Klotho/fibroblast growth factor 23- and PTH-independent estrogen receptor-α-mediated direct downregulation of NaPi-IIa by estrogen in the mouse kidney.

Estrogen treatment causes renal phosphate (Pi) wasting and hypophosphatemia in rats and humans; however, the signaling mechanisms mediating this effect are still poorly understood. To determine the specific roles of estrogen receptor isoforms (ERα and ERß) and the Klotho pathway in mediating these effects, we studied the effects of estrogen on renal Pi handling in female mice with null mutations of ERα or ERß or Klotho and their wild type (WT) using balance studies in metabolic cages. Estrogen treatment of WT and ERß knockout (KO) mice caused a significant reduction in food intake along with increased renal phosphate wasting. The latter resulted from a significant downregulation of NaPi-IIa and NaPi-IIc protein abundance. The mRNA expression levels of both transporters were unchanged in estrogen-treated mice. These effects on both food intake and renal Pi handling were absent in ERα KO mice. Estrogen treatment of Klotho KO mice or parathyroid hormone (PTH)-depleted thyroparathyroidectomized mice exhibited a significant downregulation of NaPi-IIa with no change in the abundance of NaPi-IIc. Estrogen treatment of a cell line (U20S) stably coexpressing both ERα and ERß caused a significant downregulation of NaPi-IIa protein when transiently transfected with a plasmid containing full-length or open-reading frame (ORF) 3'-untranslated region (UTR) but not 5'-UTR ORF of mouse NaPi-IIa transcript. In conclusion, estrogen causes phosphaturia and hypophosphatemia in mice. These effects result from downregulation of NaPi-IIa and NaPi-IIc proteins in the proximal tubule through the activation of ERα. The downregulation of NaPi-IIa by estrogen involves 3'-UTR of its mRNA and is independent of Klotho/fibroblast growth factor 23 and PTH signaling pathways.

Characterization of FGF23-Dependent Egr-1 Cistrome in the Mouse Renal Proximal Tubule.

Fibroblast growth factor 23 (FGF23) is a potent regulator of phosphate (Pi) and vitamin D homeostasis. The transcription factor, early growth response 1 (egr-1), is a biomarker for FGF23-induced activation of the ERK1/2 signaling pathway. We have shown that ERK1/2 signaling blockade suppresses renal egr-1 gene expression and prevents FGF23-induced hypophosphatemia and 1,25-dihydroxyvitamin D (1,25(OH)2D) suppression in mice. To test whether egr-1 itself mediates these renal actions of FGF23, we administered FGF23 to egr-1-/- and wild-type (WT) mice. In WT mice, FGF23 induced hypophosphatemia and suppressed expression of the renal Na/Pi cotransporters, Npt2a and Npt2c. In FGF23-treated egr-1-/- mice, hypophosphatemic response was greatly blunted and Na/Pi cotransporter expression was not suppressed. In contrast, FGF23 induced equivalent suppression of serum 1,25(OH)2D concentrations by suppressing renal cyp27b1 and stimulating cyp24a1 mRNA expression in both groups of mice. Thus, downstream of receptor binding and ERK1/2 signaling, we can distinguish the effector pathway that mediates FGF23-dependent inhibition of Pi transport from the pathway that mediates inhibition of 1,25(OH)2D synthesis in the kidney. Furthermore, we demonstrate that the hypophosphatemic effect of FGF23 is significantly blunted in Hyp/egr-1-/- mice; specifically, serum Pi concentrations and renal Npt2a and Npt2c mRNA expression are significantly higher in Hyp/egr-1-/- mice than in Hyp mice. We then characterized the egr-1 cistrome in the kidney using ChIP-sequencing and demonstrate recruitment of egr-1 to regulatory DNA elements in proximity to several genes involved in Pi transport. Thus, our data demonstrate that the effect of FGF23 on Pi homeostasis is mediated, at least in part, by activation of egr-1.

Regulation of renal phosphate transport by FGF23 is mediated by FGFR1 and FGFR4.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that acts on the proximal tubule to decrease phosphate reabsorption and serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2 Vitamin D3]. Abnormal FGF23 metabolism has been implicated in several debilitating hypophosphatemic and hyperphosphatemic disorders. The renal receptors responsible for the phosphaturic actions of FGF23 have not been elucidated. There are four fibroblast growth factor receptors (FGFR); 1-4 with "b" and "c" isoforms for receptors 1, 2, and 3. FGFR1, 3, and 4 are expressed in the mouse proximal tubule, and deletion of any one receptor did not affect serum phosphate levels, suggesting that more than one receptor is involved in mediating the phosphaturic actions of FGF23. To determine the receptors responsible for the phosphaturic actions of FGF23, we studied Fgfr1 (kidney conditional) and Fgfr4 (global) double mutant mice (Fgfr1⁻/⁻/Fgfr4⁻/⁻). Fgfr1⁻/⁻/Fgfr4⁻/⁻ mice have higher FGF23 levels than their wild-type counterparts (108.1 ± 7.3 vs. 4,953.6 ± 675.0 pg/ml; P < 0.001). Despite the elevated FGF23 levels, Fgfr1⁻/⁻/Fgfr4⁻/⁻ mice have elevated serum phosphorus levels, increased brush-border membrane vesicle (BBMV) phosphate transport, and increased Na-P(i) cotransporter 2c (NaPi-2c) protein expression compared with wild-type mice. These data are consistent with FGFR1 and FGFR4 being the critical receptors for the phosphaturic actions of FGF23.

Down-regulation of the Na+-coupled phosphate transporter NaPi-IIa by AMP-activated protein kinase.

BACKGROUND/AIMS: The Na(+)-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na(+) gradient across the apical cell membrane, which is maintained by Na(+) extrusion across the basolateral cell membrane through the Na(+)/K(+) ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na(+) accumulation and K(+) loss with eventual decrease of cell membrane potential, Cl(-) entry and cell swelling. Upon energy depletion, early inhibition of Na(+)-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK), a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. METHODS: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPK(γ1)-HA), of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(ß1)-Flag+AMPK(γ1)-HA) or of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPKγ1(R70Q)). NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. RESULTS: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM) to the extracellular bath solution generated a current (Ip), which was significantly decreased by coexpression of wild-type AMPK and of AMPK(γR70Q) but not of AMPK(αK45R). The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM). Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. CONCLUSION: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport. © 2013 S. Karger AG, Basel.

Genistein affects parathyroid gland and NaPi 2a cotransporter in an animal model of the andropause.

This study aimed to examine the effects of genistein on the structural and functional changes in parathyroid glands (PTG) and sodium phosphate cotransporter 2a (NaPi 2a) in orchidectomized rats. Sixteen-month-old Wistar rats were divided into sham-operated (SO), orchidectomized (Orx) and genistein-treated orchidectomized (Orx+G) groups. Genistein (30 mg/kg/day) was administered subcutaneously for 3 weeks, while the controls received vehicle alone. PTG was analyzed histomorphometrically, while the expressions of NaPi 2a mRNA/protein levels from kidneys were determined by real time PCR and Western blots. Serum and urine parameters were determined biochemically. The PTG volume in Orx rats was increased by 30% (p<0.05), compared to the SO group. Orx+G treatment increased the PTG volume by 35% and 75% (p<0.05) respectively, comparing to Orx and SO animals. Orchidectomy led to increment of serum PTH by 27% (p<0.05) compared to the SO group, Orx+G decreased it by 18% (p<0.05) comparing to Orx animals. NaPi 2a expression in Orx animals was reduced in regards to its abundance in SO animals, although it was increased in Orx+G group compared to the Orx. Phosphorus urine content of Orx animals was raised by 12% (p<0.05) compared to that for the SO group, while Orx+G induced a 17% reduction (p<0.05) in regards to Orx animals. Our study shows that Orx increases PTG volume and serum PTH level, while protein expression of NaPi 2a is reduced. Application of genistein attenuates the orchidectomy-induced changes in serum PTH level, stimulates the expression of NaPi 2a and reduces urinary Pi excretion, implying potential beneficial effects on andropausal symptoms.

Effects of phosphonoformic acid and renagel on renal type IIa sodium-dependent phosphate cotransporter mRNA expression in hyperphosphatemia rats.

OBJECTIVE: To investigate the effects of phosphonoformic acid (PFA) and sevelamer hydrochloride (Renagel) on renal type IIa sodium-dependent phosphate cotransporter (NaPi-2) mRNA expression in hyperphosphatemia rats. METHODS: Thirty rats were randomly divided into five groups based on the diet for 2 weeks after 5/6 nephrectomy (Nx): Nx + high-phosphate (HP; 1.2% P) diet; Nx + low-phosphate (LP; 0.2% P) diet; HP + PFA (injected with 0.15 g/kg PFA daily); HP + Saline (injected with the same amount of saline daily); and HP + Renagel (2%) group. Another 12 rats were sham operated and divided into Sham + HP and Sham + LP groups. Serum ionized calcium, phosphorus (P), and intact parathyroid hormone (iPTH) were measured on days 2, 7, and 14. Serum 1,25(OH)2D3 was measured on day 14 and NaPi-2 mRNA levels were assayed by RT-PCR. RESULTS: PFA decreased iPTH level but had no effect on NaPi-2 mRNA expression. Renagel decreased serum P and iPTH levels, but upregulated renal NaPi-2 mRNA expression. CONCLUSIONS: Both PFA and Renagel are effective drugs to decrease iPTH level and they might be potential candidates for treatment of clinical secondary hyperparathyroidism. Renagel can also decrease serum P and upregulate renal NaPi-2 mRNA expression.

Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function.

Sirt1, a NAD-dependent protein deacetylase, is reported to regulate intracellular metabolism and attenuate reactive oxidative species (ROS)-induced apoptosis leading to longevity and acute stress resistance. We created transgenic (TG) mice with kidney-specific overexpression of Sirt1 using the promoter sodium-phosphate cotransporter IIa (Npt2) driven specifically in proximal tubules and investigated the kidney-specific role of Sirt1 in the protection against acute kidney injury (AKI). We also elucidated the role of number or function of peroxisome and mitochondria in mediating the mechanisms for renal protective effects of Sirt1 in AKI. Cisplatin-induced AKI decreased the number and function of peroxisomes as well as mitochondria and led to increased local levels of ROS production and renal tubular apoptotic cells. TG mice treated with cisplatin mitigated AKI, local ROS, and renal tubular apoptotic tubular cells. Consistent with these results, TG mice treated with cisplatin also exhibited recovery of peroxisome number and function, as well as rescued mitochondrial function; however, mitochondrial number was not recovered. Immunoelectron microscopic findings consistently demonstrated that the decrease in peroxisome number by cisplatin in wild type mice was restored in transgenic mice. In HK-2 cells, a cultured proximal tubule cell line, overexpression of Sirt1 rescued the cisplatin-induced cell apoptosis through the restoration of peroxisome number, although the mitochondria number was not restored. These results indicate that Sirt1 overexpression in proximal tubules rescues cisplatin-induced AKI by maintaining peroxisomes number and function, concomitant up-regulation of catalase, and elimination of renal ROS levels. Renal Sirt1 can be a potential therapeutic target for the treatment of AKI.

Type 2a sodium-phosphate co-transporter serves as a histological predictor of renal dysfunction and tubular apical damage in the kidneys of septic mice.

Acute renal failure (ARF) occurs in septic patients and is histologically characterized by tubular apical damages, including brush border breakdown. Nevertheless, little information is available to identify the apical injury at a molecular level. Type 2a Na-phosphate (Pi) co-transporter (NaPiT2a) is constitutively expressed by brush borders of proximal tubules under a healthy condition. Therefore, we investigated if NaPiT2a could be used as a negative marker to predict the renal dysfunction, using an animal model of septic ARF. After the treatment of lipopolysaccharide (LPS), mice manifested the tubular apical injury and renal dysfunction, as evidenced by the increase in blood urea nitrogen (BUN) levels. Immunohistochemical examination revealed that the expression of NaPiT2a by renal proximal tubules became faint, being reciprocal to the development of tubular hypoxia during sepsis. Inversely, the loss in apical NaPiT2a was restored in a regenerating stage, associated with the recovery from renal hypoxia. Overall, there was a negative correlation between the NaPiT2a expression and BUN levels or tubular injury scores in septic mice. Our data indicate that the loss of NaPiT2a is a reliable marker for predicting the progression of septic ARF, while local hypoxia might be involved in the decrease of NaPiT2a expression.

Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion.

Renal phosphate excretion is subjected to circadian rhythmicity. The bulk of filtered inorganic phosphate (P(i)) is reabsorbed by the sodium-dependent phosphate cotransporter NaPi-IIa. The regulation of proximal tubular phosphate reabsorptive capacity is largely attributed to the altered abundance of NaPi-IIa residing in the brush border membrane (BBM) of proximal tubular cells. Therefore, we examined if the diurnal rise in renal phosphate excretion is accompanied by a corresponding change in NaPi-IIa expression. Renal phosphate excretion, creatinine clearance, and serum phosphate were determined at consecutive time points in rats, starting from 8 a.m. until 5 p.m. During this period, renal phosphate excretion (fractional P(i) excretion) increased more than eightfold until 5 p.m. compared to the morning values at 8 a.m. In addition, serum phosphate and creatinine clearance as well as the calculated tubular phosphate threshold increased. Neither immunoblot analysis of BBMs nor immunohistochemical staining for NaPi-IIa yielded evidence for a lower abundance of NaPi-IIa in kidneys collected in the afternoon compared to those in the morning. However, kidneys sampled in the afternoon showed a small decrease (14%) in (32)P uptakes into BBM vesicles (BBMVs). Thus, the diurnal rise in renal phosphate excretion was associated with a mild reduction in the sodium-dependent phosphate transport rate in proximal tubular BBMs. There was no apparent downregulation of NaPi-IIa abundance and only a small reduction in Na(+)-dependent Pi-transport activity. Thus, the diurnal changes in urinary phosphate excretion appear to be mainly related to changes in serum phosphate and tubular threshold but not to NaPi-IIa expression.

Topology of the type IIa Na+/P(i) cotransporter.

The type IIa Na(+)/P(i) cotransporter (NaPi-IIa) plays a key role in the reabsorption of inorganic phosphate (P(i)) in the renal proximal tubule. The rat NaPi-IIa isoform is a protein of 637 residues for which different algorithms predict 8-12 transmembrane domains (TMDs). Epitope tagging experiments demonstrated that both the N and the C termini of NaPi-IIa are located intracellularly. Site-directed mutagenesis revealed two N-glycosylation sites in a large putative extracellular loop. Results from structure-function studies suggested the assembly of two similar opposed regions that possibly constitute part of the substrate translocation pathway for one phosphate ion together with three sodium ions. Apart from these topological aspects, other structural features of NaPi-IIa are not known. In this study, we have addressed the topology of NaPi-IIa using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NaPi-IIa TMDs. Based on the results of in vitro transcription/translation analyses, we propose a model of NaPi-IIa comprising 12 TMDs, with both N and C termini orientated intracellularly and a large hydrophilic extracellular loop between the fifth and sixth TMDs. The proposed model is in good agreement with the prediction of the NaPi-IIa structure obtained by the hidden Markov algorithm HMMTOP.