D. Lajeunesse et al., DIRECT DEMONSTRATION OF A HUMORALLY-MEDIATED INHIBITION OF RENAL PHOSPHATE-TRANSPORT IN THE HYP MOUSE, Kidney international, 50(5), 1996, pp. 1531-1538
The murine Hyp model reproduces the characteristics of human X-linked
hypophosphatemia (XLH), an inherited disease causing renal loss of pho
sphate (Pi), severe rickets and osteomalacia. A current hypothesis con
siders that a humoral factor may be responsible for the renal Pi loss,
although in vitro experiments with renal cell models have failed to d
emonstrate the presence of such a factor in XLH or in the Hyp mouse mo
del. To test this hypothesis directly, we prepared primary mouse proxi
mal tubule cell cultures (MPTC), expressing normal features of proxima
l tubule cells. These cells possess high alkaline phosphatase activity
, and respond to human parathyroid hormone fragment 1-34 (PTH) with a
four- to sixfold increase in cAMP production but do not respond to eit
her arginine vasopressin (AVP) or to salmon calcitonin (sCT). They als
o show sodium-dependent phosphate, glucose and amino acid uptake. The
presence of 10% Hyp mouse serum in HAMF12/DMEM media (1 mM Pi) for the
last 48 hours of culture of MPTC reduced Pi uptake (0.1 mM P-32-Pi in
, the presence of 140 mM NaCl) by 45.7 +/- 3.9% (P < 0.01) as compared
to normal mouse serum. This effect of Hyp mouse serum was dose-depend
ent between 5 to 20% (final concentration) in culture media for the la
st 48 hours of culture (P < 0.01 by analysis of variance). This effect
of Hyp mouse serum was also time-dependent, with a lag time of at lea
st 12 hours. Indeed, no significant inhibition of Pi uptake could be d
etected with incubations less than 12 hours in the presence of 10% Hyp
mouse serum, whereas a maximal effect was obtained after 24 hours of
incubation and remained unchanged after 36 and 48 hours. The inhibitio
n of phosphate uptake by Hyp mouse serum was specific, since neither s
odium-dependent glucose nor alpha-aminobutyric acid uptake was modifie
d under these conditions. MPTC cells showed a very nice adaptation to
Pi concentration in the media; low Pi (0.4 mM final concentration in t
he presence of 10% serum) stimulated Pi uptake, whereas high Pi concen
tration (3 mM) reduced Pi uptake by these cells as compared to regular
HAMF12/DMEM media containing 1 mM Pi. Normal and Hyp mouse serum both
inhibited Pi uptake by MPTC following adaptation in low or normal Pi
media, however, Hyp mouse serum always showed a stronger inhibition th
an normal serum. In contrast, adaptation of MPTC in high Pi media resu
lted in no inhibition of phosphate uptake either in the presence of no
rmal or Hyp mouse serum. We next questioned whether conditioned media
from confluent Hyp mouse primary osteoblast-like cell cultures could a
ffect Pi uptake by MPTC. These osteoblast-like cells expressed high al
kaline phosphatase and produced the bone specific protein, osteocalcin
. When MPTC were treated for 48 hours with Hyp mouse bone cell media c
onditioned for the last 45 hours of cultures, Pi uptake was specifical
ly inhibited by 30.5 +/- 4.1% (P < 0.025) as compared to normal mouse
bone cell-conditioned media. This effect of primary Hyp mouse bone cel
l-conditioned media is specific for these cells since it was not obser
ved with CHO cell-conditioned media, nor with either mouse fibroblast
(NCTC), normal mouse Kupffer cell- or Hyp mouse Kupffer cell-condition
ed media. This effect also persisted through a number of passages of H
yp mouse bone cells, since conditioned-media from cells at their third
passage still resulted in a 32 +/- 9.4% inhibition (P ( 0.02). These
results are the first to show an effect of Hyp mouse serum on Pi uptak
e by primary renal cell cultures in vitro. This effect is dose- and ti
me-dependent, requiring 24 hours for maximum response, and is blocked
in Pi rich media. These results also suggest that a specific intrinsic
cellular defect, present in Hyp mouse osteoblasts, is responsible for
the release of and/or the modification of a factor that can reach the
circulation and which inhibits renal phosphate reabsorption. The mole
cular nature of this factor and its mode of action remains to be deter
mined.