphate transport The uptake of phosphate was measured for 10 min in medium containing phosphate concentrations of 10, 50, 100, 200 and 350pmol/l. Apparent affinity (ÄJ and maximal velocity (Vmax) values were calculated by Eadie-Hofstee transformation.

Phosphate deprivation Cells

were

washed and incubated in DMEM

con¬

taining phosphate concentrations from 0 (phosphate deprivation) to 2mmol/l (control cells). Phosphate uptake was measured thereafter as described above. Sodium-dependent phosphate uptake did not change significantly after incubation in DMEM without fetal calf serum for 30 min to 24 h (see Fig. 3). Alanine

uptake

Uptake of alanine was measured after 5 min incubation in the medium described for phosphate uptake except that 01 mmol L-alanine/1 replaced KH2P04 and the medium was supplemented with 1 pCi L-[3H]alanine/ml (Amersham International pic, Amersham, Bucks, U.K.; 47Ci/mmol).

=

=

Phosphate uptake The uptake of phosphate (P¡) by attached cells was measured as described by Biber, Brown & Murer (1983). Monolayers were washed three times with 1 ml of the uptake medium composed of 137 mmol NaCl/1 or 137 mmol choline chloride/1, 5-4 mmol KC1/1, 2-8 mmol CaCl2/l, 1-2 mmol MgS04/l, 14 mmol TrisHC1/1, pH 7-4, and 01 mmol KH2P04/1. The uptake of phosphate was determined at 37 °C in the same medium supplemented with 0-5 pCi (New England Nuclear; 1 Ci/mmol). At the end of incubation, cells were washed with ice-cold 137 mmol NaCl/1, 14 mmol Tris-HCl/1, pH 7-4, then solubilized in 0-5% (v/v) Triton X-100.32P was counted by liquid scintillation. The protein content of one well per dish was measured by the method of Bradford (1976) as modified by Sedmak & Grossberg, (1977) and Redinbaugh & Campbell (1985). The uptake was expressed as nmol P¡/mg protein per 10 min. The

[32P]KH2P04/ml

Cellular content of cyclic AMP (cAMP)

Production of cAMP was determined as previously described (Brooker, Harper, Terasaki & Moylan, 1979; Chabardès, Montegut, Imbert-Teboul & Morel, 1984). The culture medium was removed and replaced with 250 µ DMEM containing 0-1 % (w/v) bovine albumin (BSA), 1 mmol isobutylmethylserum xanthine/1 (Sigma Chemical Co.) and either 5 µ 0025-75 ng human parathyroid hormone (1-34) (PTH) (Sigma Chemical Co.) dissolved in 10 mmol acetic acid/1 containing 01% BSA (final concen¬ tration of PTH 0024-80nmol/1) or solvent alone. After a 10-min incubation at room temperature, the cells were washed and cellular cAMP was extracted with 15% (v/v) formic acid in ethanol. The cAMP was measured by radioimmunoassay (Brooker et al. 1979; Chabardès et al. 1984). Results are expressed as pmol cAMP/mg protein per 10 min. Statistical

analysis Experiments were performed

at least twice on each cell line, and the mean for each cell line was then calculated. Group means were calculated from the

individual

means

as means

+ s.e.m.

for cell lines. Results

Comparisons

analysis of variance. A value as indicating significance.

were