Compensatory regulation of the sodium/phosphate cotransporters NaPi-IIc (SCL34A3) and Pit-2 (SLC20A2) during Pi deprivation and acidosis.
ABSTRACT The role of four Pi transporters in the renal handling of Pi was analyzed using functional and molecular methods. The abundance of NaPi-IIa, NaPi-IIc, and Pit-2 was increased by 100% in kidney from rats on a 0.1% Pi diet, compared to a 0.6% Pi diet. Pit-1 was not modified. Type II-mediated Pi uptake in Xenopus oocytes increased as the pH of the uptake medium increased, and the opposite occurred with Pit-1 and Pit-2. At pH 6.0, Pi uptake mediated through type II was approximately 10% of the uptake at pH 7.5, but the uptake through Pit-2 was 250% of the activity at pH 7.5. Real brush-border membrane vesicles (BBMV) responded to pH changes following the same pattern as type II transporters. Adaptation to a 0.1% Pi diet was accompanied by a 65% increase in the V (max) of BBMV Pi transport at pH 7.5, compared to a 0.6% Pi diet. The increase was only 11% at pH 6.0. Metabolic acidosis increased the expression of NaPi-IIc and Pit-2 in animals adapted to a low Pi diet, and phosphaturia was only observed in control diet animals. The combination of the pH effect, Pi adaptation, and metabolic acidosis suggests very modest involvement of Pit-2 in renal Pi handling. Real-time PCR and mathematical analyses of transport findings suggest that NaPi-IIa RNA accounts for 95% of all Pi transporters and that type II handles 97% of Pi transport at pH 7.5 and 60% of Pi transport at pH 6.0, depending on the pH and the physiological conditions.
Journal of endocrinological investigation 09/2014; 37(11). DOI:10.1007/s40618-014-0158-6 · 1.65 Impact Factor
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ABSTRACT: Inorganic phosphate (Pi) transport in epithelia has both Na-dependent and Na-independent components, but so far only Na-dependent transporters have been characterized in detail and molecularly identified. Consequently, in this work we have initiated the characterization and analysis of intestinal Na-independent Pi transport using the in vitro model, Caco2BBE cells. Only Na-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when they were incubated in a high Pi DMEM (4mM) from one to several days. No response to a low Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells at 1mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4=, HCO3=, and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1mM Pi. Phosphate transport from cells maintained at 4mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that the multifunctional anion exchangers of the Slc26 family, as well as members of Slc17, Slc20, and Slc37, and the Pi exporter XPR1, are not involved.AJP Cell Physiology 10/2014; 307(12). DOI:10.1152/ajpcell.00251.2014 · 3.67 Impact Factor
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ABSTRACT: Klotho, a cofactor in suppressing 1,25(OH)2D3 formation, is a powerful regulator of mineral metabolism. Klotho-hypomorphic mice (kl/kl) exhibit excessive plasma 1,25(OH)2D3, Ca(2+), and phosphate concentrations, severe tissue calcification, volume depletion with hyperaldosteronism, and early death. Calcification is paralleled by overexpression of osteoinductive transcription factor Runx2/Cbfa1, Alpl, and senescence-associated molecules Tgfb1, Pai-1, p21, and Glb1. Here, we show that NH4Cl treatment in drinking water (0.28 M) prevented soft tissue and vascular calcification and increased the life span of kl/kl mice >12-fold in males and >4-fold in females without significantly affecting extracellular pH or plasma concentrations of 1,25(OH)2D3, Ca(2+), and phosphate. NH4Cl treatment significantly decreased plasma aldosterone and antidiuretic hormone concentrations and reversed the increase of Runx2/Cbfa1, Alpl, Tgfb1, Pai-1, p21, and Glb1 expression in aorta of kl/kl mice. Similarly, in primary human aortic smooth muscle cells (HAoSMCs), NH4Cl treatment reduced phosphate-induced mRNA expression of RUNX2/CBFA1, ALPL, and senescence-associated molecules. In both kl/kl mice and phosphate-treated HAoSMCs, levels of osmosensitive transcription factor NFAT5 and NFAT5-downstream mediator SOX9 were higher than in controls and decreased after NH4Cl treatment. Overexpression of NFAT5 in HAoSMCs mimicked the effect of phosphate and abrogated the effect of NH4Cl on SOX9, RUNX2/CBFA1, and ALPL mRNA expression. TGFB1 treatment of HAoSMCs upregulated NFAT5 expression and prevented the decrease of phosphate-induced NFAT5 expression after NH4Cl treatment. In conclusion, NH4Cl treatment prevents tissue calcification, reduces vascular senescence, and extends survival of klotho-hypomorphic mice. The effects of NH4Cl on vascular osteoinduction involve decrease of TGFB1 and inhibition of NFAT5-dependent osteochondrogenic signaling. Copyright © 2015 by the American Society of Nephrology.Journal of the American Society of Nephrology 02/2015; DOI:10.1681/ASN.2014030230 · 9.47 Impact Factor