AIM:Several Na+ -dependent phosphate cotransporters, namely NaPi-IIb/SLC34A2, Pit-1/SLC20A1 and Pit-2/SLC20A2, are expressed at the apical membrane of enterocytes but their contribution to active absorption of phosphate is unclear. The aim of this study was to compare their pattern of mRNA expression along the small and large intestine and to analyse the effect of intestinal depletion of Pit-2 on phosphate homeostasis.METHODS:Intestinal epithelial Pit-2-deficient mice were generated by crossing floxed Pit-2 with villin-Cre mice. Mice were fed 2 weeks standard or low phosphate diets. Stool, urine, plasma and intestinal and renal tissue were collected. Concentration of electrolytes and hormones, expression of mRNAs and proteins and intestinal transport of tracers were analysed.RESULTS:Intestinal mRNA expression of NaPi-IIb and Pit-1 is segment-specific, whereas the abundance of Pit-2 mRNA is more homogeneous. In ileum, NaPi-IIb mRNA expression is restricted to enterocytes, whereas Pit-2 mRNA is found in epithelial and non-epithelial cells. Overall, their mRNA expression is not regulated by dietary phosphate. The absence of Pit-2 from intestinal epithelial cells does not affect systemic phosphate homeostasis under normal dietary conditions. However, in response to dietary phosphate restriction, Pit-2-deficient mice showed exacerbated hypercalciuria and sustained elevation of 1,25(OH)2 vitamin D3 .CONCLUSIONS:In mice, the intestinal Na+ /phosphate cotransporters are not coexpressed in all segments. NaPi-IIb but not Pit-2 mRNA is restricted to epithelial cells. Intestinal epithelial Pit-2 does not contribute significantly to absorption of phosphate under normal dietary conditions. However, it may play a more significant role upon dietary phosphate restriction.

This article is part of the special issue on Phosphate transport in Pflugers Archiv – European Journal of Physiology

MCT14 is an orphan transporter belonging to the SLC16 transporter family mediating the transport of monocarboxylates, aromatic amino acids, creatine, and thyroid hormones. The expression, tissue localization, regulation, and function of MCT14 are unknown. In mouse MCT14 mRNA abundance is highest in kidney. Using a newly developed and validated antibody, MCT14 was localized to the luminal membrane of the thick ascending limb of the loop of Henle colocalizing in the same cells with uromodulin and NKCC2. MCT14 mRNA and protein was found to be highly regulated by dietary phosphate intake in mice being increased by high dietary phosphate intake at both mRNA and protein level. In order to identify the transport substrate(s), we expressed MCT14 in Xenopus laevis oocytes where MCT14 was integrated into the plasma membrane. However, no transport was discovered for the classic substrates of the SLC16 family nor for phosphate. In summary, MCT14 is an orphan transporter regulated by phosphate and highly enriched in kidney localizing to the luminal membrane of one specific nephron segment.

Background/Aims: Renal reabsorption of inorganic phosphate (Pi) is mediated by SLC34 and SLC20 Na+/Pi-cotransporters the abundance of which is under hormonal control. Extracellular Pi itself also regulates the expression of cotransporters and the concentration of Pi-regulating hormones, though the signaling pathways are largely unknown. Here, we explored the mechanisms that allow renal proximal cells to adapt to changes in the concentration of Pi. Methods: opossum kidney (OK) cells, a model of proximal epithelia, were incubated with different concentrations of Pi in the absence/presence of phosphonoformic acid (PFA), a Pi-analogue and SLC34-inhibitor, and of inhibitors of kinases involved in hormonal control of Pi-homeostasis; cells cultured in normal media were treated with uncouplers of oxidative phosphorylation. Then, the intracellular concentration of ATP and/or the Pi-transport capacity of the cultures were analyzed. Results: luminal Pi regulates the Pi-transport and the intracellular ATP levels. Changes in ATP seem secondary to alterations in Pi-transport, rather than ATP acting as a signal. Adaptation of Pi-transport to high Pi was not mimicked by PFA. Transport adaptation was blocked by PFA but not by kinase inhibitors. Conclusions: in OK cells, adaptation of Pi-transport to luminal Pi does not depend on the same signaling pathways involved in hormonal regulation.

Phosphate (Pi) homeostasis is regulated by renal, intestinal, and endocrine mechanisms through which Pi intake stimulates parathyroid hormone (PTH) and fibroblast growth factor-23 secretion, increasing phosphaturia. Mechanisms underlying the early adaptive phase and the role of the intestine, however, remain ill defined. We investigated mineral, endocrine, and renal responses during the first 4 hours after intravenous and intragastric Pi loading in rats. Intravenous Pi loading (0.5 mmol) caused a transient rise in plasma Pi levels and creatinine clearance and an increase in phosphaturia within 10 minutes. Plasma calcium levels fell and PTH levels increased within 10 minutes and remained low or high, respectively. Fibroblast growth factor-23, 1,25-(OH)2-vitamin D3, and insulin concentrations did not respond, but plasma dopamine levels increased by 4 hours. In comparison, gastric Pi loading elicited similar but delayed phosphaturia and endocrine responses but did not affect plasma mineral levels. Either intravenous or gastric loading led to decreased expression and activity of renal Pi transporters after 4 hours. In parathyroidectomized rats, however, only intravenous Pi loading caused phosphaturia, which was blunted and transient compared with that in intact rats. Intravenous but not gastric Pi loading in parathyroidectomized rats also led to higher creatinine clearance and lower plasma calcium levels but did not reduce the expression or activity of Pi transporters. This evidence suggests that an intravenous or intestinal Pi bolus causes rapid phosphaturia through mechanisms requiring PTH and downregulation of renal Pi transporters but does not support a role of the intestine in stimulating renal clearance of Pi.

Phosphate (Pi) is a vital mineral for every cell in order to maintain energy metabolism, cell signaling and structure. The kidney and intestine are two major organs in maintaining Pi homeostasis by their role in absorbing Pi from the diet and by the reabsorption of filtered Pi. Apical uptake of Pi into intestinal and proximal tubular epithelial cells is well characterized, whereas basolateral efflux mechanisms are poorly understood. The amount of Na‐Pi cotransporters (members of the Slc34 family) expressed at the apical membrane is regulated by changes in dietary Pi intake through mechanisms, which are not fully understood. The aim of this project is to identify transcripts in ileum and renal proximal tubules regulated by dietary Pi. Identified genes may be involved in the transcellular transport of Pi like the unknown basolateral transport pathway or contribute to adaption of apical Pi uptake and metabolism. C57BL/6 male mice (8 weeks old) were fed a high (1.2%) or low (0.1%) Pi diet for 3 days. Epithelial scrapings were prepared from ileum and micro‐dissected renal proximal tubules were collected. Renal and intestinal adaption was confirmed by qPCR for known Pi regulated transcripts. RNA was then analysed using the RNA sequencing service provided by the Functional Genomics Center Zurich. Comparison of the transcriptome of low and high dietary Pi conditions revealed 2029 genes to be differently (at least 2 fold changes) expressed in ileum and 327 in renal proximal tubules. Regulation of selected transcripts was confirmed by qPCR. Pathway analysis is currently being performed and selected candidates for basolateral Pi transporters will be expressed in Xenopus laevis oocytes.Funded by the Swiss National Science Foundation.

Inorganic phosphate ions (H2PO4−/ HPO42−) (abbreviated as Pi) are involved in formation of bone and generation of high-energy bonds (e.g. ATP), metabolic pathways, and regulation of cellular functions. In addition, Pi is a component of biological membranes and nucleic acids. Only about 1% of total body Pi content is present in extracellular fluids, at a plasma concentration in adults within the range 0.8–1.4 mMol/L (at pH 7.4 mostly as HPO42−), with diurnal variations of approximately 0.2 mM. A small amount of plasma Pi is bound to proteins or forms complexes with calcium. Under normal, balanced conditions, absorption of dietary Pi along the small intestine equals the output of Pi via kidney and faeces. Renal excretion of Pi represents the key determinant for the adjustment of normal Pi plasma concentrations. Renal reabsorption of Pi occurs along the proximal tubules by sodium-dependent Pi cotransporters that are strictly localized at the apical brush border membrane. Parathyroid hormone (PTH) and FGF23 are key regulators amongst a myriad of factors controlling excretion of Pi in urine, mostly by changes of the apical abundance of Na/Pi cotransporters. Hypophosphataemia may result in osteomalacia, rickets, muscle weakness, and haemolysis. Hyperphosphataemia can lead to hyperparathyroidism and severe calcifications in different tissues.

Lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), is widely used for the treatment of mood disorders. Side effects of lithium include nephrogenic diabetes insipidus, leading to renal water loss. Dehydration has in turn been shown to downregulate Klotho, which is required as co-receptor for the downregulation of 1,25(OH)2D3 formation by fibroblast growth factor 23 (FGF23). FGF23 decreases and 1,25(OH)2D3 stimulates renal tubular phosphate reabsorption. The present study explored whether lithium influences renal Klotho expression, FGF23 serum levels, 1,25(OH)2D3 formation, and renal phosphate excretion. To this end, mice were analyzed after a 14-day period of sham treatment or of treatment with lithium (200 mg/kg/day subcutaneously). Serum antidiuretic hormone (ADH), FGF23, and 1,25(OH)2D3 concentrations were determined by ELISA or EIA, renal Klotho protein abundance and GSK3 phosphorylation were analyzed by Western blotting, and serum phosphate and calcium concentration by photometry. Lithium treatment significantly increased renal GSK3 phosphorylation, enhanced serum ADH and FGF23 concentrations, downregulated renal Klotho expression, stimulated renal calcium and phosphate excretion, and decreased serum 1,25(OH)2D3 and phosphate concentrations. In conclusion, lithium treatment upregulates FGF23 formation, an effect paralleled by substantial decrease of serum 1,25(OH)2D3, and phosphate concentrations and thus possibly affecting tissue calcification.

In the kidney, both anions, phosphate and sulfate, are almost freely filtered and afterwards reclaimed (reabsorbed) to a large extent from tubular fluid along the proximal tubules. Under normal dietary conditions, fractional excretion of these anions is approximately 10%. Reabsorption of both anions occurs along the proximal tubules by active, saturable, and regulated transepithelial processes. Most of the transporters involved in renal handling of phosphate and sulfate have been identified and their transport functions as well as their cellular localizations have been described in detail. The role of these transporters in the renal handling of phosphate and sulfate has been investigated by the use of several mice knock out models and also by analysis of several inherited human diseases. Numerous hormonal and nonhormonal factors, have been described that alter renal excretion of phosphate or sulfate by mechanisms that alter the abundance of known phosphate/sulfate transporters and consequently renal excretion. These mechanisms contribute to the homeostasis of the extracellular concentrations of phosphate and sulfate.

Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2-3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/ + Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3-metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2 alpha protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action.

Renal reabsorption of inorganic phosphate (Pi) is mediated by the phosphate transporters NaPi-IIa, NaPi-IIc, and Pit-2 in the proximal tubule brush border membrane (BBM). Dietary Pi intake regulates these transporters; however, the contribution of the specific isoforms to the rapid and slow phase is not fully clarified. Moreover, the regulation of PTH and FGF23, two major phosphaturic hormones, during the adaptive phase has not been correlated. C57/BL6 and NaPi-IIa−/− mice received 5 days either 1.2 % (HPD) or 0.1 % (LPD) Pi-containing diets. Thereafter, some mice were acutely switched to LPD or HPD. Plasma Pi concentrations were similar under chronic diets, but lower when mice were acutely switched to LPD. Urinary Pi excretion was similar in C57/BL6 and NaPi-IIa−/− mice under HPD. During chronic LPD, NaPi-IIa−/− mice lost phosphate in urine compensated by higher intestinal Pi absorption. During the acute HPD-to-LPD switch, NaPi-IIa−/− mice exhibited a delayed decrease in urinary Pi excretion. PTH was acutely regulated by low dietary Pi intake. FGF23 did not respond to low Pi intake within 8 h whereas the phospho-adaptator protein FRS2α necessary for FGF-receptor cell signaling was downregulated. BBM Pi transport activity and NaPi-IIa but not NaPi-IIc and Pit-2 abundance acutely adapted to diets in C57/BL6 mice. In NaPi-IIa−/−, Pi transport activity was low and did not adapt. Thus, NaPi-IIa mediates the fast adaptation to Pi intake and is upregulated during the adaptation to low Pi despite persistently high FGF23 levels. The sensitivity to FGF23 may be regulated by adapting FRS2α abundance and phosphorylation.