Direct demonstration of a humorally-mediated inhibition of renal phosphate transport in the Hyp mouse

Abstract

The murine Hyp model reproduces the characteristics of human X-linked hypophosphatemia (XLH), an inherited disease causing renal loss of phosphate (Pi), severe rickets and osteomalacia. A current hypothesis considers that a humoral factor may be responsible for the renal Pi loss, although in vitro experiments with renal cell models have failed to demonstrate the presence of such a factor in XLH or in the Hyp mouse model. To test this hypothesis directly, we prepared primary mouse proximal tubule cell cultures (MPTC), expressing normal features of proximal 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 either arginine vasopressin (AVP) or to salmon calcitonin (sCT). They also 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 32P-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-dependent between 5 to 20% (final concentration) in culture media for the last 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 least 12 hours. Indeed, no significant inhibition of Pi uptake could be detected 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 inhibition of phosphate uptake by Hyp mouse serum was specific, since neither sodium-dependent glucose nor alpha-aminobutyric acid uptake was modified under these conditions. MPTC cells showed a very nice adaptation to Pi concentration in the media; low Pi (0.4 mM final concentration in the presence of 10% serum) stimulated Pi uptake, whereas high Pi concentration (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 than normal serum. In contrast, adaptation of MPTC in high Pi media resulted in no inhibition of phosphate uptake either in the presence of normal or Hyp mouse serum. We next questioned whether conditioned media from confluent Hyp mouse primary osteoblast-like cell cultures could affect Pi uptake by MPTC. These osteoblast-like cells expressed high alkaline phosphatase and produced the bone specific protein, osteocalcin. When MPTC were treated for 48 hours with Hyp mouse bone cell media conditioned for the last 48 hours of cultures, Pi uptake was specifically 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 cell-conditioned media is specific for these cells since it was not observed with CHO cell-conditioned media, nor with either mouse fibroblast (NCTC), normal mouse Kupffer cell- or Hyp mouse Kupffer cell-conditioned media. This effect also persisted through a number of passages of Hyp 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 uptake by primary renal cell cultures in vitro. This effect is dose- and time-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 molecular nature of this factor and its mode of action remains to be determined.