Does the composition of urinary extracellular vesicles reflect the abundance of renal Na+/phosphate transporters?

Studies addressing homeostasis of inorganic phosphate (Pi) are mostly restricted to murine models. Data provided by genetically modified mice suggest that renal Pi reabsorption is primarily mediated by the Na+/Pi cotransporter NaPi-IIa/Slc34a1, whereas the contribution of NaPi-IIc/Slc34a3 in adult animals seems negligible. However, mutations in both cotransporters associate with hypophosphatemic syndromes in humans, suggesting major inter-species heterogeneity. Urinary extracellular vesicles (UEV) have been proposed as an alternative source to analyse the intrinsic expression of renal proteins in vivo. Here, we analyse in rats whether the protein abundance of renal Pi transporters in UEV correlates with their renal content. For that, we compared the abundance of NaPi-IIa and NaPi-IIc in paired samples from kidneys and UEV from rats fed acutely and chronically on diets with low or high Pi. In renal brush border membranes (BBM) NaPi-IIa was detected as two fragments corresponding to the full-length protein and to a proteolytic product, whereas NaPi-IIc migrated as a single full-length band. The expression of NaPi-IIa (both fragments) in BBM adapted to acute as well to chronic changes of dietary Pi, whereas adaptation of NaPi-IIc was only detected in response to chronic administration. Both transporters were detected in UEV as well. UEV reflected the renal adaptation of the NaPi-IIa proteolytic fragment (but not the full-length protein) upon chronic but not acute dietary changes, while also reproducing the chronic regulation of NaPi-IIc. Thus, the composition of UEV reflects only partially changes in the expression of NaPi-IIa and NaPi-IIc at the BBM triggered by dietary Pi.

The human pathogenic 91del7 mutation in SLC34A1 has no effect in mineral homeostasis in mice.

Kidneys are key regulators of phosphate homeostasis. Biallelic mutations of the renal Na+/phosphate cotransporter SLC34A1/NaPi-IIa cause idiopathic infantile hypercalcemia, whereas monoallelic mutations were frequently noted in adults with kidney stones. Genome-wide-association studies identified SLC34A1 as a risk locus for chronic kidney disease. Pathogenic mutations in SLC34A1 are present in 4% of the general population. Here, we characterize a mouse model carrying the 91del7 in-frame deletion, a frequent mutation whose significance remains unclear. Under normal dietary conditions, 12 weeks old heterozygous and homozygous males have similar plasma and urinary levels of phosphate as their wild type (WT) littermates, and comparable concentrations of parathyroid hormone, fibroblast growth factor 23 (FGF-23) and 1,25(OH)2 vitamin D3. Renal phosphate transport, and expression of NaPi-IIa and NaPi-IIc cotransporters, was indistinguishable in the three genotypes. Challenging mice with low dietary phosphate did not result in differences between genotypes with regard to urinary and plasma phosphate. Urinary and plasma phosphate, plasma FGF-23 and expression of cotransporters were similar in all genotypes after weaning. Urinary phosphate and bone mineral density were also comparable in 300 days old WT and mutant mice. In conclusion, mice carrying the 91del7 truncation do not show signs of impaired phosphate homeostasis.

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.

RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein.

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)-mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A-NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A-NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases.

Npt2a as a target for treating hyperphosphatemia.

Hyperphosphatemia results from an imbalance in phosphate (Pi) homeostasis. In patients with and without reduced kidney function, hyperphosphatemia is associated with cardiovascular complications. The current mainstays in the management of hyperphosphatemia are oral Pi binder and dietary Pi restriction. Although these options are employed in patients with chronic kidney disease (CKD), they seem inadequate to correct elevated plasma Pi levels. In addition, a paradoxical increase in expression of intestinal Pi transporter and uptake may occur. Recently, studies in rodents targeting the renal Na+/Pi cotransporter 2a (Npt2a), responsible for ∼70% of Pi reabsorption, have been proposed as a potential treatment option. Two compounds (PF-06869206 and BAY-767) have been developed which are selective for Npt2a. These Npt2a inhibitors significantly increased urinary Pi excretion consequently lowering plasma Pi and PTH levels. Additionally, increases in urinary excretions of Na+, Cl- and Ca2+ have been observed. Some of these results are also seen in models of reduced kidney function. Responses of FGF23, a phosphaturic hormone that has been linked to the development of left ventricular hypertrophy in CKD, are ambiguous. In this review, we discuss the recent advances on the role of Npt2a inhibition on Pi homeostasis as well as other pleiotropic effects observed with Npt2a inhibition.

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.

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport.

The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na+/H+ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein-coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser162, a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser162 with alanine (S162A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser162 with aspartic acid (S162D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser162(pSer162)-PDZ2 variant. KD values determined by fluorescence anisotropy indicate that incorporation of pSer162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer162 and Asp183 of PDZ2 and Arg at position -1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser162 in PDZ2 modulates the interaction with GRK6A.

PF-06869206 is a selective inhibitor of renal Pi transport: evidence from in vitro and in vivo studies.

Plasma phosphate (Pi) levels are tightly controlled, and elevated plasma Pi levels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of Pi reabsorption: Na+-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of Pi reabsorption. The aim of the present study was to determine the in vitro effects of a novel Npt2a inhibitor (PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a-/-) mice. In OK cells, Npt2a inhibitor caused dose-dependent reductions of Na+-dependent Pi uptake (IC50: ~1.4 µmol/L), whereas the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in an ~20% stronger inhibition of Pi uptake. The dose-dependent inhibitory effects were present after 24 h of incubation with both low- and high-Pi media. Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher Km for Pi in response to Npt2a inhibition with no significant change in apparent Vmax. Higher parathyroid hormone concentrations decreased Pi uptake equivalent to the maximal inhibitory effect of Npt2a inhibitor. In vivo, the Npt2a inhibitor induced a dose-dependent increase in urinary Pi excretion in wild-type mice (ED50: ~23 mg/kg), which was completely absent in Npt2a-/- mice, alongside a lack of decrease in plasma Pi. Of note, the Npt2a inhibitor-induced dose-dependent increase in urinary Na+ excretion was still present in Npt2a-/- mice, a response possibly mediated by an off-target acute inhibitory effect of the Npt2a inhibitor on open probability of the epithelial Na+ channel in the cortical collecting duct.

Intratubular epithelial-mesenchymal transition and tubular atrophy after kidney injury in mice.

Tubular atrophy is a common pathological feature of kidney fibrosis. Although fibroblasts play a predominant role in tissue fibrosis, the role of repairing tubular epithelia in tubular atrophy is unclear. We demonstrated the essential role of focal adhesion kinase (FAK)-mediated intratubular epithelial-mesenchymal transition (EMT) in the pathogenesis of tubular atrophy after severe ischemia-reperfusion injury (IRI). Actively proliferating tubular epithelia undergoing intratubular EMT were noted in the acute phase of severe IRI, resulting in tubular atrophy in the chronic phase, reflecting failed tubular repair. Furthermore, FAK was phosphorylated in the tubular epithelia in the acute phase of severe IRI, and its inhibition ameliorated both tubular atrophy and interstitial fibrosis in the chronic phase after injury. In vivo clonal analysis of single-labeled proximal tubular epithelial cells after IRI using proximal tubule reporter mice revealed substantial clonal expansion after IRI, reflecting active epithelial proliferation during repair. The majority of these proliferating epithelia were located in atrophic and nonfunctional tubules, and FAK inhibition was sufficient to prevent tubular atrophy. In vitro, transforming growth factor-ß induced FAK phosphorylation and an EMT phenotype, which was also prevented by FAK inhibition. In an in vitro tubular epithelia gel contraction assay, transforming growth factor-ß treatment accelerated gel contraction, which was suppressed by FAK inhibition. In conclusion, injury-induced intratubular EMT is closely related to tubular atrophy in a FAK-dependent manner.

Selective pharmacological inhibition of the sodium-dependent phosphate cotransporter NPT2a promotes phosphate excretion.

The sodium-phosphate cotransporter NPT2a plays a key role in the reabsorption of filtered phosphate in proximal renal tubules, thereby critically contributing to phosphate homeostasis. Inadequate urinary phosphate excretion can lead to severe hyperphosphatemia as in tumoral calcinosis and chronic kidney disease (CKD). Pharmacological inhibition of NPT2a may therefore represent an attractive approach for treating hyperphosphatemic conditions. The NPT2a-selective small-molecule inhibitor PF-06869206 was previously shown to reduce phosphate uptake in human proximal tubular cells in vitro. Here, we investigated the acute and chronic effects of the inhibitor in rodents and report that administration of PF-06869206 was well tolerated and elicited a dose-dependent increase in fractional phosphate excretion. This phosphaturic effect lowered plasma phosphate levels in WT mice and in rats with CKD due to subtotal nephrectomy. PF-06869206 had no effect on Npt2a-null mice, but promoted phosphate excretion and reduced phosphate levels in normophophatemic mice lacking Npt2c and in hyperphosphatemic mice lacking Fgf23 or Galnt3. In CKD rats, once-daily administration of PF-06869206 for 8 weeks induced an unabated acute phosphaturic and hypophosphatemic effect, but had no statistically significant effect on FGF23 or PTH levels. Selective pharmacological inhibition of NPT2a thus holds promise as a therapeutic option for genetic and acquired hyperphosphatemic disorders.

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.

1,25-Dihydroxyvitamin D Maintains Brush Border Membrane NaPi2a and Attenuates Phosphaturia in Hyp Mice.

Phosphate homeostasis is critical for many cellular processes and is tightly regulated. The sodium-dependent phosphate cotransporter, NaPi2a, is the major regulator of urinary phosphate reabsorption in the renal proximal tubule. Its activity is dependent upon its brush border localization that is regulated by fibroblast growth factor 23 (FGF23) and PTH. High levels of FGF23, as are seen in the Hyp mouse model of human X-linked hypophosphatemia, lead to renal phosphate wasting. Long-term treatment of Hyp mice with 1,25-dihydroxyvitamin D (1,25D) or 1,25D analogues has been shown to improve renal phosphate wasting in the setting of increased FGF23 mRNA expression. Studies were undertaken to define the cellular and molecular basis for this apparent FGF23 resistance. 1,25D increased FGF23 protein levels in the cortical bone and circulation of Hyp mice but did not impair FGF23 cleavage. 1,25D attenuated urinary phosphate wasting as early as one hour postadministration, without suppressing FGF23 receptor/coreceptor expression. Although 1,25D treatment induced expression of early growth response 1, an early FGF23 responsive gene required for its phosphaturic effects, it paradoxically enhanced renal phosphate reabsorption and NaPi2a protein expression in renal brush border membranes (BBMs) within one hour. The Na-H+ exchange regulatory factor 1 (NHERF1) is a scaffolding protein thought to anchor NaPi2a to the BBM. Although 1,25D did not alter NHERF1 protein levels acutely, it enhanced NHERF1-NaPi2a interactions in Hyp mice. 1,25D also prevented the decrease in NHERF1/NaPi2a interactions in PTH-treated wild-type mice. Thus, these investigations identify a novel role for 1,25D in the hormonal regulation of renal phosphate handling.

Daidzein upregulates anti-aging protein Klotho and NaPi 2a cotransporter in a rat model of the andropause.

In a rat model of the andropause we aimed to examine the influence of daidzein, soy isoflavone, on the structure and function of parathyroid glands (PTG) and the expression levels of some of the crucial regulators of Ca2+ and Pi homeostasis in the kidney, and to compare these effects with the effects of estradiol, serving as a positive control. Middle-aged (16-month-old) male Wistar rats were divided into the following groups: sham-operated (SO), orchidectomized (Orx), orchidectomized and estradiol-treated (Orx+E; 0.625mg/kg b.w./day, s.c.) as well as orchidectomized and daidzein-treated (Orx+D; 30mg/kg b.w./day, s.c.) group. Every treated group had a corresponding control group. PTH serum concentration was decreased in Orx+E and Orx+D groups by 10% and 21% (p<0.05) respectively, in comparison with the Orx. PTG volume was decreased in Orx+E group by 16% (p<0.05), when compared to the Orx. In Orx+E group expression of NaPi 2a was lower (p<0.05), while NaPi 2a abundance in Orx+D animals was increased (p<0.05), when compared to Orx. Expression of PTH1R was increased (p<0.05) in Orx+E group, while in Orx+D animals the same parameter was decreased (p<0.05), in comparison with Orx. Klotho expression was elevated (p<0.05) in Orx+D rats, in regard to Orx. Orx+D induced reduction in Ca2+/creatinine and Pi/creatinine ratio in urine by 32% and 16% (p<0.05) respectively, in comparison with Orx. In conclusion, presented results indicate the more coherent beneficial effects of daidzein compared to estradiol, on disturbed Ca2+ and Pi homeostasis, and presumably on bone health, in the aging male rats.

Expression cloning human and rat renal cortex Na/Pi cotransporters: behind the scenes in the Murer laboratory.

In the pre-genomic era, the cloning of a cDNA represented a significant achievement, particularly if the gene of interest encoded a membrane protein. At the time, molecular probes such as partial peptide sequences, suitable nucleic acid sequences, or antibodies were unavailable for most proteins and the "sodium-phosphate transporter" was no exception. In contrast, brush-border membrane vesicles and epithelial cell culture experiments had established a reliable set of functional hallmarks that described Na-dependent phosphate transport activity in some detail. Moreover, aspects of hormonal regulation of phosphate homeostasis could be recapitulated in these model systems. Expression cloning elegantly combined functional protein expression in Xenopus laevis oocytes with molecular biology to overcome the lack of molecular probes.

Renal phosphate handling and inherited disorders of phosphate reabsorption: an update.

Renal phosphate handling critically determines plasma phosphate and whole body phosphate levels. Filtered phosphate is mostly reabsorbed by Na+-dependent phosphate transporters located in the brush border membrane of the proximal tubule: NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Here we review new evidence for the role and relevance of these transporters in inherited disorders of renal phosphate handling. The importance of NaPi-IIa and NaPi-IIc for renal phosphate reabsorption and mineral homeostasis has been highlighted by the identification of mutations in these transporters in a subset of patients with infantile idiopathic hypercalcemia and patients with hereditary hypophosphatemic rickets with hypercalciuria. Both diseases are characterized by disturbed calcium homeostasis secondary to elevated 1,25-(OH)2 vitamin D3 as a consequence of hypophosphatemia. In vitro analysis of mutated NaPi-IIa or NaPi-IIc transporters suggests defective trafficking underlying disease in most cases. Monoallelic pathogenic mutations in both SLC34A1 and SLC34A3 appear to be very frequent in the general population and have been associated with kidney stones. Consistent with these findings, results from genome-wide association studies indicate that variants in SLC34A1 are associated with a higher risk to develop kidney stones and chronic kidney disease, but underlying mechanisms have not been addressed to date.

In vivo evidence for an interplay of FGF23/Klotho/PTH axis on the phosphate handling in renal proximal tubules.

Phosphate homeostasis is primarily maintained in the renal proximal tubules, where the expression of sodium/phosphate cotransporters (Npt2a and Npt2c) is modified by the endocrine actions of both fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). However, the specific contribution of each regulatory pathway in the proximal tubules has not been fully elucidated in vivo. We have previously demonstrated that proximal tubule-specific deletion of the FGF23 coreceptor Klotho results in mild hyperphosphatemia with little to no change in serum levels of FGF23, 1,25(OH)2D3, and PTH. In the present study, we characterized mice in which the PTH receptor PTH1R was specifically deleted from the proximal tubules, either alone or in combination with Klotho ( PT-PTH1R-/- and PT-PTH1R/KL-/-, respectively). PT-PTH1R-/- mice showed significant increases in serum FGF23 and PTH levels, whereas serum phosphate levels were maintained in the normal range, and Npt2a and Npt2c expression in brush border membrane (BBM) did not change compared with control mice. In contrast, PT-PTH1R/KL-/- mice displayed hyperphosphatemia and an increased abundance of Npt2a and Npt2c in the renal BBM, along with increased circulating FGF23 levels. While serum calcium was normal, 1,25(OH)2D3 levels were significantly decreased, leading to extremely high levels of PTH. Collectively, mice with a deletion of PTH1R alone in proximal tubules results in only minor changes in phosphate regulation, whereas deletion of both PTH1R and Klotho leads to a severe disturbance, including hyperphosphatemia with increased sodium/phosphate cotransporter expression in BBM. These results suggest an important interplay between the PTH/PTH1R and FGF23/Klotho pathways to affect renal phosphate handling in the proximal tubules.

The intervention effect of zuogui pill on chronic kidney disease-mineral and bone disorder regulatory factor.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) play a critical role in the pathogenesis of cardiovascular complications in patients with chronic kidney disease (CKD). Zuogui pill as a traditional Chinese herbal drug has been used for nourish kidney essence improve bone malnutrition of renal bone disease by regulating the metabolism of calcium and phosphorus and participating in osteoblast metabolism. In the present study, 5/6 nephrectomy rat model was used to reveal the mechanism of zuogui pill in treatment of CKD-MBD. Compared with sham rats, the levels of serum phosphorus, PTH, iPTH and creatinine were significantly decreased, while the serum calcium level was significantly increased, and the Cbfa1 protein level was significantly decreased and FGF23 protein level was significantly increased by Zuogui pill treatment. Compared with model rats, the BMD of rat was significantly increased by Zuogui pill treatment. Histological analysis revealed that the kidney injury of rats with CKD was significantly reduced by zuogui pill treatment. Compared with model rats, the CYP27B1 mRNA level was significantly increased, and the PTH mRNA level and NaPiIIa protein level were significantly decreased in the kidney by zuogui pill treatment. We inferred that zuogui pill exhibited potential therapeutic effects on CKD-MBD in the rats by regulating bone metabolism and nourish kidney.

Clinical, biochemical, and pathophysiological analysis of SLC34A1 mutations.

Mutations in SLC34A1, encoding the proximal tubular sodium-phosphate transporter NaPi-IIa, may cause a range of clinical phenotypes including infantile hypercalcemia, a proximal renal Fanconi syndrome, which are typically autosomal recessive, and hypophosphatemic nephrolithiasis, which may be an autosomal dominant trait. Here, we report two patients with mixed clinical phenotypes, both with metabolic acidosis, hyperphosphaturia, and renal stones. Patient A had a single heterozygous pathogenic missense mutation (p.I456N) in SLC34A1, consistent with the autosomal dominant pattern of renal stone disease in this family. Patient B, with an autosomal recessive pattern of disease, was compound heterozygous for SLC34A1 variants; a missense variant (p.R512C) together with a relatively common in-frame deletion p.V91A97del7 (91del7). Xenopus oocyte and renal (HKC-8) cell line transfection studies of the variants revealed limited cell surface localization, consistent with trafficking defects. Co-expression of wild-type and I456N and 91del7 appeared to cause intracellular retention in HKC-8, whereas the R512C mutant had a less dominant effect. Expression in Xenopus oocytes failed to demonstrate a significant dominant negative effect for I456N and R512C; however, a negative impact of 91del7 on [32 P]phosphate transport was found. In conclusion, we have investigated pathogenic alleles of SLC34A1 which contribute to both autosomal dominant and autosomal recessive renal stone disease.

The sodium phosphate cotransporter family and nicotinamide phosphoribosyltransferase contribute to the daily oscillation of plasma inorganic phosphate concentration.

Circulating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD+ system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt+/- mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD+ system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels.

Effect of variations in dietary Pi intake on intestinal Pi transporters (NaPi-IIb, PiT-1, and PiT-2) and phosphate-regulating factors (PTH, FGF-23, and MEPE).

Hyperphosphatemia is a common condition in patients with chronic kidney disease (CKD) and can lead to bone disease, vascular calcification, and increased risks of cardiovascular disease and mortality. Inorganic phosphate (Pi) is absorbed in the intestine, an important step in the maintenance of homeostasis. In CKD, it is not clear to what extent Pi absorption is modulated by dietary Pi. Thus, we investigated 5/6 nephrectomized (Nx) Wistar rats to test whether acute variations in dietary Pi concentration over 2 days would alter hormones involved in Pi metabolism, expression of sodium-phosphate cotransporters, apoptosis, and the expression of matrix extracellular phosphoglycoprotein (MEPE) in different segments of the small intestine. The animals were divided into groups receiving different levels of dietary phosphate: low (Nx/LPi), normal (Nx/NPi), and high (Nx/HPi). Serum phosphate, fractional excretion of phosphate, intact serum fibroblast growth factor 23 (FGF-23), and parathyroid hormone (PTH) were significantly higher and ionized calcium was significantly lower in the Nx/HPi group than in the Nx/LPi group. The expression levels of NaPi-IIb and PiT-1/2 were increased in the total jejunum mucosa of the Nx/LPi group compared with the Nx/HPi group. Modification of Pi concentration in the diet affected the apoptosis of enterocytes, particularly with Pi overload. MEPE expression was higher in the Nx/HPi group than in the Nx/NPi. These data reveal the importance of early control of Pi in uremia to prevent an increase in serum PTH and FGF-23. Uremia may be a determining factor that explains the expressional modulation of the cotransporters in the small intestine segments.