Renal Na(+)-phosphate cotransport in murine X-linked hypophosphatemic rickets. Molecular characterization.

Abstract

The X-linked Hyp mouse is characterized by a specific defect in proximal tubular phosphate (Pi) reabsorption that is associated with a decrease in V .. of the high affinity Na+-Pi cotransport system in the renal brush border membrane. To understand the mechanism for V ,. reduction, we examined the effect of the Hyp mutation on renal expression of Na+-Pi cotransporter mRNA and protein. Northern hybridization of renal RNA with a rat, renal-specific Na+-Pi cotransporter cDNA probe (NaPi-2) (Magagnin et al. 1993. Proc. Nati. Acad. Sci. USA. 90:5979-5983.) demonstrated a reduction in a 2.6-kb transcript in kidneys of Hyp mice relative to normal littermates (NaPi-2/,B-actin mRNA = 57±6% of normal in Hyp mice, n = 6, P < 0.01). Na+-Pi cotransport, but not Na+-sulfate cotransport, was 50% lower in Xenopus oocytes injected with renal mRNA extracted from Hyp mice when compared with that from normal mice. Hybrid depletion experiments documented that the mRNA-dependent expression of Na+-Pi cotransport in oocytes was related to NaPi-2. Western analysis demonstrated that NaPi-2 protein is also significantly reduced in brush border membranes of Hyp mice when compared to normals. The present data demonstrate that the specific reduction in renal Na+-Pi cotransport in brush border membranes of Hyp mice can be ascribed to a proportionate decrease in the abundance of Na+-Pi cotransporter mRNA and protein. (J. Clin. Invest. 1994. 93:671-676.)

Effects of Npt2 gene ablation and low-phosphate diet on renal Na+/phosphate cotransport and cotransporter gene expression

“…One kidney from each of 3-5 mice was used for each BBM vesicle preparation, and the remaining kidney was used for the ribonuclease protection analysis described later here. The uptakes of Pi (100 µM) and glucose (10 µM), each performed in quadruplicate, were measured at 6 seconds (initial rate) and 90 minutes (steady state) in medium containing either 100 mM NaCl or 100 mM KCl by the rapid filtration technique (33). BBM proteins (10-40 µg) were fractionated on 10% SDS-PAGE gels according to the method of Laemmli (34), transferred to supported nitrocellulose membranes (Hybond-C Extra; Amersham Pharmacia Biotech, Baie d'Urfé, Quebec, Canada), and probed either sequentially or simultaneously with a rabbit polyclonal antibody raised against a COOH-terminal peptide of rat Npt2 (gift of H. Murer, University of Zurich, Zurich, Switzerland) and an mAb raised against the α subunit of rat renal endopeptidase-24.18 (meprin) (kindly provided by P. Crine, University of Montreal, Montreal, Quebec, Canada) as described previously (28).…”

Genomic Organization of the Human PEX Gene Mutated in X-Linked Dominant Hypophosphatemic Rickets

“…Because the substrate of PEX is not yet known, it is not possible to say how loss of function of PEX causes these bone and teeth defects and a renal phosphate leak. It has been shown previously that decreased phosphate reabsoption in the kidneys of Hyp mice is attributable to decreased levels of the renal type II sodium phosphate cotransporter (Npt2; Tenenhouse et al 1994). The genomic structure of human NPT2 has been described recently (Hartmann et al 1996), which will aid studies aimed at questioning the effect of PEX and its substrate in the regulation of transcription of NPT2.…”

Oncogenic osteomalacia: is there a new phosphate regulating hormone?

“…These transporters, which include NaPi2 (rat) and NaPi4 (OK cell), are largely responsible for proximal tubular phosphate reabsorption and its regulation by PTH and by phosphate deprivation . Reduced levels of NaPi2 mRNA and protein have been demonstrated in the proximal tubules of Hyp mice compared with controls (Collins & Ghishan, 1994;Tenenhouse et al, 1994). These findings were confirmed in Gy mice by one group although another group could not demonstrate reduced NaPi2 mRNA levels (Collins & Ghishan, 1996).…”