The calcium-sensing receptor has only a parathyroid hormone-dependent role in the acute response of renal phosphate transporters to phosphate intake.

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects.

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.

The Ip6k1 and Ip6k2 Kinases Are Critical for Normal Renal Tubular Function.

SIGNIFICANCE STATEMENT: Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. BACKGROUND: Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. METHODS: We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. RESULTS: In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. CONCLUSIONS: Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.

Expression of phosphate and calcium transporters and their regulators in parotid glands of mice.

The concentration of inorganic phosphate (Pi) in plasma is under hormonal control, with deviations from normal values promptly corrected to avoid hyper- or hypophosphatemia. Major regulators include parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23), and active vitamin D3 (calcitriol). This control is achieved by mechanisms largely dependent on regulating intestinal absorption and renal excretion, whose combined actions stabilise plasma Pi levels at around 1-2 mM. Instead, Pi concentrations up to 13 and 40 mM have been measured in saliva from humans and ruminants, respectively, suggesting that salivary glands have the capacity to concentrate Pi. Here we analysed the transcriptome of parotid glands, ileum, and kidneys of mice, to investigate their potential differences regarding the expression of genes responsible for epithelial transport of Pi as well as their known regulators. Given that Pi and Ca2+ homeostasis are tightly connected, the expression of genes involved in Ca2+ homeostasis was also included. In addition, we studied the effect of vitamin D3 treatment on the expression of Pi and Ca2+ regulating genes in the three major salivary glands. We found that parotid glands are equipped preferentially with Slc20 rather than with Slc34 Na+/Pi cotransporters, are suited to transport Ca2+ through the transcellular and paracellular route and are potential targets for PTH and vitamin D3 regulation.

Acute adaptation of renal phosphate transporters in the murine kidney to oral phosphate intake requires multiple signals.

AIMS: Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi-IIa and NaPi-IIc Pi transporters. Here, we aimed to clarify the role of several Pi-regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6-kinases) in the acute regulation of NaPi-IIa and NaPi-IIc. METHODS: Wildtype (WT) and PTH-deficient mice (PTH-KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12 h. RESULTS: Pi-gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1 h Within 1 h, Pi-gavage decreased NaPi-IIa abundance in WT and PTH-KO mice. NaPi-IIc was downregulated 1 h post-administration in WT and after 4 h in PTH-KO. PTH increased after 1 h in WT animals. After 4 h Pi-gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi-gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi-IIa downregulation in PTH-KO mice 4 h after Pi-gavage. Inhibition of IP6-kinases had no effect. CONCLUSIONS: (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi-sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption.

Fibroblast growth factor 23 leads to endolysosomal routing of the renal phosphate cotransporters NaPi-IIa and NaPi-IIc in vivo.

Na+-dependent phosphate cotransporters NaPi-IIa and NaPi-IIc, located at the brush-border membrane of renal proximal tubules, are regulated by numerous factors, including fibroblast growth factor 23 (FGF23). FGF23 downregulates NaPi-IIa and NaPi-IIc abundance after activating a signaling pathway involving phosphorylation of ERK1/2 (phospho-ERK1/2). FGF23 also downregulates expression of renal 1-α-hydroxylase (Cyp27b1) and upregulates 24-hydroxylase (Cyp24a1), thus reducing plasma calcitriol levels. Here, we examined the time course of FGF23-induced internalization of NaPi-IIa and NaPi-IIc and their intracellular pathway toward degradation in vivo. Mice were injected intraperitoneally with recombinant human (rh)FGF23 in the absence (biochemical analysis) or presence (immunohistochemistry) of leupeptin, an inhibitor of lysosomal proteases. Phosphorylation of ERK1/2 was enhanced 60 min after rhFGF23 administration, and increased phosphorylation was still detected 480 min after injection. Colocalization of phospho-ERK1/2 with NaPi-IIa was seen at 60 and 120 min and partly at 480 min. The abundance of both cotransporters was reduced 240 min after rhFGF23 administration, with a further reduction at 480 min. NaPi-IIa and NaPi-IIc were found to colocalize with clathrin and early endosomal antigen 1 as early as 120 min after rhFGF23 injection. Both cotransporters partially colocalized with cathepsin B and lysosomal-associated membrane protein-1, markers of lysosomes, 120 min after rhFGF23 injection. Thus, NaPi-IIa and NaPi-IIc are internalized within 2 h upon rhFGF23 injection. Both cotransporters share the pathway of clathrin-mediated endocytosis that leads first to early endosomes, finally resulting in trafficking toward the lysosome as early as 120 min after rhFGF23 administration.NEW & NOTEWORTHY The hormone fibroblast growth factor 23 (FGF23) controls phosphate homeostasis by regulating renal phosphate excretion. FGF23 acts on several phosphate transporters in the kidney. Here, we define the time course of this action and demonstrate how phosphate transporters NaPi-IIa and NaPi-IIc are internalized.

Nradd Acts as a Negative Feedback Regulator of Wnt/ß-Catenin Signaling and Promotes Apoptosis.

Wnt/ß-catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative regulator of Wnt/ß-catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/ß-catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/ß-catenin signaling during development.

Expression of NaPi-IIb in rodent and human kidney and upregulation in a model of chronic kidney disease.

Na+-coupled phosphate cotransporters from the SLC34 and SLC20 families of solute carriers mediate transepithelial transport of inorganic phosphate (Pi). NaPi-IIa/Slc34a1, NaPi-IIc/Slc34a3, and Pit-2/Slc20a2 are all expressed at the apical membrane of renal proximal tubules and therefore contribute to renal Pi reabsorption. Unlike NaPi-IIa and NaPi-IIc, which are rather kidney-specific, NaPi-IIb/Slc34a2 is expressed in several epithelial tissues, including the intestine, lung, testis, and mammary glands. Recently, the expression of NaPi-IIb was also reported in kidneys from rats fed on high Pi. Here, we systematically quantified the mRNA expression of SLC34 and SLC20 cotransporters in kidneys from mice, rats, and humans. In all three species, NaPi-IIa mRNA was by far the most abundant renal transcript. Low and comparable mRNA levels of the other four transporters, including NaPi-IIb, were detected in kidneys from rodents and humans. In mice, the renal expression of NaPi-IIa transcripts was restricted to the cortex, whereas NaPi-IIb mRNA was observed in medullary segments. Consistently, NaPi-IIb protein colocalized with uromodulin at the luminal membrane of thick ascending limbs of the loop of Henle segments. The abundance of NaPi-IIb transcripts in kidneys from mice was neither affected by dietary Pi, the absence of renal NaPi-IIc, nor the depletion of intestinal NaPi-IIb. In contrast, it was highly upregulated in a model of oxalate-induced kidney disease where all other SLC34 phosphate transporters were downregulated. Thus, NaPi-IIb may contribute to renal phosphate reabsorption, and its upregulation in kidney disease might promote hyperphosphatemia.