Phosphate transport in immortalized cell cultures from the renal proximal tubule of normal and Hyp Mice: Evidence That the HYP gene locus product is an extrarenal factor

Abstract

Whether renal phosphate wasting in X-linked hypophosphatemia (XLH) results from an intrinsic renal or humoral defect remains controversial. In studies of the murine homolog of XLH, harboring the Simian Virus 40 (SV40) large T antigen, we obviated the influence of renal cell heterogeneity and impressed memory by comparing Na(+)-phosphate cotransport in immortalized cells from the S1 segment of the proximal tubule. Cells from SV40 transgenic normal and Hyp mice exhibit characteristics of differentiated proximal tubule cells including gluconeogenesis and alkaline phosphatase activity. Surprisingly, however, we found two distinct populations of cells from the S1 proximal tubule of both normal and Hyp mice. In one, PTH treatment increases cAMP accumulation, while in the other both PTH and thyrocalcitonin enhance cAMP production. Kinetic parameters for Na(+)-phosphate cotransport were similar in both subpopulations of cells from normal (Km, 0.29 +/- 0.03 vs. 0.39 +/- 0.04 mM; Vmax, 4.6 +/- 0.6 vs. 5.2 +/- 0.4 nmol/mg/5 minutes) and Hyp mice (0.33 +/- 0.02 vs. 0.26 +/- 0.04; 6.0 +/- 0.7, 4.8 +/- 0.6). More importantly, phosphate transport in S1 cells of either subpopulation from Hyp mice is no different than that of normals. These data indicate that renal proximal tubule cells from Hyp mice have intrinsically normal phosphate transport and support the hypothesis that a humoral abnormality underlies renal phosphate wasting in XLH.

Regulation of Bone−Renal Mineral and Energy Metabolism: The PHEX, FGF23, DMP1, MEPE ASARM Pathway

“…These findings are consistent with several independent studies showing that HYP mice are hypoinsulinemic and hyperglycemic with increased gluconeogenesis in bone, liver, and kidney. 151,152,169–176 The reverse is the case for FGF23-null mice (hypoglycemic). 177 Klotho (an FGF23 coactivator), and or vitamin D likely have direct or indirect roles.…”

“…Similar findings were reported independently by researchers using immortalized HYP renal proximal cell lines. 151,152 Thus, ASARM peptides may also inhibit phosphate transport by directly binding to the phosphate transporter as demonstrated with PFA and etidronate 138,139,143–146,149,153,154 and in turn exacerbating the effects of FGF23 on NPT2 transcription. 34 …”

Sclerostin alters serum vitamin D metabolite and fibroblast growth factor 23 concentrations and the urinary excretion of calcium

“…PT cells were prepared from SV40 T-antigen mutant tsA58 ImmortoMouse (Charles River) kidneys as previously described (46,47). Kidney cortex from 12-wk-old female ImmortoMouse kidneys was harvested, minced, and digested with 1% Worthington collagenase type II.…”

Clinical Disorders of Phosphate Homeostasis

“…Current studies, however, have provided compelling evidence that the defect in renal Pi transport in XLH is secondary to the effects of a circulating hormone or metabolic factor. In this regard, immortalized cell cultures from the renal tubules of Hyp and Gy mice exhibit normal Na þ -phosphate transport, suggesting that the paradoxical effects observed in primary cultures may represent the effects of impressed memory and not an intrinsic abnormality [126,127]. Moreover, the report that cross-transplantation of kidneys in normal and Hyp mice results in neither transfer of the mutant phenotype or its correction unequivocally established the humoral basis for XLH [62].…”

“…Subsequently, several investigators identified and characterized the biological activities of a variety of phosphaturic factors (inhibitors of Na þ -dependent phosphate transport) and mineralization inhibitory factors, which may play a role in the pathogenesis of XLH (see above). Moreover, several reports have documented production of phosphaturic and mineralization inhibitory factors by Hyp mouse osteoblasts maintained in culture [61,89,93,126,129]. Therefore, as noted above, these studies argue that …”